Article

Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells

August 1996
Biophysical Journal 71(2):787-94

August 1996
71(2):787-94

Source
PubMed

Authors:

We studied monovalent permeability of Ca2+ release-activated Ca2+ channels (ICRAC) in Jurkat T lymphocytes following depletion of calcium stores. When external free Ca2+ ([Ca2+]o) was reduced to micromolar levels in the absence of Mg2+, the inward current transiently decreased and then increased approximately sixfold, accompanied by visibly enhanced current noise. The monovalent currents showed a characteristically slow deactivation (tau = 3.8 and 21.6 s). The extent of Na+ current deactivation correlated with the instantaneous Ca2+ current upon readdition of [Ca2+]o. No conductance increase was seen when [Ca2+]o was reduced before activation of ICRAC. With Na+ outside and Cs+ inside, the current rectified inwardly without apparent reversal below 40 mV. The sequence of conductance determined from the inward current at -80 mV was Na+ > Li+ = K+ > Rb+ > Cs+. Unitary inward conductance of the Na+ current was 2.6 pS, estimated from the ratios delta sigma2/delta Imean at different voltages. External Ca2+ blocked the Na+ current reversibly with an IC50 value of 4 microM. Na+ currents were also blocked by 3 mM Mg2+ or 10 microM La3+. We conclude that ICRAC channels become permeable to monovalent cations at low levels of external divalent ions. In contrast to voltage-activated Ca2+ channels, the monovalent conductance is highly selective for Na+ over Cs+. Na+ currents through ICRAC channels provide a means to study channel characteristics in an amplified current model.

Molecular Choreography and Structure of Ca2+ Release-Activated Ca2+ (CRAC) and KCa2+ Channels and Their Relevance in Disease with Special Focus on Cancer

Article

Full-text available

Dec 2020

Ca2+ ions play a variety of roles in the human body as well as within a single cell. Cellular Ca2+ signal transduction processes are governed by Ca2+ sensing and Ca2+ transporting proteins. In this review, we discuss the Ca2+ and the Ca2+-sensing ion channels with particular focus on the structure-function relationship of the Ca2+ release-activated Ca2+ (CRAC) ion channel, the Ca2+-activated K+ (KCa2+) ion channels, and their modulation via other cellular components. Moreover, we highlight their roles in healthy signaling processes as well as in disease with a special focus on cancer. As KCa2+ channels are activated via elevations of intracellular Ca2+ levels, we summarize the current knowledge on the action mechanisms of the interplay of CRAC and KCa2+ ion channels and their role in cancer cell development.

Store-Operated Calcium Channels

Article

Full-text available

Sep 2015

Store-operated cofor calcium signaling in virtually all metozoan cells and serve a wide variety of functions ranging from gene expression, motility, and secretion to tissue and organ development and the immune response. SOCs are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER), triggered physiologically through stimulation of a diverse set of surface receptors. Over 15 years after the first characterization of SOCs through electrophysiology, the identification of the STIM proteins as ER Ca2+ sensors and the Orai proteins as store-operated channels has enabled rapid progress in understanding the unique mechanism of store-operate calcium entry (SOCE). Depletion of Ca2+ from the ER causes STIM to accumulate at ER-plasma membrane (PM) junctions where it traps and activates Orai channels diffusing in the closely apposed PM. Mutagenesis studies combined with recent structural insights about STIM and Orai proteins are now beginning to reveal the molecular underpinnings of these choreographic events. This review describes the major experimental advances underlying our current understanding of how ER Ca2+ depletion is coupled to the activation of SOCs. Particular emphasis is placed on the molecular mechanisms of STIM and Orai activation, Orai channel properties, modulation of STIM and Orai function, pharmacological inhibitors of SOCE, and the functions of STIM and Orai in physiology and disease.

Store-Operated Orai Channels: Structure and Function

Article

Full-text available

Jul 2013
CURR TOP MEMBR

Murali Prakriya

In many animal cells, store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels function as an essential route for Ca(2+) entry. CRAC channels control many fundamental cellular functions including gene expression, motility, and cell proliferation, are involved in the etiology of several disease processes including a severe combined immunodeficiency syndrome, and have emerged as major targets for drug development. Although little was known of the molecular mechanisms of CRAC channel operation for several decades, the discovery of Orai1 as a prototypic CRAC channel protein and STIM1 as the endoplasmic reticulum (ER) Ca(2+) sensor has led to rapid progress in our understanding of the mechanisms and functions of CRAC channels. It is now known that activation of CRAC channels following ER Ca(2+) store depletion is governed by several events, which include the redistributions and accumulations of STIM1 and Orai1 into overlapping puncta at peripheral cellular sites, resulting in direct protein-protein interactions between the two proteins. In this chapter, I review the molecular features of the STIM and Orai proteins that regulate the gating and ion conduction mechanisms of CRAC channels.

Monovalent Permeability, Rectification, and Ionic Block of Store-operated Calcium Channels in Jurkat T Lymphocytes

Article

Full-text available

Apr 1998
J GEN PHYSIOL

We used whole-cell recording to characterize ion permeation, rectification, and block of monovalent current through calcium release-activated calcium (CRAC) channels in Jurkat T lymphocytes. Under physiological conditions, CRAC channels exhibit a high degree of selectivity for Ca2+, but can be induced to carry a slowly declining Na+ current when external divalent ions are reduced to micromolar levels. Using a series of organic cations as probes of varying size, we measured reversal potentials and calculated permeability ratios relative to Na+, PX/PNa, in order to estimate the diameter of the conducting pore. Ammonium (NH4+) exhibited the highest relative permeability (PNH4/PNa = 1.37). The largest permeant ion, tetramethylammonium with a diameter of 0.55 nm, had PTMA/PNa of 0.09. N-methyl-D-glucamine (0.50 x 0.64 x 1.20 nm) was not measurably permeant. In addition to carrying monovalent current, NH4+ reduced the slow decline of monovalent current ("inactivation") upon lowering [Ca2+]o. This kinetic effect of extracellular NH4+ can be accounted for by an increase in intracellular pH (pHi), since raising intracellular pH above 8 reduced the extent of inactivation. In addition, decreasing pHi reduced monovalent and divalent current amplitudes through CRAC channels with a pKa of 6.8. In several channel types, Mg2+ has been shown to produce rectification by a voltage-dependent block mechanism. Mg2+ removal from the pipette solution permitted large outward monovalent currents to flow through CRAC channels while also increasing the channel's relative Cs+ conductance and eliminating the inactivation of monovalent current. Boltzmann fits indicate that intracellular Mg2+ contributes to inward rectification by blocking in a voltage-dependent manner, with a z delta product of 1.88. Ca2+ block from the outside was also found to be voltage dependent with z delta of 1.62. These experiments indicate that the CRAC channel, like voltage-gated Ca2+ channels, achieves selectivity for Ca2+ by selective binding in a large pore with current-voltage characteristics shaped by internal Mg2+.

Store-dependent and -independent Modes Regulating Ca 2+ Release-activated Ca 2+ Channel Activity of Human Orai1 and Orai3

Article

Jun 2008

Critical parameters maintaining authentic CRAC channel hallmarks

Article

Full-text available

Mar 2019

Ca²⁺ ions represent versatile second messengers that regulate a huge diversity of processes throughout the cell’s life. One prominent Ca²⁺ entry pathway into the cell is the Ca²⁺ release-activated Ca²⁺ (CRAC) ion channel. It is fully reconstituted by the two molecular key players: the stromal interaction molecule (STIM1) and Orai. STIM1 is a Ca²⁺ sensor located in the membrane of the endoplasmic reticulum, and Orai, a highly Ca²⁺ selective ion channel embedded in the plasma membrane. Ca²⁺ store-depletion leads initially to the activation of STIM1 which subsequently activates Orai channels via direct binding. Authentic CRAC channel hallmarks and biophysical characteristics include high Ca²⁺ selectivity with a reversal potential in the range of + 50 mV, small unitary conductance, fast Ca²⁺-dependent inactivation and enhancements in currents upon the switch from a Na⁺-containing divalent-free to a Ca²⁺-containing solution. This review provides an overview on the critical determinants and structures within the STIM1 and Orai proteins that establish these prominent CRAC channel characteristics.

Functional Analysis of Orai1 Concatemers Supports a Hexameric Stoichiometry for the CRAC Channel

Article

Nov 2016

Store-operated Ca²⁺ entry occurs through the binding of the endoplasmic reticulum (ER) Ca²⁺ sensor STIM1 to Orai1, the pore-forming subunit of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel. Although the essential steps leading to channel opening have been described, fundamental questions remain, including the functional stoichiometry of the CRAC channel. The crystal structure of Drosophila Orai indicates a hexameric stoichiometry, while studies of linked Orai1 concatemers and single-molecule photobleaching suggest that channels assemble as tetramers. We assessed CRAC channel stoichiometry by expressing hexameric concatemers of human Orai1 and comparing in detail their ionic currents to those of native CRAC channels and channels generated from monomeric Orai1 constructs. Cell surface biotinylation results indicated that Orai1 channels in the plasma membrane were assembled from intact hexameric polypeptides and not from truncated protein products. In addition, the L273D mutation depressed channel activity equally regardless of which Orai1 subunit in the concatemer carried the mutation. Thus, functional channels were generated from intact Orai1 hexamers in which all subunits contributed equally. These hexameric Orai1 channels displayed the biophysical fingerprint of native CRAC channels, including the distinguishing characteristics of gating (store-dependent activation, Ca²⁺-dependent inactivation, open probability), permeation (ion selectivity, affinity for Ca²⁺ block, La³⁺ sensitivity, unitary current magnitude), and pharmacology (enhancement and inhibition by 2-aminoethoxydiphenyl borate). Because permeation characteristics depend strongly on pore geometry, it is unlikely that hexameric and tetrameric pores would display identical Ca²⁺ affinity, ion selectivity, and unitary current magnitude. Thus, based on the highly similar pore properties of the hexameric Orai1 concatemer and native CRAC channels, we conclude that the CRAC channel functions as a hexamer of Orai1 subunits.

Crystal Structure of the Calcium Release-Activated Calcium Channel Orai

Article

Nov 2012
SCIENCE

Architecture of a CRAC The calcium release–activated calcium (CRAC) channel generates intracellular calcium signals in response to depletion of calcium from the endoplasmic reticulum. Hou et al. (p. 1308 , published online 22 November) report a high-resolution crystal structure of Orai, the CRAC channel pore from Drosophila melanogaster. Six Orai subunits surround a central pore that extends into the cytosol. The pore is in a closed conformation that is stabilized by anions binding to a basic region near the intracellular side. A ring of glutamates on the extracellular side form a selectivity filter. The channel architecture allows calcium permeation while regulating the flow to prevent overloading the cell with calcium.

Mechanisms of ion selectivity and throughput in the mitochondrial calcium uniporter

Article

Full-text available

Dec 2022

The mitochondrial calcium uniporter, which regulates aerobic metabolism by catalyzing mitochondrial Ca ²⁺ influx, is arguably the most selective ion channel known. The mechanisms for this exquisite Ca ²⁺ selectivity have not been defined. Here, using a reconstituted system, we study the electrical properties of the channel’s minimal Ca ²⁺ -conducting complex, MCU-EMRE, from Tribolium castaneum to probe ion selectivity mechanisms. The wild-type Tc MCU-EMRE complex recapitulates hallmark electrophysiological properties of endogenous Uniporter channels. Through interrogation of pore-lining mutants, we find that a ring of glutamate residues, the “E-locus,” serves as the channel’s selectivity filter. Unexpectedly, a nearby “D-locus” at the mouth of the pore has diminutive influence on selectivity. Anomalous mole fraction effects indicate that multiple Ca ²⁺ ions are accommodated within the E-locus. By facilitating ion-ion interactions, the E-locus engenders both exquisite Ca ²⁺ selectivity and high ion throughput. Direct comparison with structural information yields the basis for selective Ca ²⁺ conduction by the channel.

The Orai Pore Opening Mechanism

Article

Full-text available

Jan 2021
INT J MOL SCI

Cell survival and normal cell function require a highly coordinated and precise regulation of basal cytosolic Ca2+ concentrations. The primary source of Ca2+ entry into the cell is mediated by the Ca2+ release-activated Ca2+ (CRAC) channel. Its action is stimulated in response to internal Ca2+ store depletion. The fundamental constituents of CRAC channels are the Ca2+ sensor, stromal interaction molecule 1 (STIM1) anchored in the endoplasmic reticulum, and a highly Ca2+-selective pore-forming subunit Orai1 in the plasma membrane. The precise nature of the Orai1 pore opening is currently a topic of intensive research. This review describes how Orai1 gating checkpoints in the middle and cytosolic extended transmembrane regions act together in a concerted manner to ensure an opening-permissive Orai1 channel conformation. In this context, we highlight the effects of the currently known multitude of Orai1 mutations, which led to the identification of a series of gating checkpoints and the determination of their role in diverse steps of the Orai1 activation cascade. The synergistic action of these gating checkpoints maintains an intact pore geometry, settles STIM1 coupling, and governs pore opening. We describe the current knowledge on Orai1 channel gating mechanisms and summarize still open questions of the STIM1–Orai1 machinery.

Cryo-EM structure of the calcium release-activated calcium channel Orai in an open conformation

Article

Full-text available

Nov 2020
eLife

The calcium release-activated calcium channel Orai regulates Ca2+ entry into non-excitable cells and is required for proper immune function. While the channel typically opens following Ca2+ release from the endoplasmic reticulum, certain pathologic mutations render the channel constitutively open. Previously, using one such mutation (H206A), we obtained low (6.7 Å) resolution X-ray structural information on Drosophila melanogaster Orai in an open conformation (Hou, Burstein, & Long, 2018). Here, we present a structure of this open conformation at 3.3 Å resolution using fiducial-assisted cryo-EM. The improved structure reveals the conformations of amino acids in the open pore, which dilates by outward movements of subunits. A ring of phenylalanine residues repositions to expose previously shielded glycine residues to the pore without significant rotational movement of the associated helices. Together with other hydrophobic amino acids, the phenylalanines act as the channel's gate. Structured M1-M2 turrets, not evident previously, form the channel's extracellular entrance.

Cryo-EM structure of the calcium release-activated calcium channel Orai in an open conformation

Preprint

Sep 2020

The calcium release-activated calcium channel Orai regulates Ca ²⁺ entry into non-excitable cells and is required for proper immune function. The channel typically opens following the release of Ca ²⁺ from the endoplasmic reticulum. Certain pathologic mutations render the channel constitutively open. Here, using one such mutation (H206A), we present a cryo-EM structure of Orai from Drosophila melanogaster in an open conformation at 3.3 Å resolution. Comparison with previous closed structures reveals that opening occurs through the outward movements of M1 helices that dilate the central pore. Repositionings of a ring of phenylalanine residues (F171) expose previously shielded glycine residues (G170) to the channel pore, despite the absence of significant rotational movement of the associated pore-lining helices. This phenylalanine ring and two rings of flanking hydrophobic amino acids act as a hydrophobic gate to control ion permeation. Extracellular M1-M2 turrets, not evident from previous Orai structures, form an electronegative pore entrance. Single sentence summary A structure of the Ca ²⁺ channel Orai in an open conformation provides insight into the opening mechanism of the channel and its role in regulating selective Ca ²⁺ entry into immune and other non-excitable cells.

Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta

Article

Full-text available

Jun 2020
J GEN PHYSIOL

Upon Ca2+ store depletion, Orai1 channels cluster and open at endoplasmic reticulum-plasma membrane (ER-PM) junctions in signaling complexes called puncta. Little is known about whether and how Orai1 channel activity may vary between individual puncta. Previously, we developed and validated optical recording of Orai channel activity, using genetically encoded Ca2+ indicators fused to Orai1 or Orai3 N or C termini. We have now combined total internal reflection fluorescence microscopy with whole-cell recording to map functional properties of channels at individual puncta. After Ca2+ store depletion in HEK cells cotransfected with mCherry-STIM1 and Orai1-GCaMP6f, Orai1-GCaMP6f fluorescence increased progressively with increasingly negative test potentials and robust responses could be recorded from individual puncta. Cell-wide fluorescence half-rise and -fall times during steps to -100 mV test potential indicated probe response times of <50 ms. The in situ Orai1-GCaMP6f affinity for Ca2+ was 620 nM, assessed by monitoring fluorescence using buffered Ca2+ solutions in "unroofed" cells. Channel activity and temporal activation profile were tracked in individual puncta using image maps and automated puncta identification and recording. Simultaneous measurement of mCherry-STIM1 fluorescence uncovered an unexpected gradient in STIM1/Orai1 ratio that extends across the cell surface. Orai1-GCaMP6f channel activity was found to vary across the cell, with inactive channels occurring in the corners of cells where the STIM1/Orai1 ratio was lowest; low-activity channels typically at edges displayed a slow activation phase lasting hundreds of milliseconds. Puncta with high STIM1/Orai1 ratios exhibited a range of channel activity that appeared unrelated to the stoichiometric requirements for gating. These findings demonstrate functional heterogeneity of Orai1 channel activity between individual puncta and establish a new experimental platform that facilitates systematic comparisons between puncta composition and activity.

Structures reveal opening of the store-operated calcium channel Orai

Article

Full-text available

Aug 2018
eLife

The store-operated calcium (Ca2+) channel Orai governs Ca2+ influx through the plasma membrane of many non-excitable cells in metazoans. The channel opens in response to the depletion of Ca2+ stored in the endoplasmic reticulum (ER). Loss- and gain-of-function mutants of Orai cause disease. Our previous work revealed the structure of Orai with a closed pore. Here, using a gain-of-function mutation that constitutively activates the channel, we present an X-ray structure of Drosophila melanogaster Orai in an open conformation. Well-defined electron density maps reveal that the pore is dramatically dilated on its cytosolic side in comparison to the slender closed pore. Cations and anions bind in different regions of the open pore, informing mechanisms for ion permeation and Ca2+ selectivity. Opening of the pore requires the release of cytosolic latches. Together with additional X-ray structures of an unlatched-but-closed conformation, we propose a sequence for store-operated activation.

Calcium signaling in brown fat cells

Thesis

Full-text available

Jan 2005

Eric Vaughan Leaver

Rat brown fat cells respond to the neurotransmitter norepinephrine in vivo through a number of signaling pathways including changes in intracellular calcium. This dissertation describes a previously unknown effect of β-adrenergic stimulation on release of calcium from endoplasmic reticulum (ER) stores. Namely, that β-adrenergic stimulation potentiates such release initiated by other receptor types such as α-adrenoceptors or P2 purinergic receptors. This finding suggests that norepinephrine elicits brown fat calcium signals in vivo through activation of both α1- and β-adrenoceptors. Simultaneous to ER calcium release, norepinephrine activates a calcium influx mechanism that contributes to intracellular calcium increases. Some have proposed that this influx is secondary to depletion of ER calcium stores – the “capacitative” calcium entry model. Pursuing this model, I have identified a store-operated calcium current (ISOC) in brown adipocytes activated by the fungal toxin cyclopiazonic acid. This current shares characteristics with the store-operated current ICRAC that has been described in other cells. The shared properties include inward rectification, voltage-dependent inactivation, blockage by lanthanides, and an apparent cation permeability sequence of Ca2+ > Na+ ≥ Cs+ based on membrane current magnitudes. Experiments characterizing ISOC also revealed two other cation currents that have not been previously described in brown fat. One current is regulated by intracellular MgATP and has characteristics nearly identical to a cation current known variously as IMIC or MagNuM and believed to be mediated by the TRPM7 gene product. The other current is a background cation conductance that is inhibited by increases in extracellular calcium, and enhanced by removal of extracellular divalents. Two other accompanying studies are previously published reports associated with my work as a trainee on the NSF Training Grant “Nonlinear Dynamics in Biology” through the UC Davis Institute of Theoretical Dynamics. The first is an experimental project describing a chloride current in endothelial cells activated by mechanical shear stress (flow). The second project is a methodological review of a particular class of spatially explicit mathematical models known as “non-local” models. This usefulness of this theoretical approach is illustrated with problems from both ecology and cell biology.

Functional Coupling of Human L-Type Ca 2+ Channels and Angiotensin AT 1A Receptors Coexpressed in Xenopus laevis Oocytes: Involvement of the Carboxyl-Terminal Ca 2+ Sensors

Article

Dec 1998

Biophysikalische Eigenschaften und Aktivierungsmechanismen des TRPC4/CCE1-Ionenkanals

Thesis

Jan 2007

Jan Warnat

The STIM-orai pathway: Orai, the pore-forming subunit of the CRAC channel

Chapter

Full-text available

Sep 2017
ADV EXP MED BIOL

This chapter focuses on the Orai proteins, Orai1–Orai3, with special emphasis on Orai1, in humans and other mammals, and on the definitive evidence that Orai is the pore subunit of the CRAC channel. It begins by reviewing briefly the defining characteristics of the CRAC channel, then discusses the studies that implicated Orai as part of the store-operated Ca²⁺ entry pathway and as the CRAC channel pore subunit, and finally examines ongoing work that is providing insights into CRAC channel structure and gating.

Physiological Characterization of a Plant Mitochondrial Calcium Uniporter in Vitro and in Vivo

Article

Full-text available

Feb 2017

Over the recent years, several proteins that make up the mitochondrial calcium uniporter complex (MCUC) mediating Ca2+ uptake into the mitochondrial matrix have been identified in mammals, including the channel-forming protein MCU. Although six MCU gene homologs are conserved in the model plant Arabidopsis in which mitochondria can accumulate Ca2+, a functional characterization of plant MCU homologs has been lacking. Using electrophysiology we show that one isoform, AtMCU1, gives rise to a Ca2+-permeable channel activity that can be observed even in the absence of accessory proteins implicated in the formation of the active mammalian channel. Furthermore, we provide direct evidence that AtMCU1 activity is sensitive to the mitochondrial calcium uniporter inhibitors Ruthenium Red (RR) and Gd3+, as well as to the Arabidopsis protein MICU, a regulatory MCUC component. AtMCU1 is prevalently expressed in roots, localizes to mitochondria and its absence causes mild changes in Ca2+ dynamics as assessed by in vivo measurements in Arabidopsis root tips. Plants either lacking or overexpressing AtMCU1 display root mitochondria with altered ultrastructure and show shorter primary roots under restrictive growth conditions. In summary, our work adds evolutionary depth to the investigation of mitochondrial Ca2+ transport, indicates that AtMCU1, together with MICU as a regulator, represents a functional configuration of the plant mitochondrial Ca2+ uptake complex with differences to the mammalian MCUC and identifies new player of the intracellular Ca2+ regulation network in plants.

Store-Operated Calcium Influx and Stimulation of Steroidogenesis in Rat Leydig Cells: Role of Ca 2+ -Activated K + Channels

Article

Sep 2001

Structural Determinants of Ion Permeation in Crac Channels

Article

Full-text available

Jan 2010

The role of Orai/STIM calcium channels in melanocytes and melanoma

Article

Feb 2016
J PHYSIOL-LONDON

Calcium signalling within normal and cancer cells regulates many important cellular functions such as migration, proliferation, differentiation and cytokine secretion. Store operated Ca(2+) entry (SOCE) via the Ca(2+) release activated Ca(2+) (CRAC) channels, which are composed of the plasma membrane based Orai channels and the endoplasmic reticulum stromal interaction molecules (STIMs), is a major Ca(2+) - entry route in many cell types. Orai and STIM have been implicated in the growth and metastasis of multiple cancers; however, while their involvement in cancer is presently indisputable, how Orai/STIM-controlled Ca(2+) signals affect malignant transformation, tumor growth, and invasion is not fully understood. Here, we review recent studies linking Orai/STIM Ca(2+) channels with cancer, with a particular focus on melanoma. We highlight and examine key molecular players and the signalling pathways regulated by Orai and STIM in normal and malignant cells, we expose discrepancies, and we reflect on the potential of Orai/STIMs as anticancer drug targets. Finally, we discuss the functional implications of future discoveries in the field of Ca(2+) signalling. This article is protected by copyright. All rights reserved.

A calcium-accumulating region, CAR, in the channel Orai1 enhances Ca2+ permeation and SOCE-induced gene transcription

Article

Dec 2015

The Ca2+ release–activated Ca2+ channel mediates Ca2+ influx in a plethora of cell types, thereby controlling diverse cellular functions. The channel complex is composed of stromal interaction molecule 1 (STIM1), an endoplasmic reticulum Ca2+-sensing protein, and Orai1, a plasma membrane Ca2+ channel. Channels composed of STIM1 and Orai1 mediate Ca2+ influx even at low extracellular Ca2+ concentrations. We investigated whether the activity of Orai1 adapted to different environmental Ca2+ concentrations. We used homology modeling and molecular dynamics simulations to predict the presence of an extracellular Ca2+-accumulating region (CAR) at the pore entrance of Orai1. Furthermore, simulations of Orai1 proteins with mutations in CAR, along with live-cell experiments, or simulations and electrophysiological recordings of the channel with transient, electrostatic loop3 interacting with loop1 (the site of CAR) determined that CAR enhanced Ca2+ permeation most efficiently at low external Ca2+ concentrations. Consistent with these results, cells expressing Orai1 CAR mutants exhibited impaired gene expression stimulated by the Ca2+-activated transcription factor nuclear factor of activated T cells (NFAT). We propose that the Orai1 channel architecture with a close proximity of CAR to the selectivity filter, which enables Ca2+-selective ion permeation, enhances the local extracellular Ca2+ concentration to maintain Ca2+-dependent gene regulation even in environments with relatively low Ca2+concentrations.

Biophysikalische Eigenschaften und Aktivierungsmechanismen des TRPC4/CCE1-Ionenkanals

Article

Oct 2007

Jan Warnat

Store-depletion activated CaT1 currents in RBL mast cells are inhibited by 2-APB Evidence for a regulatory component that controls activation of both CaT1 and CRAC channels

Article

Full-text available

The Ca2+ release-activated Ca2+ current (I(CRAC)) mediates store-operated Ca2+ entry in rat microglia

Article

Full-text available

Jan 2003

Ca2+ signaling plays a central role in microglial activation, and several studies have demonstrated a store-operated Ca2+ entry (SOCE) pathway to supply this ion. Due to the rapid pace of discovery of novel Ca2+ permeable channels, and limited electrophysiological analyses of Ca2+ currents in microglia, characterization of the SOCE channels remains incomplete. At present, the prime candidates are 'transient receptor potential' (TRP) channels and the recently cloned Orai1, which produces a Ca2+-release-activated Ca2+ (CRAC) current. We used cultured rat microglia and real-time RT-PCR to compare expression levels of Orai1, Orai2, Orai3, TRPM2, TRPM7, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 channel genes. Next, we used Fura-2 imaging to identify a store-operated Ca2+ entry pathway that was reduced by depolarization and blocked by Gd3+, SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 microM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 microM), and exhibited Ca(2+)-dependent potentiation. These properties are entirely consistent with Orai1/CRAC, rather than any known TRP channel and this conclusion was supported by patch-clamp electrophysiological analysis. We identified a store-operated Ca2+ current with the same properties, including high selectivity for Ca2+ over monovalent cations, pronounced inward rectification and a very positive reversal potential, Ca(2+)-dependent current potentiation, and block by SKF-96365, DES and 50 microM 2-APB. Determining the contribution of Orai1/CRAC in different cell types is crucial to future mechanistic and therapeutic studies; this comprehensive multi-strategy analysis demonstrates that Orai1/CRAC channels are responsible for SOCE in primary microglia

The mitochondrial calcium uniporter is a multimer that can include a dominant-negative pore-forming subunit

Article

Full-text available

Jul 2013
EMBO J

Mitochondrial calcium uniporter (MCU) channel is responsible for Ruthenium Red-sensitive mitochondrial calcium uptake. Here, we demonstrate MCU oligomerization by immunoprecipitation and Förster resonance energy transfer (FRET) and characterize a novel protein (MCUb) with two predicted transmembrane domains, 50% sequence similarity and a different expression profile from MCU. Based on computational modelling, MCUb includes critical amino-acid substitutions in the pore region and indeed MCUb does not form a calcium-permeable channel in planar lipid bilayers. In HeLa cells, MCUb is inserted into the oligomer and exerts a dominant-negative effect, reducing the [Ca(2+)]mt increases evoked by agonist stimulation. Accordingly, in vitro co-expression of MCUb with MCU drastically reduces the probability of observing channel activity in planar lipid bilayer experiments. These data unveil the structural complexity of MCU and demonstrate a novel regulatory mechanism, based on the inclusion of dominant-negative subunits in a multimeric channel, that underlies the fine control of the physiologically and pathologically relevant process of mitochondrial calcium homeostasis.

Function and expression of the epithelial Ca2+ channel family: comparison of mammalian ECaC1 and 2

Article

Dec 2001

• The epithelial Ca2+ channel (ECaC) family represents a unique group of Ca2+-selective channels that share limited homology to the ligand-gated capsaicin receptors, the osmolarity-sensitive channel OTRPC4, as well as the transient receptor potential family. Southern blot analysis demonstrated that this family is restricted to two members, ECaC1 and ECaC2 (also named CaT1). • RT-PCR analysis demonstrated that the two channels are co-expressed in calbindin-D-containing epithelia, including small intestine, pancreas and placenta, whereas kidney and brain only express ECaC1 and stomach solely ECaC2. • From an electrophysiological point of view, ECaC1 and ECaC2 are highly similar channels. Differences concern divalent cation permeability, the kinetics of Ca2+-dependent inactivation and recovery from inactivation. • Ruthenium red is a potent blocker of ECaC activity. Interestingly, ECaC2 has a 100-fold lower affinity for ruthenium red (IC50 9 ± 1 m) than ECaC1 (IC50 121 ± 13 nm). • ECaCs are modulated by intracellular Mg2+ and ATP. ECaC1 and ECaC2 activity rapidly decay in the absence of intracellular ATP. This effect is further accelerated at higher intracellular Mg2+ concentrations. • In conclusion, ECaC1 and ECaC2 are homologous channels, with an almost identical pore region. They can be discriminated by their sensitivity for ruthenium red and show differences in Ca2+-dependent regulation.

How many Orai’s does it take to make a CRAC channel?

Article

Full-text available

Jun 2013

CRAC (Calcium Release-Activated Calcium) channels represent the primary pathway for so-called "store-operated calcium entry" - the cellular entry of calcium induced by depletion of intracellular calcium stores. These channels play a key role in diverse cellular activities, most noticeably in the differentiation and activation of Tcells, and in the response of mast cells to inflammatory signals. CRAC channels are formed by members of the recently discovered Orai protein family, with previous studies indicating that the functional channel is formed by a tetramer of Orai subunits. However, a recent report has shown that crystals obtained from the purified Drosophila Orai protein display a hexameric channel structure. Here, by comparing the biophysical properties of concatenated hexameric and tetrameric human Orai1 channels expressed in HEK293 cells, we show that the tetrameric channel displays the highly calcium-selective conductance properties consistent with endogenous CRAC channels, whilst the hexameric construct forms an essentially non-selective cation channel.

Ca2+ release-activated Ca2+ (CRAC) current, structure, and function

Article

Full-text available

Jul 2012
CELL MOL LIFE SCI

Store-operated Ca(2+) entry describes the phenomenon that connects a depletion of internal Ca(2+) stores to an activation of plasma membrane-located Ca(2+) selective ion channels. Tremendous progress towards the underlying molecular mechanism came with the discovery of the two respective limiting components, STIM and Orai. STIM1 represents the ER-located Ca(2+) sensor and transmits the signal of store depletion to the plasma membrane. Here it couples to and activates Orai, the highly Ca(2+)-selective pore-forming subunit of Ca(2+) release-activated Ca(2+) channels. In this review, we focus on the molecular steps that these two proteins undergo from store-depletion to their coupling, the activation, and regulation of Ca(2+) currents.

Permeation, selectivity and gating in store-operated CRAC channels

Article

May 2012
J PHYSIOL-LONDON

Abstract Store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels are a widespread mechanism for generating cellular Ca(2+) signals and regulate many Ca(2+)-dependent functions, including transcription, motility and proliferation. The opening of CRAC channels in response to depletion of intracellular Ca(2+) stores involves a cascade of cellular events that culminate in direct interactions between STIM1, the endoplasmic reticulum Ca(2+) sensor, and the channels composed of Orai proteins. Evidence gathered over the last two decades indicates that CRAC channels display a unique functional pore fingerprint characterized by exquisite Ca(2+) selectivity, low unitary conductance, and low permeability to large cations. Here, we review the key pore properties of CRAC channels and discuss recent progress in addressing the molecular foundations of these properties. Structure-function and cysteine-scanning studies have revealed the identity and organization of pore-lining residues, including those that form the selectivity filter, providing a structural framework for understanding CRAC channel pore properties. Recent studies in pore mutants that produce STIM1-independent constitutive channel activation indicate that exquisite Ca(2+) selectivity in CRAC channels is not hardwired into Orai proteins, but is instead manifested only following the binding of STIM1 to the intrinsically poorly Ca(2+)-selective Orai channels. These findings reveal new functional aspects of CRAC channels and suggest that the selectivity filter of the CRAC channel is a dynamic structure whose conformation and functional properties are powerfully regulated by the channel activation stimulus.

Structures reveal opening of the store-operated calcium channel Orai

Preprint

Mar 2018

The store-operated calcium (Ca ²⁺ ) channel Orai governs Ca ²⁺ influx through the plasma membrane of many non-excitable cells in metazoans. The channel opens in response to depletion of Ca ²⁺ within the endoplasmic reticulum (ER). Loss- and gain-of-function mutants of Orai cause disease. Our previous work revealed the structure of Orai with a closed pore. Here, using a gain-of-function mutation that constitutively activates the channel, we present an X-ray structure of Drosophila melanogaster Orai in an open conformation. Well-defined electron density maps reveal that the open pore is dramatically dilated on its cytosolic side in comparison to the slender closed pore. Cations and anions bind in different regions of the open pore, informing mechanisms for ion permeation and the exquisite selectivity of the channel for Ca ²⁺ . Opening of the pore requires the release of cytosolic latches. Together with additional X-ray structures of an unlatched-but-closed intermediate, we propose a sequence for store-operated activation.

Review: Structure and Activation Mechanisms of CRAC Channels

Chapter

Jan 2020
ADV EXP MED BIOL

Ca²⁺ release activated Ca²⁺ (CRAC) channels represent a primary pathway for Ca²⁺ to enter non-excitable cells. The two key players in this process are the stromal interaction molecule (STIM), a Ca²⁺ sensor embedded in the membrane of the endoplasmic reticulum, and Orai, a highly Ca²⁺ selective ion channel located in the plasma membrane. Upon depletion of the internal Ca²⁺ stores, STIM is activated, oligomerizes, couples to and activates Orai. This review provides an overview of novel findings about the CRAC channel activation mechanisms, structure and gating. In addition, it highlights, among diverse STIM and Orai mutants, also the disease-related mutants and their implications.

Structural and Mechanistic Insights of CRAC Channel as a Drug Target in Autoimmune Disorder

Article

Sep 2019

Background Calcium (Ca2+) ion is a major intracellular signaling messenger, controlling a diverse array of cellular functions like gene expression, secretion, cell growth, proliferation, and apoptosis. The major mechanism controlling this Ca2+ homeostasis is store-operated Ca2+ release-activated Ca2+ (CRAC) channels. CRAC channels are integral membrane protein majorly constituted via two proteins, the stromal interaction molecule (STIM) and ORAI. Following Ca2+ depletion in the Endoplasmic reticulum (ER) store, STIM1 interacts with ORAI1 and leads to the opening of the CRAC channel gate and consequently allows the influx of Ca2+ ions. A plethora of studies report that aberrant CRAC channel activity due to Loss- or gain-of-function mutations in ORAI1 and STIM1 disturbs this Ca2+ homeostasis and cause several autoimmune disorders. Hence, it clearly says that the therapeutic target of CRAC channels provides the space for a new approach to treat autoimmune disorders. Objective This review aims to provide the key structural and mechanical insights of STIM1, ORAI1 and other molecular modulators involved in CRAC channel regulation. Results and Conclusion Understanding the structure and function of the protein is the foremost step towards improving the effective target specificity by limiting their potential side effects. Herein, the review mainly focusses on the structural underpinnings of the CRAC channel gating mechanism along with its biophysical properties that would provide the solid foundation to aid the development of novel targeted drugs for an autoimmune disorder. Finally, the immune deficiencies caused due to mutations in CRAC channel and currently used pharmacological blockers with their limitation are briefly summarized.

Peripheral myelin protein 22 modulates store operated calcium channel activity, providing insights into Charcot-Marie-Tooth disease etiology

Article

Full-text available

Jun 2019

Charcot-Marie-Tooth (CMT) disease is a peripheral neuropathy associated with gene duplication and point mutations in the peripheral myelin protein 22 (PMP22) gene. However, the role of PMP22 in Schwann cell physiology and the mechanisms by which PMP22 mutations cause CMT are not well understood. On the basis of homology between PMP22 and proteins associated with modulation of ion channels, we hypothesized that PMP22 alters ion channel activity. Using whole-cell electrophysiology we show here that heterologous PMP22 expression increases the amplitude of currents similar to those ascribed to store- operated calcium (SOC) channels, particularly those involving transient receptor canonical channel 1 (TrpC1). These channels help replenish Ca+2 in the endoplasmic reticulum (ER) following stimulus-induced depletion. Currents with similar properties were recorded in wild type but not pmp22-/- mouse Schwann cells. Heterologous expression of the CMT-associated PMP22_L16P variant, which fails to reach the plasma membrane and localizes to the ER, led to larger currents than wild type PMP22. Similarly, Schwann cells isolated from Trembler J (TrJ; PMP22_L16P) mice had larger currents than wild type littermates. Calcium imaging in live nerves and cultured Schwann cells revealed elevated intracellular Ca+2 in TrJ mice compared with wild type. Moreover, we found that PMP22 co-immunoprecipitated with stromal interaction molecule 1 (STIM1), the Ca+2 sensor SOC channel subunit in the ER. These results suggest that in the ER, PMP22 interacts with STIM1 and increases Ca+2 influx through SOC channels. Excess or mutant PMP22 in the ER may elevate intracellular Ca+2 levels, which could contribute to CMT pathology.

Molecular Dynamics Simulations of Orai Reveal How the Third Transmembrane Segment Contributes to Hydration and Ca 2+ Selectivity in CRAC Channels

Article

Mar 2018

Calcium release-activated calcium (CRAC) channels open upon depletion of Ca2+ from the endoplasmic reticulum, and when open, they are permeable to a selective flux of calcium ions. The atomic structure of Orai, the pore domain of CRAC channels, from Drosophila Melanogaster has revealed many details about conduction and selectivity in this family of ion channels. However, it is still unclear how residues on the third transmembrane helix can affect the conduction properties of the channel. Here, Molecular Dynamics and Brownian Dynamics simulations were employed to analyse how a conserved glutamate residue on the third transmembrane helix (E262) contributes to selectivity. The comparison between the wild-type and the mutant channel revealed a severe impact of the mutation on the hydration pattern of the pore domain and on the dynamics of residues K270. Brownian Dynamics simulations proved that the altered configuration of residues K270 in the mutant channel impairs selectivity to Ca2+ over Na+. The crevices of water molecules revealed by Molecular Dynamics simulations are perfectly located to contribute to the dynamics of the hydrophobic and basic gates, suggesting a possible role in channel opening and in the selectivity function.

Authentic CRAC channel activity requires STIM1 and the conserved portion of the Orai N-terminus

Article

Full-text available

Dec 2017

Calcium (Ca2+) is an essential second messenger required for diverse signaling processes in immune cells. Ca2+ release-activated Ca2+ (CRAC) channels represent one main Ca2+ entry pathway into the cell. They are fully reconstituted via two proteins, the stromal interaction molecule 1 (STIM1), a Ca2+ sensor in the endoplasmic reticulum and the Ca2+ ion channel Orai in the plasma membrane. After Ca2+ store depletion, STIM1 and Orai couple to each other allowing Ca2+ influx. CRAC-/STIM1-mediated Orai channel currents display characteristic hallmarks such as high Ca2+ selectivity, an increase in current density when switching from a Ca2+-containing solution to a divalent-free Na+ one and fast Ca2+ dependent inactivation. Here, we discovered several constitutively active Orai1 and Orai3 mutants, containing substitutions in the TM3 and/or TM4 regions, all of which displayed a loss of the typical CRAC channel hallmarks. Restoring authentic CRAC channel activity required both the presence of STIM1 and the conserved Orai N-terminal portion. Similarly these structural requisites were found in store-operated Orai channels. Key molecular determinants within the Orai N-terminus that together with STIM1 maintained the typical CRAC channel hallmarks were distinct to those which controlled store-dependent Orai activation. In conclusion, the conserved portion of the Orai N-terminus is essential for STIM1 as it fine tunes the open Orai channel gating, thereby establishing authentic CRAC channel activity.

New Perspectives on Ca2+ Influx in Mast Cells

Chapter

Jan 1999

M A McCloskey

The importance of extracellular calcium for anaphylactic release of histamine was suggested by experiments of Mongar and Schild in 1958, years before the discovery of IgE or its high-affinity receptor, the FcεRI.1 Many subsequent studies using radiotracer flux and fluorescent Ca2+ indicators revealed that multivalent binding of antigen to the IgE-FcεRI complex elicits the release of internal as well as the influx of extracellular Ca2+, and there appears to be a causal relation between these two events. For the RBL-2H3 mast cell line used in many of these studies,2 it is generally agreed that Ca2+ influx is crucial for antigen-induced secretion of preformed inflammatory mediators.3

Store-Operated Calcium Entry in Müller Glia Is Controlled by Synergistic Activation of TRPC and Orai Channels

Article

Full-text available

Mar 2016

The endoplasmic reticulum(ER) is at the epicenter of astrocyte Ca2� signaling.We sought to identify the molecular mechanism underlying store-operated calcium entry that replenishes ER stores in mouse Muller cells. Store depletion, induced through blockade of sequestration transporters in Ca2�-free saline, induced synergistic activation of canonical transient receptor potential 1 (TRPC1) and Orai channels. Store-operated TRPC1 channels were identified by their electrophysiological properties, pharmacological blockers, and ablation of the Trpc1 gene. Ca2� release-activated currents (ICRAC ) were identified by ion permeability, voltage dependence, and sensitivity to selective Orai antagonists Synta66 and GSK7975A. Depletion-evoked calcium influx was initiated at the Muller end-foot and apical process, triggering centrifugal propagation of Ca2� waves into the cell body. EM analysis of the end-foot compartment showed high-density ER cisternae that shadow retinal ganglion cell (RGC) somata and axons, protoplasmic astrocytes, vascular endothelial cells, and ER–mitochondrial contacts at the vitreal surface of the end-foot. The mouse retina expresses transcripts encoding both Stim and all known Orai genes; Muller glia predominantly express stromal interacting molecule 1 (STIM1), whereas STIM2 ismainly confined to the outer plexiform and RGC layers. Elimination of TRPC1 facilitated Muller gliosis induced by the elevation of intraocular pressure, suggesting that TRPC channels might play a neuroprotective role during mechanical stress. By characterizing the properties of store-operated signaling pathways in Muller cells, these studies expand the current knowledge about the functional roles these cells play in retinal physiology and pathology while also providing further evidence for the complexity of calcium signaling mechanisms in CNS astroglia.

The molecular mechanisms of STIM/Orai communications. A Review in the Theme: STIM and Orai Proteins in Calcium Signaling

Article

Jan 2016

Ca(2+) entry into the cell via store-operated Ca(2+) release activated Ca(2+) (CRAC) channels triggers diverse signaling cascades that affect cellular processes like cell growth, gene regulation, secretion and cell death. These store-operated Ca(2+) channels open following depletion of intracellular Ca(2+) stores and their main features are fully reconstituted by the two molecular key players: the stromal interaction molecule (STIM) and Orai. STIM represents an ER-located Ca(2+) sensor, while Orai forms a highly Ca(2+) selective ion channel in the plasma membrane. Functional as well as mutagenesis studies together with structural insights about STIM and Orai proteins provide a molecular picture of the interplay of these two key players in the CRAC signaling cascade. This review focuses on the main experimental advances in the understanding of the STIM1-Orai choreography thereby establishing a portray of key mechanistic steps in the CRAC channel signaling cascade. The focus is laid on the activation of the STIM proteins, the following coupling of STIM1 to Orai1, and the consequent, structural rearrangements that gate the Orai channels into the open state to allow Ca(2+) permeation into the cell.

STIM1-ORAI1 store-operated calcium channels

Article

Dec 2012

P.G. Hogan

Calcium release-activated calcium (CRAC) current is an established example of store-operated calcium influx, first characterized electrophysiologically in T lymphocytes and mast cells. The key proteins underlying the CRAC current are ORAI, a plasma membrane protein that forms the channel itself, and STIM, an endoplasmic reticulum (ER) membrane protein that senses ER-luminal calcium concentration and controls ORAI channel gating. The chapter summarizes the electrophysiological properties of the CRAC current and the biophysical and biochemical experiments that have provided insight into STIM-ORAI signaling.

Molecular Mechanism of Store-Operated Ca2+ Signaling and CRAC Channel Activation Mediated by STIM & Orai

Article

Dec 2010

Store-operated Ca2+ (SOC) channels in the plasma membrane (PM) open when Ca2+ is depleted within the lumen of the endoplasmic reticulum (ER). SOC channels can be highly selective Ca2+ channels that are, under physiological conditions, impermeable to monovalent cations such as sodium or potassium; or Ca2+-permeable but relatively non-selective cation channels. The former are known as Ca2+-release-activated Ca2+ (CRAC) channels, which mediate sustained Ca2+ signaling in T cells in response to antigen presentation. This chapter summarizes biophysical properties of the CRAC channel in relation to the molecular physiology of STIM and Orai coupling. It begins by describing the biophysical properties of the CRAC channel. Ion channel functional properties are generally characterized in terms of gating (modes of opening and closing), ion selectivity, single-channel conductance, modulation, and pharmacology. Under this, it considers STIM as the ER Ca2+ sensor and STIM as the messenger to the plasma membrane. Furthermore, it discusses how Orai forms the Ca2+-selective pore of the CRAC channel. Finally, it takes up the phenomenon of STIM-induced activation of Orai channels.

Store-operated Ca2+ entry (SOCE) pathways

Chapter

Jan 2012

Patrick G Hogan

This chapter focuses on the Orai proteins, Orai1-3 in humans and other mammals, and on the definitive evidence that Orai is the pore subunit of the CRAC channel. It starts by reviewing briefly the defining characteristics of the CRAC channel, then discusses the first studies that implicated Orai as part of the store-operated Ca2+ entry pathway and as the CRAC channel pore subunit, and finally examines some insights from cell biological and electrophysiological studies of recombinant Orai proteins.

CELLULAR MECHANISMS UNDERLYING GLUTAMATERGIC CALCIUM RESPONSES IN DEVELOPING AUDITORY BRAINSTEM NEURONS

Article

Dec 2003

Florenta Aura Negoita

Spontaneous and sound-driven activity, glutamatergic synaptic transmission and Ca2+ signaling are critical for formation, maturation, refinement and survival of neuronal circuits including the auditory system. The present study investigated the mechanisms by which glutamatergic inputs from the cochlear nucleus regulate intracellular calcium concentration ([Ca2+]i) in developing lateral superior olive (LSO) neurons, using Ca2+ imaging in fura-2AM labeled brainstem slices.AMPA/kainate receptors primarily mediated Ca2+ responses elicited by single stimuli and contributed to Ca2+ responses elicited by low and high frequency bursts by approximately 75% and 50% respectively. Both AMPAR and kainate receptors were Ca2+ impermeable and increased [Ca2+]i via membrane depolarization and activation of voltage gated calcium channels (VGCCs). NMDARs contributed approximately 50% to Ca2+ responses independent of the stimulus frequency. Their high contribution to Ca2+ responses was consistent with their contribution (30-60%) to EPSPs triggered by stimulation of AVCN-LSO synapses. mGluRs contributed to Ca2+ responses only under high frequency stimulation (>20Hz). Group I mGluR-mediated Ca2+ responses had two components: release from internal stores and influx from the extracellular milieu. The influx was mediated by a channel sensitive to Ni2+, La3+ and 2-APB, consistent with it being a member of the TRP family. During development, the contribution of this channel decreased and it was lost after hearing onset, suggesting that it might be downregulated by auditory experience. In summary, distinct temporal patterns of synaptic activity in the LSO activate distinct GluR types and each receptor type employs a distinct Ca2+ entry pathway. This could possibly lead to activation of distinct intracellular cascades and distinct gene expression programs (West et al., 2001) that may be involved in distinct developmental aspects.

Gating and permeation of Orai channels

Article

Jan 2012

Isabella Derler

Permeation and gating mechanisms in store-operated CRAC channels

Article

Jan 2012

Anita Engh

The neglected CRAC proteins: Orai2, Orai3, and STIM2

Article

Jul 2013
CURR TOP MEMBR

Plasma-membrane-localized Orai1 ion channel subunits interacting with ER-localized STIM1 molecules comprise the major subunit composition responsible for calcium release-activated calcium channels. STIM1 "translates" the Ca(2+) store content into Orai1 activity, making it a store-operated channel. Surprisingly, in addition to being the physical activator, STIM1 also modulates Orai1 properties, including its inactivation and permeation (see Chapter 1). STIM1 is thus more than a pure Orai1 activator. Within the past 7 years following the discovery of STIM and Orai proteins, the molecular mechanisms of STIM1/Orai1 activity and their functional importance have been studied in great detail. Much less is currently known about the other isoforms STIM2, Orai2, and Orai3. In this chapter, we summarize the current knowledge about STIM2, Orai2, and Orai3 properties and function. Are these homologues mainly modulators of predominantly STIM1/Orai1-mediated complexes or do store-dependent or -independent functions such as regulation of basal Ca(2+) concentration and activation of Orai3-containing complexes by arachidonic acid or by estrogen receptors point toward their "true" physiological function? Is Orai2 the Orai1 of neurons? A major focus of the review is on the functional relevance of STIM2, Orai2, and Orai3, some of which still remains speculative.

Characterization and regulation of rat microglial Ca2+ release‐activated Ca2+ (CRAC) channel by protein kinases

Article

Aug 2000
GLIA

We measured the activity of the Ca2+ release-activated Ca2+ (CRAC) channel present in cultured rat microglia, using the whole-cell mode of patch clamp technique. When the concentration of divalent cations in external solution was reduced to the micromolar range, and Ca2+ chelating agent BAPTA was included in the pipette solution, we were able to record Na+ current through CRAC channels in single-channel levels. The unitary Na+ conductance through CRAC channel was 42.5 pS, which was similar to that of Jurkat cell. The Na+ current activated slowly, reaching the maximal current level in about 10 min after whole-cell patches were made. The time required for the half-maximal activation of the current was 205 s (±31), while it was reduced to 84.3 s (±17.7) by including IP3 in the pipette solution as well. The peak currents ranged from 320 to 985 pA, which corresponded to 64–197 channels per cell. We studied the regulation of the current by protein kinase A (PKA) and protein kinase C (PKC). The current was enhanced by the addition of membrane-permeant analogue of cAMP, dibutyryl cAMP. Pretreating cells with PKA inhibitor, H-89, prevented the effect of dibutyryl cAMP. By contrast, the addition of PKC activator, PDBu, reduced the current. Staurosporine, a PKC inhibitor, prevented the effect of PDBu. These results suggest that CRAC channel in rat microglia is under the regulation of PKA and PKC in opposite directions. GLIA 31:118–124, 2000. © 2000 Wiley-Liss, Inc.

Store-operated calcium channels: Properties, functions and the search for a molecular mechanism

Article

Dec 2004
Adv Mol Cell Biol

This chapter presents an overview of the properties and functions of store-operated calcium channels (SOCs). A large number of Ca2+ imaging studies indicate that SOCs are ubiquitous in nonexcitable cells, where they comprise the main Ca2+ entry pathway, and some studies suggest that they may exist in excitable cells as well. Patch-clamp methods have offered the most detailed information about the biophysical properties of these channels. The first and best characterized SOC is the Ca2+ release-activated Ca2+ (CRAC) channel, expressed in mast cells, T lymphocytes, and related cell lines. While much is known about the basic biophysical properties of these channels, a critical issue remains about the identification of the molecular mechanism(s) underlying their activity. The chapter discusses those aspects of SOCs that may be especially pertinent to the search for a molecular mechanism. It highlights CRAC channels as a prototypic example of an SOC, with the expectation that many of the issues related to the molecular mechanism will apply to other SOCs as well. The chapter also discusses the known biological functions of CRAC channels and some of the intrinsic properties that are most important for carrying out these roles.

Structure, regulation and biophysics of I(CRAC), STIM/Orai1

Article

Feb 2012
ADV EXP MED BIOL

Ca(2+) release activated Ca(2+) (CRAC) channels mediate robust Ca(2+) influx when the endoplasmic reticulum Ca(2+) stores are depleted. This essential process for T-cell activation as well as degranulation of mast cells involves the Ca(2+) sensor STIM1, located in the endoplasmic reticulum and the Ca(2+) selective Orai1 channel in the plasma membrane. Our review describes the CRAC signaling pathway, the activation of which is initiated by a drop in the endoplasmic Ca(2+) level sensed by STIM1. This in term induces multimerisation and puncta-formation of STIM1 proteins is followed by their coupling to and activation of Orai channels. Consequently Ca(2+) entry is triggered through the Orai pore into the cytosol with subsequent closure of the channel by Ca(2+)-dependent inactivation. We will portray a mechanistic view of the events coupling STIM1 to Orai activation based on their structure and biophysics.

Hoth, M. & Penner, R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 355, 353-356.This is the first description of the store-operated CRAC current

Article

Full-text available

Feb 1992

In many cell types, receptor-mediated Ca2+ release from internal stores is followed by Ca2+ influx across the plasma membrane. The sustained entry of Ca2+ is thought to result partly from the depletion of intracellular Ca2+ pools. Most investigations have characterized Ca2+ influx indirectly by measuring Ca(2+)-activated currents or using Fura-2 quenching by Mn2+, which in some cells enters the cells by the same influx pathway. But only a few studies have investigated this Ca2+ entry pathway more directly. We have combined patch-clamp and Fura-2 measurements to monitor membrane currents in mast cells under conditions where intracellular Ca2+ stores were emptied by either inositol 1,4,5-trisphosphate, ionomycin, or excess of the Ca2+ chelator EGTA. The depletion of Ca2+ pools by these independent mechanisms commonly induced activation of a sustained calcium inward current that was highly selective for Ca2+ ions over Ba2+, Sr2+ and Mn2+. This Ca2+ current, which we term ICRAC (calcium release-activated calcium), is not voltage-activated and shows a characteristic inward rectification. It may be the mechanism by which electrically nonexcitable cells maintain raised intracellular Ca2+ concentrations and replenish their empty Ca2+ stores after receptor stimulation.

The Ca channel in skeletal muscle is a large pore

Article

Full-text available

Nov 1985

The permeability of Ca channels to various foreign cations has been investigated in the absence of external Ca2+. All physiological metal cations are clearly permeant, including Mg2+. The large organic cation n-butylamine+ is sparingly permeant or impermeant, but its larger derivative 1,4-diaminobutane2+ is highly permeant. Among the cations of the methylated ammonium series, permeability diminishes in a graded fashion as ion size increases. Tetramethylammonium, the largest cation found to be permeant, has a diameter of about 6 A; hence, the aqueous pore of the Ca channel at its narrowest point can be no smaller. That the pore is so large strengthens our view that, under physiologic conditions, the high selectivity of Ca channels is due to selective binding of Ca2+ rather than to rejection of other cations by, for example, a sieving mechanism.

Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells

Article

Full-text available

Oct 1986

Single channel and whole cell recordings were used to study ion permeation through Ca channels in isolated ventricular heart cells of guinea pigs. We evaluated the permeability to various divalent and monovalent cations in two ways, by measuring either unitary current amplitude or reversal potential (Erev). According to whole cell measurements of Erev, the relative permeability sequence is Ca2+ greater than Sr2+ greater than Ba2+ for divalent ions; Mg2+ is not measurably permeant. Monovalent ions follow the sequence Li+ greater than Na+ greater than K+ greater than Cs+, and are much less permeant than the divalents. These whole cell measurements were supported by single channel recordings, which showed clear outward currents through single Ca channels at strong depolarizations, similar values of Erev, and similar inflections in the current-voltage relation near Erev. Information from Erev measurements stands in contrast to estimates of open channel flux or single channel conductance, which give the sequence Na+ (85 pS) greater than Li+ (45 pS) greater than Ba2+ (20 pS) greater than Ca2+ (9 pS) near 0 mV with 110-150 mM charge carrier. Thus, ions with a higher permeability, judged by Erev, have lower ion transfer rates. In another comparison, whole cell Na currents through Ca channels are halved by less than 2 microM [Ca]o, but greater than 10 mM [Ca]o is required to produce half-maximal unitary Ca current. All of these observations seem consistent with a recent hypothesis for the mechanism of Ca channel permeation, which proposes that: ions pass through the pore in single file, interacting with multiple binding sites along the way; selectivity is largely determined by ion affinity to the binding sites rather than by exclusion by a selectivity filter; occupancy by only one Ca ion is sufficient to block the pore's high conductance for monovalent ions like Na+; rapid permeation by Ca ions depends upon double occupancy, which only becomes significant at millimolar [Ca]o, because of electrostatic repulsion or some other interaction between ions; and once double occupancy occurs, the ion-ion interaction helps promote a quick exit of Ca ions from the pore into the cell.

Calcium and barium permeation through calcium release-activated calcium (CRAC) channels

Article

Full-text available

Aug 1995

Markus Hoth

A Ca2+ current activated by store depletion has been described recently in several cell types and has been termed I CRAC (for Ca2+ release-activated Ca2+ current). In this paper, the Ca2+ and Ba2+ permeability of CRAC channels is investigated in mast cells, rat basophilic leukaemia cells (RBL) and human T-lymphocytes (Jurkat). The selectivity of CRAC channels for Ca2+ over monovalent cations is identical in all three cell types and is at least as high as that of voltage-operated Ca2+ (VOC) channels in the various tissues tested. The amplitude of Ba2+ currents relative to Ca2+ currents (I Ba/I Ca) through CRAC channels was found to be strongly dependent on the membrane potential and was much smaller in Jurkat cells compared to mast and RBL cells. An anomalous mole-fraction behavior was observed at very negative membrane potentials in all three cell types when using different mixtures of external Ca2+ and Ba2+. In contrast to VOC channels, the anomalous mole-fraction effect was not observed at potentials positive to−20 mV.

Slow Calcium-dependent Inactivation of Depletion-activated Calcium Current

Article

Full-text available

Jul 1995

Feedback regulation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole cell recording and [Ca2+]i measurement techniques. CRAC channels were activated by passively depleting intracellular Ca2+ stores in the absence of extracellular Ca2+. Under conditions of moderate intracellular Ca2+ buffering, elevating [Ca2+]o to 22 mM initiated an inward current through CRAC channels that declined slowly with a half-time of approximately 30 s. This slow inactivation was evoked by a rise in [Ca2+]i, as it was effectively suppressed by an elevated level of EFTA in the recording pipette that prevented increases in [Ca2+]i. Blockade of Ca2+ uptake into stores by thapsigargin with or without intracellular inositol 1,4,5-trisphosphate reduced the extent of slow inactivation by approximately 50%, indicating that store refilling normally contributes significantly to this process. The store-independent (thapsigargin-insensitive) portion of slow inactivation was largely prevented by the protein phosphatase inhibitor, okadaic acid, and by a structurally related compound, 1-norokadaone, but not by calyculin A nor by cyclosporin A and FK506 at concentrations that fully inhibit calcineurin (protein phosphatase 2B) in T cells. These results argue against the involvement of protein phosphatases 1, 2A, 2B, or 3 in store-independent inactivation. We conclude that calcium acts through at least two slow negative feedback pathways to inhibit CRAC channels. Slow feedback inhibition of CRAC current is likely to play important roles in controlling the duration and dynamic behavior of receptor-generated Ca2+ signals.

Ca2 Channel Selectivity at a Single Locus for High-Affinity Ca2 Interactions

Article

Full-text available

Dec 1995
NEURON

Ca2+ channels display remarkable selectivity and permeability, traditionally attributed to multiple, discrete Ca2+ binding sites lining the pore. Each of the four pore-forming segments of Ca2+ channel alpha 1 subunits contains a glutamate residue that contributes to high-affinity Ca2+ interactions. Replacement of all four P-region glutamates with glutamine or alanine abolished micromolar Ca2+ block of monovalent current without revealing any additional independent high-affinity Ca2+ binding site. Pairwise replacements of the four glutamates excluded the hypothesis that they form two independent high-affinity sites. Systematic alterations of side-chain length, charge, and polarity by glutamate replacement with aspartate, glutamine, or alanine weakened the Ca2+ interaction, with considerable asymmetry from one repeat to another. The P-region glutamate in repeat I was unusual in its sensitivity to aspartate replacement but not glutamine substitution. While all four glutamates cooperate in supporting high-affinity interactions with single Ca2+ ions, they also influence the interaction between multiple divalent cations.

Depletion-activated Ca2+ current is inhibited by protein kinase in RBL-2H3 cells

Article

Full-text available

Sep 1995

Whole-cell patch-clamp recordings and single-cell Ca2+ measurements were used to study the control of Ca2+ entry through the Ca2+ release-activated Ca2+ influx pathway (ICRAC) in rat basophilic leukemia cells. When intracellular inositol 1,4,5-trisphosphate (InsP3)-sensitive stores were depleted by dialyzing cells with high concentrations of InsP3, ICRAC inactivated only slightly in the absence of ATP. Inclusion of ATP accelerated inactivation 2-fold. The inactivation was increased further by the ATP analogue adenosine 5'-[gamma-thio]triphosphate, which is readily used by protein kinases, but not by 5'-adenylyl imidodiphosphate, another ATP analogue that is not used by kinases. Neither cyclic nucleotides nor inhibition of calmodulin or tyrosine kinase prevented the inactivation. Staurosporine and bisindolylmaleimide, protein kinase C inhibitors, reduced inactivation of ICRAC, whereas phorbol ester accelerated inactivation of the current. These results demonstrate that a protein kinase-mediated phosphorylation, probably through protein kinase C, inactivates ICRAC. Activation of the adenosine receptor (A3 type) in RBL cells did not evoke much Ca2+ influx or systematic activation of ICRAC. After protein kinase C was blocked, however, large ICRAC was observed in all cells and this was accompanied by large Ca2+ influx. The ability of a receptor to evoke Ca2+ entry is determined, at least in part, by protein kinase C. Antigen stimulation, which triggers secretion through a process that requires Ca2+ influx, activated ICRAC. The regulation of ICRAC by protein kinase will therefore have important consequences on cell functioning.

Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback

Article

Full-text available

Mar 1995

Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.

Depletion of intracellular Ca2+ stores activates a Ca2+-selective channel in vascular endothelium

Article

Full-text available

Oct 1994
Am J Physiol

The present study was designed to identify the channel responsible for Ca2+ influx after depletion of intracellular Ca2+ stores. Different maneuvers that deplete intracellular Ca2+ stores activated a Ca(2+)-selective channel. Superfusion of single bovine aortic endothelial cells with 50 nmol/l bradykinin, 10 mumol/l ATP, or 10 mumol/l 2,5-di(tert-butyl)-1,4-benzohydroquinone produced activation of channels of the same amplitude in cell-attached patches. Channel activity declined within the first minute after patch excision. The channel showed strong inward rectification and a reversal potential of 0 mV in symmetrical sodium sulfate (Na2SO4) solution. Under these conditions, the conductance was 5 pS in the inward direction. Addition of 10 mmol/l Ca2+ to the extracellular solution shifted the reversal potential to +30 +/- 5 mV, and the conductance for inward current was 11 pS. The reversal potential was used to calculate an ion permeability ratio of Ca2+/Na+ > 10:1.

Flash photolysis of caged inositol 1,4,5-trisphosphate activates plasma membrane calcium current in human T cells

Article

Full-text available

Mar 1993

Sustained elevation of intracellular Ca2+ by cell surface receptors is often dependent on influx of Ca2+ across the plasma membrane through routes not involving voltage-gated Ca2+ channels. We demonstrate that intracellular release of inositol 1,4,5-trisphosphate (InsP3), either from stimulation of transfected human muscarinic receptors or from photolytic release of caged InsP3, activates whole cell Ca2+ current in the Jurkat T cell line. Whole cell voltage clamp recordings indicate that the current is carried by a Ca(2+)-selective channel that resembles T-type voltage-gated Ca2+ channels in relative conductance of different cation species. Elevation of internal Ca2+ inactivates the channel, whereas internal perfusion with inositol 1,3,4,5-tetrakisphosphate (InsP4) does not affect it. Photolytic release of caged 1-(alpha-glycerophosphoryl)-inositol 4,5-bisphosphate, an analog of InsP3 which activates InsP3 receptors but is not readily metabolized to InsP4, also activates the current. We conclude that generation of InsP3 is sufficient to activate Ca(2+)-selective channels in the plasma membrane of T cells. InsP3 may have its effect indirectly through depletion of Ca2+ stores, or directly with a plasma membrane-associated InsP3 receptor.

A GTP-dependent step in the activation mechanism of capacitative calcium influx

Article

Full-text available

Nov 1993

Calcium influx in electrically non-excitable cells is regulated by the filling state of intracellular calcium stores. Depletion of stores activates plasma membrane channels that are voltage-independent and highly selective for Ca2+ ions. We report here that the activation of plasma membrane Ca2+ currents induced by depletion of Ca2+ stores requires a diffusible cytosolic factor that washes out with time when dialyzing cells in the whole-cell configuration of the patch-clamp technique. The activation of calcium release-activated calcium current (ICRAC) by ionomycin- or inositol 1,4,5-trisphosphate-induced store depletion is blocked by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) and guanyl-5'-yl imidodiphosphate, non-hydrolyzable analogs of GTP, suggesting the involvement of a GTP-binding protein. The inhibition by GTP gamma S occurs at a step prior to the activation of ICRAC and is prevented by the addition of GTP. We conclude that the activation mechanism of depletion-induced Ca2+ influx encompasses a GTP-dependent step, possibly involving an as yet unidentified small GTP-binding protein.

Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes

Article

Full-text available

Jun 1996

The depletion of intracellular Ca2+ stores triggers the opening of Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane of T lymphocytes. We have investigated the additional role of extracellular Ca2+ (Ca02+) in promoting CRAC channel activation in Jurkat leukemic T cells. Ca2+ stores were depleted with 1 microM thapsigargin in the nominal absence of Ca02+ with 12 mM EGTA or BAPTA in the recording pipette. Subsequent application of Ca02+ caused ICRAC to appear in two phases. The initial phase was complete within 1 s and reflects channels that were open in the absence of Ca02+. The second phase consisted of a severalfold exponential increase in current amplitude with a time constant of 5-10 s; we call this increase Ca(2+)-dependent potentiation, or CDP. The shape of the current-voltage relation and the inferred single-channel current amplitude are unchanged during CDP, indicating that CDP reflects an alteration in channel gating rather than permeation. The extent of CDP is modulated by voltage, increasing from approximately 50% at +50 mV to approximately 350% at -75 mV in the presence of 2 mM Ca02+. The voltage dependence of CDP also causes ICRAC to increase slowly during prolonged hyperpolarizations in the constant presence of Ca02+. CDP is not affected by exogenous intracellular Ca2+ buffers, and Ni2+, a CRAC channel blocker, can cause potentiation. Thus, the underlying Ca2+ binding site is not intracellular. Ba2+ has little or no ability to potentiate CRAC channels. These results demonstrate that the store-depletion signal by itself triggers only a small fraction of capacitative Ca2+ entry and establish Ca2+ as a potent cofactor in this process. CDP confers a previously unrecognized voltage dependence and slow time dependence on CRAC channel activation that may contribute to the dynamic behavior of ICRAC.

Ca 2+ Influx Pathways Mediated by Swelling or Stores Depletion in Mouse Thymocytes

Article

Full-text available

Oct 1995

We used fura-2 video imaging to characterize two Ca2+ influx pathways in mouse thymocytes. Most thymocytes (77%) superfused with hypoosmotic media (60% of isoosmotic) exhibited a sharp, transient rise in the concentration of intracellular free Ca2+ ([Ca2+]i). After a delay of approximately 70 s, these swelling-activated [Ca2+]i (SWAC) transients reached approximately 650 nM from resting levels of approximately 100 nM and declined from a time constant of 20 s. Peak [Ca2+]i during transients correlated with maximum volume during swelling. Regulatory volume decrease (RVD) was enhanced in thymocytes exhibiting SWAC transients. Three lines of evidence indicate that Ca2+ influx, and not the release of Ca2+ from intracellular stores, underlies SWAC transients in thymocytes. First, thymocytes swollen in Ca2+-free media failed to respond. Second, Gd3+ and La3+ inhibited SWAC influx with Kd's of 3.8 and 2.4 microM, respectively. Finally, the depletion of Ca2+ stores with thapsigargin (TG) before swelling did not inhibit the generation, nor decrease the amplitude, of SWAC transients. Cell phenotyping demonstrated that SWAC transients are primarily associated with immature CD4-CD8- and CD4+CD8+ thymocytes. Mature peripheral lymphocytes (mouse or human) did not exhibit SWAC transients. SWAC influx could be distinguished from the calcium release-activated Ca2+ (CRAC) influx pathway stimulated by store depletion with TG. In TG-treated thymocytes, [Ca2+]i rose steadily for approximately 100 s, peaked at approximately 900 nM, and then declined slowly. Simultaneous activation of both pathways produced an additive [Ca2+]i profile. Gd3+ and La3+ blocked Ca2+ entry during CRAC activation more potently (Kd's of 28 and 58 nM, respectively) than Ca2+ influx during SWAC transients. SWAC transients could be elicited in the presence of 1 microM Gd3+, after the complete inhibition of CRAC influx. Finally, whereas SWAC transients were principally restricted to immature thymocytes. TG stimulated the CRAC influx pathway in all four thymic CD4/CD8 subsets and in mature T cells. We conclude that SWAC and CRAC represent separate pathways for Ca2+ entry in thymocytes.

SLOW CALCIUM-DEPENDENT INACTIVATION OF DEPLETION-ACTIVATED CALCIUM CURRENT - STORE-DEPENDENT AND STORE-INDEPENDENT MECHANISMS

Article

Jun 1995

Feedback regulation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole cell recording: and [Ca2+](i) measurement techniques. CRAC channels were activated by passively depleting intracellular Ca2+ stores in the absence of extracellular Ca2+. Under conditions of moderate intracellular Ca2+ buffering, elevating [Ca2+](o) to 22 mM initiated an inward current through CRAC channels that declined slowly with a half-time of similar to 30 s. This slow inactivation was evoked by a rise in [Ca2+]i, as it was effectively suppressed by an elevated level of EGTA in the recording pipette that prevented increases in [Ca2+](i). Blockade of Ca2+ uptake into stores by thapsigargin with or without intracellular inositol 1,4,5-trisphosphate reduced the extent of slow inactivation by similar to 50%, indicating that store refilling normally contributes significantly to this process. The store-independent (thapsigargin-insensitive) portion of slow inactivation was largely prevented by the protein phosphatase inhibitor, okadaic acid, land by a structurally related compound, 1-norokadaone, but not by calyculin A nor by cyclosporin A and FK506 at concentrations that fully inhibit calcineurin (protein phosphatase 2B) in T cells. These results argue against the involvement of protein phosphatases 1, 2A, 2B, or 3 in store-independent inactivation, We conclude that calcium acts through at least two slow negative feedback pathways to inhibit CRAC channels. Slow feedback inhibition of CRAC current is likely to play important roles in controlling the duration and dynamic behavior of receptor-generated Ca2+ signals.

Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells

Article

Dec 1989
Cell Regul

A rapid rise in the level of cytosolic free calcium ([Ca2+]i) is believed to be one of several early triggering signals in the activation of T lymphocytes by antigen. Although Ca2+ release from intracellular stores and its contribution to Ca2+ signaling in many cell types is well documented, relatively little is known regarding the role and mechanism of Ca2+ entry across the plasma membrane. We have investigated mitogen-triggered Ca2+ signaling in individual cells of the human T-leukemia-derived line, Jurkat, using fura-2 imaging and patch-clamp recording techniques. Phytohemagglutinin (PHA), a mitogenic lectin, induces repetitive [Ca2+]i oscillations in these cells peaking at micromolar levels with a period of 90-120 s. The oscillations depend critically upon Ca2+ influx across the plasma membrane, as they are rapidly terminated by removal of extracellular Ca2+, addition of Ca(2+)-channel blockers such as Ni2+ or Cd2+, or membrane depolarization. Whole-cell and perforated-patch recording methods were combined with fura-2 measurements to identify the mitogen-activated Ca2+ conductance involved in this response. A small, highly selective Ca2+ conductance becomes activated spontaneously in whole-cell recordings and in response to PHA in perforated-patch experiments. This conductance has properties consistent with a role in T-cell activation, including activation by PHA, lack of voltage-dependent gating, inhibition by Ni2+ or Cd2+, and regulation by intracellular Ca2+. Moreover, a tight temporal correlation between oscillations of Ca2+ conductance and [Ca2+]i suggests a role for the membrane Ca2+ conductance in generating [Ca2+]i oscillations in activated T cells.

Current carried by monovalent cations through calcium channels in mouse neoplastic B lymphocytes

Article

Feb 1985

Membrane currents through the Ca2+ channel were studied in a hybridoma cell line (MAb-7B) constructed by fusion of S194 myeloma cells and splenic B lymphocytes from the mouse. The whole-cell variation of the patch-electrode voltage-clamp technique was used. When [Ca2+]o = 2.5 mM, [Na+]o = 150 mM and [Na+]i = 155 mM, the current reversed from inward to outward at 20.9 +/- 2.4 mV (mean +/- S.D., n = 62). Both inward and outward currents showed voltage-dependent inactivation with the same membrane potential dependence of steady-state inactivation. The decay time constant of the current decreased from about 27 ms at -44 mV to a saturation value of 16 ms at about -20 mV, and remained at this value even when the current became outward. From the above results both the inward and outward currents were considered to flow through Ca2+ channels. The inward current showed no change when the external Na+ was replaced with Cs+ or tetraethylammonium and increased when [Ca2+]o was increased. Also, the reversal potential became more positive with increasing [Ca2+]o with a slope of 29 mV/decade change of [Ca2+]o. Effects of different divalent cations examined at 10 mM concentration showed the reversal potential to become more positive in the order of Mn2+, Sr2+ approximately equal to Ba2+ and Ca2+ whereas the relative maximum amplitudes of peak inward current were 1.0 for Ca2+, 1.24 for Sr2+, 0.99 for Ba2+ and 0.07 for Mn2+. When [Ca2+]o or [Mg2+]o was reduced by chelators, monovalent cations became capable of carrying inward current through the Ca2+ channel. These monovalent currents share common kinetic properties with the Ca2+ current, as judged from the steady-state inactivation and the decay time constant of the current. The monovalent cation current was blocked by divalent cations in a voltage-dependent manner. The half-blocking concentrations of Ca2+ and Mg2+ at -45 mV were 2.0 X 10(-6) M and 3.0 X 10(-5) M respectively. The same voltage-dependent binding mechanism can explain the outward current carried by monovalent cations at large positive potentials at normal Ca2+ concentrations. The suppression of the monovalent currents by Ca2+ and Mg2+ showed different voltage dependences. The suppression by Ca2+ increased and then decreased as the membrane potential was made negative, whereas the suppression by Mg2+ increased monotonically. This difference can be explained by considering the fact the Ca2+ is permeant and Mg2+ is impermeant through the Ca2+ channel.

The influence of intracellular calcium concentration on degranulation of dialysed mast cells from rat peritoneum

Article

Feb 1988

Erwin Neher

Mast cells, isolated from rat peritoneum, were studied under tight-seal, whole-cell recording conditions. Membrane conductance, membrane capacitance and the concentration of free intracellular Ca2+, [Ca2+](i), were measured simultaneously. [Ca2+](i) could be accurately buffered to values between 0 and 1.5 μM only if relatively high concentrations of calcium buffers (in the millimolar range) were added to the pipette filling solution against which the cytoplasm was dialysed. At lower buffer concentrations [Ca2+](i) was markedly increased by hyperpolarizing the membrane. When added to the pipette, guanosine-3-thio-triphosphate (GTP-γ-S), a non-hydrolysable analogue of guanosine triphosphate, stimulated a 3.3-fold increase in membrane capacitance, which is indicative of mast cell degranulation (Fernandez, Neher & Gomperts, 1984). In weakly buffered cells, GTP-γ-S also induced a transient increase in [Ca2+](i) which, usually, preceded degranulation. Calcium buffers at 1-5 mM concentration suppressed this transient. High [Ca2+](i) alone did not induce degranulation. However, it markedly accelerated GTP-γ-S-induced degranulation. When [Ca2+](i) was buffered to zero, an appreciable fraction of cells degranulated in response to GTP-γ-S, but very slowly, and only after a long lag phase. Transient increases in [Ca2+](i), evoked either by GTP-γ-S, or by voltage changes, did not elicit capacitance changes during the lag phase, but accelerated the GTP-γ-S-induced degranulation response at later times. Internally applied inositol 1,4,5-trisphosphate (IP3) also induced transient increases in [Ca2+](i) which did not lead to secretion in the absence of GTP-γ-S. It is concluded that an increase in [Ca2+](i) is neither necessary nor sufficient for secretion from dialysed mast cells. [Ca2+](i), however, acts synergistically with other stimuli to promote secretion. It is the more efficient the more time the other stimulus has been allowed for priming the cell.

A non-selective cation conductance in frog muscle membrane blocked by micromolar external calcium ions

Article

Sep 1984

Membrane currents were recorded from voltage-clamped, EGTA-loaded muscle fibres under conditions where currents through ordinary Na+, K+ and Cl- channels were prevented by drugs or by absence of permeant ions (K+, and Cl-). At 10 mM-external [Ca2+], substitution of Na+ for the large and presumably impermeant organic cations tetramethyl- (TMA+) or tetraethylammonium (TEA+) failed to increase peak inward current. Hence the Ca2+ channel was not significantly permeable to Na+ under these conditions. When external [Ca2+] was reduced to levels below 1 microM in the presence of external Na+, step depolarizations to negative potentials produced tetrodotoxin-resistant inward currents. At -20 mV, they rose to a peak of 30-200 microA/cm2 within 150 ms and declined thereafter. Ca2+ and several other divalent cations reversibly blocked this inward current. The sequence of blocking potencies was Ca2+ greater than Sr2+ greater than or equal to Co2+ greater than Mn2+ congruent to Cd2+ greater than Ni2+ congruent to Mg2+. Large inward currents may be carried by Li+, Na+, K+, Rb+ and Cs+ but not by TMA+ and TEA+. The effect of external Ca2+ ([Ca2+]o) was explored over a 10(8)-fold range in concentrations. Na+ was present at a fixed concentration. When [Ca2+]o was gradually increased from 10(-10) to 10(-2) M, inward current first diminished 10-fold, reached a minimum at [Ca2+]o = 60 microM and then increased again as [Ca2+]o was increased further and Ca2+ itself became a current carrier. Block of inward current at [Ca2+]o less than 10(-5) M could be described by binding of a single Ca2+ to a site, with a dissociation constant of the order of 0.7 microM at -20 mV.

Mechanism of ion permeation through ion channels

Article

May 1984

Calcium channels carry out vital functions in a wide variety of excitable cells but they also face special challenges. In the medium outside the channel, Ca2+ ions are vastly outnumbered by other ions. Thus, the calcium channel must be extremely selective if it is to allow Ca2+ influx rather than a general cation influx. In fact, calcium channels show a much greater selectivity for Ca2+ than sodium channels do for Na+ despite the high flux that open Ca channels can support. Relatively little is known about the mechanism of ion permeation through Ca channels. Earlier models assumed ion independence or single-ion occupancy. Here we present evidence for a novel hypothesis of ion movement through Ca channels, based on measurements of Ca channel activity at the level of single cells or single channels. Our results indicate that under physiological conditions, the channel is occupied almost continually by one or more Ca2+ ions which, by electrostatic repulsion, guard the channel against permeation by other ions. On the other hand, repulsion between Ca2+ ions allows high throughput rates and tends to prevent saturation with calcium.

POTASSIUM and calcium channels in lymphocytes

Article

Feb 1995

Over the past decade, a variety of ion channels have been identified and characterized in lymphocytes by use of the patch-clamp technique. This review discusses biophysical and regulatory aspects of lymphocyte potassium and calcium channels with the aim of understanding the role of these channels in lymphocyte functions. Lymphocytes express both voltage-dependent potassium [K(V)] channels and calcium-activated potassium [K(Ca)] channels, and each is upregulated as cells progress toward division following mitogenic stimulation. The genes encoding two K(V) channels, Kv1.3 (type n) and Kv3.1 (type l), have been cloned. Mutational analysis is revealing functionally important regions of these channel proteins. Exogenous expression studies and the use of highly specific channel blockers have helped to establish the roles of type n K(V) channels in sustaining the resting membrane potential, in regulating cell volume, and in enabling lymphocyte activation. Blockade of K(V) and K(Ca) channels effectively inhibits the antigen-driven activation of lymphocytes, probably by inducing membrane depolarization and thereby diminishing calcium influx. A prolonged rise in intracellular calcium ([Ca2+]i) is a required signal for lymphocyte activation by antigen or mitogens. Single-cell fluorescence measurements have revealed underlying [Ca2+]i oscillations that are linked closely to the opening and closing of Ca2+ and K+ channels. Sustained Ca2+ signaling and oscillations depend absolutely on plasma-membrane Ca2+ channels that are activated by the depletion of intracellular calcium stores. Under physiological conditions these channels open as a consequence of store depletion induced by inositol 1,4,5-trisphosphate (IP3), but they can also be activated experimentally by several agents that empty the stores without generating IP3, such as the microsomal Ca(2+)-ATPase inhibitor thapsigargin. The intricate causal relationships among ion channels, membrane potential, [Ca2+]i, and lymphokine gene expression can now be pursued at the single-cell level with patch-clamp recording, calcium-dependent dyes, reporter genes, and fluorescence video techniques. These approaches will help to clarify the essential roles of ion channels in the molecular pathways subserving activation and other lymphocyte behaviors.

Primaquine, an inhibitor of vesicular transport, blocks the calcium-release-activated current in rat megakaryocytes

Article

Aug 1995

The whole-cell patch-clamp technique was used to study the effect of primaquine, an inhibitor of vesicular transport, on the calcium-release-activated current (Icrac) in rat megakaryocytes. Addition of primaquine, before emptying of internal Ca2+ stores by ionomycin, prevented the development of Icrac, with a half-maximal concentration of near 100 microM. Maximal inhibition (> or = 83%) was observed at 0.6-1 mM primaquine. At 1 mM, chloroquine, a related compound which is less effective at blocking vesicular secretion, had no effect on Icrac. Primaquine (0.8 mM) added after sustained activation of Icrac caused a gradual block of current, with maximal inhibition of 50% observed after 2-3 min. At 1 mM, internal guanosine 5'-[gamma-thio]triphosphate reduced Icrac by 65 +/- 13%. Neither 1 mM GTP nor 2 mM guanosine 5'-[beta-thio]diphosphate had any significant effect on Icrac. The recognized role of GTPases in the regulation of vesicular trafficking, together with block of Icrac activation by primaquine, provide evidence that the channels carrying Icrac may be stored in a vesicular membrane compartment and transferred to the plasma membrane following store depletion.

Immunoglobulin E receptor-activated calcium conductance in rat mast cells

Article

Feb 1995

1. The nystatin perforated-patch method was used to record macroscopic currents from anti-trinitrophenyl (TNP) immunoglobulin E (IgE)-sensitized rat basophilic leukaemia (RBL-2H3) cells at 37 degrees C. 2. An inwardly rectifying Ca2+ current (ICa) was activated upon stimulation with the multivalent antigen trinitrophenylated bovine serum albumin (TNP-BSA). Induction of ICa was not observed at room temperature. ICa was reversed and reinduced upon cyclical addition of the monovalent hapten dinitrophenyl (DNP)-lysine and multivalent antigen, indicating that a specific interaction of antigen with IgE was required to elicit ICa. 3. The antigen-induced current was also carried by Ba2+ or Sr2+, and to a lesser extent by Na+, in the nominal absence of Ca2+. ICa did not exhibit time-dependent opening (< or = 1 ms) in response to hyperpolarizing voltage steps to -100 mV, although it did accumulate steady-state inactivation of approximately 40-50% over 100 ms. 4. Two inorganic blockers of antigen-stimulated 45Ca2+ influx and secretion, La3+ and Zn2+, inhibited ICa by approximately 50% at concentrations known to produce 50% block of 45Ca2+ influx. In contrast, cromolyn sodium (0.5 mM) and the L-type Ca2+ channel antagonist nitrendipine (5 microM) had no effect on ICa. 5. ICa also was induced by the intracellular Ca2+ mobilizer thapsigargin. Because the actions of thapsigargin and antigen were not additive, IgE receptor cross-linkage appears to activate the recently described capacitative Ca2+ entry channels.

Calcium channels activated by depletion of internal calcium stores in A431 cells

Article

Aug 1994

Depletion of intracellular calcium stores induces transmembrane Ca2+ influx. We studied Ca(2+)- and Ba(2+)-permeable ion channels in A431 cells after store depletion by dialysis of the cytosol with 10 mM BAPTA solution. Cell-attached patches of cells held at low (0.5 microM) external Ca2+ exhibited transient channel activity, lasting for 1-2 min. The channel had a slope conductance of 2 pS with 200 mM CaCl2 and 16 pS with 160 mM BaCl2 in the pipette. Channel activity quickly ran down in excised inside-out patches and was not restored by InsP3 and/or InsP4. Thapsigargin induced activation in cells kept in 1 mM external Ca2+ after BAPTA dialysis. These channels represent one Ca2+ entry pathway activated by depletion of internal calcium stores and are clearly distinct from previously identified calcium repletion currents.

Intracellular calcium dependence of gene expression in single T lymphocyte

Article

Apr 1994

In T lymphocytes, intracellular Ca2+ concentration ([Ca2+]i) rises within seconds of T-cell antigen-receptor stimulation and initiates the synthesis and secretion of interleukin 2, a cytokine essential for T-cell proliferation and the immune response. Using video-imaging techniques, we tracked [Ca2+]i signals in individual T cells and measured subsequent expression of a beta-galactosidase reporter gene (lacZ) controlled by the NF-AT element of the interleukin 2 enhancer. [Ca2+]i spikes elicited by monoclonal antibody binding to the CD3 epsilon subunit of the T-cell receptor were positively correlated with gene expression, but varied widely between individual cells and were therefore difficult to relate quantitatively to lacZ expression. The [Ca2+]i dependence of NF-AT-regulated gene expression was determined by elevating [Ca2+]i with either thapsigargin or ionomycin and then "clamping" [Ca2+]i to various, stable levels by altering either extracellular [Ca2+] or extracellular [K+]. Raising [Ca2+]i from resting levels of 70 nM to between 200 nM and 1.6 microM increased the fraction of cells expressing lacZ, with Kd approximately 1 microM. Activation of protein kinase C enhanced the [Ca2+]i sensitivity of gene expression (Kd = 210 nM), whereas stimulation of protein kinase A inhibited [Ca2+]i-dependent gene expression. The experiments described here provide single-cell measurements linking a second messenger to gene expression in individual cells.

Receptor-activated Ca2+ influx: How many mechanisms for how many channels?

Article

Apr 1994
TRENDS PHARMACOL SCI

Receptors that are coupled to the production of inositol (1,4,5)-trisphosphate cause an increase in cytosolic free Ca2+ concentration as a consequence of both Ca2+ mobilization from intracellular stores and Ca2+ influx through the plasma membrane. Although this latter phenomenon appears attributable to the activation of a number of Ca(2+)-permeable channels, the channels that are controlled by the Ca2+ content of the intracellular stores have recently received much attention. In this review, Cristina Fasolato, Barbara Innocenti and Tullio Pozzan summarize the characteristics of this Ca(2+)-influx pathway and discuss the hypotheses about its mechanism of activation and its relationship with other receptor-activated Ca2+ channels.

Activation of Ca2+ currents in Jurkat T cells following the Depletion of Ca2+ stores by microsomal Ca2+-ATPase Inhibitor

Article

Jun 1994

The mechanism of TCR-stimulated Ca2+ influx was studied in the Jurkat human T cell line using Ca2+ indicator dyes and whole-cell patch clamp. Ca2+ influx induced by inositol 1,4,5-triphosphate (IP3)-coupled surface receptors (either the TCR or a heterologous muscarinic receptor) was compared with Ca2+ influx induced by inhibitors of the microsomal Ca(2+)-ATPase (thapsigargin, cyclopiazonic acid, di-tert-butylhydroquinone), which release stored Ca2+ without production of IP3. The same Ca2+ influx pathway could be activated by IP3-dependent or IP3-independent means, and therefore appeared to be regulated by the fullness of the microsomal Ca2+ stores rather than by the direct action of IP3. Depletion of stored Ca2+ by either receptor stimulation or microsomal Ca(2+)-ATPase inhibition activated a low conductance, Ca(2+)-selective, non-voltage-activated membrane current. Ca2+ currents induced by receptor stimulation and Ca(2+)-ATPase inhibition were not additive. Several properties of the depletion-activated Ca2+ current suggest that it is carried by a novel type of Ca2+ channel rather than an electrogenic carrier or pump. The conductance saturated when external Ca2+ was raised (Kd approximately 2 mM) and became highly permeable to monovalent cations when external Ca2+ was lowered to below 100 nM, much as has been observed for some voltage-gated Ca2+ channels. The Ca2+ current was reversibly blocked by > 90% with 0.3 mM Cd2+, whereas the same concentration of Ni2+ or Co2+ blocked only 50 to 60% of the current. However, the absence of voltage-dependent activation, relative conductance sequence for divalent cations (Ca2+ > Ba2+ approximately Sr2+ > Mn2+), and lack of inhibition by nifedipine, D600, diltiazem, delta-conotoxin, or aga-IVa were unlike that of voltage-gated Ca2+ channels.

Calcium release-activated calcium current (ICRAC) in rat mast cells

Article

Jul 1993

1. Whole-cell patch clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study the biophysical properties of a calcium current activated by depletion of intracellular calcium stores in rat peritoneal mast cells. 2. Calcium influx through an inward calcium release-activated calcium current (ICRAC) was induced by three independent mechanisms that result in store depletion: intracellular infusion of inositol 1,4,5-trisphosphate (InsP3) or extracellular application of ionomycin (active depletion), and intracellular infusion of calcium chelators (ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)) to prevent reuptake of leaked-out calcium into the stores (passive depletion). 3. The activation of ICRAC induced by active store depletion has a short delay (4-14 s) following intracellular infusion of InsP3 or extracellular application of ionomycin. It has a monoexponential time course with a time constant of 20-30 s and, depending on the complementary Ca2+ buffer, a mean normalized amplitude (at 0 mV) of 0.6 pA pF-1 (with EGTA) and 1.1 pA pF-1 (with BAPTA). 4. After full activation of ICRAC by InsP3 in the presence of EGTA (10 mM), hyperpolarizing pulses to -100 mV induced an instantaneous inward current that decayed by 64% within 50 ms. This inactivation is probably mediated by [Ca2+]i, since the decrease of inward current in the presence of the fast Ca2+ buffer BAPTA (10 mM) was only 30%. 5. The amplitude of ICRAC was dependent on the extracellular Ca2+ concentration with an apparent dissociation constant (KD) of 3.3 mM. Inward currents were nonsaturating up to -200 mV. 6. The selectivity of ICRAC for Ca2+ was assessed by using fura-2 as the dominant intracellular buffer (at a concentration of 2 mM) and relating the absolute changes in the calcium-sensitive fluorescence (390 nm excitation) with the calcium current integral. This relationship was almost identical to the one determined for Ca2+ influx through voltage-activated calcium currents in chromaffin cells, suggesting a similar selectivity. Replacing Na+ and K+ by N-methyl-D-glucamine (with Ca2+ ions as exclusive charge carriers) reduced the amplitude of ICRAC by only 9% further suggesting a high specificity for Ca2+ ions. 7. The current amplitude was not greatly affected by variations of external Mg2+ in the range of 0-12 mM. Even at 12 mM Mg2+ the current amplitude was reduced by only 23%. 8. ICRAC was dose-dependently inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx

Article

Sep 1993

Intracellular Ca2+ signals that last more than a few minutes after the onset of stimulation depend critically on influx of extracellular Ca2+. Such Ca2+ influx can be triggered in many cell types by depletion of intracellular Ca2+ stores without detectable elevations of known messengers. The mechanism by which store depletion can control plasma membrane Ca2+ permeability remains controversial. Here we present evidence for a novel soluble mediator. Calcium depletion of a lymphocyte cell line caused the messenger to be released from intracellular organelles into the cytoplasm and to a much lesser extent into the extracellular medium. The messenger caused Ca2+ influx when applied to macrophages, astrocytoma cells, and fibroblasts and was therefore named CIF (for Ca(2+)-influx factor). CIF appears to have hydroxyls (or hydroxyl and amino groups) on adjacent carbons, a phosphate, and a M(r) under 500.

Zweifach, A. & Lewis, R. S. Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ store. Proc. Natl Acad. Sci. USA 90, 6295-6299

Article

Jul 1993

Stimulated influx of Ca2+ across the plasma membrane of T lymphocytes is an essential triggering signal for T-cell activation by antigen. Regulation of the T-cell Ca2+ conductance is not understood; conflicting evidence supports direct activation by inositol 1,4,5-trisphosphate (IP3) or by a signal generated by the depletion of intracellular Ca2+ stores. We have used the perforated-patch recording technique to compare the biophysical properties of Ca2+ currents activated by T-cell receptor stimulation and by thapsigargin, a Ca(2+)-ATPase inhibitor that depletes intracellular stores without generating IP3. Both currents are blocked by Ni2+, are inwardly rectifying, are highly Ca(2+)-selective, and exhibit voltage-independent gating with a unitary chord conductance of approximately 24 fS in isotonic Ca2+. Fluctuation analysis suggests that the underlying Ca2+ transporter is a channel rather than an iron carrier. Thus, in terms of ion permeation, gating, and unitary conductance, the Ca2+ current activated by thapsigargin is indistinguishable from the elicited by crosslinking of T-cell receptors. Moreover, the unitary Ca2+ conductance is > 100-fold smaller than that of previously described IP3-gated, Ca(2+)-permeable channels in T cells [Kuno, M. & Gardner, P. (1987) Nature (London) 326, 301-304]. These results demonstrate that mitogen-activated Ca2+ influx is controlled by the state of intracellular Ca2+ stores rather than by the direct action of IP3 on Ca2+ channels in the plasma membrane.

Depletion of InsP3 stores activates a Ca2+ and K+ current by means of small diffusible messenger

Article

Sep 1993

In non-excitable cells, release of Ca2+ from the inositol 1,4,5-trisphosphate (InsP3)-sensitive store can activate Ca2+ entry. Very little is known about the signal mechanism relating store emptying to plasma membrane Ca2+ influx. It has been suggested that the signal may be either a diffusible messenger like an inositol phosphate, or the InsP3 receptor itself, which, by physically coupling to some component of Ca2+ entry in the plasma membrane, may link store release to Ca2+ entry. The nature of the Ca2+ entry pathway is also unclear. Only in mast cells has a very selective Ca2+ current been observed after store emptying. Activation of exogenous 5-hydroxytryptamine (5-HT) receptors expressed in Xenopus oocytes or direct injection of InsP3 evokes Ca2+ entry activated by InsP3 pool depletion. Here we investigate the nature of this influx pathway and find a current activated by pool depletion. This has an unusual selectivity in that it is more permeable to Ca2+ ions than to other divalent cations (Ba2+, Sr2+ or Mn2+). Moreover, a K+ permeability is also stimulated after pool depletion. The activation of this store depletion current involves both a phosphatase and an unidentified diffusible messenger. Both the Ca2+ entry pathway and the activating factors found here may be relevant to pool-depleted Ca2+ entry in a variety of non-excitable cells.

Two different calcium-influx pathways in melanoma cells are cell cycle-dependent Monovalent cation per-meation through stores-dependent Ca21 channels (Icrac) in Jurkat T lymphocytes

Jan 1995
152

A Lepple-Wienhues
M D Cahalan

Lepple-Wienhues, A., and M. D. Cahalan. 1995. Two different calcium-influx pathways in melanoma cells are cell cycle-dependent. Biophys. J. 68:A122. Lepple-Wienhues, A., and M. D. Cahalan. 1996. Monovalent cation per-meation through stores-dependent Ca21 channels (Icrac) in Jurkat T lymphocytes. Biophys. J. 70:A152.

Monovalent cation permeation through stores-dependent Ca21 channels (Icrac) in Jurkat T lymphocytes

Jan 1996
152

A Lepple-Wienhues
M D Cahalan

Lepple-Wienhues, A., and M. D. Cahalan. 1996. Monovalent cation permeation through stores-dependent Ca21 channels (Icrac) in Jurkat T lymphocytes. Biophys. J. 70:A152.

Conductance and permeation of monovalent cations through depletion-activated Ca2+ channels (ICRAC) in Jurkat T cells

Abstract

No full-text available

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