Monovalent Permeability, Rectification, and Ionic Block of Store-operated Calcium Channels in Jurkat T Lymphocytes
Journal of General Physiology (JGP)
Abstract and Figures
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+.
![Divalent and monovalent current through CRAC channels. CRAC channels were activated during dialysis with Na⁺ (A), Cs⁺ (B), or NMDG⁺ (C) aspartate pipette solutions. The superfusion solution was changed from 20 mM to 1 μM Ca²⁺ to induce monovalent current through CRAC channels. Currents were recorded during 200-ms voltage ramps from −120 to +50 mV delivered every second, using a holding potential of 0 mV. Sweeps depicted in A–C represent a current trace with 20 mM Ca²⁺ before CRAC channels activate, and following activation of CRAC channels using Ca²⁺ or Na⁺ as the current carrier. Ca²⁺ and Na⁺ currents through CRAC channels rectify inwardly. Small outward currents carried by Na⁺ (A) or Cs⁺ (B) can also be observed at positive voltages when [Ca²⁺]o is lowered. NMDG⁺ does not carry detectable outward currents (C). (D) Amplitude of the divalent and monovalent current through CRAC channels at −80 and +30 mV plotted against time using NMDG⁺ as internal cation. The bar indicates the main current carrier; note that the Na⁺ current declines after peaking when [Ca²⁺]o is reduced to 1 μM.](https://www.researchgate.net/publication/51317705/figure/fig1/AS:11431281177162073@1690373422088/Divalent-and-monovalent-current-through-CRAC-channels-CRAC-channels-were-activated_Q320.jpg)
![Permeability of CRAC channels to organic cations. CRAC channels were activated during whole-cell dialysis with Na⁺ aspartate. The external solution contained 1 μM Ca²⁺ and 150 mM X⁺, where X⁺ is ammonium (A and B), methylammonium (C), dimethylammonium (D), trimethylammonium (E), tetramethylammonium (F and J), ethylammonium (G), isopropylammonium (H), and NMDG⁺ (I and J). Most panels illustrate currents using voltage ramps as in Fig. 1; note that the current amplitude scales vary. For these panels, three ramp traces show, including one before activation of CRAC channels, Ca²⁺ currents through CRAC channels with 20 mM [Ca²⁺]o, and monovalent currents upon lowering [Ca²⁺]o to 1 μM. (B and J) Current amplitudes at −80 and +30 mV before and after changing the bath solution from 20 mM Ca²⁺ to 1 μM Ca²⁺ containing ammonium (B) or TMA⁺ followed by NMDG⁺ (J). The bars above the current correspond to the main external cation. Note that the NH4⁺ currents are sustained in B. In I and J, there are no detectable inward currents carried by NMDG⁺. TMA⁺ carries a small but detectable inward current (F and J).](https://www.researchgate.net/publication/51317705/figure/fig2/AS:11431281177109742@1690373422540/Permeability-of-CRAC-channels-to-organic-cations-CRAC-channels-were-activated-during_Q320.jpg)
![Effects of internal pH on current kinetics and magnitudes. CRAC channels were activated during dialysis with Na⁺ aspartate pipette solution, titrated to pH 8.2 (A and B); 7.2 (C and D), or 6.2 (E and F). Time courses of currents at −80 and +30 mV are shown on the left, and selected ramp traces illustrating currents carried by Ca²⁺ or Na⁺ are shown on the right. Changing the external solution from 20 mM to 1 μM [Ca²⁺]o induced inward currents carried by Na⁺. Note that the scale for the current varies among the individual figures and that lowering internal pH decreases the current magnitudes and makes the current decline more rapidly. This reduction in current amplitude occurred with either Tris- or HEPES-buffered pipette solution.z](https://www.researchgate.net/publication/51317705/figure/fig4/AS:11431281177133964@1690373424762/Effects-of-internal-pH-on-current-kinetics-and-magnitudes-CRAC-channels-were-activated_Q320.jpg)
![pHi dependence of “inactivation” (A) and magnitude (B) of Na⁺ current through CRAC channels. Solutions as in Fig. 4. (A) Na⁺ currents at −80 mV through CRAC channels 50 s after lowering [Ca²⁺]o (I50) were divided by the maximal Na⁺ currents (Imax) and plotted as a function of pHi. The line was drawn to the equation: I50/Imax = b + (1 − b)/[1 + (pH − pKa)], assuming a pKa of 8.3 and a baseline value b of 0.15. (B) Concentration–response relationship for the block of Ca²⁺ current through CRAC channels by internal protons. The line through the points represents a fit to the equation: I = Imax/[1 + (pH − pKa)], with a pKa of 6.8. Each data point in A and B represents between 12 and 43 cells.](https://www.researchgate.net/publication/51317705/figure/fig5/AS:11431281177171807@1690373424948/pHi-dependence-of-inactivation-A-and-magnitude-B-of-Na-current-through-CRAC_Q320.jpg)
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... Moreover, store-operated Ca 2+ entry was shown to mediate intracellular alkalinization in neutrophils 13 , and extracellular low pH was reported to inhibit I CRAC in macrophages 14 . In Jurkat T-lymphocytes, cytosolic alkalinization induces Ca 2+ release and Ca 2+ entry 15 , and acidic internal and external pH inhibit I CRAC 16 . In SH-SY5Y neuroblastoma cells, however, store-operated Ca 2+ entry was not affected by changes of intracellular pH, even though it was attenuated by low extracellular pH and potentiated by high extracellular pH 17 . ...
... in Jurkat T-lymphocytes and by external alkaline pH in macrophages has been previously reported 14,16 . Moreover, two recent studies have shown that heterologously expressed Orai1/STIM1 currents can be inhibited by external low pH 26,27 and internal low pH 27 , and enhanced by external high pH but not by internal high pH 27 . ...
... Likewise, basic pH o enhances I CRAC currents carried by both Ca 2+ ions and Na + ions (Fig. 2). Similar results have been reported for the regulation of native I CRAC currents by pH 16 . Kerschbaum and colleagues demonstrated that I CRAC currents in Jurkat T lymphocytes are inhibited by acidic extracellular and intracellular pH, and that both Ca 2+ currents and monovalent Na + currents of I CRAC can be equally inhibited by acidic pH in a voltage independent manner 16 . ...
Changes of intracellular and extracellular pH are involved in a variety of physiological and pathological processes, in which regulation of the Ca(2+) release activated Ca(2+) channel (ICRAC) by pH has been implicated. Ca(2+) entry mediated by ICRAC has been shown to be regulated by acidic or alkaline pH. Whereas several amino acid residues have been shown to contribute to extracellular pH (pHo) sensitivity, the molecular mechanism for intracellular pH (pHi) sensitivity of Orai1/STIM1 is not fully understood. By investigating a series of mutations, we find that the previously identified residue E106 is responsible for pHo sensitivity when Ca(2+) is the charge carrier. Unexpectedly, we identify that the residue E190 is responsible for pHo sensitivity when Na(+) is the charge carrier. Furthermore, the intracellular mutant H155F markedly diminishes the response to acidic and alkaline pHi, suggesting that H155 is responsible for pHi sensitivity of Orai1/STIM1. Our results indicate that, whereas H155 is the intracellular pH sensor of Orai1/STIM1, the molecular mechanism of external pH sensitivity varies depending on the permeant cations. As changes of pH are involved in various physiological/pathological functions, Orai/STIM channels may be an important mediator for various physiological and pathological processes associated with acidosis and alkalinization.
... In the meantime, ironically the founding member of the family, dTrp, was definitively shown to be store independent (313). Additional confusion arose when the use of Mg 2ϩ -free intracellular solutions inadvertently resulted in the slow induction of Mg 2ϩ -inhibited cation (MIC/TRPM7 channel) currents (140,177,301) that were initially mistaken for CRAC channel currents (169,170). ...
... Acidification of the extracellular pH strongly inhibits I CRAC in macrophages with a pK a of ϳ8.2 (217). CRAC channels are also inhibited by intracellular acidification with a pK a of 6.8, resulting in an approximately fivefold inhibition between pH 8.2 and 6.2 without a noticeable change in ion selectivity (169). Intracellular acidification also speeds the rate and extent of depotentiation of Na ϩ -I CRAC (169). ...
... CRAC channels are also inhibited by intracellular acidification with a pK a of 6.8, resulting in an approximately fivefold inhibition between pH 8.2 and 6.2 without a noticeable change in ion selectivity (169). Intracellular acidification also speeds the rate and extent of depotentiation of Na ϩ -I CRAC (169). Interestingly, the pK a of the extracellular and intracellular pH effects are very similar to those reported for L-type Ca V channels, raising the possibility that the molecular mechanisms of pH regulation could be similar in the two classes of channels. ...
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.
... There is no doubt that ionic homeostasis plays an integral role in maintaining the structural and functional integrity of both adaptive and innate cells of the immune system. There are five prevalent ionic currents corresponding to five distinct chan nels -K v 1.3 10,14-16 , K Ca 3.1 17 , CRAC channel 8 , TRPM7 [18][19][20] , and Cl Swell 21 -recorded in T cells 5 and associated with their activation 5 . Apart from Ca 2+ , which plays a predomi nant role in immune cell signaling, many other ions such as Zn 2+ , Mg 2+ , K + , Na + , and Cl − have been shown to be essential for B cell development, activation, and differ entiation into effector B cells 22 . ...
... potentiated ORAI1 and ORAI2, with marginal effect on ORAI3. Early studies, most of which predate the discovery of STIM and ORAI proteins have reported the regulation of native I CRAC by both extracellular and intracellular pH in Jurkat T-cells, macrophages and neutrophils [58][59][60][61]. These reports showed that extracellular and intracellular acidification inhibits while alkalization enhances I CRAC . ...
The ubiquitous Ca²⁺ release-activated Ca²⁺ (CRAC) channel is crucial to many physiological functions. Both gain and loss of CRAC function is linked to disease. While ORAI1 is a crucial subunit of CRAC channels, recent evidence suggests that ORAI2 and ORAI3 heteromerize with ORAI1 to form native CRAC channels. Furthermore, ORAI2 and ORAI3 can form CRAC channels independently of ORAI1, suggesting diverse native CRAC stoichiometries. Yet, most available CRAC modifiers are presumed to target ORAI1 with little knowledge of their effects on ORAI2/3 or heteromers of ORAIs. Here, we used ORAI1/2/3 triple-null cells to express individual ORAI1, ORAI2, ORAI3 or ORAI1/2/3 concatemers. We reveal that GSK-7975A and BTP2 essentially abrogate ORAI1 and ORAI2 activity while causing only a partial inhibition of ORAI3. Interestingly, Synta66 abrogated ORAI1 channel function, while potentiating ORAI2 with no effect on ORAI3. CRAC channel activities mediated by concatenated ORAI1-1, ORAI1-2 and ORAI1-3 dimers were inhibited by Synta66, while ORAI2-3 dimers were unaffected. The CRAC enhancer IA65 significantly potentiated ORAI1 and ORAI1-1 activity with marginal effects on other ORAIs. Further, we characterized the profiles of individual ORAI isoforms in the presence of Gd³⁺ (5µM), 2-APB (5µM and 50µM), as well as changes in intracellular and extracellular pH. Our data reveal unique pharmacological features of ORAI isoforms expressed in an ORAI-null background and provide new insights into ORAI isoform selectivity of widely used CRAC pharmacological compounds.
... At the same time Kerrshbaum and Cahalan (Kerschbaum and Cahalan, 1998)recorded a much larger and non-inactivating monovalent current when internal Mg 2+ was also omitted. The presumed CRAC single channel conductance in monovalent solution was 35-49 pS in size The monovalent channel conduction was 40 times higher than in the presence of [Ca 2+ ] o solution and blocked in a voltage-dependent manner. ...
The main property of neuronal and other excitable cells is their capability to transform excitatory waves into intracellular signals, where they trigger or modulate practically all cellular functions. Influx of calcium ions from the extracellular medium (“calcium signals„) plays a key role in this process. Correspondingly alterations in intracellular calcium signaling are an important component of the physiological process of aging and of the most frequent and complicated forms of pathology, and their clarification is of basic medical importance. Therefore in the present paper we will discuss the main molecular mechanisms determining such signaling as well as their possible alterations
... Two studies in Jurkat T lymphocytes suggest that extracellular pH modifies the mitochondrial control of SOCE in Jurkat cells [41,42]. Others predict a similar mechanism as assumed for L-type Ca 2+ channels via the protonation of negatively charged glutamate residues close to the channel pore [43,44]. The glutamate residue E106, which is known to contribute to the selectivity filter of multimeric Orai1 channels [45][46][47], appears to be involved in the pH dependence of heterologously expressed STIM1/Orai1 CRAC channels [48]. ...
Deviations from physiological pH (∼pH 7.2) as well as altered Ca(2+) signaling play important roles in immune disease and cancer. One of the most ubiquitous pathways for cellular Ca(2+) influx is the store-operated Ca(2+) entry (SOCE) or Ca(2+) release-activated Ca(2+) current (ICRAC), which is activated upon depletion of intracellular Ca(2+) stores. We here show that extracellular and intracellular changes in pH regulate both endogenous ICRAC in Jurkat T lymphocytes and RBL2H3 cells, and heterologous ICRAC in HEK293 cells expressing the molecular components STIM1/2 and Orai1/2/3 (CRACM1/2/3). We find that external acidification suppresses, and alkalization facilitates IP3-induced ICRAC. In the absence of IP3, external alkalization did not elicit endogenous ICRAC but was able to activate heterologous ICRAC in HEK293 cells expressing Orai1/2/3 and STIM1 or STIM2. Similarly, internal acidification reduced IP3-induced activation of endogenous and heterologous ICRAC, while alkalization accelerated its activation kinetics without affecting overall current amplitudes. Mutation of two aspartate residues to uncharged alanine amino acids (D110/112A) in the first extracellular loop of Orai1 significantly attenuated both the inhibition of ICRAC by external acidic pH as well as its facilitation by alkaline conditions. We conclude that intra- and extracellular pH differentially regulates ICRAC. While intracellular pH might affect aggregation and/or binding of STIM to Orai, external pH seems to modulate ICRAC through its channel pore, which in Orai1 is partially mediated by residues D110 and D112.
... In endogenous systems, in addition to the characteristic I/V, Ca 2+ selectivity and pharmacological tools should be used to confirm the identity of the measured currents. Sometimes small outward currents can be observed, which can either be due to some monovalent permeability through CRAC channels (Hoth 1996;Kerschbaum and Cahalan 1998) or due to other ion channels, whose amplitude varies over time. ...
Depletion of internal Ca(2+) stores activates store-operated Ca(2+) channels. The most prominent members of this class of channels are Ca(2+) release-activated Ca(2+) (CRAC) channels, which are present in a variety of cell types including immune cells. CRAC channels are composed of ORAI proteins, which are activated by endoplasmic reticulum-bound STIM proteins on Ca(2+) store depletion. The underlying Ca(2+) current is called ICRAC, which is required for many cellular functions including T-cell activation, mast cell activation, Ca(2+)-dependent gene expression, and refilling of internal Ca(2+) stores. To analyze ICRAC or the Ca(2+) current through heterologously expressed ORAI channels, whole-cell patch clamp is the technique of choice. It allows the direct analysis of ion currents through CRAC/ORAI channels. The patch-clamp technique has been used to determine selectivity, permeability, rectification, inactivation, and several other biophysical and pharmacological properties of the channels, and is the most direct and reliable technique to analyze ICRAC.
... In endogenous systems, in addition to the characteristic I/V, Ca 2+ selectivity and pharmacological tools should be used to confirm the identity of the measured currents. Sometimes small outward currents can be observed, which can either be due to some monovalent permeability through CRAC channels (Hoth 1996;Kerschbaum and Cahalan 1998) or due to other ion channels, whose amplitude varies over time. ...
Although ICRAC and other store-operated currents are often analyzed by Ca(2+) imaging, whole-cell patch clamp, described here, is the preferred technique to analyze ICRAC whenever possible. The whole-cell patch-clamp protocol can even be used to record endogenous ICRAC in primary cells. The small endogenous current size of ICRAC requires some precautions: First, it is important to inhibit potential interferences from other channels in the cell by carefully choosing the combination of pipette and bath solutions. Second, the noise should be <150 fA root mean square (RMS) when the pipette holder (with its wire) with or without a patch pipette is adjusted over (but not in!) the solution using a high amplification gain (50 mV/pA or higher) of the patch-clamp amplifier. In addition, this protocol draws attention to measures that should be considered when recording ICRAC currents from an overexpression system. This protocol also suggests sets of solutions that can be used for distinguishing ICRAC from potentially interfering currents. In addition to the solutions, the identity of ICRAC can be confirmed by the characteristic inward rectification, its high Ca(2+) selectivity, and the reversal potential of more than +50 mV. A few (mostly nonspecific) CRAC channel blockers are known, which can also be applied for characterization purposes.
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.
Signaling molecules produced in the pancreatic β-cell following mitochondrial oxidation of glycolytic intermediate metabolites and oxidative phosphorylation trigger Ca2+-dependent signaling pathways that regulate insulin exocytosis. Much is known about ATP-sensitive K+ and voltage-gated Ca2+ currents that contribute to Ca2+-dependent signal transduction in β-cells and insulin secretion, but relatively little is known about other Ca2+ channels that regulate β-cell Ca2+ signaling dynamics and insulin secretion. In a wide range of eukaryotic cells, store-operated Ca2+ entry (SOCE) plays a critical role regulating spatial and temporal changes in cytoplasmic Ca2+ concentration, endoplasmic reticulum (ER) Ca2+ homeostasis, gene expression, protein biosynthesis, and cell viability. Although SOCE has been proposed to play important roles in β-cell Ca2+ signaling and insulin secretion, the underlying molecular mechanisms remain undefined. In this chapter, we provide both an overview of our current understanding of ionic currents regulated by ER Ca2+ stores in insulin-secreting cells and a review of studies in other cell systems that have identified the molecular basis and regulation of SOCE.
We studied the blocking actions of external Ca2+, Mg2+, Ca2+, and other multivalent ions on single Ca channel currents in cell-attached patch recordings from guinea pig ventricular cells. External Cd or Mg ions chopped long-lasting unitary Ba currents promoted by the Ca agonist Bay K 8644 into bursts of brief openings. The bursts appear to arise from discrete blocking and unblocking transitions. A simple reaction between a blocking ion and an open channel was suggested by the kinetics of the bursts: open and closed times within a burst were exponentially distributed, the blocking rate varied linearly with the concentration of blocking ion, and the unblocking rate was more or less independent of the blocker concentration. Other kinetic features suggested that both Cd2+ and Mg2+ lodge within the pore. The unblocking rate was speeded by membrane hyperpolarization or by raising the Ba concentration, as if blocking ions were swept into the myoplasm by the applied electric field or by repulsive interaction with Ba2+. Ca ions reduced the amplitude of unitary Ba currents (50% inhibition at approximately 10 mM [Ca]o with 50 mM [Ba]o) without detectable flicker, presumably because Ca ions exit the pore very rapidly following Ba entry. However, Ca2+ entry and exit rates could be resolved when micromolar Ca blocked unitary Li+ fluxes through the Ca channel. The blocking rate was essentially voltage independent, but varied linearly with Ca concentration (rate coefficient, 4.5 X 10(8) M-1s-1); evidently, the initial Ca2+-pore interaction is outside the membrane field and much faster than the overall process of Ca ion transfer. The unblocking rate did not vary with [Ca]o, but increased steeply with membrane hyperpolarization, as if blocking Ca ions were driven into the cell. We suggest that Ca is both an effective permeator and a potent blocker because it dehydrates rapidly (unlike Mg2+) and binds to the pore with appropriate affinity (unlike Cd2+). There appears to be no sharp dichotomy between "permeators" and "blockers," only quantitative differences in how quickly ions enter and leave the pore.
During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca2+ homeostasis and the physiological roles of CCE.
The responses of vertebrate neurones to glutamate involve at least three receptor types. One of these, the NMDA receptor (so called because of its specific activation by N-methyl-D-aspartate), induces responses presenting a peculiar voltage sensitivity. Above resting potential, the current induced by a given dose of glutamate (or NMDA) increases when the cell is depolarized. This is contrary to what is observed at classical excitatory synapses, and recalls the properties of 'regenerative' systems like the Na+ conductance of the action potential. Indeed, recent studies of L-glutamate, L-aspartate and NMDA-induced currents have indicated that the current-voltage (I-V) relationship can show a region of 'negative conductance' and that the application of these agonists can lead to a regenerative depolarization. Furthermore, the NMDA response is greatly potentiated by reducing the extracellular Mg2+ concentration [( Mg2+]o) below the physiological level (approximately 1 mM). By analysing the responses of mouse central neurones to glutamate using the patch-clamp technique, we have now found a link between voltage sensitivity and Mg2+ sensitivity. In Mg2+-free solutions, L-glutamate, L-aspartate and NMDA open cation channels, the properties of which are voltage independent. In the presence of Mg2+, the single-channel currents measured at resting potential are chopped in bursts and the probability of opening of the channels is reduced. Both effects increase steeply with hyperpolarization, thereby accounting for the negative slope of the I-V relationship of the glutamate response. Thus, the voltage dependence of the NMDA receptor-linked conductance appears to be a consequence of the voltage dependence of the Mg2+ block and its interpretation does not require the implication of an intramembrane voltage-dependent 'gate'.
Hydrogen ions are important regulators of ion flux through voltage-gated Ca2+ channels but their site of action has been controversial. To identify molecular determinants of proton block of L-type Ca2+ channels, we combined site-directed mutagenesis and unitary current recordings from wild-type (WT) and mutant L-type Ca2+ channels expressed in Xenopus oocytes. WT channels in 150 mM K+ displayed two conductance states, deprotonated (140 pS) and protonated (45 pS), as found previously in native L-type Ca2+ channels. Proton block was altered in a unique fashion by mutation of each of the four P-region glutamates (EI-EIV) that form the locus of high affinity Ca2+ interaction. Glu(E)-->Gln(Q) substitution in either repeats I or III abolished the high-conductance state, as if the titration site had become permanently protonated. While the EIQ mutant displayed only an approximately 40 pS conductance, the EIIIQ mutant showed the approximately 40 pS conductance plus additional pH-sensitive transitions to an even lower conductance level. The EIVQ mutant exhibited the same deprotonated and protonated conductance states as WT, but with an accelerated rate of deprotonation. The EIIQ mutant was unusual in exhibiting three conductance states (approximately 145, 102, 50 pS, respectively). Occupancy of the low conductance state increased with external acidification, albeit much higher proton concentration was required than for WT. In contrast, the equilibrium between medium and high conductance levels was apparently pH-insensitive. We concluded that the protonation site in L-type Ca2+ channels lies within the pore and is formed by a combination of conserved P-region glutamates in repeats I, II, and III, acting in concert. EIV lies to the cytoplasmic side of the site but exerts an additional stabilizing influence on protonation, most likely via electrostatic interaction. These findings are likely to hold for all voltage-gated Ca2+ channels and provide a simple molecular explanation for the modulatory effect of H+ ions on open channel flux and the competition between H+ ions and permeant divalent cations. The characteristics of H+ interactions advanced our picture of the functional interplay between P-region glutamates, with important implications for the mechanism of Ca2+ selectivity and permeation.
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.
The relative permeability of endplate channels to many organic cations was determined by reversal-potential criteria. Endplate currents induced by iontophoretic "puffs" of acetylcholine were studied by a Vaseline gap, voltage clamp method in cut muscle fibers. Reversal potential changes were measured as the NaCl of the bathing medium was replaced by salts of organic cations, and permeability ratios relative to Na+ ions were calculated from the Goldman-Hodgkin-Katz equation. 40 small monovalent organic cations had permeability ratios larger than 0.1. The most permeant including NH4+, hydroxylamine, hydrazine, methylamine, guanidine, and several relatives of guanidine had permeability ratios in the range 1.3--2.0. However, even cations such as imidazole, choline, tris(hydroxymethyl)aminomethane, triethylamine, and glycine methylester were appreciably permeant with permeability ratios of 0.13--0.95. Four compounds with two charged nitrogen groups were also permeant. Molecular models of the permeant ions suggest that the smallest cross-section of the open pore must be at least as large as a square, 6.5 A x 6.5 A. Specific chemical factors seem to be less important than access or friction in determining the ionic selectivity of the endplate channel.
The past three years have seen remarkable progress in research on the molecular basis of inward rectification, with significant implications for basic understanding and pharmacological manipulation of cellular excitability. Expression cloning of the first inward rectifier K channel (Kir) genes provided the necessary break-through that has led to isolation of a family of related clones encoding channels with the essential functional properties of classical inward rectifiers, ATP-sensitive K channels, and muscarinic receptor-activated K channels. High-level expression of cloned channels led to the discovery that classical inward so-called anomalous rectification is caused by voltage-dependent block of the channel by polyamines and Mg2+ ions, and it is now clear that a similar mechanism results in inward rectification of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-kainate receptor channels. Knowledge of the primary structures of Kir channels and the ability to mutate them also has led to the determination of many of the structural requirements of inward rectification.