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Hanging Gondola Structure of the T1 Domain in a Voltage-Gated K + Channel †
Abstract
The T1 domain is a approximately 100-residue sequence in the cytoplasmic N-terminal region of K(v)-type K(+) channels. The structure of the isolated domain is known, but it is uncertain whether the structure of this domain is maintained in the fully assembled, membrane-associated, homotetrameric channel protein. We use the structure of the isolated domain as a guide for designing disulfide bonds to cross-link Shaker K(+) channels through the T1 domain. Six pairs of residues with side chains closely apposed across the T1 subunit interface were selected for replacement by cysteine. Of these, three pairs formed cross-links upon air oxidation of cysteine-substituted Shaker channels expressed in Xenopus oocyte membranes. Two of these cross-linked channels were examined electrophysiologically and were found to have gating properties only slightly altered from wild-type. The results show that the structure of the isolated T1 domain exists in the mature ion channel. They also demand that this domain is attached to the membrane-embedded part of the protein as a cytoplasmic "hanging gondola", and that ions gain access to the pore through four "windows" formed by the linker connecting T1 to the channel's first transmembrane helix.
... Our goal in this work was to test potential protein-protein contact points between Q1 and E1. Through oxidant-mediated disulfide bond formation between exogenous cysteine point mutations in a cysteine-null background (Schulteis et al., 1996;Kobertz et al., 2000), we demonstrated both proximity and juxtaposition of side chain residues between Q1 and E1 in assembled channel complexes. The particular cysteine pairings we identified occur within natively assembled and functional channel complexes on the plasma membrane, confirmed by a combination of cell-surface biotinylation, co-immunoprecipitation, PNGase F sensitivity, and I Ks -like channel function determined by whole-cell perforated patch clamp. ...
... Biochemistry -Crosslinking of Q1 and E1 point mutants was mediated through disulfide bond formation (Schulteis et al., 1996;Kobertz et al., 2000). measurable gating events (Rocheleau & Kobertz, 2008). ...
KCNQ1 is a homotetrameric voltage-gated potassium channel expressed in cardiomyocytes and epithelial tissues. However, currents arising from KCNQ1 have never been physiologically observed. KCNQ1 is able to provide the diverse potassium conductances required by these distinct cell types through coassembly with and modulation by type I transmembrane β-subunits of the KCNE gene family.
KCNQ1-KCNE K+ channels play important physiological roles. In cardiac tissues the association of KCNQ1 with KCNE1 gives rise to IKs, the slow delayed outwardly rectifying potassium current. IKs is in part responsible for repolarizing heart muscle, and is therefore crucial in maintaining normal heart rhymicity. IKs channels help terminate each action potential and provide cardiac repolarization reserve. As such, mutations in either subunit can lead to Romano-Ward Syndrome or Jervell and Lange-Nielsen Syndrome, two forms of Q-T prolongation. In epithelial cells, KCNQ1-KCNE1, KCNQ1-KCNE2 and KCNQ1-KCNE3 give rise to potassium currents required for potassium recycling and secretion. These functions arise because the biophysical properties of KCNQ1 are always dramatically altered by KCNE co-expression.
We wanted to understand how KCNE peptides are able to modulate KCNQ1. In Chapter II, we produce partial truncations of KCNE3 and demonstrate the transmembrane domain is necessary and sufficient for both assembly with and modulation of KCNQ1. Comparing these results with published results obtained from chimeric KCNE peptides and partial deletion mutants of KCNE1, we propose a bipartite modulation residing in KCNE peptides. Transmembrane modulation is either active (KCNE3) or permissive (KCNE1). Active transmembrane KCNE modulation masks juxtamembranous C-terminal modulation of KCNQ1, while permissive modulation allows C-terminal modulation of KCNQ1 to express. We test our hypothesis, and demonstrate C-terminal Long QT point mutants in KCNE1 can be masked by active trasnsmembrane modulation.
Having confirmed the importance the C-terminus of KCNE1, we continue with two projects designed to elucidate KCNE1 C-terminal structure. In Chapter III we conduct an alanine-perturbation scan within the C-terminus. C-terminal KCNE1 alanine point mutations result in changes in the free energy for the KCNQ1-KCNE1 channel complex. High-impact point mutants cluster in an arrangement consistent with an alphahelical secondary structure, "kinked" by a single proline residue. In Chapter IV, we use oxidant-mediated disulfide bond formation between non-native cysteine residues to demonstrate amino acid side chains residing within the C-terminal domain of KCNE1 are close and juxtaposed to amino acid side chains on the cytoplasmic face of the KCNQ1 pore domain. Many of the amino acids identified as high impact through alanine perturbation correspond with residues identified as able to form disulfide bonds with KCNQ1. Taken together, we demonstrate that the interaction between the C-terminus of KCNE1 and the pore domain of KCNQ1 is required for the proper modulation of KCNQ1 by KCNE1, and by extension, normal IKs function and heart rhymicity.
... 25,158 Although it is known that potassium channels confer redox sensitivity and are sensitive to changes in the concentration of reactive oxygen species (ROS) it is unclear by what mechanism low oxygen is able to decrease the conductance of these channels. 102,105,118 Volatile anaesthetics such as halothane can open potassium channels in various cell types such as TASK channels in rat carotid body. 25,143,184,140,141 At the same time, volatile anaesthetics, particularly halothane, are known to depress the acute hypoxic response, an effect that may be mediated through a preferential and potent action on the carotid bodies. ...
... Several studies have shown that potassium channels show redox sensitivity and considerable sensitivity to levels of ROS. 102,105,142,117 It is unsettled whether potassium channels possess intrinsic oxygen sensitivity, or, alternatively, are influenced or modulated by other O 2 sensing elements in the cascade, for example by (membrane associated) cytosolic redox couples . Intrinsic oxygen sensitivity could exist in the form of reduction/oxidation of thiol containing free cysteine residues in b subunits that are required for hypoxic sensitivity. ...
... Whereas the Kvα N-terminus underlies rapid channel inactivation, this segment also forms an intracellular docking platform for physical interactions with Kvβ proteins. The N-terminal domains of each α subunit assemble to form the T1 structure, a hanging gondola-like feature that protrudes into the cytoplasm and interacts with the β complex (see Figure 1) [4,[39][40][41]. Each of the four β subunits binds to N-terminal loops on the cytoplasmic face of the T1 tetramer [39]. ...
Excitable cells of the nervous and cardiovascular systems depend on an assortment of plasmalemmal potassium channels to control diverse cellular functions. Voltage-gated potassium (Kv) channels are central to the feedback control of membrane excitability in these processes due to their activation by depolarized membrane potentials permitting K+ efflux. Accordingly, Kv currents are differentially controlled not only by numerous cellular signaling paradigms that influence channel abundance and shape voltage sensitivity, but also by heteromeric configurations of channel complexes. In this context, we discuss the current knowledge related to how intracellular Kvβ proteins interacting with pore complexes of Shaker-related Kv1 channels may establish a modifiable link between excitability and metabolic state. Past studies in heterologous systems have indicated roles for Kvβ proteins in regulating channel stability, trafficking, subcellular targeting, and gating. More recent works identifying potential in vivo physiologic roles are considered in light of these earlier studies and key gaps in knowledge to be addressed by future research are described.
... The 'T1' tetramerisation domain of the mammalian voltage-gated 6TM Kv1.2 potassium channel is distinctively different from that of NaChBac. Although this tetramerisation domain is also spatially located below the cytoplasm entrance to the pore [22], it coassembles to form a globular fold and is connected to the N terminus of the S1-S4 voltage sensor by flexible linkers; this arrangement of T1 relative to the transmembrane domain resembles a 'hanging gondola' [22,23]. Here, we generated a 'T1-chimera' channel by truncating the NaChBac CTD and adding the T1domain of Kv1.2 to the N terminus and then compared expression of this construct with full-length NaChBac and the NaChBac 239D mutant (Fig. 1). ...
Cytoplasmic domains frequently promote functional assembly of multimeric ion channels. To investigate structural determinants of this process, we generated the 'T1-chimera' construct of the NaChBac sodium channel by truncating the C-terminal domain and splicing the T1-tetramerisation domain of the Kv1.2 channel to the N-terminus. Purified T1-chimera channels were tetrameric, conducted Na+ when reconstituted into proteliposomes and were blocked by the drug mibefradil. Both the T1-chimera and full-length NaChBac had comparable expression in the membrane whereas a NaChBac mutant lacking a cytoplasmic domain had greatly-reduced membranal expression. Our findings support a model whereby bringing the transmembrane regions into close proximity enables their tetramerization. This phenomemon is found with other channels and thus our findings substantiate this as a common assembly mechanism.
... Nonetheless, the most recognized accessory proteins of Kv1.2, and other Kv1 subtypes, are the Kvβ auxiliary subunits. Kvβ subunits are cytosolic proteins that assemble in a 1:1 stoichiometry with Kv1α subunits at the cytoplasmic T1 domain (Gulbis et al., 1999(Gulbis et al., , 2000Kobertz et al., 2000;Long et al., 2005). There are three mammalian Kvβ genes that encode Kvβ proteins with alternative splice variants (Kvβ1.1-1.3, ...
The voltage-gated potassium channel Kv1.2 plays a pivotal role in neuronal excitability and is regulated by a variety of known and unknown extrinsic factors. The canonical accessory subunit of Kv1.2, Kvβ, promotes N-type inactivation and cell surface expression of the channel. We recently reported that a neutral amino acid transporter, Slc7a5, alters the function and expression of Kv1.2. In the current study, we investigated the effects of Slc7a5 on Kv1.2 in the presence of Kvβ1.2 subunits. We observed that Slc7a5-induced suppression of Kv1.2 current and protein expression was attenuated with cotransfection of Kvβ1.2. However, gating effects mediated by Slc7a5, including disinhibition and a hyperpolarizing shift in channel activation, were observed together with Kvβ-mediated inactivation, indicating convergent regulation of Kv1.2 by both regulatory proteins. Slc7a5 influenced several properties of Kvβ-induced inactivation of Kv1.2, including accelerated inactivation, a hyperpolarizing shift and greater extent of steady-state inactivation, and delayed recovery from inactivation. These modified inactivation properties were also apparent in altered deactivation of the Kv1.2/Kvβ/Slc7a5 channel complex. Taken together, these findings illustrate a functional interaction arising from simultaneous regulation of Kv1.2 by Kvβ and Slc7a5, leading to powerful effects on Kv1.2 expression, gating, and overall channel function.
... The binding of Kvβ subunits to the T1 domain has been elegantly demonstrated by crystal structures of both the iso lated T1 domain as well as full-length Kv1.2 in association with Kvβs. The crystal struc tures have revealed that the T1 tetramer is separated from the intracellular pore opening as a "hanging gondola," with the Kvβ subunits attached to its lower side (Figure 3; Kobertz, Williams, & Miller, 2000;Long et al., 2005). In mammals, three Kvβ subunit genes have been identified-Kvβ1-3-and transcripts from each are subject to alternative splicing. ...
Voltage-dependent K+ (potassium; Kv) channels are ion channels that critically impact neuronal excitability and function. Four principal α subunits assemble to create a membrane-spanning pore that opens in a voltage-dependent manner to allow the selective passage of K+ ions across the cell membrane. Forty human genes encoding Kv channel α subunits have been identified, and most of them are expressed in the nervous system. The individual Kv subunits display unique cellular and subcellular expression patterns and co-assemble into distinct homo- and hetero-tetrameric channels that differ in their electrophysiological and pharmacological properties, and their sensitivity to dynamic modulation, by cellular signaling pathways. The resulting diversity allows Kv channels to impact all steps in electrical information processing, as well as numerous other aspects of neuronal functions, including those in which they appear to play a non-conducting role. This chapter reviews the current basic knowledge about this large and important family of ion channels.
... The interactions between residues of the pore and the first 2-7 residues of the inactivation gate have been previously studied by mutant cycle analysis (10) and cysteine mutagenesis followed by chemical modification and disulfide bond formation (9). Access of the tip of the N-terminus into the inner pore should occur through the side windows, located immediately above the T1 domain (55,56). Indeed, experimental data point to one specific electrostatic interaction between the positively charged residue R18 and negatively charged residues EDE161-163 in the T1 domain of Aplysia's inactivating K V channels (14), as well as their homologs R17 and EDE192-194 in Shaker K V channels (15). ...
After opening, the Shaker voltage-gated potassium (KV) channel rapidly inactivates when one of its four N-termini enters and occludes the channel pore. Although it is known that the tip of the N-terminus reaches deep into the central cavity, the conformation adopted by this domain during inactivation and the nature of its interactions with the rest of the channel remain unclear. Here, we use molecular dynamics simulations coupled with electrophysiology experiments to reveal the atomic-scale mechanisms of inactivation. We find that the first six amino acids of the N-terminus spontaneously enter the central cavity in an extended conformation, establishing hydrophobic contacts with residues lining the pore. A second portion of the N-terminus, consisting of a long 24 amino acid α-helix, forms numerous polar contacts with residues in the intracellular entryway of the T1 domain. Double mutant cycle analysis revealed a strong relationship between predicted interatomic distances and empirically observed thermodynamic coupling, establishing a plausible model of the transition of KV channels to the inactivated state.
... proposed al imited conformational change at the TM2 crossing gate withouta na ccompanying splaying of the CTD, suggesting the formation of "windows" that would enable the passage of ions [23] as postulated in some cases for Kv channels. [47,48] Later findings of CTD sensitivity to acidic pH [28,31] weres ubject to criticism that their monomer-tetramer equilibrium poorly represents the biological environmento fatetrameric channel. Electrophysiological measurements on CTD mutantsp rovided indirect evidence of CTD contribution to ap reference for the channel open state. ...
The bacterial potassium channel KcsA is gated by pH, opening for conduction under acidic conditions. Molecular determinants responsible for this effect have been identified at the extracellular selectivity filter, at the membrane–cytoplasm interface (TM2 gate), and in the cytoplasmic C‐terminal domain (CTD), an amphiphilic four‐helix bundle mediated by hydrophobic and electrostatic interactions. Here we have employed NMR and EPR to provide a structural view of the pH‐induced open‐to‐closed CTD transition. KcsA was embedded in lipoprotein nanodiscs (LPNs), selectively methyl‐protonated at Leu/Val residues to allow observation of both states by NMR, and spin‐labeled for the purposes of EPR studies. We observed a pHinduced structural change between an associated structured CTD at neutral pH and a dissociated flexible CTD at acidic pH, with a transition in the 5.0–5.5 range, consistent with a stabilization of the CTD by channel architecture. A double mutant constitutively open at the TM2 gate exhibited reduced stability of associated CTD, as indicated by weaker spin–spin interactions, a shift to higher transition pH values, and a tenfold reduction in the population of the associated “closed” channels. We extended these findings for isolated CTD‐derived peptides to full‐length KcsA and have established a contribution of the CTD to KcsA pH‐controlled gating, which exhibits a strong correlation with the state of the proximal TM2 gate.
... Several studies have attributed the inability of Kv2-related regulatory subunits to form homotetramers to their "self-incompatible" tetramerization domains (T1; Ottschytsch et al., 2002Ottschytsch et al., , 2005. The T1 domain is not required for Kv channel tetramerization per se (Kobertz and Miller, 1999), but it coassembles into a water-soluble tetramer (Kreusch et al., 1998) that hangs below the cytoplasmic S6 activation gate (Kobertz et al., 2000), preventing subunits from the different Kv (1-4) subfamilies from intermingling (Shen and Pfaffinger, 1995). Because one T1 domain interacts with T1 domains of adjacent subunits, a Kv2-related regulatory (R) subunit with a self-incompatible T1 domain could potentially give rise to functional heterotetrameric Kv2 channels with two stoichiometries: 3:1R and 2:2R with diagonally opposed regulatory subunits ( Fig. 1 A). ...
Kobertz comments on the family of “silent” Kv2-related regulatory subunits and a new study investigating their assembly idiosyncrasies.
... A structural element, termed the T1 (tetramerization) domain within the N-terminus of Shaker channels, has been suggested to play a central role in the interaction and assembly of channels into homotetrameric or heterotetrameric proteins (28)(29)(30). The T1 domain is a module of approximately 120 amino acids that adopts a 'hanging gondola' structure, and its conformation is highly conserved between Kv channel subfamilies (31,32). However, several reports have demonstrated that Kv channels can still form functional channels upon deletion of the T1 domain (33)(34)(35). ...
The voltage-gated potassium channel Kv1.5 belongs to the Shaker superfamily. Kv1.5 is composed of four subunits, each comprising 613 amino acids, which make up the N-terminus, six transmembrane segments (S1 to S6), and the C-terminus. We recently demonstrated that, in HEK cells, extracellularly applied proteinase K (PK) cleaves Kv1.5 channels at a single site in the S1-S2 linker. This cleavage separates Kv1.5 into an N-fragment (N-terminus to S1) and a C-fragment (S2 to C-terminus). Interestingly, the cleavage does not impair channel function. Here, we investigated the role of the N-terminus and S1 in Kv1.5 expression and function by creating plasmids encoding various fragments including those that mimic PK-cleaved products. Our results disclosed that while expression of the pore-containing fragment (Frag (304-613)) alone could not produce current, coexpression with Frag(1-303) generated a functional channel. Immunofluorescence and biotinylation analyses uncovered that Frag(1-303) was required for Frag(304-613) to traffic to the plasma membrane. Biochemical analysis revealed that the two fragments interacted throughout channel trafficking and maturation. In Frag(1-303)+(304-613) coassembled channels, which lack a covalent linkage between S1 and S2, amino acid residues 1-209 were important for association with Frag(304-613), and residues 210-303 were necessary for mediating trafficking of coassembled channels to the plasma membrane. We conclude that the N-terminus and S1 of Kv1.5 can attract and coassemble with the rest of the channel (i.e. Frag(304-613)) to form a functional channel independent of the S1-S2 linkage.
... In addition to the transmembrane pore, most potassium channels also possess a cytosolic pore that extends relatively far (∼60 Å) into the cytosol [144,147,192,210,249] and may contribute to the gating and permeation properties of the channels [106,191,210,250]. So far, we know nothing about the cytosolic pore of K 2P -channels, although, in view of the long C-terminal domains, the M2-M3-linker and the (usually shorter) N-terminal cytoplasmic domains, it is likely to exist and may well contribute to the regulation and the permeation properties of the channels. ...
Over the last decade, we have seen an enormous increase in the number of experimental studies on two-pore-domain potassium channels (K2P-channels). The collection of reviews and original articles compiled for this special issue of Pflügers Archiv aims to give an up-to-date summary of what is known about the physiology and pathophysiology of K2P-channels. This introductory overview briefly describes the structure of K2P-channels and their function in different organs. Its main aim is to provide some background information for the 19 reviews and original articles of this special issue of Pflügers Archiv. It is not intended to be a comprehensive review; instead, this introductory overview focuses on some unresolved questions and controversial issues, such as: Do K2P-channels display voltage-dependent gating? Do K2P-channels contribute to the generation of action potentials? What is the functional role of alternative translation initiation? Do K2P-channels have one or two or more gates? We come to the conclusion that we are just beginning to understand the extremely complex regulation of these fascinating channels, which are often inadequately described as 'leak channels'.
... Similar to the rest of the subunits, the T1 domains are identical (Kreusch et al., 1998). Since this domain is directly placed below the pore entryway to the cytoplasm, the potassium ions must flow through side portals in order for the transmembrane pore and the cytoplasm to communicate (Gulbis et al., 2000;Kobertz et al., 2000;Sokolova et al., 2001). These portals are large enough to permit the entry of the N-terminal (ball-and-chain) inactivation gate Zagotta et al., 1990). ...
... Interestingly, all these structures conserve some of the general aspects of Kv channel structure. They all contain a tetrameric assembly of subunits residing in the lipid bilayer and a cytoplasmic "hanging gondola" (Kobertz, Williams, & Miller, 2000) formed by the N and C termini containing four large windows. These findings show that, as Kv channels, TRP channels are modular proteins from which we can distinguish an N-terminal, S1-S4, a pore (S5-P domain-S6), and a C-terminal module. ...
A class of ion channels that belongs to the transient receptor potential (TRP) superfamily and is present in specialized neurons that project to the skin has evolved as temperature detectors. These channels are classified into subfamilies, namely canonical (TRPC), melastatin (TRPM), ankyrin (TRPA), and vanilloid (TRPV). Some of these channels are activated by heat (TRPM2/4/5, TRPV1-4), while others by cold (TRPA1, TRPC5, and TRPM8). The general structure of these channels is closely related to that of the voltage-dependent K(+) channels, with their subunits containing six transmembrane segments that form tetramers. Thermal TRP channels are polymodal receptors. That is, they can be activated by temperature, voltage, pH, lipids, and agonists. The high temperature sensitivity in these thermal TRP channels is due to a large enthalpy change (∼100 kcal/mol), which is about five times the enthalpy change in voltage-dependent gating. The characterization of the macroscopic currents and single-channel analysis demonstrated that gating by temperature is complex and best described by branched or allosteric models containing several closed and open states. The identification of molecular determinants of temperature sensitivity in TRPV1, TRPA1, and TRPV3 strongly suggest that thermal sensitivity arises from a specific protein domain.
... The characteristic two-layer architecture is common in a large number of tetrameric ion channels, not only of eukaryotic (Kim et al. 2004;Sokolova et al. 2001Sokolova et al. , 2012Orlova et al. 2003;Ludtke et al. 2011), but also prokaryotic (Uysal et al. 2009) origin. It is called a 'hanging gondola' and allows the large N-terminal inactivation peptide to reach the inner pore of the channel for inactivation (Kobertz et al. 2000). The classical 'hanging gondola' structure consists of two domains, separated by thin linkers that border the 'windows' between them. ...
Voltage-gated potassium Kv2.1 channels are widely distributed in the central nervous system, specifically in neuroendocrine and endocrine cells. Their cytoplasmic C-termini are large and carry out many important functions. Here we provide the first direct structural evidence that each C-terminal part within the Kv2.1 ion channel is formed by two distinct domains (Kv2 and CTA). We expressed and purified two C-terminal truncation mutants of a rat Kv2.1 channel, lacking the entire C-termini or the CTA domain. Single particle electron microscopy was used to obtain three-dimensional reconstructions of purified C-terminal Kv2.1 mutants at 2.0 and 2.4 nm resolution. Comparison of these structures to each other and to the low-resolution EM structure of the full-length Kv2.1 channel revealed the exact locations of cytoplasmic Kv2 and CTA domains within the tetramer. Four Kv2 domains envelop the N-terminal T1 domain. The tetramer of the CTA domains underlies the Kv2-T1 complex and may also affect the channel's surface expression. Subsequent molecular dynamics simulation and homology modeling produced open and closed structural models of the membrane part of the Kv2.1 channel.
... The overall height of the low-resolution TRPV1 structure is 150 Å. The smaller domain measures ~40 × 60 × 60 Å, while the larger domain, referred to as the "hanging gondola" (a term first coined for the cytoplasmic domains of a voltage-gated potassium channel (Kobertz et al., 2000)), is ~110 × 100 × 100 Å. Such a "hanging gondola" has been observed in other channels including voltage-gated potassium channels (Fig. 1). ...
Membrane proteins remain challenging targets for structural biologists, despite recent technical developments regarding sample preparation and structure determination. We review recent progress towards a structural understanding of TRP channels and the techniques used to that end. We discuss available low-resolution structures from electron microscopy (EM), X-ray crystallography, and nuclear magnetic resonance (NMR) and review the resulting insights into TRP channel function for various subfamily members. The recent high-resolution structure of TRPV1 is discussed in more detail in Chapter 11. We also consider the opportunities and challenges of using the accumulating structural information on TRPs and homologous proteins for deducing full-length structures of different TRP channel subfamilies, such as building homology models. Finally, we close by summarizing the outlook of the "holy grail" of understanding in atomic detail the diverse functions of TRP channels.
... The TRPV1 intracellular regions, which are both N-and C-terminal to the transmembrane domain in the primary sequence, come together to form an upside-down bowl-shaped structure below the ion channel pore, resembling the "hanging gondola" (Kobertz et al., 2000) described for a large number of ion channels. This bowl opens a large cavity that is accessible to the cytoplasm (Fig. 1D). ...
The first high-resolution structures of a near-full-length TRP channel were recently described, structures of the noxious heat receptor TRPV1 in the absence or presence of vanilloid agonists and a spider toxin. Here we briefly review the salient features, including the overall architecture, agonist binding sites, and conformational changes related to channel pore gating. We also discuss some of the structures' implications for the TRP channel family and a few of the many questions still left unanswered.
... It remains to be determined exactly how far the region of amplification extends and what the precise nature of the diffusion barriers might be. One possibility is that Ca V cytosolic domains may physically impede diffusion over a ∼15 nm radius (SI Appendix, Fig. S7 and Fig. 4), as would be the case if Ca 2+ ions passed through narrow Ca V cytosolic crevices or portals (13,49). Such a self-sufficient design would be robust to cellular context, and spatially isolated to the indwelling (50) and locally enriched pool (51) of CaM molecules mediating CDI and implicated in signaling to nuclear transcription factors (4,5). ...
Significance
Local Ca ²⁺ signals drive important events like synaptic transmission, neural plasticity, and cardiac contraction, yet the fundamental relationship between flux through a single channel and the Ca ²⁺ amplitude within nanometers of the channel pore has eluded direct experimental measure. Here we reverse-engineer the problem by using Ca ²⁺ -dependent inactivation of channels as a nanometer-range biosensor of Ca ²⁺ amplitude. We discovered an unexpected and dramatic boost in nanodomain Ca ²⁺ amplitude, ten-fold higher than predicted on theoretical grounds. This boost in local Ca ²⁺ signaling may act to maximize the biochemical information capacity of electrically\x{2011}active cells.
... In addition, the T1 domain restricts formation of homo-and heterotetramers by preventing tetramerization between incompatible subunits (i.e., subunits from different Kv1-Kv4 subfamilies) (29,31,47,58,74). When four compatible T1 domains assemble, they arrange into the same fourfold symmetry as the transmembrane segments, forming a "hanging gondola" structure (26). ...
Electrically silent voltage-gated potassium (KvS) α-subunits do not form homotetramers but heterotetramerize with Kv2 subunits, generating functional Kv2/KvS channel complexes in which the KvS subunits modulate the Kv2 current. This poses intriguing questions into the molecular mechanisms by which these KvS subunits cannot form functional homotetramers, why they only interact with Kv2 subunits, and how they modulate the Kv2 current.
... Thus, in contrast with the well defined position of the ball plugging the conduction pathway inside the pore to inactivate the channel, the resting position of the chain segments and their dynamic rearrangements during the closed to open and inactive transitions are not well known. There is indirect evidence supporting the interpretation that these structures might wrap around the scaffold provided by the T1 tetramer hanging below the channel core (Figure 3A ), so that upon depolarization they would snake through the windows lined by the S1–T1 linkers to reach a pocket on the upper T1 domain surface and subsequently the channel cavity below the selectivity filter (Gulbis et al., 2000; Kobertz et al., 2000; Zhou et al., 2001; Baker et al., 2006 ). However, it has also been proposed that the ball-andchain combination is confined to the space between the T1 domain and the transmembrane portion of the channel (Varhsney et al., 2004). ...
The basic architecture of the voltage-dependent K⁺ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered.
... Indeed, intrinsic flexibility of the S4–S5 linker, one of the partners of the N-terminus/core interacting pair proposed here, has recently been recognized as an essential modulatory factor of hERG gating [46]. Poor structuralization and flexibility of the N-terminal most segment of the amino terminus has been recognized in Shaker-like channels as an important factor for allowing this protein segment to snake its way to reaching its interaction site near the channel and produce N-type inactivation515253. If this characteristic is similarly involved in docking the initial segment of the hERG amino end with its interaction site in the channel core (e.g. to the S4–S5 linker) remains an interesting possibility. ...
A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG) potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4-S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET) analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF) conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4-S5 linker.
... In excised membrane patches, this hypoxia-induced depolarization is still present, but in the course of time a significant rundown occurs, indicating that O 2 sensing may be a membrane-delimited process requiring cytosolic factors to be preserved for longer time periods (800,842). Whether or not potassium channels possess structural components required for direct oxygen sensing, many of them appear to be redox sensitive (392,409,468,603), but it is unlikely that hypoxia inhibits the current through these channels by oxidizing or reducing functional channel protein components (reviewed in Ref. 282). Generally, reductants and oxidants decrease and increase, respectively, the open probability of rapidly inactivating O 2sensitive potassium channels in particular, but the effects on maxi-K channels are more variable (references in Ref. 282). ...
The respiratory response to hypoxia in mammals develops from an inhibition of breathing movements in utero into a sustained increase in ventilation in the adult. This ventilatory response to hypoxia (HVR) in mammals is the subject of this review. The period immediately after birth contains a critical time window in which environmental factors can cause long-term changes in the structural and functional properties of the respiratory system, resulting in an altered HVR phenotype. Both neonatal chronic and chronic intermittent hypoxia, but also chronic hyperoxia, can induce such plastic changes, the nature of which depends on the time pattern and duration of the exposure (acute or chronic, episodic or not, etc.). At adult age, exposure to chronic hypoxic paradigms induces adjustments in the HVR that seem reversible when the respiratory system is fully matured. These changes are orchestrated by transcription factors of which hypoxia-inducible factor 1 has been identified as the master regulator. We discuss the mechanisms underlying the HVR and its adaptations to chronic changes in ambient oxygen concentration, with emphasis on the carotid bodies that contain oxygen sensors and initiate the response, and on the contribution of central neurotransmitters and brain stem regions. We also briefly summarize the techniques used in small animals and in humans to measure the HVR and discuss the specific difficulties encountered in its measurement and analysis.
... The assumption by analogy is that the MA helices of all members of this family of receptors will share this common structure. Similar structures have been found in a growing number of ion channels with disparate gating mechanisms, such as the Shaker potassium channel (40,41), the bacterial mechanosensitive channel MscL (42), a cyclic-nucleotide gated channel (43), and the voltage-activated sodium channel (44). It is conceivable, therefore, that protein interactions involving the gondola domains of these receptors could potentially alter ion-channel properties. ...
Native GABA(A) channels display a single-channel conductance ranging between approximately 10 and 90 pS. Diazepam increases the conductance of some of these native channels but never those of recombinant receptors, unless they are coexpressed with GABARAP. This trafficking protein clusters recombinant receptors in the membrane, suggesting that high-conductance channels arise from receptors that are at locally high concentrations. The amphipathic (MA) helix that is present in the large cytoplasmic loop of every subunit of all ligand-gated ion channels mediates protein-protein interactions. Here we report that when applied to inside-out patches, a peptide mimicking the MA helix of the gamma2 subunit (gamma(381-403)) of the GABA(A) receptor abrogates the potentiating effect of diazepam on both endogenous receptors and recombinant GABA(A) receptors coexpressed with GABARAP, by substantially reducing their conductance. The protein interaction disrupted by the peptide did not involve GABARAP, because a shorter peptide (gamma(386-403)) known to compete with the gamma2-GABARAP interaction did not affect the conductance of recombinant alphabetagamma receptors coexpressed with GABARAP. The requirement for receptor clustering and the fact that the gamma2 MA helix is able to self-associate support a mechanism whereby adjacent GABA(A) receptors interact via their gamma2-subunit MA helices, altering ion permeation through each channel. Alteration of ion-channel function arising from dynamic interactions between ion channels of the same family has not been reported previously and highlights a novel way in which inhibitory neurotransmission in the brain may be differentially modulated.
Ion channels are highly diverse in the cnidarian model organism Nematostella vectensis (Anthozoa), but little is known about the evolutionary origins of this channel diversity and its conservation across Cnidaria. Here we examined the evolution of voltage-gated K+ channels in Cnidaria by comparing genomes and transcriptomes of diverse cnidarian species from Anthozoa and Medusozoa. We found an average of over 40 voltage-gated K+ channel genes per species, and phylogenetic reconstruction of the Kv, KCNQ and EAG gene families identified 28 voltage-gated K+ channels present in the last common ancestor of Anthozoa and Medusozoa (23 Kv, 1 KCNQ and 4 EAG). Thus, much of the diversification of these channels took place in the stem cnidarian lineage prior to the emergence of modern cnidarian classes. In contrast, the stem bilaterian lineage, from which humans evolved, contained no more than 9 voltage-gated K+ channels. These results hint at a complexity to electrical signaling in all cnidarians that contrasts with the perceived anatomical simplicity of their neuromuscular systems. These data provide a foundation from which the function of these cnidarian channels can be investigated, which will undoubtedly provide important insights into cnidarian physiology.
The osmotic activity produced by internal, non‐permeable, anionic nucleic acids and metabolites causes a persistent and life‐threatening cell swelling, or cellular edema, produced by the Gibbs‐Donnan effect. This evolutionary‐critical osmotic challenge must have been resolved by LUCA or its ancestors, but we lack a cell‐physiology look into the biophysical constraints to the solutions. Like mycoplasma, early cells conceivably preserved their volume with Cl−, Na+, and K+‐channels, Na+/H+‐exchangers, and a light‐dependent bacteriorhodopsin‐like H+‐pump. Here, I simulated protocells having these ionic‐permeabilities and inhabiting an oceanic pond before the Great‐Oxygenation‐Event. Protocells showed better volume control and stable resting potentials at lower external pH and higher temperatures, favoring a certain type of extremophile life. Prevention of Na+‐influx at night, with low bacteriorhodopsin activity, required deep shutdown of highly voltage‐sensitive Na+‐channels and extremely selective K+‐channels, two conserved features essential for modern neuronal encoding. The Gibbs‐Donnan effect universality implies that extraterrestrial cells, if they exist, may reveal similar volume‐controlling mechanisms. A scenario for the origin of life: In a pre‐GOE oceanic pond, an early cell or protocell, filled with negative macromolecules and metabolites, controls its natural tendency to inflate, due to the Gibbs‐Donnan effect, with a bacteriorhodopsin‐like proton‐pump together with other ion‐transporting proteins, producing a negative resting‐potential and internal alkalinization .
Membrane proteins, including ion channels, became the focus of structural proteomics midway through the 20th century. Methods for studying ion channels are diverse and include structural (X-ray crystallography, cryoelectron microscopy, currently X-ray free electron lasers) and functional (e.g., patch clamp) approaches. This review highlights the evolution of approaches to study of the structure of cardiac ion channels, provides an overview of new techniques of structural biology concerning ion channels, including the use of lipo- and nanodiscs, and discusses the contribution of electrophysiological studies and molecular dynamics to obtain a complete picture of the structure and functioning of cardiac ion channels. Electrophysiological studies have become a powerful tool for deciphering the mechanisms of ion conductivity and selectivity, gating and regulation, as well as testing molecules of pharmacological interest. Obtaining the atomic structure of ion channels became possible by the active development of X-ray crystallography and cryoelectron micro-scopy, and, recently, with the use of XFEL.
The structures of an increasing number of channels and other a-helical membrane proteins have been determined recently, including the KcsA potassium channel, the MscL mechanosensitive channel, and the AQP1 and GlpF members of the aquaporin family. In this chapter, the orientation and packing characteristics of bilayer-spanning helices are surveyed in integral membrane proteins. In the case of channels, a-helices create the scaled barrier that separates the hydrocarbon region of the bilayer from the permeation pathway for solutes. The helices surrounding the permeation pathway tend to be rather steeply tilted relative to the membrane normal and are consistently arranged in a right-handed bundle. The helical framework further provides a supporting scaffold for nonmembrane-spanning structures associated with channel selectivity. Although structural details remain scarce, the conformational changes associated with gating transitions between closed and open states of channels are reviewed, emphasizing the potential roles of helix-helix interactions in this process.
Regulation of intracellular elemental ion levels is essential for normal cellular homeostasis. Regulation of potassium ion flux impacts many biological processes, such as proliferation and cell survival, therefore it is of particular importance and the link between its dysregulation and disease is unsurprising. Abundant evidence demonstrates that cancer cells hijack the normal physiologic regulation of potassium channels to promote tumor pathogenesis. As potassium channel expression and function is relatively unexplored in cancer, many gaps exist in our understanding of the mechanisms underlying their aberrant regulation. This thesis details a novel mechanism, comprised of two epigenetic components, underlying repression of the Kv1.5 potassium channel-encoding gene, KCNA5, in the pediatric cancers, Ewing Sarcoma and Neuroblastoma. Furthermore, it elucidates the consequence of KCNA5 repression on cancer cell survival and proliferation.
Cancer cells are able to resist cell death despite exposure to cell intrinsic and extrinsic stress. This ability to survive is linked to the concentration of intracellular potassium, as high levels of intracellular potassium inhibit caspase activation and promote cell survival. Given the role of potassium in regulating apoptosis, we reasoned that repression of potassium channel genes might play a role in cancer cell survival. In this thesis, we describe our novel finding that polycomb-dependent epigenetic repression of KCNA5 is a mechanism by which cancer cells resist physiological stressors (i.e. hypoxia and growth factor deprivation) and promote cell survival.
Potassium flux regulates cell-cycle progression and its dysregulation allows cancer cells to proliferate. In cancer, this dysregulation promotes advancement through cell-cycle checkpoints and sustains proliferative signaling. In this work, we show that the hyper-proliferative phenotype in cancer is partially dependent on silencing of KCNA5, which is caused by DNA hypermethylation. Interestingly, promoters of PcG targets are often subject to aberrant DNA hypermethylation, and this is true of KCNA5.
This thesis reveals a key role for suppression of Kv1.5 in cancer pathogenesis and identifies epigenetic mechanisms as mediators of repression. Furthermore, it demonstrates that inhibition of this epigenetic repression sensitizes cancer cells to stress-induced death signals and prevents their hyper-proliferative phenotype. Thus, this thesis supports the potential use of epigenetic modifiers in adjunct cancer therapy.
In our previous study, a set of homology models of the tetramerization (T1) domain of six eukaryotic potassium channels Kv1.1-Kv1.6 from Homo Sapiens was constructed based on the crystal structure of the Shaker T1 domain from Aplysia californica. The results reveal that the T1 domains of these Kv channels exhibit similar folds as those of Shaker K + channel. In this study, several molecular dynamics (MD) simulations towards the Shaker and Kv1.1 T1 domains were conducted at various temperatures. Our results show that the Shaker T1 domain exhibit higher structural integrity than the Kv1.1 T1 domain at all temperatures examined. In addition, the thermal unfolding of the Shaker T1 domain begins at layer 3. In contrast, layers 1 and 2 exhibit higher structural stability because layer 1 remains more hydrogen bonding interactions at elevated temperatures and layer 2 is located in the highly conserved hydrophobic core. Ile121 in the Shaker T1 domain plays an important role in disrupting the loop between helices 4 and 5. During the thermal unfolding process, the newly formed hydrophobic interactions between A1a120, Ile121, Leu122, Leu131, and Leu151 may distort the native contact between layers 2 and 3 of the T1 domain.
Vascular contractility is closely related to the structural and functional integrity of K⁺ channels in vascular smooth muscle cells (SMCs). SMCs have a high input resistance. K⁺ efflux resulting from the activation of even a small number of K⁺ channels will hyperpolarize cell membrane, which in turn inhibits the agonistinduced increase in inositol triphosphate (IP 3), reduces Ca²⁺ sensitivity and resting Ca²⁺ levels, inactivates voltage-dependent Ca²⁺ channels, and relaxes vascular SMCs. 1 Conversely, the closing of K⁺ channels causes vasoconstriction by the depolarization of cell membrane.
Nicotinic pharmacology goes back to the 1844 discovery by Claude Bernard that curare could paralyze rabbit skeletal muscles without affecting the heart (Bennett, 2000; see Chapter 4 A). John Langley developed the concept of transmitter receptors through studies of neuromuscular transmission in 1905 to 1907. Otto Loewi demonstrated chemical transmission on heart muscle in 1921, and Dale and coworkers identifi ed this transmitter as acetylcholine in 1936.
The aim of this book is to present a detailed review of physiological and pathophysiological concepts related to the QT interval. Because much of the confusion in the QT interval prediction power and its use as a risk assessment tool is primarily resulting from the inappropriate correction of the QT interval for heart rate, this work also aims to revisit the QT/RR dynamics as the HR shifts from one zone to another (bradycardia, normal, and tachycardia). This work represents an excellent and up-to-date reference for researchers and clinicians interested in the science of electrocardiology in general and in the science of ventricular repolarization in specific. Several factors make this book unique and exquisite. First, the high quality of the material presented which are based on deep understanding of the science of ventricular repolarization, long years of experience, and careful review of hundreds of research articles related to this field of study. Second, this book is written using an easygoing approach even for the in-experienced reader. The reader finds no need to consult other references to understand the concepts presented. Third, this work eliminates much of the effort and hassle that fellow and colleague researchers would encounter if they were left alone with the literature without appropriate guidance and assistance.
This book is arranged into three organizing chapters. These organizing chapters furnish the theoretical background for understanding the QT interval. Chapter 1, the QT interval and its prognostic significance, introduces the QT interval as an index of ventricular depolarization and repolarization duration and describes several of its interesting features. A complete section discussing the history of the QT interval and its prognostic significance is included. This chapter also discusses the consequences of an excessively long QT interval, either because of genetic factors or because of an acquired abnormality such as a drug induced long QT interval. Torsade de pointes is the major arrhythmic event associated with long QT interval. This ventricular arrhythmia and the underlying mechanism leading to its occurrence are also discussed.
The QT interval is long when the ventricular action potential is long. In chapter 2, the cellular and molecular biology of a long QT interval is discussed. I explore at the molecular level ion channel abnormalities that lengthen the ventricular action potential. At the cellular level, I describe the different types of ventricular myocardial cells that participate in carving the QT interval on the body surface ECG, with special emphasis on the midmyocardial cells (M cells). When the M cell action potential duration is selectively longer, ventricular transmural dispersion of repolarization is increased and the risk for developing ventricular arrhythmias is substantially increased. In this chapter, the same concepts discussed in chapter 1 will be reviewed but with a deeper and a wider understanding. At the end of this chapter, a greater emphasis on the role of the autonomic nervous system in regulating the QT interval is introduced.
QT interval correction is one of the most challenging issues in electrocardiology. Chapter 3, correction of the QT interval, discusses most of the major issues related to this topic. This chapter deals with issues related to QT interval correction such as rate and hysteresis correction. QT dynamicity is discussed at length in this chapter to provide the reader with a basic understanding of QT/RR dynamics.
Undoubtedly, the reader will enjoy moving from one section to another in this book while exploring the amazing science of ventricular repolarization as manifested by the QT interval on the body surface ECG and its potential power as a risk prediction tool.
The cytoplasmic domain of the bacterial mechanosensitive (MS) channel of small conductance (MscS) is shaped by its C‐termini forming a large chamber filled with water. Studies indicate that the chamber is a dynamic structure that undergoes severe conformational changes on the channel gating. Various electrophysiological and biochemical methods combined with molecular biology have been used to investigate this phenomenon and the results are presented in this chapter. The size of the chamber and its shape resemble cytoplasmic domains from eukaryotic non‐MS channels whose function in stabilization of the channel closed state is established. Analogous role of the MscS cytoplasmic chamber is discussed. Bacterial MS channels protect these cells against hypoosmotic shock. Two types of MS channels from the cytoplasmic membrane of Escherichia coli, MscL and MscS (the large and small conductance MS channel, respectively), play an essential role in the physiology of this bacterium, allowing efflux of solutes from the cytoplasm when osmolarity of the external medium decreases.
Dielectrophoresis (DEP) can be used to noninvasively measure the dielectric state of the cell, and this data can be used to monitor cell health or apoptosis. In this study, we followed events associated with cytosine arabinoside (Ara-C)-induced apoptosis in NB4 cells using DEP analysis. Our data showed that the membrane capacitance of NB4 cells decreases from 9.42 to 7.63 mF/m² in the first 2 hours following treatment with Ara-C, and that this decreased capacitance persists for >12 hours. Additionally, cytoplasmic conductivity decreases from 0.217 to 0.190 S/m within 2 hours of Ara-C treatment; this level is maintained for a short period of time before decreasing. We also investigated these events molecularly at the level of gene expression using microarray analysis and showed that the expression of genes related to membrane capacitance and cytoplasmic conductivity change dramatically as early as 2 hours post-Ara-C treatment, and further demonstrated a temporal relationship between the dielectric properties and key events in apoptosis. This study, integrating physical electrical properties of the cell membrane and cytoplasm with those of conductivity-related gene networks, provides new insights into the molecular mechanisms underlying the initiation of apoptosis, establishing a systematic foundation for DEP application in follow-up drug screening and development of medicines for treating leukemia.
Voltage-sensitive K+ channels (Kv) serve numerous important roles, e.g. in the control of neuron excitability and the patterns of synaptic activity. Here, we use electron microscopy (EM) and single particle analysis to obtain the first, complete structure of Kv1 channels, purified from rat brain, which contain four transmembrane channel-forming α-subunits and four cytoplasmically-associated β-subunits. The 18Å resolution structure reveals an asymmetric, dumb-bell-shaped complex with 4-fold symmetry, a length of 140Å and variable width. By fitting published X-ray data for recombinant components to our EM map, the modulatory (β)4 was assigned to the innermost 105Å end, the N-terminal (T1)4 domain of the α-subunit to the central 50Å moiety and the pore-containing portion to the 125Å membrane part. At this resolution, the selectivity filter could not be localised. Direct contact of the membrane component with the central (T1)4 domain occurs only via peripheral connectors, permitting communication between the channel and β-subunits for coupling of responses to changes in excitability and metabolic status of neurons.
We studied the structure of the C terminus of the Shaker potassium channel. The 3D structures of the full-length and a C-terminal deletion (Delta C) mutant of Shaker were determined by electron microscopy and single-particle analysis. The difference map between the full-length and the truncated channels clearly shows a compact density, located on the sides of the T1 domain, that corresponds to a large part of the C terminus. We also expressed and purified both WT and Delta C Shaker, assembled with the rat KvBeta2-subunit. By using a difference map between the full-length and truncated Shaker alpha-beta complexes, a conformational change was identified that shifts a large part of the C terminus away from the membrane domain and into close contact with the Beta-subunit. This conformational change, induced by the binding of the KvBeta2-subunit, suggests a possible mechanism for the modulation of the K+ voltage-gated channel function by its Beta-subunit.
Potassium (KC) channels are largely responsible for shaping the electrical behavior of cell membranes. KC channel currents set the resting membrane potential, control action potential duration, control the rate of action potential
firing, control the spread of excitation and Ca2C influx, and provide active opposition to excitation. To support these varied functions, there are a large number of KC channel types, with a great deal of phenotypic diversity, whose properties can be modified by many different accessory proteins
and biochemical modulators.
To operate in the extreme cold, ion channels from psychrophiles must have evolved structural changes to compensate for their
thermal environment. A reasonable assumption would be that the underlying adaptations lie within the encoding genes. Here,
we show that delayed rectifier K+ channel genes from an Antarctic and a tropical octopus encode channels that differ at only four positions and display very
similar behavior when expressed in Xenopus oocytes. However, the transcribed messenger RNAs are extensively edited, creating functional diversity. One editing site,
which recodes an isoleucine to a valine in the channel’s pore, greatly accelerates gating kinetics by destabilizing the open
state. This site is extensively edited in both Antarctic and Arctic species, but mostly unedited in tropical species. Thus
adenosine-to-inosine RNA editing can respond to the physical environment.
P2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain. The x-ray structure of the P2X4 receptor from zebrafish (zfP2X4) receptor reveals that the extracellular vestibule above the gate opens to the outside through lateral fenestrations, providing a potential pathway for ions to enter and exit the pore. The extracellular region also contains a void at the central axis, providing a second potential pathway. To investigate the energetics of each potential ion permeation pathway, we calculated the electrostatic free energy by solving the Poisson-Boltzmann equation along each of these pathways in the zfP2X4 crystal structure and a homology model of rat P2X2 (rP2X2). We found that the lateral fenestrations are energetically favorable for monovalent cations even in the closed-state structure, whereas the central pathway presents strong electrostatic barriers that would require structural rearrangements to allow for ion accessibility. To probe ion accessibility along these pathways in the rP2X2 receptor, we investigated the modification of introduced Cys residues by methanethiosulfonate (MTS) reagents and constrained structural changes by introducing disulfide bridges. Our results show that MTS reagents can permeate the lateral fenestrations, and that these become larger after ATP binding. Although relatively small MTS reagents can access residues in one of the vestibules within the central pathway, no reactive positions were identified in the upper region of this pathway, and disulfide bridges that constrain movements in that region do not prevent ion conduction. Collectively, these results suggest that ions access the pore using the lateral fenestrations, and that these breathe as the channel opens. The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function.
KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K(+) channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein-protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4-S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1-KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K(+) channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1-KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1-KCNE1 cytoplasmic region where these protein-protein interactions are poised to slow activation gate opening.
The transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel responsive to various stimuli including cell swelling, warm temperatures (27-35 degrees C), and chemical compounds such as phorbol ester derivatives. Here we report the three-dimensional structure of full-length rat TRPV4 purified from baculovirus-infected Sf9 cells. Hexahistidine-tagged rat TRPV4 (His-rTRPV4) was solubilized with detergent and purified through affinity chromatography and size-exclusion chromatography. Chemical cross-linking analysis revealed that detergent-solubilized His-rTRPV4 was a tetramer. The 3.5-nm structure of rat TRPV4 was determined by cryoelectron microscopy using single-particle reconstruction from Zernike phase-contrast images. The overall structure comprises two distinct regions; a larger dense component, likely corresponding to the cytoplasmic N- and C-terminal regions, and a smaller component corresponding to the transmembrane region.
The pulmonary vasculature has the unique ability to undergo vasoconstriction in response to acute hypoxia, a physiological mechanism known as hypoxic pulmonary vasoconstriction (HPV). Sustained or chronic hypoxia, however, leads to proliferation of vascular smooth muscle cells of pulmonary arterioles, which causes a permanent increase in pulmonary vascular resistance, and may lead to right heart dysfunction. The underlying mechanisms of vascular proliferation under chronic hypoxia have not been fully defined. The NADPH oxidases are one family of recently discovered molecules which generate reactive oxygen species (ROS) and have been suggested to be important for cellular signaling under physiological conditions. However, NADPH oxidase generated oxidative stress can also lead to inflammation, vascular smooth muscle cell proliferation and endothelial damage under pathological conditions. Many homologs of NADPH oxidases exist, the classical homolog is gp91phox or NOX2, and the recently discovered homologs include NOX1, NOX3, NOX4, NOX5, DUOX1 and DUOX2. Superoxide production by classical gp91phox is induced by assembly of the cytosolic subunits such as p40phox, p47phox and p67phox with membrane-bound gp91phox complex. A previous report from our laboratory has shown that the knockout mice of p47phox subunit exhibit reduced acute HPV as compared to wild type mice suggesting an essential role of NADPH oxidases in regulation of vascular tone in acute hypoxia. Against this background, the current thesis aimed to elucidate the role of NADPH oxidases in vascular remodeling in chronic hypoxia, and its possible downstream mediators. Screening of NADPH oxidase expression revealed that all subunits were expressed in the lung homogenate and that NOX4 was prominently up-regulated under chronic hypoxia. The NOX4 mRNA was also up-regulated in the microdissected vessels of mice exposed up to three weeks of chronic hypoxia. In addition, a functional interference with NOX4 using NOX4 siRNA resulted in reduced ROS production and reduced proliferation of pulmonary arterial smooth muscle cells (PASMC) revealing an important contribution of NOX4 in PASMC proliferation and particularly in hypoxia induced pulmonary hypertension. Intriguingly, a similar reflection was found in lungs of patients with idiopathic pulmonary hypertension that underwent lung transplantation. Further experiments demonstrated that NOX4 inhibited voltage-gated delayed rectifier K+ channels (KDR) under hypoxia. Pharmacological inhibition with apocynin and genetic ablation with NOX4siRNA resulted in increased KDR current under hypoxia. In addition, the current study demonstrated that NOX4 is essential for ET-1 mediated calcium influx in PASMC as NOX4 knockdown using NOX4 siRNA abolished the ET-1 mediated calcium influx under chronic hypoxia. Thus, the NOX4-ROS-KDR-[Ca2+] pathway may contribute to the development of pulmonary hypertension. Das pulmonale Gefäßsystem besitzt die besondere Eigenschaft, auf akute Hypoxie mit einer Vasokonstriktion zu reagieren. Dies ist ein physiologischer Mechanismus, der als hypoxische pulmonale Vasokonstriktion (HPV) bezeichnet wird. Anhaltende oder chronische Hypoxie führt darüberhinaus zu einer Proliferation der Media der Lungenarteriolen, das einen permanenten Anstieg des pulmonalen Gefäßwiderstandes auslöst, der im weiteren zu einem Cor pulmonale führen kann. Die der HPV zu Grunde liegenden Mechanismen sind bisher noch nicht umfassend geklärt worden. NADPH-Oxidasen sind eine vor kurzem entdeckte Proteinfamilie, die reaktive Sauerstoffspezies (ROS) generieren können und unter physiologischen Bedingungen für die zelluläre Signaltransduktion wichtig sind. Andererseits können die von NADPH-Oxidasen stammenden ROS unter pathologischen Bedingungen zu Entzündung und Proliferation von vaskulären glatten Muskelzellen und zur Schädigung des Endothels führen. In der Literatur sind mehrere Homologe von NADPH-Oxidasen beschrieben, wobei das klassische Homolog als gp91phox oder NOX2 bekannt ist. Weitere, erst kürzlich entdeckte Homologe umfassen NOX1, NOX3, NOX4, NOX5, DUOX1 und DUOX2. Die Superoxidproduktion der klassischen gp91phox wird durch die Anlagerung der zytosolischen Untereinheiten p40phox, p47phox und p67phox an gp91phox induziert. Unsere bisherigen Daten zeigen, dass Mäuse mit einer p47phox-Defizienz im Vergleich zu Wildtyp-Mäusen eine geringere akute HPV aufweisen, das auf eine essentielle Rolle der NADPH-Oxidasen für die Regulation des vaskulären Tonus unter akuter Hypoxie hinweist. Ziel der vorliegenden Arbeit war, die Rolle der NADPH-Oxidasen für den vaskulären Umbauprozess unter chronischer Hypoxie und deren Signaltransduktionsmechanismen aufzuklären. Untersuchungen der NADPH-Oxidasen Expression zeigten, dass alle Untereinheiten im Lungenhomogenat exprimiert waren und die Expression von NOX4 unter chronischer Hypoxie stark hochreguliert war. Darüberhinaus war die NOX4-mRNA auch in den durch Mikrodissektion gewonnenen Gefäßen der Mäuse, die 3 Wochen unter chronischer Hypoxie gehalten wurden, hochreguliert. Zusätzlich bewirkte eine funktionelle Interferenz von NOX4 mit NOX4-siRNA eine reduzierte ROS-Produktion und eine verringerte Proliferation der pulmonalarteriellen glatten Muskelzellen (PASMC). Dies weist auf eine bedeutungsvolle Rolle von NOX4 für die Proliferation von PASMC, insbesondere bei Hypoxia-induzierter pulmonaler Hypertonie, hin. Interessanterweise wurden ähnliche Ergebnisse in Lungen von Patienten mit idiopatischer pulmonaler Hypertonie nach Transplantation der Lunge gewonnen. Weitere Experimente zeigten, dass NOX4 unter chronischer Hypoxie die voltage gated delayed rectifier K+-Kanäle (KDR) hemmt. Eine pharmakologische Inhibition von NOX4 mittels Apocynin oder eine genetische Ablation durch siRNA erhöhten den KDR-Strom unter chronischer Hypoxie. Zusätzlich ist in der vorliegenden Arbeit gezeigt, dass NOX4 essentiell für den ET-1 vermittelten Kalziumeinstrom ist, da durch Reduzierung von NOX4 mittels NOX4 siRNA der ET-1 vermittelte Kalziumeinstrom unter chronischer Hypoxie nicht mehr vorhanden war. Zusammengefasst gesagt, könnte der NOX4-ROS-KDR-[Ca2+]-Signalweg somit zur Entstehung der pulmonalen Hypertonie beitragen.
Das Öffnen und Schließen von Kaliumkanälen kann durch Spannung und/oder Liganden gesteuert werden. In dieser Arbeit wurde das 'Gating' zweier Kanaltypen auf molekularer Ebene untersucht: die Aktivierung bzw. Deaktivierung von Ca2+-gesteuerten SK-Kanälen sowie die Inaktivierung von spannungsgesteuerten Kv-Kanälen. Die einzelnen Projekte wurden in interdisziplinärer Zusammenarbeit mit Wissenschaftlern aus derselben und anderen Arbeitsgruppen durchgeführt.
Der Schwerpunkt lag auf der Etablierung einer Methode, mit deren Hilfe direkte und/oder indirekte Interaktionspartner der cytoplasmatischen N- und C-terminalen Domänen der alpha-Untereinheiten von SK-Kanälen identifiziert werden können. Calmodulin diente als Positivkontrolle und wurde spezifisch von den C-Termini der SK-Kanäle gebunden. Von den vielen potentiellen Interaktionspartnern wurde Caseinkinase II (CK2) zweifelsfrei als Protein bestätigt, das direkt mit den SK2 alpha-Untereinheiten assoziiert ist. Zusätzlich gibt es Hinweise für eine spezifische Interaktion mit Proteinphosphatase 2A (PP2A).
Durch In-vitro-Phosphorylierung und Koexpressions-Experimente wurde gezeigt, dass die CK2-vermittelte Phosphorylierung von SK-gebundenem Calmodulin an einem Threonin-Rest im zentralen Linker die Ca2+-Sensitivität der Kanäle durch Erhöhung der Deaktivierungsrate herabsetzt. Das 'Gating' von SK-Kanälen kann daher in vivo vermutlich durch reversible Phosphorylierung und Dephosphorylierung von Calmodulin moduliert werden.
Anhand in Bakterien exprimierter und gereinigter Peptide wurden mit Hilfe der NMR-Spektroskopie die Lösungsstrukturen der intrazellulären 'Gating'-Domänen von SK- und Kv-Kanälen ermittelt. Sowohl für die Calmodulin-Bindungsdomäne von SK2 als auch für die Tandem-Inaktivierungsdomäne von Kv1.4 wurde ein unmittelbarer Zusammenhang zwischen Struktur und Funktion festgestellt.
Voltage-gated potassium (Kv) channels are transmembrane tetramers of individual alpha-subunits. Eight different Shaker-related Kv subfamilies have been identified in which the tetramerization domain T1, located on the intracellular N terminus, facilitates and controls the assembly of both homo- and heterotetrameric channels. Only the Kv2 alpha-subunits are able to form heterotetramers with members of the silent Kv subfamilies (Kv5, Kv6, Kv8, and Kv9). The T1 domain contains two subdomains, A and B box, which presumably determine subfamily specificity by preventing incompatible subunits to assemble. In contrast, little is known about the involvement of the A/B linker sequence. Both Kv2 and silent Kv subfamilies contain a fully conserved and negatively charged sequence (CDD) in this linker that is lacking in the other subfamilies. Neutralizing these aspartates in Kv2.1 by mutating them to alanines did not affect the gating properties, but reduced the current density moderately. However, charge reversal arginine substitutions strongly reduced the current density of these homotetrameric mutant Kv2.1 channels and immunocytochemistry confirmed the reduced expression at the plasma membrane. Förster resonance energy transfer measurements using confocal microscopy showed that the latter was not due to impaired trafficking, but to a failure to assemble the tetramer. This was further confirmed with co-immunoprecipitation experiments. The corresponding arginine substitution in Kv6.4 prevented its heterotetrameric interaction with Kv2.1. These results indicate that these aspartates (especially the first one) in the A/B box linker of the T1 domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/silent Kv6.4 channels.
Fast inactivation in voltage-gated potassium channels has traditionally been associated exclusively with the N-terminus. Here, we explore the role of the T1 domain using a series of chimeric channels. A chimeric channel, 4N/2, (N-terminus from the rapidly inactivating hKv1.4, and the channel body from the non-inactivating hKv1.2), exhibited slower and incomplete inactivation as compared to the wild-type hKv1.4. Replacing the T1 domain of 4N2 with that from hKv1.2 (4N/2T1/2), restored inactivation, while that from hKv1.1 (4N/1T1/2) completely abolished inactivation. Based on these observations, we hypothesize a correlation between the tetramerization domain and the putative inactivation domain receptor in the process of rapid inactivation of hKv1 channels.
Cavernous chambers, intricate passages, a gate with a curious lock - the structure of an ATP-activated ion channel reveals its architecture. And this intriguing interior design is found in another type of ion channel too.
KcsA is a proton-activated, voltage-modulated K(+) channel that has served as the archetype pore domain in the Kv channel superfamily. Here, we have used synthetic antigen-binding fragments (Fabs) as crystallographic chaperones to determine the structure of full-length KcsA at 3.8 A, as well as that of its isolated C-terminal domain at 2.6 A. The structure of the full-length KcsA-Fab complex reveals a well-defined, 4-helix bundle that projects approximately 70 A toward the cytoplasm. This bundle promotes a approximately 15 degree bending in the inner bundle gate, tightening its diameter and shifting the narrowest point 2 turns of helix below. Functional analysis of the full-length KcsA-Fab complex suggests that the C-terminal bundle remains whole during gating. We suggest that this structure likely represents the physiologically relevant closed conformation of KcsA.
In Shaker K+ channels depolarization displaces outwardly the positively charged residues of the S4 segment. The amount of this displacement is unknown, but large movements of the S4 segment should be constrained by the length and flexibility of the S3–S4 linker. To investigate the role of the S3–S4 linker in the ShakerH4Δ(6–46) (ShakerΔ) K+ channel activation, we constructed S3–S4 linker deletion mutants. Using macropatches of Xenopus oocytes, we tested three constructs: a deletion mutant with no linker (0 aa linker), a mutant containing a linker 5 amino acids in length, and a 10 amino acid linker mutant. Each of the three mutants tested yielded robust K+ currents. The half-activation voltage was shifted to the right along the voltage axis, and the shift was +45 mV in the case of the 0 aa linker channel. In the 0 aa linker, mutant deactivation kinetics were sixfold slower than in ShakerΔ. The apparent number of gating charges was 12.6 ± 0.6 eo in ShakerΔ, 12.7 ± 0.5 in 10 aa linker, and 12.3 ± 0.9 in 5 aa linker channels, but it was only 5.6 ± 0.3 eo in the 0 aa linker mutant channel. The maximum probability of opening (Pomax) as measured using noise analysis was not altered by the linker deletions. Activation kinetics were most affected by linker deletions; at 0 mV, the 5 and 0 aa linker channels' activation time constants were 89× and 45× slower than that of the ShakerΔ K+ channel, respectively. The initial lag of ionic currents when the prepulse was varied from −130 to −60 mV was 0.5, 14, and 2 ms for the 10, 5, and 0 aa linker mutant channels, respectively. These results suggest that: (a) the S4 segment moves only a short distance during activation since an S3–S4 linker consisting of only 5 amino acid residues allows for the total charge displacement to occur, and (b) the length of the S3–S4 linker plays an important role in setting ShakerΔ channel activation and deactivation kinetics.
Six transmembrane segments, S1–S6, cluster around the central pore-forming region in voltage-gated K⁺ channels. To investigate the structural characteristics of the S2 segment in the Shaker K⁺ channel, we replaced each residue in S2 singly with tryptophan (or with alanine for the native tryptophan). All but one of the 23 Trp mutants expressed voltage-dependent K⁺ currents in Xenopus oocytes. The effects of the mutations were classified as being of low or high impact on channel gating properties. The periodicity evident in the effects of these mutations supports an α-helical structure for the S2 segment. The high- and low-impact residues cluster onto opposite faces of a helical wheel projection of the S2 segment. The low-impact face is also tolerant of single mutations to asparagine. All results are consistent with the idea that the low-impact face projects toward membrane lipids and that changes in S2 packing occur upon channel opening. We conclude that the S2 segment is a transmembrane α helix and that the high-impact face packs against other transmembrane segments in the functional channel.
Two monoclonal antibodies designated as rhodopsin (rho) 1D4 and rho 4A2 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of a mouse immunized with bleached bovine rod outer segment disk membranes. These antibodies were specific for rhodopsin as determined by radioimmune labeling of bovine rod outer segment disk membrane proteins electrophoretically transferred from sodium dodecyl sulfate gels to CNBr-activated paper. Limited proteolytic digestion of rhodopsin in sealed disk membranes in conjunction with radioimmune assays indicated that the rho 1D4 antibody bound to the carboxyl-terminal segment of rhodopsin on the cytoplasmic side of disk membranes, whereas the rho 4A2 antibody bound to a determinant along the amino-terminal third of the rhodopsin polypeptide chain. Binding of the rho 4A2 antibody was sensitive to solubilization and photobleaching of rhodopsin. The rho 4A2 antibody did not bind to rhodopsin in sealed membrane disks but did bind to detergent-solubilized rhodopsin. Detergent-solubilized bleached rhodopsin was 13 times more antigenic than unbleached rhodopsin. Rhodopsin solubilized in Triton X-100 was more antigenic than rhodopsin solubilized in cholate. These results indicate that the 4A2 antibody serves as a sensitive immunological probe for structural changes of rhodopsin caused by solubilization and photobleaching. Both the rho 1D4 and 4A2 antibodies were also found to cross-react with frog rhodopsin but not Halobacterium halobium bacteriorhodopsin. The rho 4A2 antibody bound to the three forms of frog rhodopsin resolved by sodium dodecyl sulfate gel electrophoresis whereas rho 1D4 bound to only the two higher molecular weight frog rhodopsins. Finally, lectin inhibition studies using 125I-labeled succinyl-Con A and antibody inhibition studies confirmed previous results indicating freshly prepared bovine disks were sealed with the lectin binding sites oriented toward the inside of the disk, whereas frozen-thawed disks were predominantly unsealed with both membrane surfaces exposed. Frog disk membrane vesicles were shown to have the same orientation.
Site-directed mutagenesis experiments have suggested a model for the inactivation mechanism of Shaker potassium channels from
Drosophila melanogaster. In this model, the first 20 amino acids form a cytoplasmic domain that interacts with the open channel
to cause inactivation. The model was tested by the internal application of a synthetic peptide, with the sequence of the first
20 residues of the ShB alternatively spliced variant, to noninactivating mutant channels expressed in Xenopus oocytes. The
peptide restored inactivation in a concentration-dependent manner. Like normal inactivation, peptide-induced inactivation
was not noticeably voltage-dependent. Trypsin-treated peptide and peptides with sequences derived from the first 20 residues
of noninactivating mutants did not restore inactivation. These results support the proposal that inactivation occurs by a
cytoplasmic domain that occludes the ion-conducting pore of the channel.
We report here the high-level expression of a synthetic gene for bovine rhodopsin in transfected monkey kidney COS-1 cells. Rhodopsin is produced in these cells to a level of 0.3% of the cell protein, and it binds exogenously added 11-cis-retinal to generate the characteristic rhodopsin absorption spectrum. We describe a one-step immunoaffinity procedure for purification of the rhodopsin essentially to homogeneity. The COS-1 cell rhodopsin activates the GTPase activity of bovine transducin in a light-dependent manner with the same specific activity as that of purified bovine rhodopsin. Electron microscopy of immunogold-stained cells indicates that rhodopsin is located in the plasma membrane of the transfected cells and is oriented with the amino terminus on the extracellular side of the membrane. This orientation is analogous to that of rhodopsin in the disk membranes of photoreceptor cells in the bovine retina.
The potassium channel T1 domain plays an important role in the regulated assembly of subunit proteins. We have examined the assembly properties of the Shaker channel T1 domain to determine if the domain can self-assemble, the number of subunits in a multimer, Ns and the mechanism of assembly. High pressure liquid chromatography (HPLC) size exclusion chromotography (SEC) separates T1 domain proteins into two peaks. By co-assembly assays, these peaks are identified to be a high molecular weight assembled form and a low molecular weight monomeric form. To determine the Ns of the assembled protein peak on HPLC SEC, we first cross-linked the T1 domain proteins and then separated them on HPLC. Four evenly spaced bands co-migrate with the assembled protein peak; thus, the T1 domain assembles to form a tetramer. The absence of separate dimeric and trimeric peaks of assembled T1 domain protein suggests that the tetramer is the stable assembled state, most probably a closed ring structure.
Mechanosensitive ion channels play a critical role in transducing physical stresses at the cell membrane into an electrochemical response. The MscL family of large-conductance mechanosensitive channels is widely distributed among prokaryotes and may participate in the regulation of osmotic pressure changes within the cell. In an effort to better understand the structural basis for the function of these channels, the structure of the MscL homolog from Mycobacterium tuberculosis was determined by x-ray crystallography to 3.5 angstroms resolution. This channel is organized as a homopentamer, with each subunit containing two transmembrane alpha helices and a third cytoplasmic alpha helix. From the extracellular side, a water-filled opening approximately 18 angstroms in diameter leads into a pore lined with hydrophilic residues which narrows at the cytoplasmic side to an occluded hydrophobic apex that may act as the channel gate. This structure may serve as a model for other mechanosensitive channels, as well as the broader class of pentameric ligand-gated ion channels exemplified by the nicotinic acetylcholine receptor.
The T1 domain, a highly conserved cytoplasmic portion at the N-terminus of the voltage-dependent K+ channel (Kv) alpha-subunit, is responsible for driving and regulating the tetramerization of the alpha-subunits. Here we report the identification of a set of mutations in the T1 domain that alter the gating properties of the Kv channel. Two mutants produce a leftward shift in the activation curve and slow the channel closing rate while a third mutation produces a rightward shift in the activation curve and speeds the channel closing rate. We have determined the crystal structures of T1 domains containing these mutations. Both of the leftward shifting mutants produce similar conformational changes in the putative membrane facing surface of the T1 domain. These results suggest that the structure of the T1 domain in this region is tightly coupled to the channel's gating states.
The intracellular segment of the Shaker K+ channel between transmembrane domains S4 and S5 has been proposed to form at least part of the receptor for the tethered N-type inactivation "ball." We used the approach of cysteine substitution mutagenesis and chemical modification to test the importance of this region in N-type inactivation. We studied N-type inactivation or the block by a soluble inactivation peptide ("ball peptide") before and after chemical modification by methanethiosulfonate reagents. Particularly at position 391, chemical modification altered specifically the kinetics of ball peptide binding without altering other biophysical properties of the channel. Results with reagents that attach different charged groups at 391 C suggested that there are both electrostatic and steric interactions between this site and the ball peptide. These findings identify this site to be in or near the receptor site for the inactivation ball. At many of the other positions studied, modification noticeably inhibited channel current. The accessible cysteines varied in the state-dependence of their modification, with five- to tenfold changes in reactions rate depending on the gating state of the channel.
The subunit stoichiometry of the mammalian K+ channel KV1.1 (RCK1) was examined by linking together the coding sequences of 2-5 K+ channel subunits in a single open reading frame and tagging the expression of individual subunits with a mutation (Y379K or Y379R) that altered the sensitivity of the channel to block by external tetraethylammonium ion. Two lines of evidence argue that these constructs lead to K+ channel expression only through the formation of functional tetramers. First, currents expressed by tetrameric constructs containing a single mutant subunit have a sensitivity to tetraethylammonium that is well fitted by a single site binding isotherm. Second, a mutant subunit (Y379K) that expresses only as part of a heteromultimer contributes to the expression of functional channels when coexpressed with a trimeric construct but not a tetrameric construct.
The functional heterogeneity of potassium channels in eukaryotic cells arises not only from the multiple potassium channel
genes and splice variants but also from the combinatorial mixing of different potassium channel polypeptides to form heteromultimeric
channels with distinct properties. One structural element that determines the compatibility of different potassium channel
polypeptides in subunit assembly has now been localized to the hydrophilic amino-terminal domain. A Drosophila Shaker B (ShB)
potassium channel truncated polypeptide that contains only the hydrophilic amino-terminal domain can form a homomultimer;
the minimal requirement for the homophilic interaction has been localized to a fragment of 114 amino acids. Substitution of
the amino-terminal domain of a distantly related mammalian potassium channel polypeptide (DRK1) with that of ShB permits the
chimeric DRK1 polypeptide to coassemble with ShB.
Inactivation of ion channels is important in the control of membrane excitability. For example, delayed-rectifier K+ channels, which regulate action potential repolarization, are inactivated only slowly, whereas A-type K+ channels, which affect action potential duration and firing frequency, have both fast and slow inactivation. Fast inactivation of Na+ and K+ channels may result from the blocking of the permeation pathway by a positively charged cytoplasmic gate such as the one encoded by the first 20 amino acids of the Shaker B (ShB) K+ channel. We report here that mutation of five highly conserved residues between the proposed membrane-spanning segments S4 and S5 (also termed H4) of ShB affects the stability of the inactivated state and alters channel conductance. One such mutation stabilizes the inactivated state of ShB as well as the inactivated state induced in the delayed-rectifier type K+ channel drk1 by the cytoplasmic application of the ShB N-terminal peptide. The S4-S5 loop, therefore, probably forms part of a receptor for the inactivation gate and lies near the channel's permeation pathway.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
The voltage-sensitive gates regulating the permeability of ionic channels in nerve membrane were originally thought to be insensitive to the concentration1 and species2,3 of readily permeant cations. In contrast, blocking cations-tetraethyl-ammonium4, N-methylstrychnine5 or pancuronium6-when present in the inner end of potassium or sodium channels, hinder closing of the gates by a `foot in the door' effect. Recent work on acetylcholine channels has clearly shown that the lifetime of the conducting state is prolonged by certain species of permeant cations7,8. In this case, the presence of a permeant ion near the inner end of the channel is thought to prevent closing. Somewhat similar effects have recently been reported for the K+ channels of nerve membrane9-11. We report here unequivocal evidence that the permeant cations K+ and Rb+ when present externally, slow the closing of K+ channels in squid axon, supporting the view that gating is indeed sensitive to the presence and species of permeant cations in the channels.
Voltage-gated K+ channels were expressed in COS cells transiently transfected with a plasmid carrying a cDNA for an inactivation-removed Shaker K+ channel driven by an adenovirus promoter. Channel expression was followed by immunological detection, binding of radioactive charybdotoxin (CTX), and functional reconstitution into planar lipid bilayers. About 10(7) channels per transfected cell are expressed on the plasma membrane. The expressed channels are glycosylated and competent to bind CTX with the expected characteristics. Channels observed after insertion into planar lipid bilayers displayed the voltage-dependent gating, conduction, and ion selectivity behavior expected for this channel. Channels were solubilized in several detergents without loss of CTX binding activity. The results make plausible a systematic attack on the purification of milligram-level amounts of functional K+ channels from a heterologous expression system.
Voltage-gated ion channels are membrane proteins that play a central role in the propagation and transduction of cellular signals (Hille, 1992). Calcium ions entering cells through voltage-gated calcium channels serve as the trigger for neurotransmitter release, muscle contraction, and the release of hormones. Voltage-gated sodium channels initiate the nerve action potential and provide for its rapid propagation because the ion fluxes through these channels regeneratively cause more channels to open.
We are analyzing features of the K+ channel subunit proteins that are critical for function and regulation of these proteins. Our studies show biochemically that subunit proteins from the Shaker and Shaw subfamilies fail to assemble into a heteromultimer. The basis for this incompatibility is the sequences contained within the T1 assembly domain. For a subunit protein to heteromultimerize with a Shaker subunit protein, two regions within the T1 domain, A and B, must be of the Shaker subtype. Finally, we show that the incompatibility of a Shaw A region for assembly with a Shaker protein depends upon the composition of a 30 amino acid conserved sequence in the A region.
The galactose--glucose binding protein possesses two structural domains bordering a ligand binding cleft, with three polypeptide strands serving as a flexible hinge connecting the two domains. The hinge is known to bend, enabling the cleft to open by an angle of at least 18 degrees. Here the twisting motions of the hinge were examined by placing pairs of engineered cysteines on the perimeter of the cleft to generate six stable di-cysteine proteins. Each cysteine pair introduced reactive sulfhydryls into both rims of the cleft, one in the N-terminal domain and the other in the C-terminal domain. Collisions between sulfhydryls in different domains were trapped by disulfide formation, yielding sensitive detection of large amplitude domain rotations. When the cleft was occupied by the ligand D-glucose, counterclockwise hinge twist rotations were detected with amplitudes up to 36 degrees, and frequencies ranging from 10(1) to 10(3) collisions s-1. Removal of ligand from the cleft increased the range of twist angles 3-fold and the frequency of motions up to 10(2)-fold. Thus, in this representative hinged cleft protein, large amplitude hinge twist motions occur on biologically relevant timescales. The functional implications of such motions are discussed.
We have studied the glycosylation of Shaker K+ channel protein made in two expression systems: an insect cell culture line and amphibian oocytes. In both systems, two potential sites for N-linked glycosylation were modified. The modified sites were located between the first and second putative transmembrane segments, S1 and S2. Although the same sites appeared to be glycosylated in both systems, the fraction of protein glycosylated and the size, structure, or composition of the oligosaccharide chains added were quite different. The results indicate that the S1-S2 loop is extracellular, consistent with a cytoplasmic location for the N-terminus and a transmembrane disposition for hydrophobic segment S1. We have also shown that glycosylation occurs in two stages in oocytes, generating an immature and a mature form of Shaker protein. However, glycosylation is not required either for the assembly of functional channels or for their transport to the cell surface.
Cardiac beta-adrenergic receptors accelerate heart rate by modulating ionic currents through a pathway involving cyclic AMP-dependent protein kinase A (PKA). Previous studies have focused on the regulation of Ca2+ channels by PKA; however, due to the heterogeneity of K+ channels expressed within the heart, little is known about the mechanism by which PKA modulates individual K+ channels. Here we report that PKA strongly enhanced the activity of a cloned delayed rectifier K+ channel that is normally expressed in cardiac atria. This effect required a single PKA consensus phosphorylation site located near the amino terminus of the channel protein. Furthermore, patch clamp analysis revealed that PKA phosphorylation increased the open time that single channels spend in higher conductance states. These studies provide evidence that hormonal modulation of a cardiac K+ channel involves direct phosphorylation by PKA.
Voltage-activated potassium (Kv) channels from mammalian brain are hetero-oligomers containing alpha and beta subunits. Coexpression of Kv1 alpha and Kv beta 1 subunits confers rapid A-type inactivation on noninactivating potassium channels (delayed rectifiers) in expression systems in vitro. We have delineated a Kv1.5 aminoterminal region of up to 90 amino acids (residues 112-201) that is sufficient for interactions of Kv1.5 alpha and Kv beta 1 subunits. Within this region of the Kv1.5 amino terminus (residues 193-201), a Kv beta 1 interaction site necessary for Kv beta 1-mediated rapid inactivation of Kv1.5 currents was detected. This interaction site motif (FYE/QLGE/DEAM/L) is found exclusively in the Shaker-related subfamily (Kv1). The results show that hetero-oligomerization between alpha and Kv beta 1 subunits is restricted to Shaker-related potassium channel alpha subunits.
Shaker potassium (K+) channels normally lack intrasubunit and intersubunit disulfide bonds. However, disulfide bonds are formed between Shaker subunits in intact cells exposed to oxidizing conditions. Upon electrophoresis under nonreducing conditions, intersubunit disulfide bond formation was detected by the presence of four high molecular weight adducts of Shaker protein. This result suggests that intracellular cysteine residues are in sufficiently close proximity in the native structure of the Shaker channel to form intersubunit disulfide bonds. To test this hypothesis, wild-type and mutant Shaker proteins were exposed to oxidizing conditions in intact cells. Intersubunit disulfide bond formation was eliminated upon serine substitution of either C96 in the amino terminal or C505 in the carboxyl terminal of the protein. In contrast, disulfide bond formation was not eliminated upon serine substitution of both C301 and C308 in the cytoplasmic loop between transmembrane segments S2 and S3. Exposure of Shaker-expressing cells to oxidizing conditions did not significantly alter the amplitude, kinetics, or voltage dependence of the Shaker current, demonstrating that the native tertiary and quaternary structures of the channel were maintained under oxidizing conditions. These results indicate that intersubunit disulfide bonds form between C96 and C505, providing evidence that the amino- and carboxyl-terminal regions of adjacent subunits are in proximity in the native structure of the channel. The disulfide-bonded adducts were found to represent a dimer, a trimer, and two forms of tetramer, one linear and one circular, containing one, two, three, or four disulfide bonds, respectively. These results provide a direct biochemical demonstration that Shaker K+ channels contain four pore-forming subunits.
The nicotinic acetylcholine (ACh) receptor is the neurotransmitter-gated ion channel responsible for the rapid propagation of electrical signals between cells at the nerve/muscle synapse. We report here the 4.6 A structure of this channel in the closed conformation, determined by electron microscopy of tubular crystals of Torpedo postsynaptic membranes embedded in amorphous ice. The analysis was conducted on images recorded at 4 K with a 300 kV field emission source, by combining data from four helical families of tubes (-16,6; -18,6; -15,7; -17,5), and applying three-dimensional corrections for lattice distortions. The study extends earlier work on the same specimen at 9 A resolution. Several features having functional implications now appear with better definition. The gate of the channel forms a narrow bridge, consisting of no more than one or two rings of side-chains, across the middle portion of the membrane-spanning pore. Tunnels, framed by twisted beta-sheet strands, are resolved in the extracellular wall of the channel connecting the water-filled vestibule to the putative ACh-binding pockets. A set of narrow openings through which ions can flow are resolved between alpha-helical segments forming part of the cytoplasmic wall of the channel. It is suggested that the extracellular tunnels are access routes to the binding pockets for ACh, and that the cytoplasmic openings serve as filters to exclude anions and other impermeant species from the vicinity of the pore. Both transverse pathways are likely to be important in achieving a rapid postsynaptic response.
Ion channels, like many other proteins, have moving parts that perform useful functions. The channel proteins contain an aqueous, ion-selective pore that crosses the plasma membrane, and they use a number of distinct ‘gating’ mechanisms to open and close this pore in response to biological stimuli such as the binding of a ligand or a change in the transmembrane voltage.
This review is written at a watershed in our understanding of ion channels.
1. INTRODUCTION 240
1.1 Basic structure of voltage-activated channels 241
1.2 What are the physical motions of the channel protein during gating? 243
1.3 Gating involves several distinct mechanisms of activation and inactivation 246
2. ACTIVATION GATING 246
2.1 Early evidence for an activation gate at the intracellular mouth 247
2.1.1 Open channel blockade 247
2.1.2 The ‘ foot-in-the-door’ effect 249
2.1.3 Trapping of blockers behind closed activation gates 249
2.2 Site-directed mutagenesis and the difficulty of inferring structural roles from functional effects 250
2.3 State-dependent cysteine modification as a reporter of position and motion 251
2.4 Localization of activation gating 254
2.4.1 The trapping cavity 254
2.4.2 The activation gate 255
2.4.3 Is there more than one site of activation gating? 258
3. INACTIVATION GATING 259
3.1 Ball-and-chain ( N-type ) inactivation 261
3.1.1 Nature of the ‘ball’ – a tethered blocking particle 262
3.1.2 The ball receptor 263
3.1.3 The chain 264
3.1.4 Variations on the N-type inactivation theme: multiple balls, foreign balls, anti-balls 265
3.2 C-type inactivation 266
3.2.1 C-type inactivation and the outer mouth of the K ⁺ channel 266
3.2.2 The selectivity filter participates in C-type inactivation 267
3.2.3 A consistent structural picture of C-type inactivation 269
3.3 By what mechanism do other voltage-gated channels inactivate? 272
4. THE VOLTAGE SENSOR 273
4.1 Quantitative principles of voltage-dependent gating 276
4.2 S 4 ( and its neighbours ) as the principal voltage sensor 277
4.2.1 Mutational effects on voltage-dependence and charge movement 277
4.2.2 Evidence for the translocation of S 4 279
4.2.3 Real-time monitoring of S 4 motion by fluorescence 282
4.3 Coupling between the voltage sensor and gating 283
5. CONCLUSION 284
6. ACKNOWLEDGEMENTS 287
7. REFERENCES 287
1. The whole-cell configuration of the patch-clamp technique and immunoprecipitation experiments were used to investigate the effects of tyrosine kinases on voltage-dependent K+ channel gating in cultured mouse Schwann cells. 2. Genistein, a broad-spectrum tyrosine kinase inhibitor, markedly reduced the amplitude of a slowly inactivating delayed-rectifier current (IK) and, to a lesser extent, that of a transient K+ current (IA). Similar results were obtained on IK with another tyrosine kinase inhibitor, herbimycin A. Daidzein, the inactive analogue of genistein, was without effect. 3. Unlike herbimycin A, genistein produced additional effects on IA by profoundly affecting its gating properties. These changes consisted of slower activation kinetics with an increased time to peak, a positive shift in the voltage dependence of activation (by +30 mV), a decrease in the steepness of activation gating (9 mV per e-fold change) and an acceleration of channel deactivation. 4. The steepness of the steady-state inactivation was increased by genistein treatment, while the recovery from inactivation was not significantly altered. 5. The action of genistein on voltage-dependent K+ (Kv) currents was accompanied by a decrease in tyrosine phosphorylation of Kv1.4 as well as Kv1.5 and Kv2.1 encoding transient and slowly inactivating delayed-rectifier K+ channel alpha subunits, respectively. 6. In conclusion, the present study shows that tyrosine kinases markedly affect the amplitude of voltage-dependent K+ currents in Schwann cells and finely tune the gating properties of the transient K+ current component IA. These modulations may be functionally relevant in the control of K+ channel activity during Schwann cell development and peripheral myelinogenesis.
Electrical excitability is a fundamental property of the neuromuscular systems of metazoans. The varied response of neurons to electrical excitation is largely accounted for by a diverse set of voltage-gated potassium (KV) channels in the excitable membrane. The complete structure of a KV channel is not yet available. However, recent structural biological experiments have begun to provide new insight into how specific KV channels are formed and regulated, and how they function and interact with other proteins. In particular, the selectivity of KV channels for K+ and suggestions as to how these structural elements might assemble into a functional KV channel are discussed.
The structure of the cytoplasmic assembly of voltage-dependent K+ channels was solved by x-ray crystallography at 2.1 angstrom resolution. The assembly includes the cytoplasmic (T1) domain of the integral membrane alpha subunit together with the oxidoreductase beta subunit in a fourfold symmetric T1(4)beta4 complex. An electrophysiological assay showed that this complex is oriented with four T1 domains facing the transmembrane pore and four beta subunits facing the cytoplasm. The transmembrane pore communicates with the cytoplasm through lateral, negatively charged openings above the T1(4)beta4 complex. The inactivation peptides of voltage-dependent K(+) channels reach their site of action by entering these openings.