Gating modifier toxins reveal a conserved structural motif in voltage-gated Ca2+ and K+ channels

Protein toxins from venomous animals exhibit remarkably specific and selective interactions with a wide variety of ion channels. Hanatoxin and grammotoxin are two related protein toxins found in the venom of the Chilean Rose Tarantula, Phrixotrichus spatulata. Hanatoxin inhibits voltage-gated K⁺ channels and grammotoxin inhibits voltage-gated Ca²⁺ channels. Both toxins inhibit their respective channels by interfering with normal operation of the voltage-dependent gating mechanism. The sequence homology of hanatoxin and grammotoxin, as well as their similar mechanism of action, raises the possibility that they interact with the same region of voltage-gated Ca²⁺ and K⁺ channels. Here, we show that each toxin can interact with both voltage-gated Ca²⁺ and K⁺ channels and modify channel gating. Moreover, mutagenesis of voltage-gated K⁺ channels suggests that hanatoxin and grammotoxin recognize the same structural motif. We propose that these toxins recognize a voltage-sensing domain or module present in voltage-gated ion channels and that this domain has a highly conserved three-dimensional structure.     

Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo)

Large conductance calcium- and voltage-sensitive K⁺ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca²⁺ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca²⁺ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4–5 elementary charges.     

Toward a Molecular Understanding of Voltage-Gated Potassium Channels

Voltage-Gated Potassium Channels . Many different types of potassium (K⁺) channels exist and they play a central role in the fine tuning of excitability properties. Of the distinct subpopulations of K⁺ channels expressed in different cells, voltage-gated K⁺ channels have been studied most thoroughly at a molecular level. Over the last few years, the joint application of recombinant DNA technology together with electrophysiology, such as the voltage clamp and the patch clamp techniques, has produced a wealth of information. We have begun to unravel the genetic basis of ion channel diversity. In particular, the Xenopus oocyte expression system has turned out to be of enormous experimental value. Oocytes microinjected with “cloned” mRNA have been used to gain insight into biophysical and pharmacologic properties of voltage-gated K⁺, Na⁺, and Ca²⁺ channels. Here, we will review our understanding of K⁺ channel diversity based upon the fact that ion channels are encoded as a large multigene family. We have caught a first glimpse at possible molecular mechanisms underlying several biophysical properties characteristic for voltage-gated ion channels: voltage dependence of activation and inactivation, and ion permeation and selectivity. We will discuss molecular mechanisms of K⁺ channel activation and inactivation. We will also describe experiments that led to the identification of the “pore region,” and we will present a model of a potassium selective ion channel pore.     

Importance of lipid–pore loop interface for potassium channel structure and function

Potassium (i.e., K⁺) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K⁺ channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K⁺ channel inactivation and exhibits a remarkable structural plasticity that correlates to K⁺ channel inactivation. The transmembrane helix 1 unwinds when the K⁺ channel enters the inactivated state and rewinds during the transition to the closed state. In addition to well-characterized changes at the K⁺ ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K⁺ channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K⁺ channel pore.     

Molecular basis of plant-specific acid activation of K+ uptake channels

During stomatal opening potassium uptake into guard cells and K⁺ channel activation is tightly coupled to proton extrusion. The pH sensor of the K⁺ uptake channel in these motor cells has, however, not yet been identified. Electrophysiological investigations on the voltage-gated, inward rectifying K⁺ channel in guard cell protoplasts from Solanum tuberosum (KST1), and the kst1 gene product expressed in Xenopus oocytes revealed that pH dependence is an intrinsic property of the channel protein. Whereas extracellular acidification resulted in a shift of the voltage-dependence toward less negative voltages, the single-channel conductance was pH-insensitive. Mutational analysis allowed us to relate this acid activation to both extracellular histidines in KST1. One histidine is located within the linker between the transmembrane helices S3 and S4 (H160), and the other within the putative pore-forming region P between S5 and S6 (H271). When both histidines were substituted by alanines the double mutant completely lost its pH sensitivity. Among the single mutants, replacement of the pore histidine, which is highly conserved in plant K⁺ channels, increased or even inverted the pH sensitivity of KST1. From our molecular and biophysical analyses we conclude that both extracellular sites are part of the pH sensor in plant K⁺ uptake channels.     

Large conductance voltage- and calcium-dependent K+ channel, a distinct member of voltage-dependent ion channels with seven N-terminal transmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C terminus

Large conductance voltage- and Ca²⁺-dependent K⁺ (MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. At the N terminus, we have recently proposed a new model where MaxiK channels have an additional transmembrane region (S0) that confers β subunit regulation. Using transient expression of epitope tagged MaxiK channels, in vitro translation, functional, and “in vivo” reconstitution assays, we now show that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Further, our results indicate that hydrophobic segments S9-S10 in the C terminus are cytoplasmic and unequivocally demonstrate that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus.     

Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2

The BCL-2 family of proteins is composed of both pro- and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-X_L monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion efflux. Release is maximum at ≈pH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4.0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl⁻ selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl⁻-selective (P_K/P_Cl = 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K⁺-selective (P_K/P_Cl = 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K⁺ selectivity (P_K/P_Cl = 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.     

Two functionally distinct subsites for the binding of internal blockers to the pore of voltage-activated K+ channels

Many blockers of Na⁺ and K⁺ channels act by blocking the pore from the intracellular side. For Shaker K⁺ channels, such intracellular blockers vary in their functional effect on slow (C-type) inactivation: Some blockers interfere with C-type inactivation, whereas others do not. These functional differences can be explained by supposing that there are two overlapping “subsites” for blocker binding, only one of which inhibits C-type inactivation through an allosteric effect. We find that the ability to bind to these subsites depends on specific structural characteristics of the blockers, and correlates with the effect of mutations in two distinct regions of the channel protein. These interactions are important because they affect the ability of blockers to produce use-dependent inhibition.     

Structural determinants of ion permeation in CRAC channels

CRAC channels generate Ca²⁺ signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca²⁺ selectivity, low Cs⁺ permeability, and small unitary conductance. To identify the ion conduction pathway and gain insight into the structural bases of these permeation characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiol-reactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca²⁺ selectivity is conferred mainly by E106. The ion conduction pathway is sufficiently narrow along much of its length to permit stable coordination of Cd²⁺ by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. These results provide a structural framework to understand the unique permeation properties of CRAC channels.     

Rearrangement of a unique Kv1.3 selectivity filter conformation upon binding of a drug

We report two structures of the human voltage-gated potassium channel (Kv) Kv1.3 in immune cells alone (apo-Kv1.3) and bound to an immunomodulatory drug called dalazatide (dalazatide–Kv1.3). Both the apo-Kv1.3 and dalazatide–Kv1.3 structures are in an activated state based on their depolarized voltage sensor and open inner gate. In apo-Kv1.3, the aromatic residue in the signature sequence (Y447) adopts a position that diverges 11 Å from other K⁺ channels. The outer pore is significantly rearranged, causing widening of the selectivity filter and perturbation of ion binding within the filter. This conformation is stabilized by a network of intrasubunit hydrogen bonds. In dalazatide–Kv1.3, binding of dalazatide to the channel’s outer vestibule narrows the selectivity filter, Y447 occupies a position seen in other K⁺ channels, and this conformation is stabilized by a network of intersubunit hydrogen bonds. These remarkable rearrangements in the selectivity filter underlie Kv1.3’s transition into the drug-blocked state.

Potassium channels form K⁺-selective pores that span cell membranes in virtually all living organisms. In humans, a family of 78 genes encodes four classes of K⁺ channels (voltage-gated, calcium-activated, inward rectifier, and two-pore channels), which are involved in a multitude of physiological functions in both electrically excitable and nonexcitable cells (1). All four classes of channels conduct K⁺ ions selectively and rapidly, but they differ in how they are gated. The selectivity filter is the structural element responsible for the exquisitely K⁺-selective pore (2–5). It is the narrowest part of the ion conduction pathway and connects a water-filled cavity in the center of the protein with an outer vestibule in the extracellular solution. The filter accommodates K⁺ ions at four sites called S1, S2, S3, and S4 starting at the extracellular side. The signature sequence G(Y/F)G in the selectivity filter plays a critical role in making the pore K⁺ selective (6, 7). In all K⁺ channel structures determined, both bacterial and eukaryotic, the aromatic residue (Y or F) in the signature sequence is nearly identical in position, although these channels differ in the conformation (closed or open) of the S6 helical inner gate (8). In the hERG/Kv11.1 channel, a subtle deviation in the position of F627 in the signature sequence causes a slight widening of the selectivity filter, which has been suggested to underlie the channel’s transition into the C-type inactivated state (8).The voltage-gated potassium channel (Kv) Kv1.3–Kvβ2 in lymphocytes and microglia provides the counterbalancing cation efflux to promote calcium entry necessary for calcium signaling (9, 10). Selective blockers of Kv1.3–Kvβ2 treat diverse autoimmune and neuroinflammatory diseases in rodent models (9, 10), highlighting the channel’s physiological and pharmacological importance. Here, we determined structures of Kv1.3 complexed to its accessory subunit Kvβ2 alone (apo-Kv1.3) and bound to dalazatide (dalazatide–Kv1.3), a potent and selective peptide inhibitor of Kv1.3 in clinical trials for autoimmune and neuroinflammatory diseases (10–14). Both apo-Kv1.3 and dalazatide–Kv1.3 are in the activated state based on the depolarized voltage sensor and open S6 helical inner gate. Comparison of the two structures reveals substantial conformational changes in the selectivity filter. In apo-Kv1.3, Y447 in the signature sequence diverges more than 11 Å from the position of corresponding aromatic residues in other K⁺ channels, both in eukaryotes and bacteria. The outer pore is wider at S1 and S2 and narrowed at S0 K⁺-binding sites, resulting in loss of the K⁺ ion from site S2. A network of intrasubunit hydrogen bonds (H451–Y447, H451–D449) stabilizes this unique conformation of the selectivity filter of apo-Kv1.3, and, interestingly, the intrasubunit hydrogen bond (W436–D449) that prevents C-type inactivation (15) is absent. Apo-Kv1.3’s selectivity filter and voltage-sensing domain (VSD) differ significantly from two structures of Kv1.3 that were recently described (16). In dalazatide–Kv1.3, dalazatide’s interaction with H451 disrupts the H451–Y447 hydrogen bond, freeing Y447 to swing back into the interior of the selectivity filter and adopt a position seen in other K⁺ channels. The selectivity filter is narrower, and K⁺ ions are present at sites S2–S4 but not at S1. This conformation is stabilized by a network of intersubunit hydrogen bonds (Y447–W437, Y447–T441, and H451–D449), but the intrasubunit hydrogen bond (W436–D449) that prevents C-type inactivation (15) is likely absent. Our structures provide a basis for the design of Kv1.3 inhibitors for use as immunomodulatory therapeutics.     

Family of prokaryote cyclic nucleotide-modulated ion channels

Cyclic nucleotide-modulated ion channels are molecular pores that mediate the passage of ions across the cell membrane in response to cAMP or GMP. Structural insight into this class of ion channels currently comes from a related homolog, MloK1, that contains six transmembrane domains and a cytoplasmic cyclic nucleotide binding domain. However, unlike eukaryote hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels, MloK1 lacks a C-linker region, which critically contributes to the molecular coupling between ligand binding and channel opening. In this study, we report the identification and characterization of five previously unidentified prokaryote homologs with high sequence similarity (24–32%) to eukaryote HCN and CNG channels and that contain a C-linker region. Biochemical characterization shows that two homologs, termed AmaK and SthK, can be expressed and purified as detergent-solubilized protein from Escherichia coli membranes. Expression of SthK channels in Xenopus laevis oocytes and functional characterization using the patch-clamp technique revealed that the channels are gated by cAMP, but not cGMP, are highly selective for K⁺ ions over Na⁺ ions, generate a large unitary conductance, and are only weakly voltage dependent. These properties resemble essential properties of various eukaryote HCN or CNG channels. Our results contribute to an understanding of the evolutionary origin of cyclic nucleotide-modulated ion channels and pave the way for future structural and functional studies.Hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels belong to the superfamily of voltage-gated K⁺ channels. Both types of channels share a similar domain topology with six transmembrane domains, a C-linker region, and a cyclic nucleotide binding domain (CNBD). The S5–S6 segment forms the channel pore, including the selectivity filter for cations. The S4 segment contains several positively charged amino acids, suggesting that it acts as voltage sensor. Despite these similarities in sequence, the function of HCN and CNG channels is noticeably different: HCN channels activate upon membrane hyperpolarization and can be modulated by cyclic nucleotides. They are weakly selective for K⁺ over Na⁺ ions (for reviews, see refs. 1–3). In contrast, CNG channels are activated by the binding of cyclic nucleotides solely and their activity depends only weakly on voltage. The ionic current is carried by both monovalent and divalent cations (for reviews, see refs. 4 and 5).Insight into the structure of HCN channels has been gained only from crystal structures of the isolated intracellular C-linker and CNBD of mammalian HCN1, HCN2, HCN4, and invertebrate spHCN1. These parts of the channel assemble into tetramers (6–9). Further structural information comes from prokaryote ion channels that are homologous to HCN and CNG channels, such as the bacterial cyclic nucleotide-regulated K⁺ channel MloK1 (10–13). MloK1 lacks a C-linker region, but has a CNBD with an overall structure that is remarkably similar to the CNBD of eukaryote HCN channels (10). Based on the dimer assembly of the MloK1 CNBD in the crystal structure, a gating mechanism has been proposed in which the pore opening in the tetrameric channel arises from the action of the four CNBDs as a dimer of dimers (10). The crystal structure of the MloK1 transmembrane domain (11) reveals a domain topology that resembles that of the voltage-gated K⁺ channel Kv1.2 (14), but with important differences. The MloK1 structure suggests that the S1–S4 domain and its associated linker in MloK1 can serve as a clamp to constrain the gate and possibly function in concert with the CNBD to regulate channel opening (11). Additionally, crystal structures have also been determined for the C-linker and cyclic nucleotide binding homology domain (CNBHD) of related ion channels, including the zebrafish EAG-like (ELK) K⁺ channel (15), the mosquito ERG K⁺ channel (16), and the mouse EAG1 K⁺ channel (17). Structural insight into the mechanism of ion permeation has been derived from a prokaryote ion channel NaK (18), which was mutated to mimic the CNG channel pore region (19). Collectively, these structural data have brought valuable information about the determinants of ion permeation, domain assembly, ligand recognition, channel gating and regulation, as well as effects of disease-causing mutations (20).Despite this tremendous progress, crystal structures for whole-eukaryote HCN and CNG channels are still not available at present, and structural insight into fundamental aspects of ion channel function is still lacking, such as the inverse voltage sensitivity in HCN channels and the coupling between cyclic nucleotide binding and channel opening by the C-linker domain, which is, as mentioned, absent in the MloK1 channel (10). In contrast, a putative voltage-gated K⁺ channel containing a C-linker region and CNBD similar to eukaryote channels was identified in the genome of the cyanobacterium Trichodesmium erythraeum (21), here termed TerK, and it was suggested to possibly represent an ancestral HCN or CNG channel (21). However, neither structure nor function of this prokaryote homolog is known. In this study, we report the characterization of TerK and four additional prokaryote ion channels, which all contain six putative transmembrane domains, a C-linker region, and a CNBD, and apparently form a family of prokaryote ion channels with close similarity to eukaryote HCN and CNG channels. We describe the expression in Escherichia coli, detergent screening and biochemical purification of these different homologs. Moreover, we identified two homologs, SthK (from Spirochaeta thermophila) and AmaK (from Arthrospira maxima), which can be stably extracted with detergents and purified in sufficiently high amounts for biochemical and structural studies. Using confocal fluorescence microscopy and electrophysiological recordings, we describe essential functional properties of one homolog, SthK. We find that SthK has electrophysiological properties that closely resemble those of eukaryote CNG channels as it is gated by intracellular cAMP and produces large unitary currents, whereas its activity is relatively insensitive to voltage. However, unlike CNG channels, SthK contains the selectivity filter sequence -TIGYGD-, which is more similar to HCN channels and other K⁺ selective channels. We could experimentally demonstrate that SthK channels are highly selective for K⁺ over Na⁺ ions. Importantly, SthK has several sequence features that closely resemble eukaryote cyclic nucleotide-modulated channels, including a C-linker region, which is missing in previously studied prokaryote homologs, such as MloK1 (10, 12, 13) and MmaK (22). Together, these data make the SthK channel a promising candidate for future structural analysis to learn more about how mammalian CNG and HCN channels work.     

Sodium channel selectivity filter regulates antiarrhythmic drug binding

Local anesthetic antiarrhythmic drugs block Na⁺ channels and have important clinical uses. However, the molecular mechanism by which these drugs block the channel has not been established. The family of drugs is characterized by having an ionizable amino group and a hydrophobic tail. We hypothesized that the charged amino group of the drug may interact with charged residues in the channel’s selectivity filter. Mutation of the putative domain III selectivity filter residue of the adult rat skeletal muscle Na⁺ channel (μ1) K1237E increased resting lidocaine block, but no change was observed in block by neutral analogs of lidocaine. An intermediate effect on the lidocaine block resulted from K1237S and there was no effect from K1237R, implying an electrostatic effect of Lys. Mutation of the other selectivity residues, D400A (domain I), E755A (domain II), and A1529D (domain IV) allowed block by externally applied quaternary membrane-impermeant derivatives of lidocaine (QX314 and QX222) and accelerated recovery from block by internal QX314. Neo-saxitoxin and tetrodotoxin, which occlude the channel pore, reduced the amount of QX314 bound in D400A and A1529D, respectively. Block by outside QX314 in E755A was inhibited by mutation of residues in transmembrane segment S6 of domain IV that are thought to be part of an internal binding site. The results demonstrate that the Na⁺ channel selectivity filter is involved in interactions with the hydrophilic part of the drugs, and it normally limits extracellular access to and escape from their binding site just within the selectivity filter. Participation of the selectivity ring in antiarrhythmic drug binding and access locates this structure adjacent to the S6 segment.     

Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K+ channels

K⁺ channels distinguish K⁺ from Na⁺ in the selectivity filter, which consists of four ion-binding sites (S1–S4, extracellular to intracellular) that are built mainly using the carbonyl oxygens from the protein backbone. In addition to ionic discrimination, the selectivity filter regulates the flow of ions across the membrane in a gating process referred to as C-type inactivation. A characteristic of C-type inactivation is a dependence on the permeant ion, but the mechanism by which permeant ions modulate C-type inactivation is not known. To investigate, we used amide-to-ester substitutions in the protein backbone of the selectivity filter to alter ion binding at specific sites and determined the effects on inactivation. The amide-to-ester substitutions in the protein backbone were introduced using protein semisynthesis or in vivo nonsense suppression approaches. We show that an ester substitution at the S1 site in the KcsA channel does not affect inactivation whereas ester substitutions at the S2 and S3 sites dramatically reduce inactivation. We determined the structure of the KcsA S2 ester mutant and found that the ester substitution eliminates K⁺ binding at the S2 site. We also show that an ester substitution at the S2 site in the K_vAP channel has a similar effect of slowing inactivation. Our results link C-type inactivation to ion occupancy at the S2 site. Furthermore, they suggest that the differences in inactivation of K⁺ channels in K⁺ compared with Rb⁺ are due to different ion occupancies at the S2 site.Potassium channels are a ubiquitous family of integral membrane proteins that facilitate the selective conduction of K⁺ ions across cellular membranes (1). K⁺ selectivity is achieved by a structural element in the K⁺ channel pore called the selectivity filter (2). The selectivity filter consists of four sequential ion-binding sites (labeled S1–S4, from the outside to inside) that are built using protein backbone carbonyl oxygen atoms and the threonine side chain from the protein sequence T-V-G-Y-G (Fig. 1A) (4, 5).Open in a separate windowFig. 1.Ester substitutions in the selectivity filter of the KcsA channel. (A) Close-up view of the selectivity filter of the wild-type KcsA channel [Protein Data Bank (PDB): 1K4C]. Two opposite subunits are shown in stick representation, and the K⁺ ions bound are shown as purple spheres. The amide bonds (1′–4′) and the ion-binding sites in the selectivity filter (S1–S4) are labeled. (B) Macroscopic currents for the KcsA channels were elicited at +100 mV by a rapid change in pH from 7.5 to 3.0. (C) Single-channel currents recorded at steady-state conditions at pH 3.0. The pH 3.0 solution is 10 mM succinate, 200 mM KCl, and the pH 7.5 solution is 10 mM Hepes–KOH, 200 mM KCl. The data for KcsA^WT are from ref. 3.In addition to selective conduction of K⁺, the selectivity filter acts as a gate to regulate the flow of ions through the pore (6–8). During this gating process, conformational changes at the selectivity filter convert it from a conductive to a nonconductive state. In voltage-gated K⁺ (K_v) channels, this gating process is referred to as C-type inactivation (9). C-type inactivation is a physiologically important process as it plays a direct role in regulating neuronal firing and in pacing cardiac action potentials (8). The KcsA K⁺ channel from Streptomyces lividans undergoes an inactivation process that is functionally similar to C-type inactivation in a eukaryotic K_v channel (10–13). As the KcsA channel is easily amenable to structural studies, it has become an important model system for understanding the structure of the selectivity filter in the C-type–inactivated state and the forces that drive inactivation (14, 15).One of the hallmarks of C-type inactivation is a dependence on the permeant ion (6, 7). The rate of C-type inactivation decreases when the K⁺ concentration is increased or when the permeant ion is changed from K⁺ to Rb⁺ (16, 17). Crystallographic studies on K⁺ channels have shown that a change in the permeant ion or its concentration results in changes in the ion occupancy at the binding sites in the selectivity filter (18, 19). For example, K⁺ and Rb⁺ at similar concentrations show different occupancies at the ion-binding sites, and the channel exhibits different rates of inactivation in K⁺ compared with Rb⁺ (3, 16, 20), which suggests a link between ion occupancy at the selectivity filter and inactivation (21, 22). The influence of permeant ions on inactivation has been proposed to arise from a “foot in the door”-like effect in which ion binding at a specific site prevents inactivation, similar to the presence of a foot in the doorway that prevents a door from closing (16, 23). The binding site responsible for the foot in the door effect is suspected to be at the extracellular side of the channel, but the exact location of the binding site, whether in the selectivity filter or at the extracellular mouth of the filter, is not known (6).In this study, we investigate this link between ion binding at the selectivity filter and inactivation. The approach that we use is to alter ion binding at the selectivity filter sites and to determine the effect on inactivation. The S1–S3 ion-binding sites in the selectivity filter are constructed by backbone carbonyl oxygens. Therefore, conventional site-directed mutagenesis does not allow us to alter these sites. Instead, we use chemical synthesis and nonsense suppression approaches to introduce amide-to-ester substitutions in the protein backbone to perturb ion binding to specific sites in the selectivity filter (24, 25).Amide-to-ester substitutions have previously been used to engineer the protein backbone for studies on protein stability and folding (26). Ester bonds are isosteric to amide bonds but have altered hydrogen-bonding properties and reduced electronegativity at the carbonyl oxygen (27). This reduction in the electronegativity of the carbonyl oxygen, by roughly one-half compared with an amide bond, perturbs ion binding to the selectivity filter. Amide-to-ester substitutions have previously been reported in the selectivity filters of the Kir2.1 and the KcsA K⁺ channels (28, 29). In the Kir2.1 channel, an ester substitution for the 3′ amide bond (see Fig. 1A for nomenclature) was found to reduce channel conductance and to produce distinct subconductance levels. In the KcsA channel, an ester substitution for the 1′ amide bond was found to reduce channel conductance, and a crystal structure of the ester mutant showed that the ester substitution decreased ion occupancy at the S1 site. Neither of these studies examined the effect of the ester substitutions on inactivation.Here we substitute the 1′, 2′, and 3′ amide bonds in the selectivity filter of the KcsA K⁺ channel with esters and investigate the effect on inactivation. We determine the crystal structure of the 2′ ester mutant of the KcsA channel to examine the effect of the ester substitution on the structure and ion occupancy of the selectivity filter. We also investigate the effect of an ester substitution at the 2′ amide bond in the selectivity filter on inactivation in the voltage-gated K⁺ channel, K_vAP. Our results show that the S1 and S2 sites in the selectivity filter do not act as the foot in the door sites to prevent inactivation. Unexpectedly, we find that a lack of ion binding at the S2 site reduces inactivation.     

Filter gate closure inhibits ion but not water transport through potassium channels

The selectivity filter of K⁺ channels is conserved throughout all kingdoms of life. Carbonyl groups of highly conserved amino acids point toward the lumen to act as surrogates for the water molecules of K⁺ hydration. Ion conductivity is abrogated if some of these carbonyl groups flip out of the lumen, which happens (i) in the process of C-type inactivation or (ii) during filter collapse in the absence of K⁺. Here, we show that K⁺ channels remain permeable to water, even after entering such an electrically silent conformation. We reconstituted fluorescently labeled and constitutively open mutants of the bacterial K⁺ channel KcsA into lipid vesicles that were either C-type inactivating or noninactivating. Fluorescence correlation spectroscopy allowed us to count both the number of proteoliposomes and the number of protein-containing micelles after solubilization, providing the number of reconstituted channels per proteoliposome. Quantification of the per-channel increment in proteoliposome water permeability with the aid of stopped-flow experiments yielded a unitary water permeability p_f of (6.9 ± 0.6) × 10⁻¹³ cm³⋅s⁻¹ for both mutants. “Collapse” of the selectivity filter upon K⁺ removal did not alter p_f and was fully reversible, as demonstrated by current measurements through planar bilayers in a K⁺-containing medium to which K⁺-free proteoliposomes were fused. Water flow through KcsA is halved by 200 mM K⁺ in the aqueous solution, which indicates an effective K⁺ dissociation constant in that range for a singly occupied channel. This questions the widely accepted hypothesis that multiple K⁺ ions in the selectivity filter act to mutually destabilize binding.     

Killing K Channels with TEA+

Tetraethylammonium (TEA⁺) is widely used for reversible blockade of K channels in many preparations. We noticed that intracellular perfusion of voltage-clamped squid giant axons with a solution containing K⁺ and TEA⁺ irreversibly decreased the potassium current when there was no K⁺ outside. Five minutes of perfusion with 20 mM TEA⁺, followed by removal of TEA⁺, reduced potassium current to <5% of its initial value. The irreversible disappearance of K channels with TEA⁺ could be prevented by addition of ≥ 10 mM K⁺ to the extracellular solution. The rate of disappearance of K channels followed first-order kinetics and was slowed by reducing the concentration of TEA⁺. Killing is much less evident when an axon is held at −110 mV to tightly close all of the channels. The longer-chain TEA⁺ derivative decyltriethylammonium (C10⁺) had irreversible effects similar to TEA⁺. External K⁺ also protected K channels against the irreversible action of C10⁺. It has been reported that removal of all K⁺ internally and externally (dekalification) can result in the disappearance of K channels, suggesting that binding of K⁺ within the pore is required to maintain function. Our evidence further suggests that the crucial location for K⁺ binding is external to the (internal) TEA⁺ site and that TEA⁺ prevents refilling of this location by intracellular K⁺. Thus in the absence of extracellular K⁺, application of TEA⁺ (or C10⁺) has effects resembling dekalification and kills the K channels.     

Site-specific, photochemical proteolysis applied to ion channels in vivo

A method for site-specific, nitrobenzyl-induced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K⁺ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the “signature” Cys128–Cys142 disulfide loop of the nAChR α subunit.     

The selectivity of the hair cell’s mechanoelectrical-transduction channel promotes Ca2+ flux at low Ca2+ concentrations

The mechanoelectrical-transduction channel of the hair cell is permeable to both monovalent and divalent cations. Because Ca²⁺ entering through the transduction channel serves as a feedback signal in the adaptation process that sets the channel’s open probability, an understanding of adaptation requires estimation of the magnitude of Ca²⁺ influx. To determine the Ca²⁺ current through the transduction channel, we measured extracellular receptor currents with transepithelial voltage-clamp recordings while the apical surface of a saccular macula was bathed with solutions containing various concentrations of K⁺, Na⁺, or Ca²⁺. For modest concentrations of a single permeant cation, Ca²⁺ carried much more receptor current than did either K⁺ or Na⁺. For higher cation concentrations, however, the flux of Na⁺ or K⁺ through the transduction channel exceeded that of Ca²⁺. For mixtures of Ca²⁺ and monovalent cations, the receptor current displayed an anomalous mole-fraction effect, which indicates that ions interact while traversing the channel’s pore. These results demonstrate not only that the hair cell’s transduction channel is selective for Ca²⁺ over monovalent cations but also that Ca²⁺ carries substantial current even at low Ca²⁺ concentrations. At physiological cation concentrations, Ca²⁺ flux through transduction channels can change the local Ca²⁺ concentration in stereocilia in a range relevant for the control of adaptation.     

Association of beta-catenin with the alpha-subunit of neuronal large-conductance Ca2+-activated K+ channels

The association of Ca²⁺-activated K⁺ channels with voltage-gated Ca²⁺ channels at the presynaptic active zones of hair cells, photoreceptors, and neurons contributes to rapid repolarization of the membrane after excitation. Ca²⁺ channels have been shown to bind to a large set of synaptic proteins, but the proteins interacting with Ca²⁺-activated K⁺ channels remain unknown. Here, we report that the large-conductance Ca²⁺-activated K⁺ channel of the chicken's cochlear hair cell interacts with β-catenin. Yeast two-hybrid assays identified the S10 region of the K⁺ channel's α-subunit and the ninth armadillo repeat and carboxyl terminus of β-catenin as necessary for the interaction. An antiserum directed against the α-subunit specifically coprecipitated β-catenin from brain synaptic proteins. β-Catenin is known to associate with the synaptic protein Lin7/Velis/MALS, whose interaction partner Lin2/CASK also binds voltage-gated Ca²⁺ channels. β-Catenin may therefore provide a physical link between the two types of channels at the presynaptic active zone.     

Characterization of a calmodulin-binding transporter from the plasma membrane of barley aleurone

We have used Arabidopsis calmodulin (CaM) covalently coupled to horseradish peroxidase to screen a barley aleurone cDNA expression library for CaM binding proteins. The deduced amino acid sequence of one cDNA obtained by this screen was shown to be a unique protein of 702 amino acids with CaM and cyclic nucleotide binding domains at the carboxyl terminus and high similarity to olfactory and K⁺ channels. This cDNA was designated HvCBT1 (Hordeum vulgare CaM binding transporter). Hydropathy plots of HvCBT1 showed the presence of six putative transmembrane domains, but sequence alignment indicated a pore domain that was unlike the consensus domains in K⁺ and olfactory channels. Expression of a subclone of amino acids 482–702 in Escherichia coli generated a peptide that bound CaM. When a fusion protein of HvCBT1 and green fluorescent protein was expressed in barley aleurone protoplasts, fluorescence accumulated in the plasma membrane. Expression of HvCBT1 in the K⁺ transport deficient Saccharomyces cerevisiae mutant CY162 showed no rescue of the mutant phenotype. However, growth of CY162 expressing HvCBT1 with its pore mutated to GYGD, the consensus sequence of K⁺ channels, was compromised. We interpret these data as indicating that HvCBT1 acts to interfere with ion transport.     

Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice

ATP-sensitive K⁺ (K_ATP) channels regulate many cellular functions by linking cell metabolism to membrane potential. We have generated K_ATP channel-deficient mice by genetic disruption of Kir6.2, which forms the K⁺ ion-selective pore of the channel. The homozygous mice (Kir6.2^−/−) lack K_ATP channel activity. Although the resting membrane potential and basal intracellular calcium concentrations ([Ca²⁺]_i) of pancreatic beta cells in Kir6.2^−/− are significantly higher than those in control mice (Kir6.2^+/+), neither glucose at high concentrations nor the sulfonylurea tolbutamide elicits a rise in [Ca²⁺]_i, and no significant insulin secretion in response to either glucose or tolbutamide is found in Kir6.2^−/−, as assessed by perifusion and batch incubation of pancreatic islets. Despite the defect in glucose-induced insulin secretion, Kir6.2^−/− show only mild impairment in glucose tolerance. The glucose-lowering effect of insulin, as assessed by an insulin tolerance test, is increased significantly in Kir6.2^−/−, which could protect Kir6.2^−/− from developing hyperglycemia. Our data indicate that the K_ATP channel in pancreatic beta cells is a key regulator of both glucose- and sulfonylurea-induced insulin secretion and suggest also that the K_ATP channel in skeletal muscle might be involved in insulin action.