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.     

Potassium channels: Structure-function relationships, diversity, and pharmacology

There are many different types of potassium (K⁺) channels: A good number are voltage-dependent, others are activated by variations of intracellular concentrations of Ca²⁺ and the activity of others is controlled by cytoplasmic variations of the ATP/ADP ratio or by variations of intracellular Na⁺ or arachidonic acid and other fatty acids; a large number are modulated by phosphorylation and/or interaction with G proteins. Considerable progress has been made in the past few years in the molecular knowledge of some of these channels. Some of the voltage-dependent K⁺ channels have been cloned. In each tissue several genes encode several different K⁺ channel subunits that assemble to form large families of voltage-dependent K⁺ channels with different biophysical properties (different voltage dependence, different time course), which are associated with different physiological functions. The molecular structure of other types of K⁺ channels is not yet solved. Investigation of the molecular pharmacology of K⁺ channels has also made tremendous progress recently. High-affinity ligands are now available for some of the voltage-dependent K⁺ channels, Ca²⁺-activated K⁺ channels, and ATP-sensitive K⁺ channels.     

Tetrameric assembly of KvLm K+ channels with defined numbers of voltage sensors

Voltage-gated K⁺ (Kv) channels are tetrameric assemblies in which each modular subunit consists of a voltage sensor and a pore domain. KvLm, the voltage-gated K⁺ channel from Listeria monocytogenes, differs from other Kv channels in that its voltage sensor contains only three out of the eight charged residues previously implicated in voltage gating. Here, we ask how many sensors are required to produce a functional Kv channel by investigating heterotetramers comprising combinations of full-length KvLm (FL) and its sensorless pore module. KvLm heterotetramers were produced by cell-free expression, purified by electrophoresis, and shown to yield functional channels after reconstitution in droplet interface bilayers. We studied the properties of KvLm channels with zero, one, two, three, and four voltage sensors. Three sensors suffice to promote channel opening with FL₄-like voltage dependence at depolarizing potentials, but all four sensors are required to keep the channel closed during membrane hyperpolarization.     

Intractable hyperkalemia due to nicorandil induced potassium channel syndrome

Nicorandil is a commonly used antianginal agent, which has both nitrate-like and ATP-sensitive potassium (K_ATP) channel activator properties. Activation of potassium channels by nicorandil causes expulsion of potassium ions into the extracellular space leading to membrane hyperpolarization, closure of voltage-gated calcium channels and finally vasodilatation. However, on the other hand, being an activator of K_ATP channel, it can expel K⁺ ions out of the cells and can cause hyperkalemia. Here, we report a case of nicorandil induced hyperkalemia unresponsive to medical treatment in a patient with diabetic nephropathy.     

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.     

Stabilization of ion selectivity filter by pore loop ion pairs in an inwardly rectifying potassium channel

Ion selectivity is critical for the biological functions of voltage-dependent cation channels and is achieved by specific ion binding to a pore region called the selectivity filter. In voltage-gated K⁺, Na⁺ and Ca²⁺ channels, the selectivity filter is formed by a short polypeptide loop (called the H5 or P region) between the fifth and sixth transmembrane segments, donated by each of the four subunits or internal homologous domains forming the channel. While mutagenesis studies on voltage-gated K⁺ channels have begun to shed light on the structural organization of this pore region, little is known about the physical and chemical interactions that maintain the structural stability of the selectivity filter. Here we show that in an inwardly rectifying K⁺ (IRK) channel, IRK1, short range interactions of an ion pair in the H5 pore loop are crucial for pore structure and ion permeation. The two residues, a glutamate and an arginine, appear to form exposed salt bridges in the tetrameric channel. Alteration or disruption of such ion pair interactions dramatically alters ion selectivity and permeation. Since this ion pair is conserved in all IRK channels, it may constitute a general mechanism for maintaining the stability of the pore structure in this channel superfamily.     

Heme impairs the ball-and-chain inactivation of potassium channels

Fine-tuned regulation of K⁺ channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K⁺ channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K⁺ channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K⁺ current with an apparent EC₅₀ value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.A-type K⁺ channels, a family of voltage-gated K⁺ (Kv) channels, play a vital role in the control of neuronal excitability, regulation of presynaptic spike duration, Ca²⁺ entry, and neurotransmitter release (1). One of the prominent features of A-type K⁺ channels is their inactivation, which is mediated by two structurally distinct processes (2, 3). The fast inactivation is initiated by the N-terminal protein structure, thereby termed N-type inactivation, whereas the slow C-type inactivation is related to the pore structure (2, 3). N-type inactivation proceeds according to a “ball-and-chain” mechanism; the positive charges of the N-terminal ball domains bring the structures to the pore domain of the channel and the distal segment of one of the four intrinsically disordered N-terminal ball domains enters the hydrophobic central cavity/vestibule of the inner pore of the channel thus obstructing the flow of K⁺ (2–5).Acute enzymatic or mutational removal of the distal N terminus eliminates N-type inactivation, and in such inactivation-removed channels, intracellular application of peptides corresponding to the N-terminal sequence restores inactivation (4, 6, 7). Structural analysis suggests that the N-terminal inactivation structure needs to be flexible or even intrinsically disordered to reach the receptor in the channel’s cavity (8, 9).“Tuning” of rapid N-type inactivation is an effective way of adapting cells to specific needs. For example, molecular processes affecting the speed and degree of N-type inactivation in Kv1.4 (KCNA4) channels include redox regulation of a cysteine residue in the N-terminal ball structure (C13) (10), protonation of histidine at position 16 (11), interaction with membrane lipids (12), and Ca²⁺-dependent phosphorylation (13). Furthermore, low-molecular-weight compounds affecting N-type inactivation (N-type disinactivators) have been discussed as potential drugs regulating cellular excitability (14).Heme [Fe(II) protoporphyrin-IX] is well known as a protein cofactor, often conferring gas sensitivity as exemplified in hemoglobin, cytochromes, myoglobin, and soluble guanylyl cyclase. In many heme proteins including soluble guanylyl cyclase, heme is bound or coordinated in part by an amino acid sequence typically containing a histidine or cysteine residue, which acts as an axial fifth ligand (in addition to the four bonds provided by the nitrogen atoms of the protoporphyrin-IX ring to the iron center) to the redox-sensitive iron center, and water or a bound gas molecule acts as the sixth ligand (15). However, recent advances revealed a novel role of heme as a nongenomic modulator of ion channel functions, first exemplified for the large-conductance voltage- and Ca²⁺-dependent K⁺ channel (Slo1 BK) (16) and later for the epithelial Na⁺ channel (17). Detailed analysis of the biophysical action of heme [ferrous iron (Fe²⁺)] or hemin [ferric iron (Fe³⁺)] on the Slo1 BK channel demonstrated that hemin is a potent modulator of the allosteric gating mechanism of the channel (18), and mutagenesis studies have indicated the sequence CKACH located in the cytoplasmic C terminus of the channel plays a critical role (16, 19). However, neither for Slo1 BK channels nor for epithelial Na⁺ channels, the interaction of heme with the ion channel protein is structurally resolved. In this study, we found that the fast N-type inactivation of Kv1.4 A-type K⁺ channels is potently modulated by heme/hemin. Furthermore, we provide structural insight into heme interaction with a channel explaining how heme prevents A-type channels from entering an inactivated state.     

Voltage dependence of K channels in guard-cell protoplasts

Stomatal pores in leaves enable plants to regulate the exchange of gases with their environment. Variations of the pore aperture are mediated by controlled changes of potassium salt concentrations in the surrounding guard cells. The voltage-dependent gating of K⁺-selective channels in the plasma membrane (plasmalemma) of cell-wall-free guard cells (protoplasts) was studied at the molecular level in order to investigate the regulation of K⁺ fluxes during stomatal movements. Inward and outward K⁺ currents across the plasmalemma of guard cells were identified by using the whole-cell configuration of the patch-clamp technique. Depolarizations of the membrane potential from a holding potential of -60 mV to values more positive than -40 mV produced outward currents that were shown to be carried by K⁺. Hyperpolarizations elicited inward K⁺ currents. Inward and outward currents were selective for K⁺ over Na⁺ and could be partially blocked by exposure to extracellular Ba²⁺. In cell-attached and excised membrane patches, previously identified K⁺-selective single channels in guard cells were studied. Averaging of single-channel currents during voltage pulses resulted in activation and deactivation kinetics that were similar to corresponding kinetics of inward and outward currents in whole cells, showing that K⁺-selective channels were the molecular pathways for the K⁺ currents recorded across the plasmalemma of single guard-cell protoplasts. Estimates demonstrate that K⁺ currents through the voltage-gated K⁺ channels recorded in whole guard cells can account for physiological K⁺ fluxes reported to occur during stomatal movements in leaves.     

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.     

Initial steps of inactivation at the K+ channel selectivity filter

K⁺ efflux through K⁺ channels can be controlled by C-type inactivation, which is thought to arise from a conformational change near the channel’s selectivity filter. Inactivation is modulated by ion binding near the selectivity filter; however, the molecular forces that initiate inactivation remain unclear. We probe these driving forces by electrophysiology and molecular simulation of MthK, a prototypical K⁺ channel. Either Mg²⁺ or Ca²⁺ can reduce K⁺ efflux through MthK channels. However, Ca²⁺, but not Mg²⁺, can enhance entry to the inactivated state. Molecular simulations illustrate that, in the MthK pore, Ca²⁺ ions can partially dehydrate, enabling selective accessibility of Ca²⁺ to a site at the entry to the selectivity filter. Ca²⁺ binding at the site interacts with K⁺ ions in the selectivity filter, facilitating a conformational change within the filter and subsequent inactivation. These results support an ionic mechanism that precedes changes in channel conformation to initiate inactivation.Potassium (K⁺) channels are activated and opened by a variety of stimuli, including ligand binding and transmembrane voltage, to enable K⁺ efflux and thus, modulate physiological processes related to electrical excitability, such as regulation of action potential firing, smooth muscle contraction, and hormone secretion (1). In addition, many K⁺ channels are further controlled by a gating phenomenon known as C-type inactivation, in which K⁺ conduction is stopped, despite the continued presence of an activating stimulus (2). The mechanisms underlying C-type inactivation in voltage-gated K⁺ channels (Kv channels) are linked to both intracellular and extracellular permeant ion concentrations, and several lines of evidence have suggested that C-type inactivation is associated with a conformational change near the external mouth of the K⁺ channel pore (i.e., at the canonical K⁺ channel selectivity filter) (3–11).In Shaker Kv channels, C-type inactivation is known to be enhanced and recovery from inactivation is slowed by impermeant cations accessing the cytoplasmic side of the channel (5, 6, 10). Enhancement of inactivation by these cations suggests a working hypothesis, in which the impermeant ion prevents refilling of the selectivity filter with K⁺ (6). Thus, K⁺ presumably dissociates from the filter to the external solution, and this vacancy leaves the filter susceptible to a conformational change that underlies the nonconducting, inactivated state. However, the physical basis for the relation between ion movements and C-type inactivation as well as the structural underpinnings of the mechanism remain unclear.Here, we use divalent metal cations (Mg²⁺, Ca²⁺, and Sr²⁺) as probes of inactivation mechanisms in MthK, a model K⁺ channel of known structure (Fig. 1) (12–14). Specifically, we analyze conduction and gating of single MthK channels by electrophysiology combined with analysis of ion and protein movements by molecular simulation. Our electrophysiological experiments indicate that, although each of these divalent metal ions can reduce the size of single channel currents, only Ca²⁺ and Sr²⁺ can enhance inactivation, whereas Mg²⁺ does not. Using molecular simulation and potential of mean force (PMF) calculations, we find that Ca²⁺, but not Mg²⁺, can shed its hydration shell waters to access a site, termed S5, at the entry to the channel’s selectivity filter (Fig. 1C) after displacement of K⁺ ions to the extracellular side of the channel. Subsequent dissociation of a K⁺ ion from the filter, in turn, favors a conformational change within the selectivity filter, contributing to enhanced inactivation. These results support a working hypothesis that directly relates dissociation of K⁺ with a structural change in the selectivity filter to initiate inactivation of K⁺ channels.Open in a separate windowFig. 1.Structure and activation properties of MthK. (A) Presumed biological structure of MthK shown as a Cα-trace [Protein Data Bank (PDB) ID code 3RBZ]. The channel consists of a transmembrane pore domain tethered to a ring of RCK domains, which mediate channel activation by cytoplasmic Ca²⁺ (green spheres). The gray-shaded region represents the presumed plasma membrane; dashed lines represent the linker region between the pore and RCK gating ring that is unresolved in the crystal structure. (B) High-resolution structure of the MthK pore domain, with the selectivity filter shown in ball and stick representation (PDB ID code 3LDC). Subunits in the front and back have been removed for clear visualization of the conduction pathway (inside dashed rectangle), with K⁺ ions shown as purple spheres and ordered water molecules shown as red spheres. (C) –Magnified view of the MthK conduction pathway (boxed region in B) with potential ion binding sites (S0–S_cav) indicated. (D) Po vs. [Ca²⁺] (black symbols) and [Cd²⁺] (red symbols) from currents recorded at −100 mV. MthK activation requires ∼20-fold lower [Cd²⁺] compared with [Ca²⁺]. Curves represent fits with a Hill equation with the following parameters: EC₅₀ = 1.0 mM and n_H = 9.5 for Ca²⁺; EC₅₀ = 49 μM and n_H = 8.4 for Cd²⁺. (E) Representative single channel currents from reconstituted MthK at depolarized voltages with 200 mM KCl at both sides of the membrane and Ca²⁺ or Cd²⁺ at the cytoplasmic side of the channel as indicated. Cd²⁺ can fully activate MthK at concentrations that produce much less fast blockade than Ca²⁺. O and C indicate open and closed current levels, respectively. (F) Unitary current vs. voltage for MthK channels activated with 30 and 100 μM Cd²⁺ (green and red, respectively) and 2 mM Ca²⁺ (black). Smooth curves are drawn for display only; 100 μM Cd²⁺ results in nominal levels of fast blockade, yielding large outward current.     

Protonation state of E71 in KcsA and its role for channel collapse and inactivation

The prototypical prokaryotic potassium channel KcsA alters its pore depending on the ambient potassium; at high potassium, it exists in a conductive form, and at low potassium, it collapses into a nonconductive structure with reduced ion occupancy. We present solid-state NMR studies of KcsA in which we test the hypothesis that an important channel-inactivation process, known as C-type inactivation, proceeds via a state similar to this collapsed state. We test this using an inactivation-resistant mutant E71A, and show that E71A is unable to collapse its pore at both low potassium and low pH, suggesting that the collapsed state is structurally similar to the inactivated state. We also show that E71A has a disordered selectivity filter. Using site-specific K⁺ titrations, we detect a local change at E71 that is coupled to channel collapse at low K⁺. To gain more insight into this change, we site specifically measure the chemical shift tensors of the side-chain carboxyls of E71 and its hydrogen bond partner D80, and use the tensors to assign protonation states to E71 and D80 at high K⁺ and neutral pH. Our measurements show that E71 is protonated at pH 7.5 and must have an unusually perturbed pK_a (> 7.5) suggesting that the change at E71 is a structural rearrangement rather than a protonation event. The results offer new mechanistic insights into why the widely used mutant KcsA–E71A does not inactivate and establish the ambient K⁺ level as a means to populate the inactivated state of KcsA in a controlled way.     

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.     

Modulation of Kv Channel α/β Subunit Interactions

Voltage-gated K⁺ channels comprise the largest and most diverse class of ion channels. These channels establish the resting membrane potential and modulate the frequency and duration of action potentials in nerve and muscle, as well as being the targets of several antiarrhythmic drugs in the heart. The multiplicity of Kv channel function is further enhanced through modulation by accessory β subunits, which confer rapid inactivation, alter current amplitudes, and promote cell surface expression. In addition, α/β interactions are also influenced by second messenger pathways. Recent evidence demonstrates that phosphorylation of Kv channel α and/or β subunits may dramatically affect channel properties. The functional response of different K⁺ channel subunits to activation of protein kinases represents not only a means to modulate subunit interactions, but also another mechanism for K⁺ channel diversity in vivo.     

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.     

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.     

The mode of action of tedisamil on voltage-dependent K+ channels

The mechanism of the interaction of tedisamil with voltage-dependent K⁺ channels was studied using whole-cell and single-channel recordings in a variety of species and cell types. In K⁺ channels with rapid activation kinetics (I_to of rat ventricular myocytes; I_A of mouse astroglial cells), tedisamil enhanced the kinetics of inactivation of the current without significantly suppressing the amplitude of the initial current. In K⁺ channels with slower activation/inactivation kinetics, tedisamil had a divergent effect. On I_K of the glial cells, which have slow activation and inactivation kinetics, the kinetics of inactivation were enhanced and the initial peak current was reduced. On the other hand, in I_K of guinea-pig ventricular myocytes, which have even slower activation kinetics with no inactivation, tedisamil slowed or completely suppressed the activation of the current. Finally, in K⁺ channels with rapid activation but slow inactivation kinetics (pedestal-type current of rat ventricular myocytes), tedisamil accelerated the inactivation without affecting the initial current. Thus, the prime determinant of the blocking mode of tedisamil appeared to be the kinetics of activation of the K⁺ channel; that is, the slower the kinetics of activation of the channel, the greater the initial block by the drug. Unitary I_to currents recorded in rat ventricular myocytes showed that tedisamil induced a rapid flicker block of the open channel and prolonged the time between the burst of openings without any effect on the unitary conductance. These effects were modeled by assuming that the drug bound to the open channel at a finite rate. Thus, tedisamil appears to decrease K⁺ currents by interacting uniformly with the open state of the channel.     

Pharmacological Profile of U‐37883A,a Channel Blocker of Smooth Muscle‐Type ATP‐Sensitive K+ Channels

U‐37883A (PNU‐37883A, guanidine; 4‐morpholinecarboximidine‐N‐1‐adamantyl‐N′‐cyclohexyl hydrochloride) was originally developed as a potential diuretic with specific binding in kidney and vascular smooth muscle rather than in brain or pancreatic β cells. U‐37883A inhibits ATP‐sensitive K⁺ channels (K_ATP channels) in vascular smooth muscle at submicromolar concentrations whilst even at high concentrations (≥10 μM) it has no inhibitory effect at pancreatic, cardiac or skeletal K_ATP channels. Thus, it is generally thought that U‐37883A is a selective inhibitor of vascular smooth muscle K_ATP channels. Approximately one decade ago, K_ATP channels were cloned and found to consist of at least two subunits: an inwardly‐rectifying K⁺ channel six family (K_ir6.x; K_ir6.1 and K_ir6.2) which forms the ion conducting pore and a modulatory sulphonylurea receptor (SUR.x; SUR1, SUR2A, and SUR2B) that accounts for several pharmacological properties. It is generally believed that different combinations of K_ir6.x and SUR.x determine the molecular properties of K_ATP channels. Thus, K_ir6.2/SUR1 channel represents the pancreatic β‐cell K_ATP channel, K_ir6.2/SUR2A channel is thought to represent the cardiac K_ATP channel, whereas K_ir6.1/SUR2B channel is likely to represent the vascular smooth muscle K_ATP channel. Recent molecular studies have shown that U‐37883A selectively suppresses the activity of recombinant K_ATP channels which contain K_ir6.1 subunits in the channel pore unit. It was thus thought that U‐37883A was a selective pharmacological tool which could be used to investigate the activity of vascular smooth muscle K_ATP channels. However, due to its multiple pharmacological actions on several ion channels and poor tissue selectivity, U‐37883A should not be viewed as a selective blocker of smooth muscle K_ATP 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.     

Semisynthetic K+ channels show that the constricted conformation of the selectivity filter is not the C-type inactivated state

C-type inactivation of K⁺ channels plays a key role in modulating cellular excitability. During C-type inactivation, the selectivity filter of a K⁺ channel changes conformation from a conductive to a nonconductive state. Crystal structures of the KcsA channel determined at low K⁺ or in the open state revealed a constricted conformation of the selectivity filter, which was proposed to represent the C-type inactivated state. However, structural studies on other K⁺ channels do not support the constricted conformation as the C-type inactivated state. In this study, we address whether the constricted conformation of the selectivity filter is in fact the C-type inactivated state. The constricted conformation can be blocked by substituting the first conserved glycine in the selectivity filter with the unnatural amino acid d-Alanine. Protein semisynthesis was used to introduce d-Alanine into the selectivity filters of the KcsA channel and the voltage-gated K⁺ channel K_vAP. For semisynthesis of the K_vAP channel, we developed a modular approach in which chemical synthesis is limited to the selectivity filter whereas the rest of the protein is obtained by recombinant means. Using the semisynthetic KcsA and K_vAP channels, we show that blocking the constricted conformation of the selectivity filter does not prevent inactivation, which suggests that the constricted conformation is not the C-type inactivated state.The ability of K⁺ channels to selectively conduct K⁺ ions is accomplished by a structural unit called the selectivity filter (1). The selectivity filter consists of four K⁺ binding sites built using the main chain carbonyl oxygens and the threonine side chain from the protein sequence, which is typically T-V-G-Y-G (Fig. 1A) (2, 3). This sequence, referred to as the signature sequence, is highly conserved among K⁺ channels (2). The high degree of conservation of the signature sequence indicates a similar structure for the selectivity filter of all K⁺ channels, which is in fact observed in the K⁺ channel structures presently available (4).Open in a separate windowFig. 1.The conductive and constricted conformations of the K⁺ selectivity filter. (A) Close-up view of the selectivity filter of wild-type KcsA channel at high K⁺ concentration [K⁺] (PDB ID code: 1k4c). Two diagonally opposite subunits are shown in stick representation. K⁺ ions are shown as purple spheres. (B) Macroscopic currents of the wild-type KcsA channel elicited by a pH jump show inactivation. Currents were elicited at +100 mV by a rapid change of solution pH, at the arrow, from pH 7.5 (10 mM Hepes-KOH, 200 mM KCl) to pH 3.0 (10 mM succinate, 200 mM KCl). The selectivity filter of the KcsA channels at low [K⁺] (C, PDB ID code: 1k4d) and in the 32-Ǻ open structure (D, PDB ID code: 3f5w) show the constricted conformation. A rotation of the Val76–Gly77 bond causes constriction of the pore. The Gly77 Cα–Cα distance in the opposite subunits is 8.1 Å for the conductive conformation and 5.4–5.5 Å for the constricted conformation at low [K⁺] or in the 32-Å open state. (E) Structure of the selectivity filter of KcsA^G77dA at high [K⁺] (PDB ID code: 2ih3). (F) A hypothetical structure of the KcsA^G77dA selectivity filter in the constricted conformation. Two adjacent subunits are shown. The methyl side chain of d-Ala77 of one subunit and the carbonyl oxygen atoms of the Val76 and d-Ala77 in the adjacent subunit that clash are shown in van der Waals (VDW) representation. (G) Structure of the selectivity filter of KcsA^G77dA at low [K⁺] (PDB ID code: 2ih1). (H) Superposition of the selectivity filter of the KcsA^G77dA in high [K⁺] (blue) and low [K⁺] (red) shows that the d-Ala substitution in the selectivity filter blocks the constricted conformation.In addition to ion discrimination, the selectivity filter participates in a gating process referred to as C-type inactivation, during which the channel transitions from the conductive state to a nonconductive state (5). C-type inactivation has been extensively investigated in voltage-gated K⁺ (K_v) channels and is observed on prolonged opening of K_v channels by a sustained membrane depolarization (4, 6). C-type inactivation is an effective mechanism to control K_v channel activity and to regulate action-potential frequency in an excitable cell (7). An inactivation process, which is similar to C-type inactivation, is also observed in K⁺ channels that do not belong to the K_v family, such as the bacterial K⁺ channel KcsA. The KcsA channel is gated by pH (8). A decrease in the intracellular pH causes channel opening by conformational changes at the bundle crossing of the pore lining helices. In the closed state, the bundle crossing of the pore lining helices acts as a barrier for the movement of ions across the membrane (9). Activation of the KcsA channel is followed by inactivation during which the current decreases (Fig. 1B) (10, 11). Inactivation in the KcsA channel is proposed to be C-type as it shares a number of functional similarities with C-type inactivation in K_v channels (12–14). This similarity, coupled with the amenability of KcsA to structural studies, has made it an attractive system for elucidating the structure of the selectivity filter in the C-type inactivated state.Models for the selectivity filter in the C-type inactivated state have been proposed based on structures of the KcsA channel at low K⁺ or in the open state. The selectivity filter of the KcsA channel undergoes a conformational change from the conductive state at high K⁺ to a nonconductive state at low K⁺ (Fig. 1C) (3, 15). In the low K⁺ conformation, there is a rotation around the Gly77–Val76 peptide bond that causes the α-carbon of Gly77 to twist inwards and constrict the pore. This rotation disrupts the second and third ion binding sites in the selectivity filter and renders the channel nonconductive (Fig. 1 A, C, and D). As the rate of C-type inactivation increases at low K⁺, the conformation of the selectivity filter at low K⁺ was proposed to represent the C-type inactivated state (16). Recently, a series of structures with varying degrees of opening at the bundle crossing of the pore lining helices were obtained by using a constitutively open mutant of the KcsA channel (17). Higher degrees of opening at the bundle crossing (25–32 Å) were accompanied by a conformational change in the selectivity filter that was presumed to be nonconductive (Fig. 1D). This nonconductive conformation of the selectivity filter was proposed to represent the C-type inactivated state. The conformations of the selectivity filter in low K⁺ or in the open-channel structure are quite similar except for slight differences toward the lower half of the selectivity filter and the orientation of the Thr75 side chain. Due to their similarity, we jointly refer to these conformations as the “constricted” conformation of the selectivity filter. Changes in the conformation of the KcsA selectivity filter at low K⁺ or low pH have also been detected by solution and solid-state NMR and are consistent with the constricted conformation of the selectivity filter (18–20).However, does the constricted conformation represent the selectivity filter in the C-type inactivated state? An important caveat of the structural studies is that the C-type inactivated state must be accurately captured by the conditions used for structure determination. Experimental validation is therefore necessary before the constricted conformation can be conclusively assigned as the C-type inactivated state. Here, we used unnatural amino acid mutagenesis to test whether the constricted conformation of the selectivity filter of the KcsA channel corresponds to the C-type inactivated state. We also used unnatural amino acid mutagenesis on the archaebacterial K_v channel K_vAP, to test whether the constricted conformation is relevant during C-type inactivation in a K_v channel. We show that inactivation in the K_vAP channel is functionally similar to C-type inactivation in a eukaryotic K_v channel. To carry out unnatural amino acid mutagenesis, we developed a modular semisynthesis of the K_vAP channel that allowed us to use chemical synthesis to modify the selectivity filter. Our results on the KcsA and the K_vAP channels show that the constricted conformation of the selectivity filter is not the C-type inactivated state.