Coupling of activation and inactivation gate in a K+‐channel: potassium and ligand sensitivity

Potassium (K⁺)‐channel gating is choreographed by a complex interplay between external stimuli, K⁺ concentration and lipidic environment. We combined solid‐state NMR and electrophysiological experiments on a chimeric KcsA–Kv1.3 channel to delineate K⁺, pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH‐induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K⁺ and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K⁺ ion on the inactivation gate modulate activation‐gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K⁺‐channel pore.     

Electrostatic state of the cytoplasmic domain influences inactivation at the selectivity filter of the KcsA potassium channel

KcsA is a proton-activated K⁺ channel that is regulated at two gates: an activation gate located in the inner entrance of the pore and an inactivation gate at the selectivity filter. Previously, we revealed that the cytoplasmic domain (CPD) of KcsA senses proton and that electrostatic changes of the CPD influences the opening and closing of the activation gate. However, our previous studies did not reveal the effect of CPD on the inactivation gate because we used a non-inactivating mutant (E71A). In the present study, we used mutants that did not harbor the E71A mutation, and showed that the electrostatic state of the CPD influences the inactivation gate. Three novel CPD mutants were generated in which some negatively charged amino acids were replaced with neutral amino acids. These CPD mutants conducted K⁺, but showed various inactivation properties. Mutants carrying the D149N mutation showed high open probability and slow inactivation, whereas those without the D149N mutation showed low open probability and fast inactivation, similar to wild-type KcsA. In addition, mutants with D149N showed poor K⁺ selectivity, and permitted Na⁺ to flow. These results indicated that electrostatic changes in the CPD by D149N mutation triggered the loss of fast inactivation and changes in the conformation of selectivity filter. Additionally, the loss of fast inactivation induced by D149N was reversed by R153A mutation, suggesting that not only the electrostatic state of D149, but also that of R153 affects inactivation.     

Regulation of Ion Channel Function by the Host Lipid Bilayer Examined by a Stopped-Flow Spectrofluorometric Assay

To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl⁺) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it's noninactivating (E71A) KcsA mutant, by extravesicular protons (H⁺). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is <10%. The activation curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology, and the activation curves for wild-type and E71A KcsA are indistinguishable, indicating that channel activation and inactivation are separate processes. We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. Increasing the acyl chain length (from C_18:1 to C_22:1 in diacylphosphatidylcholine), but not the mole fraction of POPG (>0.25) in the bilayer-forming phospholipid mixture, alters KcsA H⁺ gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H⁺ affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels’ lipid bilayer environment or other regulatory processes.     

Structures of Gating Intermediates in a K+ channel

Regulation of ion conduction through the pore of a K⁺ channel takes place through the coordinated action of the activation gate at the bundle crossing of the inner helices and the inactivation gate located at the selectivity filter. The mechanism of allosteric coupling of these gates is of key interest. Here we report new insights into this allosteric coupling mechanism from studies on a W67F mutant of the KcsA channel. W67 is in the pore helix and is highly conserved in K⁺ channels. The KcsA W67F channel shows severely reduced inactivation and an enhanced rate of activation. We use continuous wave EPR spectroscopy to establish that the KcsA W67F channel shows an altered pH dependence of activation. Structural studies on the W67F channel provide the structures of two intermediate states: a pre- open state and a pre-inactivated state of the KcsA channel. These structures highlight key nodes in the allosteric pathway. The structure of the KcsA W67F channel with the activation gate open shows altered ion occupancy at the second ion binding site (S2) in the selectivity filter. This finding in combination with previous studies strongly support a requirement for ion occupancy at the S2 site for the channel to inactivate.     

Dynamics, Energetics, and Selectivity of the Low-K KcsA Channel Structure

Potassium channels are a diverse family of integral membrane proteins through which K⁺ can pass selectively. There is ongoing debate about the nature of conformational changes associated with the opening/closing and conductive/nonconductive states of potassium channels. The channels partly exert their function by varying their conductance through a mechanism known as C-type inactivation. Shortly after the activation of K⁺ channels, their selectivity filter stops conducting ions at a rate that depends on various stimuli. The molecular mechanism of C-type inactivation has not been fully understood yet. However, the X-ray structure of the KcsA channel obtained in the presence of low K⁺ concentration is thought to be representative of a K⁺ channel in the C-type inactivated state. Here, extensive, fully atomistic molecular dynamics and free-energy simulations of the low-K⁺ KcsA structure in an explicit lipid bilayer are performed to evaluate the stability of this structure and the selectivity of its binding sites. We find that the low-K⁺ KcsA structure is stable on the timescale of the molecular dynamics simulations performed, and that ions preferably remain in S1 and S4. In the absence of ions, the selectivity filter evolves toward an asymmetric architecture, as already observed in other computations of the high-K⁺ structure of KcsA and KirBac. The low-K⁺ KcsA structure is not permeable by Na⁺, K⁺, or Rb⁺, and the selectivity of its binding sites is different from that of the high-K⁺ structure.     

Regulation of Ion Channel Function by the Host Lipid Bilayer Examined by a Stopped-Flow Spectrofluorometric Assay

To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl⁺) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it''s noninactivating (E71A) KcsA mutant, by extravesicular protons (H⁺). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is <10%. The activation curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology, and the activation curves for wild-type and E71A KcsA are indistinguishable, indicating that channel activation and inactivation are separate processes. We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. Increasing the acyl chain length (from C_18:1 to C_22:1 in diacylphosphatidylcholine), but not the mole fraction of POPG (>0.25) in the bilayer-forming phospholipid mixture, alters KcsA H⁺ gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H⁺ affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels’ lipid bilayer environment or other regulatory processes.     

Functional Equilibrium of the KcsA Structure Revealed by NMR

KcsA is a tetrameric K⁺ channel that is activated by acidic pH. Under open conditions of the helix bundle crossing, the selectivity filter undergoes an equilibrium between permeable and impermeable conformations. Here we report that the population of the permeable conformation (p_perm) positively correlates with the tetrameric stability and that the population in reconstituted high density lipoprotein, where KcsA is surrounded by the lipid bilayer, is lower than that in detergent micelles, indicating that dynamic properties of KcsA are different in these two media. Perturbation of the membrane environment by the addition of 1–3% 2,2,2-trifluoroethanol increases p_perm and the open probability, revealed by NMR and single-channel recording analyses. These results demonstrate that KcsA inactivation is determined not only by the protein itself but also by the surrounding membrane environments.     

Changing Val-76 towards Kir channels drastically influences the folding and gating properties of the bacterial potassium channel KcsA

All K⁺-channels are stabilized by K⁺-ions in the selectivity filter. However, they differ from each other with regard to their selectivity filter. In this study, we changed specific residue Val-76 in the selectivity filter of KcsA to its counterpart Ile in inwardly rectifying K⁺-channels (Kir). The tetramer was exclusively converted into monomers as determined by conventional gel electrophoresis. However, by perfluoro-octanoic acid (PFO) gel electrophoresis mutant channel was mostly detected as tetramer. Tryptophan fluorescence and acrylamide quenching experiments demonstrated significant alteration in channel folding properties via increase in hydrophilicity of local environment. Furthermore, in planar lipid bilayer experiments V76I exhibited drastically lower conductance and decreased channel open time as compared to the unmodified KcsA. These studies suggest that V76I might contribute to determine the stabilizing, folding and channel gating properties in a selective K⁺-channel.     

A multipoint hydrogen-bond network underlying KcsA C-type inactivation

In the prokaryotic potassium channel KcsA activation gating at the inner bundle gate is followed by C-type inactivation at the selectivity filter. Entry into the C-type inactivated state has been directly linked to the strength of the H-bond interaction between residues Glu-71 and Asp-80 behind the filter, and is allosterically triggered by the rearrangement of the inner bundle gate. Here, we show that H-bond pairing between residues Trp-67 and Asp-80, conserved in most K⁺ channels, constitutes another critical interaction that determines the rate and extent of KcsA C-type inactivation. Disruption of the equivalent interaction in Shaker (Trp-434-Asp-447) and Kv1.2 (Trp-366-Asp-379) leads also to modulation of the inactivation process, suggesting that these residues also play an analogous role in the inactivation gating of Kv channels. The present results show that in KcsA C-type inactivation gating is governed by a multipoint hydrogen-bond network formed by the triad Trp-67-Glu71-Asp-80. This triad exerts a critical role in the dynamics and conformational stability of the selectivity filter and might serve as a general modulator of selectivity filter gating in other members of the K⁺ channel family.     

Mutations in the K+-Channel KcsA Toward Kir Channels Alter Salt-Induced Clusterization and Blockade by Quaternary Alkylammonium Ions

Protein aggregation is a result of malfunction in protein folding, assembly, and transport, caused by protein mutation and/or changes in the cell environment, thus triggering many human diseases. We have shown that bacterial K⁺-channel KcsA, which acts as a representative model for ion channels, forms salt-induced large conductive complexes in a particular environment. In the present study, we investigated the effects of point mutations in the selectivity filter of KcsA on intrinsic stability, aggregation, and channel blocking behavior. First, we found that a low sodium chloride concentration in potassium-containing media induced fast transfer of single channels to a planar lipid bilayer. Second, increasing the sodium chloride concentration drastically increased the total channel current, indicating enhanced vesicle fusion and transfer of multiple channels to a planar lipid bilayer. However, such complexes exhibited high conductance as well as higher open probability compared to the unmodified KcsA behavior shown previously. Interestingly, the affinity of aggregated complexes for larger symmetric quaternary alkylammonium ions (QAs) was found to be much higher than that for tetraethylammonium, a classical blocker of the K⁺ channel. Based on these findings, we propose that mutant channel complexes exhibit larger pore dimensions, thus resembling more the topological properties of voltage-gated and inwardly rectifying K⁺ channels.     

Changes in single K(+) channel behavior induced by a lipid phase transition

We show that the activity of an ion channel is correlated with the phase state of the lipid bilayer hosting the channel. By measuring unitary conductance, dwell times, and open probability of the K⁺ channel KcsA as a function of temperature in lipid bilayers composed of POPE and POPG in different relative proportions, we obtain that all those properties show a trend inversion when the bilayer is in the transition region between the liquid-disordered and the solid-ordered phase. These data suggest that the physical properties of the lipid bilayer influence ion channel activity likely via a fine-tuning of its conformations. In a more general interpretative framework, we suggest that other parameters such as pH, ionic strength, and the action of amphiphilic drugs can affect the physical behavior of the lipid bilayer in a fashion similar to temperature changes resulting in functional changes of transmembrane proteins.     

Transferring knowledge towards understanding the pore stabilizing variations in K+ channels

Recent advances in structural biology underlying mechanisms of channel gating have strengthened our knowledge about how K⁺ channels can be inter-convertible between conductive and non-conductive states. We have reviewed and combined mutagenesis with biochemical, biophysical and structural information in order to understand the critical roles of the pore residues in stabilizing the pore structure and channel open state. We also discuss how the latest knowledge on the K⁺ channel KcsA may provide a step towards better understanding of distinct pore stabilizing differences among diversified K⁺ channels.     

Conformational changes in the selectivity filter of the open-state KcsA channel: an energy minimization study

Potassium channels switch between closed and open conformations and selectively conduct K⁺ ions. There are at least two gates. The TM2 bundle at the intracellular site is the primary gate of KcsA, and rearrangements at the selectivity filter (SF) act as the second gate. The SF blocks ion flow via an inactivation process similar to C-type inactivation of voltage-gated K⁺ channels. We recently generated the open-state conformation of the KcsA channel. We found no major, possibly inactivating, structural changes in the SF associated with this massive inner-pore rearrangement, which suggests that the gates might act independently. Here we energy-minimize the open state of wild-type and mutant KcsA, validating in silico structures of energy-minimized SFs by comparison with crystallographic structures, and use these data to gain insight into how mutation, ion depletion, and K⁺ to Na⁺ substitution influence SF conformation. Both E71 or D80 protonations/mutations and the presence/absence of protein-buried water molecule(s) modify the H-bonding network stabilizing the P-loops, spawning numerous SF conformations. We find that the inactivated state corresponds to conformations with a partially unoccupied or an entirely empty SF. These structures, involving modifications in all four P-loops, are stabilized by H-bonds between amide H and carbonyl O atoms from adjacent P-loops, which block ion passage. The inner portions of the P-loops are more rigid than the outer parts. Changes are localized to the outer binding sites, with innermost site S4 persisting in the inactivated state. Strong binding by Na⁺ locally contracts the SF around Na⁺, releasing ligands that do not participate in Na⁺ coordination, and occluding the permeation pathway. K⁺ selectivity primarily appears to arise from the inability of the SF to completely dehydrate Na⁺ ions due to basic structural differences between liquid water and the “quasi-liquid” SF matrix.     

Mixed modes in opening of KcsA potassium channel from a targeted molecular dynamics simulation

Potassium channels conduct K⁺ flow selectively across the membrane through a central pore. During a process called gating, the potassium channels undergo a conformational change that opens or closes the ion-conducting pore. The potassium channel KcsA has been structurally determined in its closed state. However, the dynamic mechanism of the gating transition of the KcsA channel is still being investigated. Here, a targeted molecular dynamics simulation up to 150 ns is performed to investigate the detailed opening process of the KcsA channel with an open Kv1.2 structure serving as the target. The channel arrived at a self-determined quasi-stable state within 60 ns. The rigid-body and hinge-bending modes are observed mixed together in the remaining 90 ns long quasi-stable state. The mixed-mode movement seems come from the competition between the helix rigidity and the biased-applied gating force.     

Formation of Very Large Conductance Channels by Bacillus cereus Nhe in Vero and GH4 Cells Identifies NheA + B as the Inherent Pore-Forming Structure

Mutation E71A in the bacterial K⁺-channel KcsA has been shown to abolish the activation-coupled inactivation of KcsA via significant alterations of the peptide backbone in the vicinity of the selectivity filter. In the present study, we examined channel-blocking behavior of KcsA-E71A by tetraethylammonium (TEA) from both the extra- and the intracellular sides. First, we found that E71A is inserted either in cis or trans orientation in a planar lipid bilayer; however, it exhibits only one orientation in proteoliposomes as determined by extravesicular partial chymotrypsin digestion. Second, E71A exhibits a lower extracellular TEA affinity and is more sensitive to intracellular TEA compared to wild-type KcsA, which apparently has >50-fold higher affinity for extracellular TEA and ~2.5-fold lower affinity for intracellular TEA compared to E71A. In additional experiments, we investigated the influence of negatively charged phosphatidylglycerol (PG) on channel-gating properties in phosphatidylcholine lipid bilayers. It was found that high PG content decreases the single-channel conductance and increases the channel open time and open probability. Taken together, our data suggest that the “flipped” conformation of the selectivity filter present in E71A allows weaker extracellular and stronger intracellular TEA binding, whereas higher PG content decreases channel conductivity and stabilizes the channel open “flipped” state via electrostatic interaction in the proximity of the channel pore.     

Diverse gating in K channels: Differential role of the pore-helix glutamate in stabilizing the channel pore

The selectivity filter and adjacent regions in the bacterial KcsA and inwardly rectifying K⁺ (Kir) channels reveal significant conformational changes that cause the channel pore to transition from an activated to inactive state (C-type inactivation) once the channel is open. The meshwork of residues stabilizing the pore of KcsA involves Glu71–Asp80 carboxyl–carboxylate interaction ‘behind’ the selectivity filter. Interestingly, the Kir channels do not have this exact interaction, but instead have a Glu–Arg salt bridge where the Glu is in the same position but the Arg is one position N-terminal compared to the Asp in KcsA. Also, the Kir channels lack the Trp that hydrogen bonds to Asp80 in KcsA. Here, the sequence and structural information are combined to understand the dissimilarity in the role of the pore-helix Glu in stabilizing the pore structure in KcsA and Kir channels. This review illustrates that although Glu is quite conserved among both types of channels, the network of interactions is not translatable from one channel to the other; thereby suggesting a unique phenomenon of diverse gating patterns in K⁺ channels.     

Muscarinic K+ Channel in the Heart : Modal Regulation by G Protein βγ Subunits

The membrane-delimited activation of muscarinic K⁺ channels by G protein βγ subunits plays a prominent role in the inhibitory synaptic transmission in the heart. These channels are thought to be heterotetramers comprised of two homologous subunits, GIRK1 and CIR, both members of the family of inwardly rectifying K⁺ channels. Here, we demonstrate that muscarinic K⁺ channels in neonatal rat atrial myocytes exhibit four distinct gating modes. In intact myocytes, after muscarinic receptor activation, the different gating modes were distinguished by differences in both the frequency of channel opening and the mean open time of the channel, which accounted for a 76-fold increase in channel open probability from mode 1 to mode 4. Because of the tetrameric architecture of the channel, the hypothesis that each of the four gating modes reflects binding of a different number of Gβγ subunits to the channel was tested, using recombinant Gβ₁γ₅. Gβ₁γ₅ was able to control the equilibrium between the four gating modes of the channel in a manner consistent with binding of Gβγ to four equivalent and independent sites in the protein complex. Surprisingly, however, Gβ₁γ₅ lacked the ability to stabilize the long open state of the channel that is responsible for the augmentation of the mean open time in modes 3 and 4 after muscarinic receptor stimulation. The modal regulation of muscarinic K⁺ channel gating by Gβγ provides the atrial cells with at least two major advantages: the ability to filter out small inputs from multiple membrane receptors and yet the ability to create the gradients of information necessary to control the heart rate with great precision.     

Dissimilarity in the channel intrinsic stability among the bacterial KcsA and the inwardly rectifying potassium channel ROMK1

In this study, we compared the channel intrinsic stability of the bacterial K⁺-channel KcsA and the inwardly rectifying potassium channel (Kir) ROMK1. ROMK1 was successfully cloned, expressed and purified from Saccharomyces cerevisae. By conventional gel electrophoresis, ROMK1 was detected in monomeric form running exclusively at ～45 kDa either in its oxidized or reduced form. By perfluoro-octanoic acid (PFO)-PAGE, the reduced ROMK1 was identified as tetrameric as well as oligomeric complex. However, in its oxidized form ROMK1 was exclusively detected in oligomeric form thus indicating the role of intrinsic cysteine residues and formation of disulfide bonds in stabilizing the oligomeric ROMK1. On the other hand, KcsA purified from E. coli was detected as an extremely stable tetramer both in its oxidized or reduced forms as determined by conventional or PFO-PAGE. Furthermore, in planar lipid bilayer ROMK1 exhibited prominent inward rectification, low single channel conductance and high channel open probability as compared to the KcsA channel which showed typically slight outward rectification and low open probability under similar conditions. Our experiments clearly indicate that KcsA and ROMK1 channels differ with regard to their intrinsic stability which might be related to their structural and functional differences.     

Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel

C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K⁺ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K⁺ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.