The voltage-sensing domain of a phosphatase gates the pore of a potassium channel

The modular architecture of voltage-gated K⁺ (Kv) channels suggests that they resulted from the fusion of a voltage-sensing domain (VSD) to a pore module. Here, we show that the VSD of Ciona intestinalis phosphatase (Ci-VSP) fused to the viral channel Kcv creates Kv_Synth1, a functional voltage-gated, outwardly rectifying K⁺ channel. Kv_Synth1 displays the summed features of its individual components: pore properties of Kcv (selectivity and filter gating) and voltage dependence of Ci-VSP (V_1/2 = +56 mV; z of ∼1), including the depolarization-induced mode shift. The degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition. This highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains.     

Salt bridges in the miniature viral channel Kcv are important for function

The viral potassium channel Kcv comprises only 94 amino acids, which represent the pore module of more complex K⁺ channels. As for Kir-type channels, Kcv also has a short N-terminal helix exposed to the cytoplasm, upstream of the first transmembrane domain. Here we show that this helix is relevant for Kcv function. The presence of charged amino acids, which form dynamic inter- and intra-subunit salt bridges is crucial. Electrophysiological measurements, yeast rescue experiments and molecular dynamics simulations show that mutants in which the critical salt bridge formation is impaired have no or reduced channel activity. We conclude that these salt bridges destabilise the complexation of K⁺ ions by negative charges on the inner transmembrane domain at the entrance into the cavity. This feature facilitates a continuous and coordinated transfer of ions between the cavity and the cytoplasm for channels without the canonical bundle crossing.     

Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K⁺ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K⁺ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K⁺, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.     

In vitro synthesis, tetramerization and single channel characterization of virus-encoded potassium channel Kcv

Chlorella virus-encoded membrane protein Kcv represents a new class of potassium channel. This 94-amino acids miniature K(+) channel consists of two trans-membrane alpha-helix domains intermediated by a pore domain that contains a highly conserved K(+) selectivity filter. Therefore, as an archetypal K(+) channel, the study of Kcv may yield valuable insights into the structure-function relationships underlying this important class of ion channel. Here, we report a series of new properties of Kcv. We first verified Kcv can be synthesized in vitro. By co-synthesis and assembly of wild-type and the tagged version of Kcv, we were able to demonstrate a tetrameric stoichiometry, a molecular structure adopted by all known K(+) channels. Most notably, the tetrameric Kcv complex retains its functional integrity in SDS (strong detergent)-containing solutions, a useful feature that allows for direct purification of protein from polyacrylamide gel. Once purified, the tetramer can form single potassium-selective ion channels in a lipid bilayer with functions consistent to the heterologously expressed Kcv. These finding suggest that the synthetic Kcv can serve as a model of virus-encoded K(+) channels; and its newly identified properties can be applied to the future study on structure-determined mechanisms such as K(+) channel functional stoichiometry.     

Possible function for virus encoded K+ channel Kcv in the replication of chlorella virus PBCV-1

The K⁺ channel Kcv is encoded by the chlorella virus PBCV-1. There is evidence that this channel plays an essential role in the replication of the virus, because both PBCV-1 plaque formation and Kcv channel activity in Xenopus oocytes have similar sensitivities to inhibitors. Here we report circumstantial evidence that the Kcv channel is important during virus infection. Recordings of membrane voltage in the host cells Chlorella NC64A reveal a membrane depolarization within the first few minutes of infection. This depolarization displays the same sensitivity to cations as Kcv conductance; depolarization also requires the intact membrane of the virion. Together these data are consistent with the idea that the virus carries functional K⁺ channels in the virion and inserts them into the host cell plasma membrane during infection.     

Secrets of the fungus-specific potassium channel TOK family

Several families of potassium (K⁺) channels are found in membranes of all eukaryotes, underlining the importance of K⁺ uptake and redistribution within and between cells and organs. Among them, TOK (tandem-pore outward-rectifying K⁺) channels consist of eight transmembrane domains and two pore domains per subunit organized in dimers. These channels were originally studied in yeast, but recent identifications and characterizations in filamentous fungi shed new light on this fungus-specific K⁺ channel family. Although their actual function in vivo is often puzzling, recent works indicate a role in cellular K⁺ homeostasis and even suggest a role in plant–fungus symbioses. This review aims at synthesizing the current knowledge on fungal TOK channels and discussing their potential role in yeasts and filamentous fungi.     

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.     

Stabilization of the Conductive Conformation of a Voltage-gated K+ (Kv) Channel: THE LID MECHANISM*

Voltage-gated K⁺ (Kv) channels are molecular switches that sense membrane potential and in response open to allow K⁺ ions to diffuse out of the cell. In these proteins, sensor and pore belong to two distinct structural modules. We previously showed that the pore module alone is a robust yet dynamic structural unit in lipid membranes and that it senses potential and gates open to conduct K⁺ with unchanged fidelity. The implication is that the voltage sensitivity of K⁺ channels is not solely encoded in the sensor. Given that the coupling between sensor and pore remains elusive, we asked whether it is then possible to convert a pore module characterized by brief openings into a conductor with a prolonged lifetime in the open state. The strategy involves selected probes targeted to the filter gate of the channel aiming to modulate the probability of the channel being open assayed by single channel recordings from the sensorless pore module reconstituted in lipid bilayers. Here we show that the premature closing of the pore is bypassed by association of the filter gate with two novel open conformation stabilizers: an antidepressant and a peptide toxin known to act selectively on Kv channels. Such stabilization of the conductive conformation of the channel is faithfully mimicked by the covalent attachment of fluorescein at a cysteine residue selectively introduced near the filter gate. This modulation prolongs the occupancy of permeant ions at the gate. It is this longer embrace between ion and gate that we conjecture underlies the observed stabilization of the conductive conformation. This study provides a new way of thinking about gating.     

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.     

Long distance interactions within the potassium channel pore are revealed by molecular diversity of viral proteins

Kcv is a 94-amino acid protein encoded by chlorella virus PBCV-1 that corresponds to the pore module of K(+) channels. Therefore, Kcv can be a model for studying the protein design of K(+) channel pores. We analyzed the molecular diversity generated by approximately 1 billion years of evolution on kcv genes isolated from 40 additional chlorella viruses. Because the channel is apparently required for virus replication, the Kcv variants are all functional and contain multiple and dispersed substitutions that represent a repertoire of allowed sets of amino acid substitutions (from 4 to 12 amino acids). Correlations between amino acid substitutions and the new properties displayed by these channels guided site-directed mutations that revealed synergistic amino acid interactions within the protein as well as previously unknown interactions between distant channel domains. The effects of these multiple changes were not predictable from a priori structural knowledge of the channel pore.     

Using mutagenesis to study potassium channel mechanisms

The voltage-activated K⁺ channels are members of an ion channel family that includes the voltage-activated Na⁺ and Ca²⁺ channels. These ion channels mediate the transmembrane ionic currents that are responsible for the electrical signals produced by cells. The recent cloning of numerous voltage-activated K⁺ channels has made it possible to combine molecular-genetic and biophysical methods to study K⁺ channel mechanisms. These mutagenesis-function studies are beginning to provide new information about the architecture of K⁺ channel proteins and how they form a voltage-gated, K⁺-selective pore.     

Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History

Voltage-gated potassium (K⁺) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K⁺ channel type segregates into at least two main functional categories—hyperpolarization-activated, inward-rectifying (K_in) and depolarization-activated, outward-rectifying (K_out) channels. Voltage-gated K⁺ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in K_in and K_out channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K⁺ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between K_in and K_out channels. Among them is a loop between TMD S5 and the pore that is very short in animal K_out, longer in plant and animal K_in and the longest in plant K_out channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom.     

The viral potassium channel Kcv: structural and functional features

The chlorella virus PBCV-1 was the first virus found to encode a functional potassium channel protein (Kcv). Kcv is small (94 aa) and basically consists of the M1-P-M2 (membrane-pore-membrane) module typical of the pore regions of all known potassium channels. Kcv forms functional channels in three heterologous systems. This brief review discusses the gating, permeability and modulation properties of Kcv and compares them to the properties of bacterial and mammalian K+ channels.     

Membrane anchoring and interaction between transmembrane domains are crucial for K+ channel function

The small viral channel Kcv is a Kir-like K(+) channel of only 94 amino acids. With this simple structure, the tetramer of Kcv represents the pore module of all complex K(+) channels. To examine the structural contribution of the transmembrane domains (TMDs) to channel function, we performed Ala scanning mutagenesis of the two domains and tested the functionality of the mutants in a yeast complementation assay. The data reveal, in combination with computational models, that the upper halves of both TMDs, which face toward the external medium, are rather rigid, whereas the inner parts are more flexible. The rigidity of the outer TMD is conferred by a number of essential aromatic amino acids that face the membrane and probably anchor this domain in the bilayer. The inner TMD is intimately connected with the rigid part of the outer TMD via π···π interactions between a pair of aromatic amino acids. This structural principle is conserved within the viral K(+) channels and also present in Kir2.2, implying a general importance of this architecture for K(+) channel function.     

Potassium channel regulator KCNRG regulates surface expression of Shaker-type potassium channels

Besides their role in the generation of action potentials, voltage-gated potassium channels are implicated in cellular processes ranging from cell division to cell death. The K⁺ channel regulator protein (KCNRG), identified as a putative tumor suppressor, reduces K⁺ currents through human K⁺ channels hKv1.1 and hKv1.4 expressed in Xenopus oocytes. Current attenuation requires the presence of the N-terminal T1 Domain and immunoprecipitation experiments suggest association of KCNRG with the N-terminus of the channel. Our data indicates that KCNRG is an ER-associated protein, which we propose regulates Kv1 family channel proteins by retaining a fraction of channels in endomembranes.     

Interaction of local anesthetics with the K+ channel pore domain

Local anesthetics and related drugs block ionic currents of Na⁺, K⁺ and Ca²⁺ conducted across the cell membrane by voltage-dependent ion channels. Many of these drugs bind in the permeation pathway, occlude the pore and stop ion movement. However channel-blocking drugs have also been associated with decreased membrane stability of certain tetrameric K⁺ channels, similar to the destabilization of channel function observed at low extracellular K⁺ concentration. Such drug-dependent stability may result from electrostatic repulsion of K⁺ from the selectivity filter by a cationic drug molecule bound in the central cavity of the channel. In this study we used the pore domain of the KcsA K⁺ channel protein to test this hypothesis experimentally with a biochemical assay of tetramer stability and theoretically by computational simulation of local anesthetic docking to the central cavity. We find that two common local anesthetics, lidocaine and tetracaine, promote thermal dissociation of the KcsA tetramer in a K⁺-dependent fashion. Docking simulations of these drugs with open, open-inactivated and closed crystal structures of KcsA yield many energetically favorable drug-channel complexes characterized by nonbonded attraction to pore-lining residues and electrostatic repulsion of K⁺. The results suggest that binding of cationic drugs to the inner cavity can reduce tetramer stability of K⁺ channels.     

Distinct lipid bilayer compositions have general and protein-specific effects on K+ channel function

It has become increasingly apparent that the lipid composition of cell membranes affects the function of transmembrane proteins such as ion channels. Here, we leverage the structural and functional diversity of small viral K⁺ channels to systematically examine the impact of bilayer composition on the pore module of single K⁺ channels. In vitro–synthesized channels were reconstituted into phosphatidylcholine bilayers ± cholesterol or anionic phospholipids (aPLs). Single-channel recordings revealed that a saturating concentration of 30% cholesterol had only minor and protein-specific effects on unitary conductance and gating. This indicates that channels have effective strategies for avoiding structural impacts of hydrophobic mismatches between proteins and the surrounding bilayer. In all seven channels tested, aPLs augmented the unitary conductance, suggesting that this is a general effect of negatively charged phospholipids on channel function. For one channel, we determined an effective half-maximal concentration of 15% phosphatidylserine, a value within the physiological range of aPL concentrations. The different sensitivity of two channel proteins to aPLs could be explained by the presence/absence of cationic amino acids at the interface between the lipid headgroups and the transmembrane domains. aPLs also affected gating in some channels, indicating that conductance and gating are uncoupled phenomena and that the impact of aPLs on gating is protein specific. In two channels, the latter can be explained by the altered orientation of the pore-lining transmembrane helix that prevents flipping of a phenylalanine side chain into the ion permeation pathway for long channel closings. Experiments with asymmetrical bilayers showed that this effect is leaflet specific and most effective in the inner leaflet, in which aPLs are normally present in plasma membranes. The data underscore a general positive effect of aPLs on the conductance of K⁺ channels and a potential interaction of their negative headgroup with cationic amino acids in their vicinity.     

The ligand-sensitive gate of a potassium channel lies close to the selectivity filter

Potassium channels selectively conduct K⁺ ions across cell membranes and have key roles in cell excitability. Their opening and closing can be spontaneous or controlled by membrane voltage or ligand binding. We used Ba²⁺ as a probe to determine the location of the ligand-sensitive gate in an inwardly rectifying K⁺ channel (Kir6.2). To a K⁺ channel, Ba²⁺ and K⁺ are of similar sizes, but Ba²⁺ blocks the pore by binding within the selectivity filter. We found that internal Ba²⁺ could still access its binding site when the channel was shut, which indicates that the ligand-sensitive gate lies above the Ba²⁺-block site, and thus within or above the selectivity filter. This is in marked contrast to the voltage-dependent gate of K_V channels, which is located at the intracellular mouth of the pore.     

Functional role of the N-terminus of Na,K-ATPase α-subunit as an inactivation gate of palytoxin-induced pump channel

The N-terminus of the Na⁺,K⁺-ATPase α-subunit shows some homology to that of Shaker-B K⁺ channels; the latter has been shown to mediate the N-type channel inactivation in a ball-and-chain mechanism. When the Torpedo Na⁺,K⁺-ATPase is expressed in Xenopus oocytes and the pump is transformed into an ion channel with palytoxin (PTX), the channel exhibits a time-dependent inactivation gating at positive potentials. The inactivation gating is eliminated when the N-terminus is truncated by deleting the first 35 amino acids after the initial methionine. The inactivation gating is restored when a synthetic N-terminal peptide is applied to the truncated pumps at the intracellular surface. Truncated pumps generate no electrogenic current and exhibit an altered stoichiometry for active transport. Thus, the N-terminus of the α-subunit appears to act like an inactivation gate and performs a critical step in the Na⁺,K⁺-ATPase pumping function.     

Hydration valve controlled non-selective conduction of Na and K in the NaK channel

The Na⁺ and K⁺ channels are essential to neural signaling, but our current knowledge at the atomic level is mainly limited to the conducting mechanism of K⁺. Unlike a K⁺ channel having four equivalent K⁺-binding sites in its selectivity filter, a NaK channel has a vestibule in the middle part of its selectivity filter, and can conduct both Na⁺ and K⁺ ions. However, the underlying mechanism for non-selective ion conduction in NaK remains elusive. Here we find four small grottos connecting with the vestibule of the NaK selectivity filter, which form a vestibule-grotto complex perpendicular to the filter pore with a few water molecules within it. It is shown that two or more of the water molecules coming to the vestibule to coordinate the cation are necessary for conducting both Na⁺ and K⁺ ions, while only one water molecule in the vestibule will obstruct ion permeation. Thus, the complex with the aid of interior water movement forms a dynamic hydration valve which is flexible in conveying different cations through the vestibule. Similar exquisite hydration valve mechanisms are expected to be utilized by other non-selective cation channels, and the results should shed new light on the importance of water in neural signaling.