Voltage-dependent Gating of Single Wild-Type and S4 Mutant KAT1 Inward Rectifier Potassium Channels

The voltage-dependent gating mechanism of KAT1 inward rectifier potassium channels was studied using single channel current recordings from Xenopus oocytes injected with KAT1 mRNA. The inward rectification properties of KAT1 result from an intrinsic gating mechanism in the KAT1 channel protein, not from pore block by an extrinsic cation species. KAT1 channels activate with hyperpolarizing potentials from −110 through −190 mV with a slow voltage-dependent time course. Transitions before first opening are voltage dependent and account for much of the voltage dependence of activation, while transitions after first opening are only slightly voltage dependent. Using burst analysis, transitions near the open state were analyzed in detail. A kinetic model with multiple closed states before first opening, a single open state, a single closed state after first opening, and a closed-state inactivation pathway accurately describes the single channel and macroscopic data. Two mutations neutralizing charged residues in the S4 region (R177Q and R176L) were introduced, and their effects on single channel gating properties were examined. Both mutations resulted in depolarizing shifts in the steady state conductance–voltage relationship, shortened first latencies to opening, decreased probability of terminating bursts, and increased burst durations. These effects on gating were well described by changes in the rate constants in the kinetic model describing KAT1 channel gating. All transitions before the open state were affected by the mutations, while the transitions after the open state were unaffected, implying that the S4 region contributes to the early steps in gating for KAT1 channels.     

Probing the Transition State of the Allosteric Pathway of the Shaker Kv Channel Pore by Linear Free-Energy Relations

Long-range coupling between distant functional elements of proteins may rely on allosteric communication trajectories lying along the protein structure, as described in the case of the Shaker voltage-activated potassium (Kv) channel model allosteric system. Communication between the distant Kv channel activation and slow inactivation pore gates was suggested to be mediated by a network of local pairwise and higher-order interactions among the functionally unique residues that constitute the allosteric trajectory. The mechanism by which conformational changes propagate along the Kv channel allosteric trajectory to achieve pore opening, however, remains unclear. Such conformational changes may propagate in either a concerted or a sequential manner during the reaction coordinate of channel opening. Residue-level structural information on the transition state of channel gating is required to discriminate between these possibilities. Here, we combine patch-clamp electrophysiology recordings of Kv channel gating and analysis using linear free-energy relations, focusing on a select set of residues spanning the allosteric trajectory of the Kv channel pore. We show that all allosteric trajectory residues tested exhibit an open-like conformation in the transition state of channel opening, implying that coupling interactions occur along the trajectory break in a concerted manner upon moving from the closed to the open state. Energetic coupling between the Kv channel gates thus occurs in a concerted fashion in both the spatial and the temporal dimensions, strengthening the notion that such trajectories correspond to pathways of mechanical deformation along which conformational changes propagate.     

Ion channel gating: insights via molecular simulations

Ion channels are gated, i.e. they can switch conformation between a closed and an open state. Molecular dynamics simulations may be used to study the conformational dynamics of ion channels and of simple channel models. Simulations on model nanopores reveal that a narrow (<4 A) hydrophobic region can form a functionally closed gate in the channel and can be opened by either a small (approximately 1 A) increase in pore radius or an increase in polarity. Modelling and simulation studies confirm the importance of hydrophobic gating in K channels, and support a model in which hinge-bending of the pore-lining M2 (or S6 in Kv channels) helices underlies channel gating. Simulations of a simple outer membrane protein, OmpA, indicate that a gate may also be formed by interactions of charged side chains within a pore, as is also the case in ClC channels.     

Inter-Subunit Interactions across the Upper Voltage Sensing-Pore Domain Interface Contribute to the Concerted Pore Opening Transition of Kv Channels

The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.     

The pore helix is involved in stabilizing the open state of inwardly rectifying K+ channels

Ion channels can be gated by various extrinsic cues, such as voltage, pH, and second messengers. However, most ion channels display extrinsic cue-independent transitions as well. These events represent spontaneous conformational changes of the channel protein. The molecular basis for spontaneous gating and its relation to the mechanism by which channels undergo activation gating by extrinsic cue stimulation is not well understood. Here we show that the proximal pore helix of inwardly rectifying (Kir) channels is partially responsible for determining spontaneous gating characteristics, affecting the open state of the channel by stabilizing intraburst openings as well as the bursting state itself without affecting K(+) ion-channel interactions. The effect of the pore helix on the open state of the channel is qualitatively similar to that of two well-characterized mutations at the second transmembrane domain (TM2), which stabilize the channel in its activated state. However, the effects of the pore helix and the TM2 mutations on gating were additive and independent of each other. Moreover, in sharp contrast to the two TM2 mutations, the pore helix mutation did not affect the functionality of the agonist-responsive gate. Our results suggest that in Kir channels, the bottom of the pore helix and agonist-induced conformational transitions at the TM2 ultimately stabilize via different pathways the open conformation of the same gate.     

The molecular mechanism by which PIP(2) opens the intracellular G-loop gate of a Kir3.1 channel

Inwardly rectifying potassium (Kir) channels are characterized by a long pore comprised of continuous transmembrane and cytosolic portions. A high-resolution structure of a Kir3.1 chimera revealed the presence of the cytosolic (G-loop) gate captured in the closed or open conformations. Here, we conducted molecular-dynamics simulations of these two channel states in the presence and absence of phosphatidylinositol bisphosphate (PIP(2)), a phospholipid that is known to gate Kir channels. Simulations of the closed state with PIP(2) revealed an intermediate state between the closed and open conformations involving direct transient interactions with PIP(2), as well as a network of transitional inter- and intrasubunit interactions. Key elements in the G-loop gating transition involved a PIP(2)-driven movement of the N-terminus and C-linker that removed constraining intermolecular interactions and led to CD-loop stabilization of the G-loop gate in the open state. To our knowledge, this is the first dynamic molecular view of PIP(2)-induced channel gating that is consistent with existing experimental data.     

Ion effects on gating of the Ca(2+)-activated K+ channel correlate with occupancy of the pore.

We studied the effects of permeant ions on the gating of the large conductance Ca(2+)-activated K+ channel from rat skeletal muscle. Rb+ blockade of inward K+ current caused an increase in the open probability as though Rb+ occupancy of the pore interferes with channel closing. In support of this hypothesis, we directly measured the occupancy of the pore by the impermeant ion Cs+ and found that it strongly correlates with its effect on gating. This is consistent with the "foot-in-the-door" model of gating, which states that channels cannot close with an ion in the pore. However, because Rb+ and Cs+ not only slow the closing rate (as predicted by the model), but also speed the opening rate, our results are more consistent with a modified version of the model in which the channel can indeed close while occupied, but the occupancy destabilizes the closed state. Increasing the occupancy of the pore by the addition of other permeant (K+ and Tl+) and impermeant (tetraethylammonium) ions did not affect the open probability. To account for this disparity, we used a two-site permeation model in which only one of the sites influenced gating. Occupancy of this "gating site" interferes with channel closing and hastens opening. Ions that directly or indirectly increase the occupancy of this site will increase the open probability.     

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.     

Basis for allosteric open-state stabilization of voltage-gated potassium channels by intracellular cations

The open state of voltage-gated potassium (Kv) channels is associated with an increased stability relative to the pre-open closed states and is reflected by a slowing of OFF gating currents after channel opening. The basis for this stabilization is usually assigned to intrinsic structural features of the open pore. We have studied the gating currents of Kv1.2 channels and found that the stabilization of the open state is instead conferred largely by the presence of cations occupying the inner cavity of the channel. Large impermeant intracellular cations such as N-methyl-d-glucamine (NMG⁺) and tetraethylammonium cause severe slowing of channel closure and gating currents, whereas the smaller cation, Cs⁺, displays a more moderate effect on voltage sensor return. A nonconducting mutant also displays significant open state stabilization in the presence of intracellular K⁺, suggesting that K⁺ ions in the intracellular cavity also slow pore closure. A mutation in the S6 segment used previously to enlarge the inner cavity (Kv1.2-I402C) relieves the slowing of OFF gating currents in the presence of the large NMG⁺ ion, suggesting that the interaction site for stabilizing ions resides within the inner cavity and creates an energetic barrier to pore closure. The physiological significance of ionic occupation of the inner cavity is underscored by the threefold slowing of ionic current deactivation in the wild-type channel compared with Kv1.2-I402C. The data suggest that internal ions, including physiological concentrations of K⁺, allosterically regulate the deactivation kinetics of the Kv1.2 channel by impairing pore closure and limiting the return of voltage sensors. This may represent a primary mechanism by which Kv channel deactivation kinetics is linked to ion permeation and reveals a novel role for channel inner cavity residues to indirectly regulate voltage sensor dynamics.     

Does the Na channel conduct ions through a water-filled pore or a condensed-state pathway?

Many investigators assert that the ion-conducting pathway of the Na channel is a water-filled pore. This assertion must be reevaluated to clear the way for more productive approaches to channel gating. The hypothesis of an aqueous pore leaves the questions of voltage-dependent gating and ion selectivity unexplained because a column of water can neither serve as a switch nor provide the necessary selectivity. The price of believing in an aqueous pore therefore is a futile search for separate ad hoc mechanisms for gating and selectivity. The fallacy is to assume that only water is available to carry ions rapidly, ignoring the role of the glycoprotein, which can form an elastomeric phase with water. The elastomer is a state of matter, neither liquid nor solid, in which the molecules of a liquid are threaded together with cross-linked polymer chains; it supports fast ion motion (Owen, 1989). An alternative hypothesis for channel gating, based on condensed-state materials science, already exists (Leuchtag, 1988, 1991a). The ferroelectric-superionic transition hypothesis (FESITH) postulates that the Na channel exists in a metastable ordered (closed) state at resting potential and, on threshold depolarization, undergoes a reversible order-disorder phase transition to a less-ordered, ion-conducting (open) state. The ordered state is ferroelectric; the disordered state is a fast ion conductor selective for Li+ and Na+. The basis of the voltage dependence is elevation of transition temperature with electric field, well established in ferroelectrics. FESITH is consistent with single-channel transitions, gating currents, heat and cold block, and other phenomena observed at channel or membrane level.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Link between Ion Permeation and Inactivation Gating of Kv4 Potassium Channels

Kv4 potassium channels undergo rapid inactivation but do not seem to exhibit the classical N-type and C-type mechanisms present in other Kv channels. We have previously hypothesized that Kv4 channels preferentially inactivate from the preopen closed state, which involves regions of the channel that contribute to the internal vestibule of the pore. To further test this hypothesis, we have examined the effects of permeant ions on gating of three Kv4 channels (Kv4.1, Kv4.2, and Kv4.3) expressed in Xenopus oocytes. Rb⁺ is an excellent tool for this purpose because its prolonged residency time in the pore delays K⁺ channel closing. The data showed that, only when Rb⁺ carried the current, both channel closing and the development of macroscopic inactivation are slowed (1.5- to 4-fold, relative to the K⁺ current). Furthermore, macroscopic Rb⁺ currents were larger than K⁺ currents (1.2- to 3-fold) as the result of a more stable open state, which increases the maximum open probability. These results demonstrate that pore occupancy can influence inactivation gating in a manner that depends on how channel closing impacts inactivation from the preopen closed state. By examining possible changes in ionic selectivity and the influence of elevating the external K⁺ concentration, additional experiments did not support the presence of C-type inactivation in Kv4 channels.     

Free-energy landscapes of ion-channel gating are malleable: changes in the number of bound ligands are accompanied by changes in the location of the transition state in acetylcholine-receptor channels

Acetylcholine-receptor channels (AChRs) are allosteric membrane proteins that mediate synaptic transmission by alternatively opening and closing ("gating") a cation-selective transmembrane pore. Although ligand binding is not required for the channel to open, the binding of agonists (for example, acetylcholine) increases the closed right harpoon over left harpoon open equilibrium constant because the ion-impermeable --> ion-permeable transition of the ion pathway is accompanied by a low-affinity --> high-affinity change at the agonist-binding sites. The fact that the gating conformational change of muscle AChRs can be kinetically modeled as a two-state reaction has paved the way to the experimental characterization of the corresponding transition state, which represents a snapshot of the continuous sequence of molecular events separating the closed and open states. Previous studies of fully (di) liganded AChRs, combining single-channel kinetic measurements, site-directed mutagenesis, and data analysis in the framework of the linear free-energy relationships of physical organic chemistry, have suggested a transition-state structure that is consistent with channel opening being an asynchronous conformational change that starts at the extracellular agonist-binding sites and propagates toward the intracellular end of the pore. In this paper, I characterize the gating transition state of unliganded AChRs, and report a remarkable difference: unlike that of diliganded gating, the unliganded transition state is not a hybrid of the closed- and open-state structures but, rather, is almost indistinguishable from the open state itself. This displacement of the transition state along the reaction coordinate obscures the mechanism underlying the unliganded closed right harpoon over left harpoon open reaction but brings to light the malleable nature of free-energy landscapes of ion-channel gating.     

The mammalian and yeast translocon complexes comprise a characteristic Sec61 channel

In eukaryotes, protein translocation across and insertion into the membrane of the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex or translocon. In our previous electrophysiological studies, we characterized the mammalian Sec61 channel from Canis familiaris. Here we extended these initial results to the Sec61 channel from the yeast Saccharomyces cerevisiae and compared the basic electrophysiological properties of both channel preparations with respect to the gating behaviour, distribution of channel open states, ionic conductance, approximated pore dimensions, reversal potential and selectivity as well as voltage-dependent open probability. We found that the Sec61 complexes from both species displayed conformable characteristics of the highly dynamic channel in an intrinsically open state. In contrast, the bacterial Sec61-homologue, the SecYEG complex from Escherichia coli, displayed under the same experimental conditions significantly different properties residing in an intrinsically closed state. We therefore propose that considerable differences between the respective eukaryote and prokaryote protein-conducting channel units and their regulation exist.     

Toward elucidating the heat activation mechanism of the TRPV1 channel gating by molecular dynamics simulation

As a key cellular sensor, the TRPV1 cation channel undergoes a gating transition from a closed state to an open state in response to various physical and chemical stimuli including noxious heat. Despite years of study, the heat activation mechanism of TRPV1 gating remains enigmatic at the molecular level. Toward elucidating the structural and energetic basis of TRPV1 gating, we have performed molecular dynamics (MD) simulations (with cumulative simulation time of 3 μs), starting from the high‐resolution closed and open structures of TRPV1 solved by cryo‐electron microscopy. In the closed‐state simulations at 30°C, we observed a stably closed channel constricted at the lower gate (near residue I679), while the upper gate (near residues G643 and M644) is dynamic and undergoes flickery opening/closing. In the open‐state simulations at 60°C, we found higher conformational variation consistent with a large entropy increase required for the heat activation, and both the lower and upper gates are dynamic with transient opening/closing. Through ensemble‐based structural analyses of the closed state versus the open state, we revealed pronounced closed‐to‐open conformational changes involving the membrane proximal domain (MPD) linker, the outer pore, and the TRP helix, which are accompanied by breaking/forming of a network of closed/open‐state specific hydrogen bonds. By comparing the closed‐state simulations at 30°C and 60°C, we observed heat‐activated conformational changes in the MPD linker, the outer pore, and the TRP helix that resemble the closed‐to‐open conformational changes, along with partial formation of the open‐state specific hydrogen bonds. Some of the residues involved in the above key hydrogen bonds were validated by previous mutational studies. Taken together, our MD simulations have offered rich structural and dynamic details beyond the static structures of TRPV1, and promising targets for future mutagenesis and functional studies of the TRPV1 channel. Proteins 2016; 84:1938–1949. © 2016 Wiley Periodicals, Inc.     

A Leucine Zipper Motif Essential for Gating of Hyperpolarization-activated Channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are pacemakers in cardiac myocytes and neurons. Although their membrane topology closely resembles that of voltage-gated K⁺ channels, the mechanism of their unique gating behavior in response to hyperpolarization is still poorly understood. We have identified a highly conserved leucine zipper motif in the S5 segment of HCN family members. In order to study the role of this motif for channel function, the leucine residues of the zipper were individually mutated to alanine, arginine, or glutamine residues. Leucine zipper mutants traffic to the plasma membrane, but the channels lose their sensitivity to open upon hyperpolarization. Thus, our data indicate that the leucine zipper is an important molecular determinant for hyperpolarization-activated channel gating. Residues of the leucine zipper interact with the adjacent S6 segment of the channel. This interaction is essential for voltage-dependent gating of the channel. The lower part of the leucine zipper, at the intracellular mouth of the channel, is important for stabilizing the closed state. Mutations at these sites increase current amplitudes or result in channels with deficient closing and increased min-P_o. Our data are further supported by homology models of the open and closed state of the HCN2 channel pore. Thus, we conclude that the leucine zipper of HCN channels is a major determinant for hyperpolarization-activated channel gating.     

Voltage-dependent conformational changes in connexin channels

Channels formed by connexins display two distinct types of voltage-dependent gating, termed V(j)- or fast-gating and loop- or slow-gating. Recent studies, using metal bridge formation and chemical cross-linking have identified a region within the channel pore that contributes to the formation of the loop-gate permeability barrier. The conformational changes are remarkably large, reducing the channel pore diameter from 15 to 20? to less than 4?. Surprisingly, the largest conformational change occurs in the most stable region of the channel pore, the 3(10) or parahelix formed by amino acids in the 42-51 segment. The data provide a set of positional constraints that can be used to model the structure of the loop-gate closed state. Less is known about the conformation of the V(j)-gate closed state. There appear to be two different mechanisms; one in which conformational changes in channel structure are linked to a voltage sensor contained in the N-terminus of Cx26 and Cx32 and a second in which the C-terminus of Cx43 and Cx40 may act either as a gating particle to block the channel pore or alternatively to stabilize the closed state. The later mechanism utilizes the same domains as implicated in effecting pH gating of Cx43 channels. It is unclear if the two V(j)-gating mechanisms are related or if they represent different gating mechanisms that operate separately in different subsets of connexin channels. A model of the V(j)-closed state of Cx26 hemichannel that is based on the X-ray structure of Cx26 and electron crystallographic structures of a Cx26 mutation suggests that the permeability barrier for V(j)-gating is formed exclusively by the N-terminus, but recent information suggests that this conformation may not represent a voltage-closed state. Closed state models are considered from a thermodynamic perspective based on information from the 3.5? Cx26 crystal structure and molecular dynamics (MD) simulations. The applications of computational and experimental methods to define the path of allosteric molecular transitions that link the open and closed states are discussed. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.     

Molecular dynamics simulations of Piezo1 channel opening by increases in membrane tension

Piezo1 is a mechanosensitive channel involved in many cellular functions and responsible for sensing shear stress and pressure forces in cells. Piezo1 has a unique trilobed topology with a curved membrane region in the closed state. It has been suggested that upon activation Piezo1 adopts a flattened conformation, but the molecular and structural changes underpinning the Piezo1 gating and opening mechanisms and how the channel senses forces in the membrane remain elusive. Here, we used molecular dynamics simulations to reveal the structural rearrangements that occur when Piezo1 moves from a closed to an open state in response to increased mechanical tension applied to a model membrane. We find that membrane stretching causes Piezo1 to flatten and expand its blade region, resulting in tilting and lateral movement of the pore-lining transmembrane helices 37 and 38. This is associated with the opening of the channel and movement of lipids out of the pore region. Our results reveal that because of the rather loose packing of Piezo1 pore region, movement of the lipids outside the pore region is critical for the opening of the pore. Our simulations also suggest synchronous flattening of the Piezo1 blades during Piezo1 activation. The flattened structure lifts the C-terminal extracellular domain up, exposing it more to the extracellular space. Our studies support the idea that it is the blade region of Piezo1 that senses tension in the membrane because pore opening failed in the absence of the blades. Additionally, our simulations reveal that upon opening, water molecules occupy lateral fenestrations in the cytosolic region of Piezo1, which might be likely paths for ion permeation. Our results provide a model for how mechanical force opens the Piezo1 channel and thus how it might couple mechanical force to biological response.     

Nonequilibrium gating and voltage dependence of the ClC-0 Cl- channel

The gating of ClC-0, the voltage-dependent Cl- channel from Torpedo electric organ, is strongly influenced by Cl- ions in the external solution. Raising external Cl- over the range 1-600 mM favors the fast- gating open state and disfavors the slow-gating inactivated state. Analysis of purified single ClC-0 channels reconstituted into planar lipid bilayers was used to identify the role of Cl- ions in the channel's fast voltage-dependent gating process. External, but not internal, Cl- had a major effect on the channel's opening rate constant. The closing rate was more sensitive to internal Cl- than to external Cl-. Both opening and closing rates varied with voltage. A model was derived that postulates (a) that in the channel's closed state, Cl- is accessible to a site located at the outer end of the conduction pore, where it binds in a voltage-independent fashion, (b) that this closed conformation can open, whether liganded by Cl- or not, in a weakly voltage-dependent fashion, (c) that the Cl(-)-liganded closed channel undergoes a conformational change to a different closed state, such that concomitant with this change, Cl- ion moves inward, conferring voltage-dependence to this step, and (d) that this new Cl(-)- liganded closed state opens with a very high rate. According to this picture, Cl- movement within the pre-open channel is the major source of voltage dependence, and charge movement intrinsic to the channel protein contributes very little to voltage-dependent gating of ClC-0. Moreover, since the Cl- activation site is probably located in the ion conduction pathway, the fast gating of ClC-0 is necessarily coupled to ion conduction, a nonequilibrium process.     

Ion channel gates: comparative analysis of energy barriers

The energetic profile of an ion translated along the axis of an ion channel should reveal whether the structure corresponds to a functionally open or closed state of the channel. In this study, we explore the combined use of Poisson–Boltzmann electrostatic calculations and evaluation of van der Waals interactions between ion and pore to provide an initial appraisal of the gating state of a channel. This approach is exemplified by its application to the bacterial inward rectifier potassium channel KirBac3.1, where it reveals the closed gate to be formed by a ring of leucine (L124) side chains. We have extended this analysis to a comparative survey of gating profiles, including model hydrophobic nanopores, the nicotinic acetylcholine receptor, and a number of potassium channel structures and models. This enables us to identify three gating regimes, and to show the limitation of this computationally inexpensive method. For a (closed) gate radius of 0.4 nm < R < 0.8 nm, a hydrophobic gate may be present. For a gate radius of 0.2 nm < R < 0.4 nm, both electrostatic and van der Waals interactions will contribute to the barrier height. Below R = 0.2 nm, repulsive van der Waals interactions are likely to dominate, resulting in a sterically occluded gate. In general, the method is more useful when the channel is wider; for narrower channels, the flexibility of the protein may allow otherwise-unsurmountable energetic barriers to be overcome.     

Allosteric Gating of a Large Conductance Ca-activated K+ Channel

Large-conductance Ca-activated potassium channels (BK channels) are uniquely sensitive to both membrane potential and intracellular Ca²⁺. Recent work has demonstrated that in the gating of these channels there are voltage-sensitive steps that are separate from Ca²⁺ binding steps. Based on this result and the macroscopic steady state and kinetic properties of the cloned BK channel mslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca²⁺ in the gating of the mslo channel (Cui, J., D.H. Cox, and R.W. Aldrich. 1997. J. Gen. Physiol. In press.). This scheme supposes that the channel exists in two main conformations, closed and open. The conformational change between closed and open is voltage dependent. Ca²⁺ binds to both the closed and open conformations, but on average binds more tightly to the open conformation and thereby promotes channel opening. Here we describe the basic properties of models of this form and test their ability to mimic mslo macroscopic steady state and kinetic behavior. The simplest form of this scheme corresponds to a voltage-dependent version of the Monod-Wyman-Changeux (MWC) model of allosteric proteins. The success of voltage-dependent MWC models in describing many aspects of mslo gating suggests that these channels may share a common molecular mechanism with other allosteric proteins whose behaviors have been modeled using the MWC formalism. We also demonstrate how this scheme can arise as a simplification of a more complex scheme that is based on the premise that the channel is a homotetramer with a single Ca²⁺ binding site and a single voltage sensor in each subunit. Aspects of the mslo data not well fitted by the simplified scheme will likely be better accounted for by this more general scheme. The kinetic schemes discussed in this paper may be useful in interpreting the effects of BK channel modifications or mutations.