Mechanism Of C-type Inactivation Research Articles

A distinct mechanism of C-type inactivation in the Kv-like KcsA mutant E71V

C-type inactivation is of great physiological importance in voltage-activated K+ channels (Kv), but its structural basis remains unresolved. Knowledge about C-type inactivation has been largely deduced from the bacterial K+ channel KcsA, whose selectivity filter constricts under inactivating conditions. However, the filter is highly sensitive to its molecular environment, which is different in Kv channels than in KcsA. In particular, a glutamic acid residue at position 71 along the pore helix in KcsA is substituted by a valine conserved in most Kv channels, suggesting that this side chain is a molecular determinant of function. Here, a combination of X-ray crystallography, solid-state NMR and MD simulations of the E71V KcsA mutant is undertaken to explore inactivation in this Kv-like construct. X-ray and ssNMR data show that the filter of the Kv-like mutant does not constrict under inactivating conditions. Rather, the filter adopts a conformation that is slightly narrowed and rigidified. On the other hand, MD simulations indicate that the constricted conformation can nonetheless be stably established in the mutant channel. Together, these findings suggest that the Kv-like KcsA mutant may be associated with different modes of C-type inactivation, showing that distinct filter environments entail distinct C-type inactivation mechanisms.

A different mechanism of C-type inactivation in the Kv-like KcsA mutant E71V

C-type inactivation is of great physiological importance particularly in voltage-activated K+ channels (Kv), affecting the firing patterns of neurons and shaping cardiac action potentials. While understanding the molecular basis of inactivation has a direct impact on human health, its structural basis remains unresolved. Knowledge about C-type inactivation has been largely deduced from the pH-activated bacterial K+ channel KcsA, whose selectivity filter under inactivating conditions adopts a constricted conformation at the level of the central glycine (TTVGYGD) that is stabilized by tightly bound water molecules.

Mechanism of C-type inactivation in the hERG potassium channel.

The fast C-type inactivation displayed by the voltage-activated potassium channel hERG plays a critical role in the repolarization of cardiac cells, and malfunction caused by nonspecific binding of drugs or naturally occurring missense mutations affecting inactivation can lead to pathologies. Because of its impact on human health, understanding the molecular mechanism of C-type inactivation in hERG represents an advance of paramount importance. Here, long-time scale molecular dynamics simulations, free energy landscape calculations, and electrophysiological experiments are combined to address the structural and functional impacts of several disease-associated mutations. Results suggest that C-type inactivation in hERG is associated with an asymmetrical constricted-like conformation of the selectivity filter, identifying F627 side-chain rotation and the hydrogen bond between Y616 and N629 as key determinants. Comparison of hERG with other K+ channels suggests that C-type inactivation depends on the degree of opening of the intracellular gate via the filter-gate allosteric coupling.

Molecular Mechanism of Depolarization-Dependent Inactivation in W366F Mutant of Kv1.2.

Voltage-gated potassium channels play crucial roles in regulating membrane potential. They are activated by membrane depolarization, allowing the selective permeation of K+ ions across the plasma membrane, and enter a nonconducting state after lasting depolarization, a process known as inactivation. Inactivation in voltage-activated potassium channels occurs through two distinct mechanisms, N-type and C-type inactivation. C-type inactivation is caused by conformational changes in the extracellular mouth of the channel, whereas N-type inactivation is elicited by changes in the cytoplasmic mouth of the protein. The W434F-mutated Shaker channel is known as a nonconducting mutant and is in a C-type inactivation state at a depolarizing membrane potential. To clarify the structural properties of C-type inactivated protein, we performed molecular dynamics simulations of the wild-type and W366F (corresponding to W434F in Shaker) mutant of the Kv1.2-2.1 chimera channel. The W366F mutant was in a nearly nonconducting state with a depolarizing voltage and recovered from inactivation with a reverse voltage. Our simulations and three-dimensional reference interaction site model analysis suggested that structural changes in the selectivity filter upon membrane depolarization trap K+ ions around the inner mouth of the selectivity filter and prevent ion permeation. This pore restriction is involved in the molecular mechanism of C-type inactivation.

Crystal structure of an inactivated mutant mammalian voltage-gated K+ channel.

C-type inactivation underlies important roles played by voltage-gated K+ (Kv) channels. Functional studies have provided strong evidence that a common underlying cause of this type of inactivation is an alteration near the extracellular end of the channel's ion-selectivity filter. Unlike N-type inactivation, which is known to reflect occlusion of the channel's intracellular end, the structural mechanism of C-type inactivation remains controversial and may have many detailed variations. Here we report that in voltage-gated Shaker K+ channels lacking N-type inactivation, a mutation enhancing inactivation disrupts the outermost K+ site in the selectivity filter. Furthermore, in a crystal structure of the Kv1.2-2.1 chimeric channel bearing the same mutation, the outermost K+ site, which is formed by eight carbonyl-oxygen atoms, appears to be slightly too small to readily accommodate a K+ ion and in fact exhibits little ion density; this structural finding is consistent with the functional hallmark of C-type inactivation.

The Conformation of KcsA's Selectivity Filter Influences the Opening of its Activation Gate

Our knowledge about structural-function relationships in K+ channels has grown exponentially, mostly because of the use of the prokaryotic version of these membrane proteins. This is especially true for the KcsA K+ channel, a two transmembrane, proton-gated K+ channel from Streptomyces lividans, for which the structures, for different kinetic states, are known1,2. This minimal version of a K+ channel faithfully imitates the most important functional features of its eukaryotic and more complex relatives, the Kv channels. Previously, it has been shown that in KcsA, C-type inactivation is allosterically coupled to activation gating, as well as in other K+ channels. This means that the conformational changes associated to the opening of the activation gate (AG) propagate to the channel's selectivity filter (SF), triggering its collapse conformation. It has been shown before that the more open is the channel's AG, the deeper inactivates. By extension, the structural conformation of the SF should influence the AG opening. Thus to investigate this possibility, we have systematically studied the opening of the channel's AG by spectroscopic approaches, in conditions that relieve or promote C-inactivation. Our results suggest that the conformational state of the channel's SF indeed strongly influence the AG opening.Reference:1. Cuello, L. G., Jogini, V., Cortes, D. M. & Perozo, E. Structural mechanism of C-type inactivation in K(+) channels. Nature 466, 203-208, doi:10.1038/nature09153 (2010).2. Zhou, Y., Morais-Cabral, J. H., Kaufman, A. & MacKinnon, R. Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution.[see comment]. Nature 414, 43-48 (2001).Funding: NIH 1R01GM097159-01A1; Welch Foundation BI-1757

Ion Permeation in the MthK Potassium Channel

KcsA from S.lividans is well established as an archetype model for the whole family of potassium channels. However, MthK from M. thermoautotrophicum, another model potassium channel, behaves quite differently in many respects. Comparison of functional and structural data of both channels reveals important similitudes, but also key differences. An important but not well understood feature of potassium channels is their C-type inactivation mechanism. The structure of the inactivated state of KcsA was first solved by crystallization under conditions of low potassium concentration. It revealed conformational changes in the selectivity filter region preventing the permeation of both K(+) and Na(+) ions. Surprisingly, x-ray crystallography experiments under similar low potassium conditions revealed that the structure of MthK remains in its canonical conducting state even when it is deprived of K(+) ions. Functional studies nevertheless show that MthK undergoes C-type inactivation like KcsA and most eukaryotic K(+) channels. To understand the mechanisms underlying this apparent discrepancy, we investigated on the permeation and gating mechanisms in the MthK channel using molecular dynamic simulations and free energy calculations.

Structural mechanism of C-type inactivation in K+ channels

Interconversion between conductive and non-conductive forms of the K(+) channel selectivity filter underlies a variety of gating events, from flicker transitions (at the microsecond timescale) to C-type inactivation (millisecond to second timescale). Here we report the crystal structure of the Streptomyces lividans K(+) channel KcsA in its open-inactivated conformation and investigate the mechanism of C-type inactivation gating at the selectivity filter from channels 'trapped' in a series of partially open conformations. Five conformer classes were identified with openings ranging from 12 A in closed KcsA (Calpha-Calpha distances at Thr 112) to 32 A when fully open. They revealed a remarkable correlation between the degree of gate opening and the conformation and ion occupancy of the selectivity filter. We show that a gradual filter backbone reorientation leads first to a loss of the S2 ion binding site and a subsequent loss of the S3 binding site, presumably abrogating ion conduction. These structures indicate a molecular basis for C-type inactivation in K(+) channels.

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.

On the Structure-Function correlates of Ion Occupancy and modulation of C-type Inactivation

KcsA, a proton activated K channel, has served as an archetypical K pore providing molecular insights into understanding selectivity, ion-permeation, gating and pore-blocking. A recent set of crystal structures describing the mechanism of C-type inactivation in this channel now allows for an understanding this mechanism at atomistic level. Our results show that KcsA inactivation is strongly coupled to the opening of the activation gates and is modulated by the amount and direction of current passing through the channels. As also implicated by studies in eukaryotic channels, C-type inactivation in KcsA involves an intimate interplay between the selectivity-filter region and permeant-ions. This study attempts at further understanding this close association between the ion and filter by correlating high resolution structures with macroscopic and single- channel functional data. We have obtained several KcsA crystal structures of the closed and the open mutant channel, in the presence of different permeant ions (K+, Rb+, Cs+ and NH4+) and blockers (Ba2+ and TEA). These structures reveal different ion occupancies depending upon the nature of the permeant ion, blocker and the extent of channel opening. Analyzed in the light of extensive functional evidence, these results uncover several important features of the interplay between ion interactions and the evolution of C-type inactivation.

Stability And Conductance Assessment Of A Putative Low-k+ Inactivated State Of The Kcsa Channel

Potassium channels constitute a large family of proteins, notably involved in the regulation of the activity of excitable cells. The channels partly exert that function by varying their conductance through a mechanism known as C-type inactivation: Shortly after the activation of K+ channels, their selectivity filter stop conducting ions at a rate that depends on various stimuli. This inactivation process plays a critical role in controlling the length and frequency of cardiac action potentials, as well as the firing patterns in neurons. The molecular process underlying the C-type inactivation mechanism remains unexplained despite the accumulation of experimental evidences showing the key role played by the channels' selectivity filter and some neighboring residues.It's been recently shown that the prokaryotic KcsA channel undergoes C-type inactivation like its eukaryotic counterparts (Gao et al., PNAS, 102:17630 (2005)), establishing KcsA as a perfect prototypic model to study the structural basis of the inactivation mechanism. An X-ray structure of the KcsA channel obtained in presence of low K+ concentration (Zhou et al., Nature 414:43 (2001), pdb code 1K4D) has since then been postulated to correspond to the C-type inactivated state of the channel. While the structural analysis of this static conformation suggests that pore lining amide hydrogens would prevent the permeation of ions, uncertainties remain about its stability under physiological conditions and its ion occupancy state. These questions are of primary importance to better understand the relevance of this structure to the physiological regulation of ion permeation in K+ channels. Using molecular dynamics simulations and free energy calculations, we investigated on the stability, selectivity, and conductance of the selectivity filter of KcsA in this putative inactivated state.

Effect of propafenone on Kv1.4 inactivation

Interactions between antiarrhythmic drugs and ion channels are important subjects in the field of cardiovascular electro-pharmacology. This study explores the relationship between propafenone and C-type inactivation of Kv1.4 channel. fKvl.4deltaN, a ferret Kv1.4 N-terminal deleted mutant, was employed in this study. fKvl.4deltaN cRNA was injected into Xenopus oocytes to express fKvl.4deltaN channel and two electrode voltage clamp technique was used to record the current. We found that fKvl.4deltaN channel current was rapidly depressed in a frequency-dependent manner and meanwhile, C-type inactivation in this channel was increased more than 7 folds in the presence of 100 microM propafenone. While propafenone has no effect on Kv1.4deltaN recovery. All the results indicate that propafenone blocks Kvl.4deltaN channel through intracellular bindings and that binding of propafenone with Kvl.4deltaN channel leads to a conformational change on the extracellular site which accelerates C-type inactivation, suggesting that propafenone, as an open channel blocker, may affect the mechanism of C-type inactivation.

K+ Currents Activated by Depolarization in Cardiac Fibroblasts

K(+) currents expressed in freshly dispersed rat ventricular fibroblasts have been studied using whole-cell patch-clamp recordings. Depolarizing voltage steps from a holding potential of -90 mV activated time- and voltage-dependent outward currents at membrane potentials positive to approximately -30 mV. The relatively slow activation kinetics exhibited strong dependence on the membrane potential. Selected changes in extracellular K(+) concentration ([K(+)](o)) revealed that the reversal potentials of the tail currents changed as expected for a K(+) equilibrium potential. The activation and inactivation kinetics of this K(+) current, as well as its recovery from inactivation, were well-fitted by single exponential functions. The steady-state inactivation was well described by a Boltzmann function with a half-maximal inactivation potential (V(0.5)) of -24 mV. Increasing [K(+)](o) (from 5 to 100 mM) shifted this V(0.5) in the hyperpolarizing direction by -11 mV. Inactivation was slowed by increasing [K(+)](o) to 100 mM, and the rate of recovery from inactivation was decreased after increasing [K(+)](o). Block of this K(+) current by extracellular tetraethylammonium also slowed inactivation. These [K(+)](o)-induced changes and tetraethylammonium effects suggest an important role for a C-type inactivation mechanism. This K(+) current was sensitive to dendrotoxin-I (100 nM) and rTityustoxin Kalpha (50 nM).

External pore collapse as an inactivation mechanism for Kv4.3 K+ channels.

Kv4 channels are thought to lack a C-type inactivation mechanism (collapse of the external pore) and to inactivate as a result of a concerted action of cytoplasmic regions of the channel. To investigate whether Kv4 channels have outer pore conformational changes during the inactivation process, the inactivation properties of Kv4.3 were characterized in 0 mM and in 2 mM external K+ in whole-cell voltage-clamp experiments. Removal of external K+ increased the inactivation rates and favored cumulative inactivation by repetitive stimulation. The reduction in current amplitude during repetitive stimulation and the faster inactivation rates in 0 mM external K+ were not due to changes in the voltage dependence of channel opening or to internal K+ depletion. The extent of the collapse of the K+ conductance upon removal of external K+ was more pronounced in NMG+-than in Na+-containing solutions. The reduction in the current amplitude during cumulative inactivation by repetitive stimulation is not associated with kinetic changes, suggesting that it is due to a diminished number of functional channels with unchanged gating properties. These observations meet the criteria for a typical C-type inactivation, as removal of external K+ destabilizes the conducting state, leading to the collapse of the pore. A tentative model is presented, in which K+ bound to high-affinity K+-binding sites in the selectivity filter destabilizes an outer neighboring K+ modulatory site that is saturated at approximately 2 mM external K+. We conclude that Kv4 channels have a C-type inactivation mechanism and that previously reported alterations in the inactivation rates after N- and C- termini mutagenesis may arise from secondary changes in the electrostatic interactions between K+-binding sites in the selectivity filter and the neighboring K+-modulatory site, that would result in changes in its K+ occupancy.

Inactivation and Pharmacological Properties of sqKv1A Homotetramers in Xenopus Oocytes Cannot Account for Behavior of the Squid “Delayed Rectifier” K + Conductance

Considerable published evidence suggests that α-subunits of the cloned channel sqKv1A compose the “delayed rectifier” in the squid giant axon system, but discrepancies regarding inactivation properties of cloned versus native channels exist. In this paper we define the mechanism of inactivation for sqKv1A channels in Xenopus oocytes to investigate these and other discrepancies. Inactivation of sqKv1A in Xenopus oocytes was found to be unaffected by genetic truncation of the N-terminus, but highly sensitive to certain amino acid substitutions around the external mouth of the pore. External TEA and K + ions slowed inactivation of sqKv1A channels in oocytes, and chloramine T (Chl-T) accelerated inactivation. These features are all consistent with a C-type inactivation mechanism as defined for Shaker B channels. Treatment of native channels in giant fiber lobe neurons with TEA or high K + does not slow inactivation, nor does Chl-T accelerate it. Pharmacological differences between the two channel types were also found for 4-aminopyridine (4AP). SqKv1A’s affinity for 4AP was poor at rest and increased after activation, whereas 4AP block occurred much more readily at rest with native channels than when they were activated. These results suggest that important structural differences between sqKv1A homotetramers and native squid channels are likely to exist around the external and internal mouths of the pore.

State-dependent barium block of wild-type and inactivation-deficient HERG channels in Xenopus oocytes.

The effects of Ba2+ on current resulting from the heterologous expression of the human ether-à-go-go related gene (HERG) (IHERG) was studied with two-electrode voltage clamp techniques in Xenopus oocytes. Ba2+ produced time- and voltage-dependent block of IHERG. Significant inhibition was seen at concentrations as low as 1 microM. Inhibition was greatest at step potentials between -40 and 0 mV; at more positive potentials, inhibition decreased in association with time-dependent unblocking of channels. An inactivation-attenuated mutant of HERG (S631A) was prepared and expressed in Xenopus oocytes. Ba2+ block of S631A differed from that of HERG in that extensive unblocking was no longer seen at positive potentials and the voltage dependence of step current block was greatly attenuated. A mathematical model was applied to analyse quantitatively the inhibitory effects of Ba2+ on IHERG. The model suggested similar voltage-dependent affinity of Ba2+ for the open and closed states, along with absence of binding to the inactivated state, and accounted well for Ba2+ effects on both wild-type and S631A channels. We conclude that Ba2+ potently inhibits IHERG in a characteristic state-dependent fashion, with strong unblocking at positive potentials related to the presence of an intact C-type inactivation mechanism.

Functional Analysis of a Mouse Brain Elk-Type K+Channel

Members of the Ether à go-go (Eag) K+ channel subfamilies Eag, Erg, and Elk are widely expressed in the nervous system, but their neural functions in vivo remain largely unknown. The biophysical properties of channels from the Eag and Erg subfamilies have been described, and based on their characteristic features and expression patterns, Erg channels have been associated with native currents in the heart. Little is known about the properties of channels from the Elk subfamily. We have identified a mouse gene, Melk2, that encodes a predicted polypeptide with 48% amino acid identity to Drosophila Elk but only 40 and 36% identity with mouse Erg (Merg) and Eag (Meag), respectively. Melk2 RNA appears to be expressed at high levels only in brain tissue. Functional expression of Melk2 in Xenopus oocytes reveals large, transient peaks of current at the onset of depolarization. Like Meag currents, Melk2 currents activate relatively quickly, but they lack the nonsuperimposable Cole-Moore shift characteristic of the Eag subfamily. Melk2 currents are insensitive to E-4031, a class III antiarrhythmic compound that blocks the Human Ether-à-go-go-Related Gene (HERG) channel and its counterpart in native tissues, IKr. Melk2 channels exhibit inward rectification because of a fast C-type inactivation mechanism, but the slower rate of inactivation and the faster rate of activation results in less inward rectification than that observed in HERG channels. This characterization of Melk currents should aid in identification of native counterparts to the Elk subfamily of channels in the nervous system.

Transfer of rapid inactivation and sensitivity to the class III antiarrhythmic drug E-4031 from HERG to M-eag channels.

The gating behaviour and pharmacological sensitivity of HERG are remarkably different from the corresponding properties of M-eag, a structurally similar member of the Eag family of potassium channels. In contrast to HERG, M-eag exhibits no apparent inactivation and little rectification, and is insensitive to the class III antiarrhythmic drug E-4031. We generated chimeric channels of HERG and M-eag sequences and made point mutations to identify the region necessary for rapid inactivation in HERG. This region includes the P region and half of the S6 putative transmembrane domain, including sites not previously associated with inactivation and rectification in HERG. Transfer of a small segment of the HERG polypeptide to M-eag, consisting largely of the P region and part of the S6 transmembrane domain, is sufficient to confer rapid inactivation and E-4031 sensitivity to M-eag. This region differs from the corresponding region in M-eag by only fifteen residues. Previous hypotheses that rapid inactivation of HERG channels occurs by a C-type inactivation mechanism are supported by the parallel effects on rates of HERG inactivation and Shaker C-type inactivation by a series of mutations at two equivalent sites in the polypeptide sequences. In addition to sites homologous to those previously described for C-type inactivation in Shaker, inactivation in HERG involves a residue in the upstream P region not previously associated with C-type inactivation. Although this site is equivalent to one implicated in P-type inactivation in Kv2.1 channels, our data are most consistent with a single, C-type inactivation mechanism.