Inactivation In KcsA Research Articles

The Constricted Filter Conformation: A General Property for C-Type Inactivation of Different Potassium Channels?

C-type inactivation of K+ channels is a molecular process of great physiological significance. It affects the firing patterns of neurons in the central nervous system, and the repolarization of cardiac cells in the heart. The most widely held hypothesis is that C-type inactivation involves a constriction of the selectivity filter, which has been observed in crystal structures of the bacterial KcsA channel. However, crystal structures of no other K+ channels have been captured in a similar constricted conformation, and the significance of the KcsA structures remains controversial. Our recent study of the KcsA channel explained the origin of the extremely long timescale for recovery from inactivation in KcsA (∼10 sec) on the basis of the constricted filter conformation. From this result, we now are in a position to systematically test the hypothesis that C-type inactivation is universally caused by a conformational transition toward a KcsA-like constricted filter. Using all-atom molecular dynamics simulations, we have modeled the Kv1.2, Shaker, and MthK channels with their selectivity filter in the constricted conformation by combining the high-resolution crystal structure of these channels and the coordinates of the pinched KcsA filter (1K4D). Extended molecular dynamics simulations were performed for each channel to determine the stability of the constricted conformation. The goal is to determine why some channels can inactivate (KcsA, KcsA D-ala77, MthK, Shaker) whereas some channels cannot (Kv1.2), and the effect of critical mutations on C-type inactivation in KcsA, Shaker and Kv1.2.

NMR Structural Studies of the Binding of Activating Mamba Toxin Tx7335 on the Potassium Channel KcsA

We have recently identified a novel 63 amino acid residue three-finger toxin (called Tx7335) from eastern green mamba snake (Dendroaspis angusticeps) which interacts with KcsA and induces an increase in frequency and duration of individual channel openings when added to the outside of the channel. The toxin exerts this activating effect both on wild-type KcsA as well as on an agitoxin2-sensitive mutant form of the channel, indicating a mode of action and binding site that are different from the classic pore-blocker toxins. We are currently using NMR spectroscopy to unravel the structural underpinnings of this mechanism of action. High yield of purified 15N labeled KcsA and excellent NMR spectral quality have been achieved. Currently the characterization of toxin binding using 1H15N correlation spectra of 15N labeled KcsA in the absence and presence of toxin is ongoing. Experiments are conducted in different membrane mimetics including DMPC/DHPC bicelles and DPC or DM micelles at different pH, temperature and salt concentration. Some chemical shifts and peak intensity changes upon toxin addition have been observed. Continuing NMR structural studies will further elucidate the mechanism of how Tx7335 interacts with KcsA and shed light on the conformational and dynamic changes of C-type inactivation in KcsA and on a novel mechanism of ion channel regulation.

Thermodynamic coupling between activation and inactivation gating in potassium channels revealed by free energy molecular dynamics simulations

The amount of ionic current flowing through K+ channels is determined by the interplay between two separate time-dependent processes: activation and inactivation gating. Activation is concerned with the stimulus-dependent opening of the main intracellular gate, whereas inactivation is a spontaneous conformational transition of the selectivity filter toward a nonconductive state occurring on a variety of timescales. A recent analysis of multiple x-ray structures of open and partially open KcsA channels revealed the mechanism by which movements of the inner activation gate, formed by the inner helices from the four subunits of the pore domain, bias the conformational changes at the selectivity filter toward a nonconductive inactivated state. This analysis highlighted the important role of Phe103, a residue located along the inner helix, near the hinge position associated with the opening of the intracellular gate. In the present study, we use free energy perturbation molecular dynamics simulations (FEP/MD) to quantitatively elucidate the thermodynamic basis for the coupling between the intracellular gate and the selectivity filter. The results of the FEP/MD calculations are in good agreement with experiments, and further analysis of the repulsive, van der Waals dispersive, and electrostatic free energy contributions reveals that the energetic basis underlying the absence of inactivation in the F103A mutation in KcsA is the absence of the unfavorable steric interaction occurring with the large Ile100 side chain in a neighboring subunit when the intracellular gate is open and the selectivity filter is in a conductive conformation. Macroscopic current analysis shows that the I100A mutant indeed relieves inactivation in KcsA, but to a lesser extent than the F103A mutant.

The Second Highly-Conserved Threonine Residue of the Potassium Channel Signature Sequence Mediates Activation-Coupled to C-Type Inactivation

In the amino-acid sequence of a K+ channel pore domain are encoded most of the structural elements necessary to 1) catalyze the permeation of K+ ions and 2) execute activation and C-type inactivation gating. In KcsA, a C-type inactivation process similar to that found in eukaryotic channels has been identified [1] and crystallographic evidences indicate that the collapse of the channel selectivity filter (SF) is responsible for it [2]. It was also established that an allosteric communication between the channel activation gate and the SF underlies activation coupled to C-type inactivation in KcsA and other K+ channels [3]. In KcsA, F103 seems to mediate this allosteric communication by interacting with T75 of the same subunit, the second highly conserved Threonine in the SF of K+ channels (TTXGYGD). In order to gain functional and structural insights regarding this allosteric communication, we have mutated KcsA-T75 to a Glycine or an Alanine and evaluated the functional consequences of severing this molecular interaction. The resulting phenotype for these perturbations was a non-inactivating one, albeit having altered conduction properties (smaller single-channel conductance). Additionally, a similar phenotype was observed for the Shaker K+ channel when the equivalent position T442 was mutated to either Gly or Ala, suggesting that a common mechanism underlies activation coupled to C-type inactivation in K+ channels. Finally, the X-ray solution structure for these KcsA mutants trapped in the open state, will be presented and discussed in the context of activation coupled to C-type inactivation in K+ channels.“DGG is supported by FRSQ and CIHR fellowships.”1)Cordero-Morales, J.F., et al. Nat Struct Mol Biol, 2006. 13(4): p. 311-8.2)Cuello, L.G., et al. Nature, 2010. 466(7303): p. 203-8.3)Cuello, L.G., et al.. Nature, 2010. 466(7303): p. 272-5.

Structural basis for the coupling between activation and inactivation gates in K+ channels

The coupled interplay between activation and inactivation gating is a functional hallmark of K(+) channels. This coupling has been experimentally demonstrated through ion interaction effects and cysteine accessibility, and is associated with a well defined boundary of energetically coupled residues. The structure of the K(+) channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation-inactivation gating. Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe 103 tilts towards residues Thr 74 and Thr 75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp 67, Glu 71 and Asp 80 to destabilize the selectivity filter, allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe 103 and the surrounding residues. We probed similar interactions in the Shaker K(+) channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K(+) channels.

On the Nature of hERG Inactivation using KcsA, Shaker and Kv1.2 as Structural and Functional Models

In K+ channels, C-type inactivation locks the selectivity filter in a non-conductive state. The prokaryotic proton-gated potassium channel KcsA undergoes a time dependent inactivation process. The interaction between Glu71 and Asp80 is one of the key driving forces that promote filter instability and inactivation. This interaction promotes a compression of the selectivity filter parallel to the permeation pathway, which biases it towards the inactivated conformation. Since KcsA represents a structural model for the pore domain of K+ channels, it is obvious that a detailed understanding of the molecular basis of inactivation is not limited to this prokaryotic channel, but offers new directions into how inactivation gating might proceed in other K+ channels. Here, using patch clamp experiments, EPR spectroscopy, functional assays, molecular dynamics, and X-ray crystallography we show that interactions involving residues Trp67, Tyr78, and Asp80 in KcsA, conserved in most potassium channels, also constitute critical contacts between the selectivity filter and its adjacent pore helix which determines the rate and extent of C-type inactivation in Shaker, Kv1.2, and hERG. Substitution of a tryptophan or tyrosine at the pore helix to phenylalanine in these channels decreases the rate and extent of inactivation, pointing this position as key modulator of gating. Furthermore, by substituting equivalent amino acids critical for hERG inactivation in KcsA we were able to create a non-conducting KcsA mutant with normal pH activated lower gate. These results suggest commonalities in inactivation gating mechanism of eukaryotic channels, and provide evidence that the hydrogen bond network and Van der Waals interactions between the pore helix, selectivity filter, and external vestibule serve as the basis for C-type inactivation in the K+ channel family.

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.

Free Energy Landscape for the Inactivation of the KcsA Potassium Channel

The potassium ion channel, KcsA, gates the passage of ions through cell membranes in response to a change in pH. Recent experimental results have demonstrated the existence of two gates in KcsA: an intracellular gate and a gate at the selectivity filter. Lowering the pH opens the intracellular gate allowing ions to pass. After a period of time, however, the channel inactivates by constricting the selectivity filter and impeding the flow of ions even though the bottom gate remains open. We have used path-based molecular dynamics simulations to probe the detailed mechanism of this phenomenon by finding dynamical pathways to inactivation in KcsA. We have computed free energies and rates of inactivation that agree with recent experimental results. We also provide a molecular rationalization for the coupling between the opening and closing of the lower gate and the inactivation of the selectivity filter.

N-type Inactivation of the Potassium Channel KcsA by the Shaker B “Ball” Peptide: MAPPING THE INACTIVATING PEPTIDE-BINDING EPITOPE

The effects of the inactivating peptide from the eukaryotic Shaker BK(+) channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr(8), which exhibit the strongest STD effects; the C4H in the imidazole ring of His(16); the methyl protons of Val(4), Leu(7), and Leu(10) and the side chain amine protons of one, if not both, the Lys(18) and Lys(19) residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val(4) and Leu(10), whereas His(16) seems similarly affected as before. Conversely, STD effects on Tyr(8) are strongly diminished, and those on Lys(18) and/or Lys(19) are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of (13)C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a beta-hairpin structure when bound to the KcsA channel. Indeed, docking such a beta-hairpin structure into an open pore model for K(+) channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations.

A Quantitative Description of KcsA Gating I: Macroscopic Currents

The prokaryotic K+ channel KcsA is activated by intracellular protons and its gating is modulated by transmembrane voltage. Typically, KcsA functions have been studied under steady-state conditions, using macroscopic Rb+-flux experiments and single-channel current measurements. These studies have provided limited insights into the gating kinetics of KcsA due to its low open probability, uncertainties in the number of channels in the patch, and a very strong intrinsic kinetic variability. In this work, we have carried out a detailed analysis of KcsA gating under nonstationary conditions by examining the influence of pH and voltage on the activation, deactivation, and slow-inactivation gating events. We find that activation and deactivation gating of KcsA are predominantly modulated by pH without a significant effect of voltage. Activation gating showed sigmoidal pH dependence with a pKa of ∼4.2 and a Hill coefficient of ∼2. In the sustained presence of proton, KcsA undergoes a time-dependent decay of conductance. This inactivation process is pH independent but is modulated by voltage and the nature of permeant ion. Recovery from inactivation occurs via deactivation and also appears to be voltage dependent. We further find that inactivation in KcsA is not entirely a property of the open-conducting channel but can also occur from partially “activated” closed states. The time course of onset and recovery of the inactivation process from these pre-open closed states appears to be different from the open-state inactivation, suggesting the presence of multiple inactivated states with diverse kinetic pathways. This information has been analyzed together with a detailed study of KcsA single-channel behavior (in the accompanying paper) in the framework of a kinetic model. Taken together our data constitutes the first quantitative description of KcsA gating.