A Quantitative Description of KcsA Gating I: Macroscopic Currents

Chakrapani, Sudha; Cordero-Morales, Julio F.; Perozo, Eduardo

doi:10.1085/jgp.200709843

Cited by 106 publications

(169 citation statements)

References 59 publications

(95 reference statements)

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“…Analysis of the dwell-time histograms revealed that both open and closed durations were composed of multiple kinetic components, which is in good agreement with previous studies (40,41). Under all experimentally tested phospholipid conditions, dwell-time histograms for closed durations were best fit with three exponentials corresponding to three nonconductive states of the KcsA channel (Fig.…”

Section: Resultssupporting

confidence: 90%

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Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Cruijsen

Prokofyev

Pongs

et al. 2017

Biophysical Journal

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Ion conduction across the cellular membrane requires the simultaneous opening of activation and inactivation gates of the K þ channel pore. The bacterial KcsA channel has served as a powerful system for dissecting the structural changes that are related to four major functional states associated with K þ gating. Yet, the direct observation of the full gating cycle of KcsA has remained structurally elusive, and crystal structures mimicking these gating events require mutations in or stabilization of functionally relevant channel segments. Here, we found that changes in lipid composition strongly increased the KcsA open probability. This enabled us to probe all four major gating states in native-like membranes by combining electrophysiological and solid-state NMR experiments. In contrast to previous crystallographic views, we found that the selectivity filter and turret region, coupled to the surrounding bilayer, were actively involved in channel gating. The increase in overall steady-state open probability was accompanied by a reduction in activation-gate opening, underscoring the important role of the surrounding lipid bilayer in the delicate conformational coupling of the inactivation and activation gates.

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Section: Resultssupporting

confidence: 90%

“…S1 in the Supporting Material). Closed times representing mainly inactivated states under steady-state conditions (40) were sensitive to changes in the anionic phospholipid environment. The total mean closed time (t mean ) was~8 fold shorter in the presence of DOPA and CL in comparison with DOPG and DOPS (Fig S1; Table S1).…”

Section: Resultsmentioning

confidence: 99%

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Cruijsen

Prokofyev

Pongs

et al. 2017

Biophysical Journal

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“…In contrast, we observed significant structural differences in the turret region, including an elongation of the TM1 helix for bilayer embedded WT and Chim channels. Notably, our ssNMR results are in good agreement with earlier electron paramagnetic resonance (EPR) work on WT KcsA in asolectin liposomes (31). The EPR data indicated that TM1 helix extends until WT Arg52.…”

Section: Pore Loop Structure Is Defined By a Combination Of Protein-lsupporting

confidence: 91%

“…Similar to WT KcsA (30), this state can be induced for the Chim channel by triggering inner gate bundle opening at low pH and reducing K + concentrations to less than 20 mM (8,9). Notably, further reduction to pH 3.7 did not change our Chim ssNMR spectra, even though macroscopic and single channel measurements suggest that the apparent activation pK a is closer to pH 4.2 for WT KcsA (31). For the analysis of the ssNMR data, an initial 3D structural model was constructed by using torsion angle restraints in channel regions known to undergo conformational changes after inactivation (8,9).…”

Section: Resultsmentioning

confidence: 76%

Importance of lipid–pore loop interface for potassium channel structure and function

Cruijsen

Nand

Weingarth

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Potassium (i.e., K + ) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K + channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K + channel inactivation and exhibits a remarkable structural plasticity that correlates to K + channel inactivation. The transmembrane helix 1 unwinds when the K + channel enters the inactivated state and rewinds during the transition to the closed state. In addition to wellcharacterized changes at the K + ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K + channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K + channel pore. membrane protein | ion channel | solid-state NMR spectroscopy P otassium (i.e., K + ) channels are embedded in the plasma membrane to control the selective passage of potassium ions across the lipid bilayer. The channels open and close their conduction pathway by sensing changes in physicochemical parameters such as pH, ligand concentration, and membrane voltage (1). Structure-function studies on voltage-gated K + (Kv) channels suggested that lipid molecules are an integral part of the voltage-sensing domains, which transfer during the gating process electrical charges across the cell membrane (2-4). In some of the available Kv channel crystal structures, lipid molecules appear most densely packed against the pore domain, presumably providing an appropriate environment for the stability and the operation of the gating machinery to open and close the conduction pathway. In general, the activity of Kv pore domains is thought to be determined by the activity of two gates in series, one for activation and one for inactivation. These gates jointly control the conduction of ions through the pore (5-11). The activation gate is located at the intracellular entrance of the pore and the inactivation gate is situated toward the extracellular entrance at the selectivity filter (i.e., C-type inactivation). In addition, some potassium channels possess close to the activation gate a receptor for an N-terminal inactivating domain (i.e., N-type inactivation).The K + channel pore domain is conserved across all K + channels. It comprises a tetrameric assembly of t...

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“…1 B and C). These two gates are functionally coupled as demonstrated by C-type inactivation, in which channel opening triggers loss of conduction at the selectivity filter (1)(2)(3)(4). A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4)(5)(6)(7)(8)(9)(10).…”

mentioning

confidence: 99%

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Brettmann

Urusova

Tonelli

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassiumselective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.solution NMR | ion channels | membrane protein | protein dynamics

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A Quantitative Description of KcsA Gating I: Macroscopic Currents

Cited by 106 publications

References 59 publications

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

Importance of lipid–pore loop interface for potassium channel structure and function

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

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