Sequential interaction of chloride and proton ions with the fast gate steer the voltage‐dependent gating in ClC‐2 chloride channels

Sánchez-Rodríguez, Jorge E.; Santiago-Castillo, Jose A. De; Contreras-Vite, Juan A.; Nieto-Delgado, Pablo G.; Castro-Chong, Alejandra; Arreola, Jorge

doi:10.1113/jphysiol.2012.232660

Cited by 21 publications

(40 citation statements)

References 38 publications

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“…The second major motion describes the flip of the GLU ex side chain on the extracellular vestibule. This flip of the GLU ex side chain displays a sensitivity to residue protonation that correlates well with experimental evidence that this conserved glutamate residue regulates the CLC "fast" gate in CLC-2 [71,72]. These observed gating motions are all consistent with data in the literature.…”

Section: Introductionsupporting

confidence: 87%

“…The second additional highlight of our kinetic analysis is that the transition rate from O to 10/25 C oi is slowed when GLU ex is protonated. While this result is uncertain due to the magnitude of the error bars of our calculations, it is worth highlighting given its consistency with experimental results, which demonstrate that the CLC-2 closing rate decreases with increasing extracellular [H + ] [72].…”

Section: Kinetic Modeling and Comparison To Experimental Datamentioning

confidence: 55%

“…Additional work is necessary to determine the dynamic details of how conformational changes at the inner and outer gates are linked to opening CLC channel pores. Because CLC channel homologs differ in the degree to which the pore is gated by Cl − , H + , transmembrane voltage, and other physiological variables [71,72,[78][79][80][81][82][83][84], the details of pore gating are likely to vary subtly between the homologs, despite the overall similarity in their structures. In this work, we sought specifically to gain insight into pore behavior in the CLC-2 homolog.…”

Section: Resultsmentioning

confidence: 99%

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Dynamical model of the CLC-2 ion channel exhibits a two-step gating mechanism

McKiernan

Koster

Maduke

et al. 2017

Preprint

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AbstractThis work reports a dynamical Markov state model of CLC-2 “fast” (pore) gating, based on 600 microseconds of molecular dynamics (MD) simulation. In the starting conformation of our CLC-2 model, both outer and inner channel gates are closed. The first conformational change in our dataset involves rotation of the inner-gate backbone along residues S168-G169-I170. This change is strikingly similar to that observed in the cryo-EM structure of the bovine CLC-K channel, though the volume of the intracellular (inner) region of the ion conduction pathway is further expanded in our model. From this state (inner gate open and outer gate closed), two additional states are observed, each involving a unique rotameric flip of the outer-gate residue GLUex. Both additional states involve conformational changes that orient GLUex away from the extracellular (outer) region of the ion conduction pathway. In the first additional state, the rotameric flip of GLUex results in an open, or near-open, channel pore. The equilibrium population of this state is low (∼1%), consistent with the low open probability of CLC-2 observed experimentally in the absence of a membrane potential stimulus (0 mV). In the second additional state, GLUex rotates to occlude the channel pore. This state, which has a low equilibrium population (∼1%), is only accessible when GLUex is protonated. Together, these pathways model the opening of both an inner and outer gate within the CLC-2 selectivity filter, as a function of GLUex protonation. Collectively, our findings are consistent with published experimental analyses of CLC-2 gating and provide a high-resolution structural model to guide future investigations.Author summaryIn the brain, the roles and mechanisms of sodium-, potassium-, and calcium-selective ion channels are well established. In contrast, chloride-selective channels have been studied much less and are not sufficiently understood, despite known associations of chloride-channel defects with brain disorders. The most broadly expressed voltage-activated chloride channel in the brain is CLC-2 (one of 9 human CLC homologs). In this work, we use simulations to model the conformational dynamics of the CLC-2 chloride ion channel selectivity filter (SF), which is the part of the protein that controls whether the channel is in an ion-conducting or non-conducting state. Our analysis identifies four primary conformational states and a specific progression through these states. Our results are consistent with structural and functional data in the literature and provide a high-resolution model for guiding further studies of CLC-2. These results will inform our understanding of how CLC-2 governs electrical activity and ion homeostasis in the brain.

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Section: Introductionsupporting

confidence: 87%

Section: Kinetic Modeling and Comparison To Experimental Datamentioning

confidence: 55%

Section: Resultsmentioning

confidence: 99%

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Dynamical model of the CLC-2 ion channel exhibits a two-step gating mechanism

McKiernan

Koster

Maduke

et al. 2017

Preprint

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“…In native cells as well as in heterologous expression systems, ClC-2 is closed at positive potentials and slowly opens upon membrane hyperpolarization resulting in a strong rectification of chloride currents [6, 16, 27, 34, 37]. We here show that the kinetics of ClC-2 activation and deactivation are significantly altered by intracellular ATP (Figs.…”

Section: Discussionmentioning

confidence: 66%

Regulation of ClC-2 gating by intracellular ATP

Stölting

Teodorescu

Begemann

et al. 2013

Pflugers Arch - Eur J Physiol

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ClC-2 is a voltage-dependent chloride channel that activates slowly at voltages negative to the chloride reversal potential. Adenosine triphosphate (ATP) and other nucleotides have been shown to bind to carboxy-terminal cystathionine-ß-synthase (CBS) domains of ClC-2, but the functional consequences of binding are not sufficiently understood. We here studied the effect of nucleotides on channel gating using single-channel and whole-cell patch clamp recordings on transfected mammalian cells. ATP slowed down macroscopic activation and deactivation time courses in a dose-dependent manner. Removal of the complete carboxy-terminus abolishes the effect of ATP, suggesting that CBS domains are necessary for ATP regulation of ClC-2 gating. Single-channel recordings identified long-lasting closed states of ATP-bound channels as basis of this gating deceleration. ClC-2 channel dimers exhibit two largely independent protopores that are opened and closed individually as well as by a common gating process. A seven-state model of common gating with altered voltage dependencies of opening and closing transitions for ATP-bound states correctly describes the effects of ATP on macroscopic and microscopic ClC-2 currents. To test for a potential pathophysiological impact of ClC-2 regulation by ATP, we studied ClC-2 channels carrying naturally occurring sequence variants found in patients with idiopathic generalized epilepsy, G715E, R577Q, and R653T. All naturally occurring sequence variants accelerate common gating in the presence but not in the absence of ATP. We propose that ClC-2 uses ATP as a co-factor to slow down common gating for sufficient electrical stability of neurons under physiological conditions.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-013-1286-0) contains supplementary material, which is available to authorized users.

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“…To search for a kinetic scheme able to describe the time- and V m -dependencies of I Cl collected under a variety of experimental conditions we used Markov chain kinetic models [9] because they provide a good description of gating properties for different channels [24, 26, 28, 43, 48]. Different models with various discrete closed (C) and open (O) states were built using IChMASCOT software [10, 42]. Experimental and model I Cl vs V m , G Norm vs [Ca 2+ ] i , and EC 50 were compared.…”

Section: Methodsmentioning

confidence: 99%

Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models

Contreras-Vite

Cruz-Rangel

Jesús-Pérez

et al. 2016

Pflugers Arch - Eur J Physiol

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TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth, and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca2+]i), membrane depolarization, extracellular Cl− or permeant anions, and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. a) TMEM16A is activated by voltage in the absence of intracellular Ca2+. b) The Cl− conductance is decreased after reducing extracellular Cl− concentration ([Cl−]o). c) ICl is regulated by physiological concentrations of [Cl−]o. d) In cells dialyzed with 0.2 µM [Ca2+]i, Cl− has a bimodal effect: at [Cl−]o < 30 mM TMEM16A current activates with a monoexponential time course, but above 30 mM [Cl−]o ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca2+ and Cl− to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca2+ ions coupled to a Vm-dependent binding of an external Cl− ion, with Vm-dependent transitions between states. Our model predicts that extracellular Cl− does not alter the apparent Ca2+ affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl− acts by stabilizing the open configuration induced by Ca2+ and by contributing to the Vm dependence of activation.

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Sequential interaction of chloride and proton ions with the fast gate steer the voltage‐dependent gating in ClC‐2 chloride channels

Cited by 21 publications

References 38 publications

Dynamical model of the CLC-2 ion channel exhibits a two-step gating mechanism

Dynamical model of the CLC-2 ion channel exhibits a two-step gating mechanism

Regulation of ClC-2 gating by intracellular ATP

Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models

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