Gating the glutamate gate of CLC-2 chloride channel by pore occupancy

Jesús-Pérez, José J. De; Castro-Chong, Alejandra; Shieh, Ru‐Chi; Hernández-Carballo, Carmen Y.; Santiago-Castillo, Jose A. De; Arreola, Jorge

doi:10.1085/jgp.201511424

Cited by 34 publications

(47 citation statements)

References 47 publications

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“…Consistently with other CLC-2 channels, this can be explained by the lower permeability of gluconate [33] with respect to chloride through the channel. The lower gluconate permeability shifts the reversal potential (from a few mV, see S2B Fig) towards more positive values, determining a greater driving force and a consequent larger chloride current flowing out of the cell.…”

Section: Resultssupporting

confidence: 84%

Osmoregulated Chloride Currents in Hemocytes from Mytilus galloprovincialis

et al. 2016

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We investigated the biophysical properties of the transport mediated by ion channels in hemocytes from the hemolymph of the bivalve Mytilus galloprovincialis. Besides other transporters, mytilus hemocytes possess a specialized channel sensitive to the osmotic pressure with functional properties similar to those of other transport proteins present in vertebrates. As chloride fluxes may play an important role in the regulation of cell volume in case of modifications of the ionic composition of the external medium, we focused our attention on an inwardly-rectifying voltage-dependent, chloride-selective channel activated by negative membrane potentials and potentiated by the low osmolality of the external solution. The chloride channel was slightly inhibited by micromolar concentrations of zinc chloride in the bath solution, while the antifouling agent zinc pyrithione did not affect the channel conductance at all. This is the first direct electrophysiological characterization of a functional ion channel in ancestral immunocytes of mytilus, which may bring a contribution to the understanding of the response of bivalves to salt and contaminant stresses.

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Section: Resultssupporting

confidence: 84%

Osmoregulated Chloride Currents in Hemocytes from Mytilus galloprovincialis

et al. 2016

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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%

“…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%

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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“…Our idea describes the effect of external Cl − onTMEM16A based on what has been proposed for other Cl − channels. For example, in CLC-0 and CLC-1 external anions have a profound facilitating effect on gating [7, 14, 37] and in CLC-2 V m -dependence is conferred by permeant anions that occupy and then exit the pore [25, 41]. Thus, the role of permeant anions in gating the Cl − channels may be more important that thought in the past.…”

Section: Discussionmentioning

confidence: 99%

“…Gating of Cl − channels such as ClC-2, volume-sensitive channels, and CFTR are reported to be dependent on permeant anion [23, 25, 41, 51]. CaCCs are not the exception.…”

Section: Introductionmentioning

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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Gating the glutamate gate of CLC-2 chloride channel by pore occupancy

Abstract: Intracellular permeant anions, and not extracellular protons, are the predominant driver of fast gating in the hyperpolarization-activated CLC-2 chloride channel.

Cited by 34 publications

References 47 publications

Osmoregulated Chloride Currents in Hemocytes from Mytilus galloprovincialis

Osmoregulated Chloride Currents in Hemocytes from Mytilus galloprovincialis

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

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

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