Electro-steric opening of the clc-2 chloride channel gate

Jesús-Pérez, José J. De; Méndez-Maldonado, Gloria Arlette; López-Romero, Ana E.; Esparza-Jasso, David; González-Hernández, Irma L; Rosa, Víctor De la; Gastélum-Garibaldi, Roberto; Sánchez-Rodríguez, Jorge E.; Arreola, Jorge

doi:10.1038/s41598-021-92247-3

Cited by 10 publications

(11 citation statements)

References 55 publications

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“…In contrast, the protopore gates are located in each protomer. Similar to other ClC proteins, three residues were identified in ClC-2 in the potential chloride binding sites along the channel (S162, E205 and Y553 in human ClC-2; S168, E211 and Y559 in rat ClC-2; S170, E213 and Y561 in mouse ClC-2) 18 , 41 , 42 . Structural modeling and electrophysiological recording experiments demonstrated that E213 and Y561 in mouse ClC-2 are energetically coupled in the closed state 41 .…”

Section: Introductionmentioning

confidence: 63%

“…Similar to other ClC proteins, three residues were identified in ClC-2 in the potential chloride binding sites along the channel (S162, E205 and Y553 in human ClC-2; S168, E211 and Y559 in rat ClC-2; S170, E213 and Y561 in mouse ClC-2) 18 , 41 , 42 . Structural modeling and electrophysiological recording experiments demonstrated that E213 and Y561 in mouse ClC-2 are energetically coupled in the closed state 41 . Upon occupied by Cl − ion, the Y561-E213 coupling is disrupted by electrostatic and steric repulsion, E213 is then repelled outward to adopt an outward-facing conformation 41 .…”

Section: Introductionmentioning

confidence: 63%

“…Structural modeling and electrophysiological recording experiments demonstrated that E213 and Y561 in mouse ClC-2 are energetically coupled in the closed state 41 . Upon occupied by Cl − ion, the Y561-E213 coupling is disrupted by electrostatic and steric repulsion, E213 is then repelled outward to adopt an outward-facing conformation 41 . This observation supports the idea that the pore occupancy of permeant anions regulates the activation of ClC-2 43 , 44 .…”

Section: Introductionmentioning

confidence: 99%

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Cryo-EM structures of ClC-2 chloride channel reveal the blocking mechanism of its specific inhibitor AK-42

Ma,

Wang,

Chai

et al. 2023

Nat Commun

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ClC-2 transports chloride ions across plasma membranes and plays critical roles in cellular homeostasis. Its dysfunction is involved in diseases including leukodystrophy and primary aldosteronism. AK-42 was recently reported as a specific inhibitor of ClC-2. However, experimental structures are still missing to decipher its inhibition mechanism. Here, we present cryo-EM structures of apo ClC-2 and its complex with AK-42, both at 3.5 Å resolution. Residues S162, E205 and Y553 are involved in chloride binding and contribute to the ion selectivity. The side-chain of the gating glutamate E205 occupies the putative central chloride-binding site, indicating that our structure represents a closed state. Structural analysis, molecular dynamics and electrophysiological recordings identify key residues to interact with AK-42. Several AK-42 interacting residues are present in ClC-2 but not in other ClCs, providing a possible explanation for AK-42 specificity. Taken together, our results experimentally reveal the potential inhibition mechanism of ClC-2 inhibitor AK-42.

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Section: Introductionmentioning

confidence: 63%

Section: Introductionmentioning

confidence: 63%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cryo-EM structures of ClC-2 chloride channel reveal the blocking mechanism of its specific inhibitor AK-42

Ma,

Wang,

Chai

et al. 2023

Nat Commun

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“…For example, in voltage-gated Cl − channels CLC-0 and CLC-1, a Cl − ion could be responsible for opening a glutamate gate protruding the permeation pathway ( Pusch et al, 1995 ; Chen and Miller, 1996 ; Chen, 2003 ; Pusch, 1996 ; Rychkov et al, 2001 ). Furthermore, we showed that the interaction between the hyperpolarization-propelled intracellular Cl − and the pore gate triggers CLC-2 gating ( De Jesús-Pérez et al, 2016 , 2021 ; Sánchez-Rodríguez et al, 2010 , 2012 ). In TMEM16B, highly permeable anions facilitated the voltage-gated opening and increased the apparent Ca 2+ sensitivity ( Betto et al, 2014 ).…”

Section: Discussionmentioning

confidence: 96%

Gating and anion selectivity are reciprocally regulated in TMEM16A (ANO1)

Jesús-Pérez

López-Romero

Posadas

et al. 2022

Journal of General Physiology

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Numerous essential physiological processes depend on the TMEM16A-mediated Ca2+-activated chloride fluxes. Extensive structure–function studies have helped to elucidate the Ca2+ gating mechanism of TMEM16A, revealing a Ca2+-sensing element close to the anion pore that alters conduction. However, substrate selection and the substrate–gating relationship in TMEM16A remain less explored. Here, we study the gating–permeant anion relationship on mouse TMEM16A expressed in HEK 293 cells using electrophysiological recordings coupled with site-directed mutagenesis. We show that the apparent Ca2+ sensitivity of TMEM16A increased with highly permeant anions and SCN− mole fractions, likely by stabilizing bound Ca2+. Conversely, mutations at crucial gating elements, including the Ca2+-binding site 1, the transmembrane helix 6 (TM6), and the hydrophobic gate, impaired the anion permeability and selectivity of TMEM16A. Finally, we found that, unlike anion-selective wild-type channels, the voltage dependence of unselective TMEM16A mutant channels was less sensitive to SCN−. Therefore, our work identifies structural determinants of selectivity at the Ca2+ site, TM6, and hydrophobic gate and reveals a reciprocal regulation of gating and selectivity. We suggest that this regulation is essential to set ionic selectivity and the Ca2+ and voltage sensitivities in TMEM16A.

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“…On the other hand, there are substantial differences between CLC-2’s “slow” gating mode and CLC-0’s common gating in terms of kinetics and temperature dependence. Moreover, unlike CLC-0 (Lin et al, 1999), CLC-2 slow and fast gating cannot be separately affected by mutation (Zuniga et al, 2004; de Santiago et al, 2005; Yusef et al, 2006; De Jesus-Perez et al, 2021). Finally, in contrast to CLC-0, it has not been demonstrated that the “slow” gate in CLC-2 corresponds to a common gating process operating on both pores simultaneously.…”

Section: Discussionmentioning

confidence: 99%

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

Xu,

Neelands,

Powers

et al. 2023

Preprint

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CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different mammalian tissues and cell types. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating mechanisms among closely related CLC homologs has been a long-standing mystery, in part because few CLC channel structures are available, and those that exist exhibit high conformational similarity. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the potent and selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl--permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct apo conformations of CLC-2 involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl--permeation pathway from the intracellular side. This peptide is highly conserved among species variants of CLC-2 but is not present in any other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a ball-and-chain gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we show that loss of this short sequence increases the magnitude and decreases the rectification of CLC-2 currents expressed in mammalian cells. Furthermore, we show that with repetitive hyperpolarization WT CLC-2 currents increase in resemblance to the hairpin-deleted CLC-2 currents. These functional results combined with our structural data support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl--permeation pathway.

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Electro-steric opening of the clc-2 chloride channel gate

Cited by 10 publications

References 55 publications

Cryo-EM structures of ClC-2 chloride channel reveal the blocking mechanism of its specific inhibitor AK-42

Cryo-EM structures of ClC-2 chloride channel reveal the blocking mechanism of its specific inhibitor AK-42

Gating and anion selectivity are reciprocally regulated in TMEM16A (ANO1)

CryoEM structures of the human CLC-2 voltage gated chloride channel reveal a ball and chain gating mechanism

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