Scientific Reports (Jun 2021)

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

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

DOI
https://doi.org/10.1038/s41598-021-92247-3
Journal volume & issue
Vol. 11, no. 1
pp. 1 – 14

Abstract

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Abstract The widely expressed two-pore homodimeric inward rectifier CLC-2 chloride channel regulates transepithelial chloride transport, extracellular chloride homeostasis, and neuronal excitability. Each pore is independently gated at hyperpolarized voltages by a conserved pore glutamate. Presumably, exiting chloride ions push glutamate outwardly while external protonation stabilizes it. To understand the mechanism of mouse CLC-2 opening we used homology modelling-guided structure–function analysis. Structural modelling suggests that glutamate E213 interacts with tyrosine Y561 to close a pore. Accordingly, Y561A and E213D mutants are activated at less hyperpolarized voltages, re-opened at depolarized voltages, and fast and common gating components are reduced. The double mutant cycle analysis showed that E213 and Y561 are energetically coupled to alter CLC-2 gating. In agreement, the anomalous mole fraction behaviour of the voltage dependence, measured by the voltage to induce half-open probability, was strongly altered in these mutants. Finally, cytosolic acidification or high extracellular chloride concentration, conditions that have little or no effect on WT CLC-2, induced reopening of Y561 mutants at positive voltages presumably by the inward opening of E213. We concluded that the CLC-2 gate is formed by Y561-E213 and that outward permeant anions open the gate by electrostatic and steric interactions.