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.
Highlights
The widely expressed two-pore homodimeric inward rectifier CLC-2 chloride channel regulates transepithelial chloride transport, extracellular chloride homeostasis, and neuronal excitability
We performed a functional analysis combined with site-directed mutagenesis of critical residues for gating located within the pore to determine the molecular entities involved in the opening of the pore gate in CLC-2, a C l− channel highly expressed throughout the central nervous system that controls chloride transport, extracellular chloride homeostasis, neuronal excitability, aldosterone secretion and heart rate[10,28,30]
We constructed CLC-2CLC-K based on bovine CLC-K structure (PDB: 5TQQ14), and CLC-2CLC-1 based on human CLC-1 structures (PDBs: 6COY, 6QVB, 6QV6, 6 QVU13,31) available later. mCLC-2 is 48.77% identical to CLC-K within the transmembrane domain (TMD; Fig. 1A)
Summary
The widely expressed two-pore homodimeric inward rectifier CLC-2 chloride channel regulates transepithelial chloride transport, extracellular chloride homeostasis, and neuronal excitability. Extracellular protonation cannot explain CLC-2 activation because gating happens under unfavourable protonation conditions[16,17] Given these observations, we proposed that hyperpolarization drives Cl− inside the pore causing E213 to open by electrostatic and steric repulsion[15,17], extracellular protonation of E213 residue stabilizes the open c onformation[16]. We performed a functional analysis combined with site-directed mutagenesis of critical residues for gating located within the pore to determine the molecular entities involved in the opening of the pore gate in CLC-2, a C l− channel highly expressed throughout the central nervous system that controls chloride transport, extracellular chloride homeostasis, neuronal excitability, aldosterone secretion and heart rate[10,28,30]
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