Probing coordinated gating of inner and outer gates in a CLC antiporter through mutational and structural analyses

초록

CLC family proteins regulate chloride (Cl⁻) homeostasis across cellular membranes and are classified into two functional classes: Cl⁻ channels and Cl⁻/H⁺ antiporters. While channels mediate passive Cl⁻ transport, antiporters stoichiometrically exchange two Cl⁻ for one H⁺. In both cases, ion transport is regulated by inner and outer gates, which respond to stimuli such as voltage, pH, and permeant ions. Structural and functional studies have identified a key glutamate residue (Glugate or Gluex) acting as the outer gate, which undergoes multiple conformational changes during pH- and voltage-dependent gating in both CLC channels and antiporters. In contrast, the inner gate, formed by two hydroxyl-bearing residues, conserved serine and tyrosine at central positions (Sercen and Tyrcen), has been considered rather a static kinetic barrier. However, this kinetic barrier model appears inefficient to achieve 2:1 Cl⁻/H⁺ coupling, compared to the classical alternating access mechanism of other coupled transporters. To test whether the inner gate actively gates and coordinates with the outer gate, we introduced mutations at pore-lining residues and examined their effects on ion transport rates, chloride-binding affinity, and structure. In this conference, I will present the functional and structural impact of two key mutations: one stabilizes the outer gate while destabilizing the inner gate, and the other selectively stabilizes the inner gate. These findings support a model in which the CLC antiporter exchanges Cl⁻ and H⁺ through a semi-alternating access mechanism involving coordinated gating of the inner and outer gates.