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 (the gating glutamate, 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 a static kinetic barrier. However, this kinetic barrier model appears inefficient to achieve 2:1 Cl⁻/H⁺ coupling, compared to the classical “alternating access model” used to describe conventional transporter mechanisms. 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. We identified two critical residues through mutational screening: one stabilizes the outer gate while destabilizing the inner gate, and the other selectively stabilizes the inner gate. These findings support a model wherein the CLC antiporter mediates Cl⁻/H⁺ exchange via a semi-alternating access mechanism involving coordinated gating of the inner and outer gates.