Mechanism of Anion Conduction in the Calcium-Activated Chloride Channel TMEM16A

Abstract

The calcium-activated chloride channel TMEM16A mediates selective anion conduction upon activation by intracellular calcium. Using the cryo-EM structure of mouse TMEM16A as a template, we have investigated the mechanism of anion conduction by patch-clamp electrophysiology. In the structure, an hourglass-shaped, protein-enclosed aqueous conduit populated by basic residues is found in each subunit in a homodimer. We analysed the conduction characteristics of alanine mutants of these basic residues and found a position-dependent effect on ion conduction that is manifested in the rectification of current-voltage relationships. This underlines their importance for the electrostatics in the narrow neck region of the pore creating a favourable environment for anion conduction. The compromised conduction characteristics of the cysteine mutant of a basic residue located on α-helix 5 at the intracellular end of the narrow neck can be reverted to the wild-type phenotype upon reaction with the small, positively charged MTSEA, indicating the intracellular accessibility of the site and reinforcing the role of the positive potential for anion conduction. The equivalent mutation of a residue located one helix turn towards the extracellular side is inaccessible, consistent with its location in the constricted part of the pore. When analysed in the context of a phenomenological model of ion permeation, it appears that the positively charged residues in the aqueous pore facilitate anion conduction by lowering local energy barrier(s) according to their specific position in the pore. Because the major energy barriers are located at the inner and outer ends of the pore, it is plausible that anions dehydrate as they enter the narrow conduit for conduction, a process that might be facilitated by the resident basic residues.