The ClC‐5 N‐terminus confers plasma membrane Cl−/H+ transport capacity to ClC‐3

Abstract

ClC family Cl−/H+ exchangers are primarily endosomal proteins that support vesicle acidification and reactive O2 production. When expressed in HEK293 cells, WT ClC‐5 localizes to the plasma membrane and produces outwardly rectifying whole‐cell ion currents, whereas WT ClC‐3 localizes intracellularly and ion currents are absent. We utilized N‐terminus mutations and ClC‐5/3 chimeras in combination with confocal imaging and whole‐cell patch clamp recordings, to show that this region regulates ClC‐5 and ‐3 trafficking and functional ion currents in HEK cells. Disruption of an N‐terminal clathrin‐ binding motif (LLDLLD to LLALLA) localized ClC‐3 to the plasma membrane, but yielded no ion current. By contrast, replacing the ClC‐3 N‐terminus (M1‐ E59) with that of ClC‐5 (M1‐A46) resulted in both plasma membrane localization of “N‐ClC‐5/3”, and rapidly activated, strongly rectifying (ClC‐5‐like) ion currents. Cellular pH changes were quantified with BCECF fluorescence and revealed coupled Cl−/H+ transport during both ClC‐5‐ and N‐ClC‐5/3 currents. These findings directly demonstrate for the first time that ClC‐3 is a functional Cl−/H+ exchanger, and support the hypothesis that the N‐terminus of these proteins directs membrane localization and modulates ion current gating.