Cl^- and H⁺ coupling properties and subcellular localizations of wildtype and disease-associated variants of the voltagegated Cl^-/H⁺ exchanger ClC-5

Dent disease 1 (DD1) is caused by mutations in the CLCN5 gene encoding a voltage-gated electrogenic nCl^-/H⁺ exchanger ClC-5. Using ion-selective microelectrodes and Xenopus oocytes, here we studied Cl^-/H⁺ coupling properties ofWTClC-5 and four DD1-associated variants (S244L, R345W, Q629*, and T657S), along with trafficking and localization of ClC-5. WT ClC-5 had a 2Cl^-/H⁺ exchange ratio at a Vh of +40 mVwith a [Cl^-]_out of 104mM, but the transport direction did not reverse with a [Cl^-]_out of 5 mM, indicating that ClC-5-mediated exchange of two Cl^- out for one H⁺ in is not permissible. We hypothesized that ClC-5 and H⁺-ATPase are functionally coupled during H⁺-ATPase-mediated endosomal acidification, crucial for ClC-5 activation by depolarizing endosomes. ClC-5 transport that provides three net negative charges appeared selfinhibitory because of ClC-5's voltage-gated properties, but shunt conductance facilitated further H⁺-ATPase-mediated endosomal acidification. Thus, an on-and-off "burst" of ClC-5 activity was crucial for preventing Cl^- exit from endosomes. The subcellular distribution of the ClC-5:S244L variant was comparable with that of WT ClC-5, but the variant had a much slower Cl^- and H⁺ transport and displayed an altered stoichiometry of 1.6:1. The ClC-5:R345W variant exhibited slightly higher Cl^-/H⁺ transport than ClC-5:S244L, but co-localized with early endosomes, suggesting decreased ClC-5:R345W membrane trafficking is perhaps in a fully functional form. The truncated ClC-5:Q629∗ variant displayed the lowest Cl^-/H⁺ exchange and was retained in the endoplasmic reticulum and cis-Golgi, but not in early endosomes, suggesting the nonsense mutation affects ClC-5 maturation and trafficking.