Cl? and H+ coupling properties and subcellular localizations of wildtype and disease-associated variants of the voltage-gated 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 of WT ClC-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 V-h of +40 mV with a [Cl-?](out) of 104 mm, 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 self-inhibitory 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.