Functional analysis of acid-activated Cl− channels: Properties and mechanisms of regulation

Abstract Cl − channels activated by acidic extracellular pH have been observed in various mammalian cells but their molecular identity and mechanisms of regulation are unknown. The aim of this study was to analyse the acid-activated Cl − current (I Cl(H) ) by elucidating its functional properties and mechanisms of regulation in three different cell types: primary human bronchial epithelial (HBE) cells, neuroblastoma SK-N-MC cells and HEK-293 cells. We found that outward rectification, sensitivity to acidic pH (50% activation at pH 5.15), permeability sequence (SCN −  > I −  > Br −  > Cl −  > gluconate), voltage dependence and sensitivity to blockers of I Cl(H) were identical in all cells. These findings suggest a common molecular basis for I Cl(H) . We analysed the possible relationship of I Cl(H) with members of ClC and TMEM16 protein families. By gene silencing, validated using RT-PCR, we found that I Cl(H) is unrelated to ClC-3, ClC-7, TMEM16A, TMEM16D, TMEM16F, TMEM16H and TMEM16K. Analysis of possible mechanisms of regulation indicate that Ca 2 + , ATP and phosphorylation by PKA or PKC do not seem to be implicated in channel activation. Instead, the inhibition of I Cl(H) by genistein and wortmannin suggest regulation by other kinases, possibly a tyrosine kinase and a phosphatidylinositol-3-kinase. Moreover, by using dynasore, the dynamin inhibitor, we found indications that exo/endocytosis is a mechanism responsible for I Cl(H) regulation. Our results provide the first evidence about acid-activated Cl − channel regulation and, thus, could open the way for a better understanding of the channel function and for the molecular identification of the underlying protein.