ClC‐3B, a novel ClC‐3 splicing variant that interacts with EBP50 and facilitates expression of CFTR‐regulated ORCC

ABSTRACT We have cloned ClC‐3B, a novel alternative splicing variant of ClC‐3 (ClC‐3A) that is expressed predominantly in epithelial cells. ClC‐3B has a different, slightly longer C‐terminal end than ClC‐3A and contains a consensus motif for binding to the second PDZ (PSD95/Dlg/ZO‐1) domain of the epithelium‐specific scaffolding protein EBP50. Both in vitro and in vivo binding assays demonstrate interaction between ClC‐3B and EBP50. C127 mouse mammary epithelial cells transfected with ClC‐3B alone showed diffuse immunoreactivity for ClC‐3B in the cytoplasmic region. In contrast, when EBP50 was cotransfected with ClC‐3B, strong immunoreactivity for ClC‐3B appeared at the leading edges of membrane ruffles. Patch‐clamp experiments revealed that cotransfection of ClC‐3B and EBP50 resulted in a remarkable increase in outwardly rectifying Cl– channel (ORCC) activities at the leading edges of membrane ruffles in C127 cells. The electrophysiological properties of the ClC‐3B‐induced ORCCs are similar to those of ORCCs described in native epithelial cells. When cystic fibrosis transmembrane conductance regulator (CFTR) was cotransfected with ClC‐3B and EBP50, ClC‐3B‐dependent ORCCs were activated via the protein kinase A‐dependent pathway. These findings indicate that ClC‐3B is itself a CFTR‐regulated ORCC molecule or its activator.