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.

Plasmodium falciparum-activated Chloride Channels Are Defective in Erythrocytes from Cystic Fibrosis Patients

“…Moreover, our results also indicate that CFTR is additionally required for the activation of a host cell-encoded inwardly rectifying chloride current by either cell shrinkage or P. falciparum infection. Because the inwardly rectifying conductance differs from the CFTR-chloride channel with respect to several major electrophysical, regulatory, and pharmacological properties (anion permeability sequence, rectifying behavior, sustained opening in the absence of ATP, and furosemide inhibition), our data collectively support a dual channel model in which CFTR and the inward rectifier constitute separate molecular identities but are tightly coupled functionally, analogous to the well established relationship between CFTR and the outwardly rectifying chloride channel (23). However, in view of recent data suggesting that ion selectivity and activation modes of CFTR in native tissues may (n ϭ 6).…”

Anion Channels, Including ClC-3, Are Required for Normal Neutrophil Oxidative Function, Phagocytosis, and Transendothelial Migration

“…ClC-3 has been proposed to be a cell-surface channel regulated by: 1) swelling (34), 2) calcium-calmodulin-dependent protein kinase II (35), or 3) the cystic fibrosis transmembrane regulator (36). Localization of ClC-3 protein to intracellular vesicles has supported an alternative hypothesis that ClC-3 is predominantly an intracellular anion channel, located in vesicular membranes, that regulates acidification of synaptic vesicles and lysosomes (37,38).…”

The ClC-3 Chloride Transport Protein Traffics through the Plasma Membrane via Interaction of an N-terminal Dileucine Cluster with Clathrin

“…ClC-3 has been shown to be involved in acidification of synaptic vesicles and endosomes (18 -20), but its deficiency is associated with lysosomal defects as well (21). It exists in several different isoforms that result from splicing variations (22,23), and the factors that control ClC protein localization are incompletely understood.…”