ClC-3B, a novel ClC-3 splicing variant that interacts with EBP50 and facilitates expression of CFTR-regulated ORCC.
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.
Ogura
Takehiko
T
Department of Pharmacology, Chiba University Graduate School of Medicine, Chiba, Japan.
Furukawa
Tetsushi
T
Toyozaki
Tetsuya
T
Yamada
Katsuya
K
Zheng
Ya-Juan
YJ
Katayama
Yoshifumi
Y
Nakaya
Haruaki
H
Inagaki
Nobuya
N
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D016475:3T3 Cells; D017398:Alternative Splicing; D000818:Animals; D016466:CHO Cells; D000001:Calcimycin / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D014162:Transfection
C505032:CFTR protein, human; D002352:Carrier Proteins; D018118:Chloride Channels; C115376:ClC-3 channel; D007476:Ionophores; D008565:Membrane Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000001:Calcimycin; D015056:1-Methyl-3-isobutylxanthine