Characterization of CLCA protein expressed in ductal cells of rat salivary glands.
A molecular entity for Ca2+-dependent Cl- transport has not been well characterized in salivary cells. Here, we identify a rat CLCA homologue (rCLCA1) using a polymerase chain reaction (PCR)-based strategy. The full length of the isoform was 3.3 kb, and the predicted open reading frame encoded a 903-amino acid protein. Immunoblotting using a specific anti-rCLCA antibody recognizing near the amino-terminus showed the expression of N-glycosylated 120- and 86-kDa proteins in the membrane fraction of rCLCA1-transfected HEK293 cells. Reverse transcription-PCR results showed mRNA expressions in rat submandibular gland (SMG), ileum, and lung. Intense immunostaining was detected in the striated ducts, but not in the acinar cells, of SMG. Immunoblot for the membrane fraction of SMG revealed the existence of 137- and 90-kDa protein species. N-glycosidase F reduced the size of these bands toward those of the deglycosylated forms in the transfected HEK293 cells. A marked ionomycin-induced Cl- conductance was observed in the transfected cells. The current was Ca2+-dependent and sensitive to niflumic acid and DIDS. rCLCA1 proteins are probably responsible for modulation of Ca2+-dependent Cl- transport in salivary ductal cells, where the 137- and 90-kDa proteins may be modified posttranslationally in a manner similar to those in the heterologous expression system.
Yamazaki
Jun
J
Department of Physiological Science and Molecular Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan. junyama@college.fdcnet.ac.jp
Okamura
Kazuhiko
K
Ishibashi
Kazunari
K
Kitamura
Kenji
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D006367:HeLa Cells; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012469:Salivary Glands / Q000166:cytology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D018118:Chloride Channels; C504023:Clca5 protein, rat; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins