Physiological function and localization of a novel rCLCA molecule related to Cl-flux in salivary tissues

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Epithelial Cl^- transport is known to be important for saliva production although a molecular entity for Ca^<2+>-dependent C1^- transport has not been well characterized. We identified a new member of the CLCA family, rCLCA, which is strongly expressed in the submandibular gland (SMG). The predicted ORF encodes a 903-a.a. protein containing an N-terminal region located extracellularly. Transient and stable transfection of HEK293 cells with rCLCA cDNA resulted in expression of 120^- and 86-kDa proteins in the membrane fraction. The latter appeared to be a proteolytic product of the former. Streptavidin recognized the 86-kDa protein immunoprecipitated from surface-biotinylated cells using the anti-rCLCA antibody, suggesting that the proteolytic product locates on the plasma membrane. The transfection with rCLCA revealed a marked niflumic acid-sensitive Ca^<2+>-dependent Cl^- conductance evoked by ionomycin with an outward rectification in the I-V relationship. This molecule was also found to be expressed in early and recycling endosomes labeled by their specific markers. RT-PCR revealed expression of rCLCA in the stomach, lung and liver as well as the SMG. Intense immunostaining was detected in the striated ducts of SMG, but not in the acinar cells. Immunoblot of the membrane fraction yielded N-glycan-linked 120-130- and 90-kDa bands. To address the question as to whether rCLCA is involved in Cl^- reabsorption in rat SMG during muscarinic stimulation, we evaluated the electrolyte content of saliva from the glands pretreated with rCLCA siRNA. Retrograde injection into a given submandibular duct with the siRNA significantly increased Cl^- concentration in saliva collected after administration of pilocarpine. These results represent a physiological significance of rCLCA in transepithelial Ca^<2+>-dependent Cl^- transport in the ductal system of rat SMG.