H-K-ATPase in the RCCT-28A rabbit cortical collecting duct cell line

In the present study, we demonstrate that the rabbit cortical collecting duct cell line RCCT-28A possesses three distinct H-K-ATPase catalytic subunits (HK alpha). Intracellular measurements of RCCT-28A cells using the pH-sensitive dye 2',7' -bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) indicated that the mechanism accounting for recovery from an acid load exhibited both K(+) dependence and sensitivity to Sch-28080 characteristic of H-K-ATPases. Recovery rates were 0.022 +/- 0.005 pH units/ min in the presence of K(+), 0.004 +/- 0.002 in the absence of K(+), and 0.002 +/- 0.002 in the presence of Sch-28080. The mRNAs encoding the HK alpha(1) subunit and the H-K-ATPase beta-subunit (HK beta) were detected by RT-PCR. In addition, two HK alpha(2) species were found by RT-PCR and 5' rapid amplification of cDNA ends (5'-RACE) in the rabbit renal cortex. One was homologous to HK alpha(2) cDNAs generated from other species, and the second was novel. The latter, referred to as HK alpha(2c) encoded an apparent 61-residue amino-terminal extension that bore no homology to reported sequences. Antipeptide antibodies were designed on the basis of this extension, and these antibodies recognized a protein of the appropriate mass in both rabbit renal tissue samples and RCCT-28A cells. Such findings constitute very strong evidence for expression of the HK alpha(2c) subunit in vivo. The results suggest that the rabbit kidney and RCCT-28A cells express at least three distinct H-K-ATPases.