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). 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 HK₁ subunit and the H-K-ATPase -subunit (HK) were detected by RT-PCR. In addition, two HK₂ species were found by RT-PCR and 5' rapid amplification of cDNA ends (5'-RACE) in the rabbit renal cortex. One was homologous to HK₂ cDNAs generated from other species, and the second was novel. The latter, referred to as HK_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_2c subunit in vivo. The results suggest that the rabbit kidney and RCCT-28A cells express at least three distinct H-K-ATPases.

renal cell line; potassium homeostasis; acid-base balance; alternative splicing

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