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

Abstract

In the present study, we demonstrate that the rabbit cortical collecting duct cell line RCCT-28A possesses three distinct H-K-ATPase catalytic subunits (HKα). 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 HKα1 subunit and the H-K-ATPase β-subunit (HKβ) were detected by RT-PCR. In addition, two HKα2 species were found by RT-PCR and 5′ rapid amplification of cDNA ends (5′-RACE) in the rabbit renal cortex. One was homologous to HKα2 cDNAs generated from other species, and the second was novel. The latter, referred to as HKα2c, 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 HKα2c subunit in vivo. The results suggest that the rabbit kidney and RCCT-28A cells express at least three distinct H-K-ATPases.