A novel splicing variant of ATP1AL1, a human nongastric proton pump

Abstract

ATP1AL1 is a human nongastric H⁺,K⁺-ATPase. The physiological function of ATP1AL1 has not been clarified yet. Herein we have cloned a wild type and a novel splicing valiant deleting exon 4 of ATP1AL1 from human colorectum. Then, the stable cell lines expressing gastric H⁺,K⁺-ATPase β-subunit were transfected with the pcDNA4/His-ATP1AL1 cDNA (wild type or the variant) construct. Exon4 encodes amino acids 77-144 of ATP1AL1, and this region includes the M1 transmembrane domain and the extracellular loop between M1 and M2. Messenger RNA of the splicing variant deleting exon 4 of ATP1AL1 was expressed in human colorectum, kidney and brain. Protein expression of ATP1AL1 in the cell lines was monitored by using anti-Xpress antibody which reacts with the epitope at the N-terminal of the construct. Interestingly, the variant protein could be expressed in the membrane fraction of the cells. The enzyme activity of ATP1AL1 in the transfecting cells was estimated by subtracting 5 μM ouabain-sensitive activity from 1 mM ouabain-sensitive activity. The variant seems to be ouabain-insensitive, because exon 4 contains the region which is one of the important ouabain-binding sites of Na⁺,K⁺-ATPase. The enzyme activity of both wild type and the variant were sensitive to 100 μM SCH28080, an inhibitor of gastric H⁺,K⁺-ATPase. These results suggest that the splicing variant of ATP1AL1 may be functional in the human colorectum. [Jpn J Physiol 54 Suppl:S80 (2004)]