The K⁺-dependent ATPase gene family is divided into three subgroups including Na⁺,K⁺-ATPase, gastric H⁺,K⁺-ATPase and non-gastric H⁺,K⁺-ATPases. ATP1AL1 is a human nongastric H⁺,K⁺-ATPase, and its gene has been reported to be expressed in brain, skin and kidney (Grishin et al. 1994). However, the identification of physiological roles of ATP1AL1 is still in its infancy. Recently, we found that ATP1AL1 mRNA was overexpressed in 12 out of 20 human colorectal carcinomas compared with the level in the accompanying normal mucosa (Takahashi et al. 2002).

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 (HEK-293) 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. The specimens of colorectal adenocarcinomas and accompanying normal mucosa were obtained from surgical resection of patients in accordance with the recommendations of the Declaration of Helsinki. Informed consents were obtained from all patients at Toyama Medical and Pharmaceutical University Hospital. Data are shown as means ± S.E.M.

Messenger RNA of the splicing variant deleting the exon 4 of ATP1AL1 was expressed in both the colorectal adenocarcinomas and accompanying normal mucosa. The mRNA was also expressed in human 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-terminus of the construct. Interestingly, the variant protein could be expressed in the membrane fraction of the cells, although the level of expression was (0.28 ± 0.05)-fold that of wild-type protein (n = 3) in the same experimental condition. 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 enzyme activities of wild-type and the variant were 0.45 ± 0.09 and 0.04 ± 0.04 µmol Pi (mg protein)^-1 h^-1, respectively (n = 3). The variant seems to be ouabain-insensitive, because exon 4 contains the region which is one of the important major ouabain-binding sites of Na⁺,K⁺-ATPase.

These results suggest that the splicing variant of ATP1AL1 which is resistant to ouabain may be functional in the human colorectum, kidney and brain.