FUNCTIONAL EXPRESSION OF THE CDNA ENCODED BY THE HUMAN ATP1AL1 GENE

The human ATP1AL1 gene encodes a protein expressed in brain, kidney, a nd skin and that is highly homologous to the recently cloned nongastri c isoforms of H-K-adenosinetriphosphatase (H-K-ATPase). We have genera ted polyclonal antibodies against the protein encoded by ATP1AL1 and u sed them to monitor the protein's expression and distribution in trans fection studies. The protein was retained in the endplasmic reticulum when it was transiently expressed alone in COS cells. In COS cells cot ransfected with ATP1AL1 plus gastric H-K-ATPase beta-subunit cDNAs (AT P1AL1-gH-K beta), both proteins reached the surface. Stably transfecte d lines of HEK 293 cells expressing both of these proteins demonstrate a Rb-86(+) uptake activity sensitive to both 2-methyl,8-(phenylmethox y)imidazo(1,2-a)pyridine 3-acetonitrile (SCH-28080) and ouabain (inhib itory constants of similar to 131 and 42 mu M, respectively). Outward proton fluxes were measured in the same cells as the spontaneous intra cellular pH (pH(i)) recovery in cells loaded with a pH-sensitive dye [ 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] and subjected to acid loading through an NH4Cl pulse. The cells expressing both the ATP1AL1 -encoded protein and the gastric H-K-ATPase beta-subunit possess a net acid extrusion activity that can be inhibited by 1 mM ouabain. Compar ison of the Rb-86(+) influx and proton efflux, however, does not suppo rt equal H+/Rb+ exchange mediated by this pump under the conditions of pH(i)-monitoring experiments. Moreover, whereas the acid extrusion ac tivity mediated by the pump shows a marked pH dependence, the Rb-86(+) uptake activity present in the cells expressing the ATP1AL1-gH-K beta complex cannot be stimulated by acute lowering of pH(i). These data s uggest that the ATP1AL1-encoded protein is the catalytic alpha-subunit of a human K+-dependent ATPase. The possible implications of the disc repancy between Rb-86(+) uptake and pH(i) monitoring data are discusse d.