Functional expression of the cdna encoded by the human atp1al1
gene.
Grishin, Alexander V., Mark O. Bevensee, Nikolai N. Modyanov, Vanathy
Rajendran, Walter F. Boron, Michael J. Caplan.
Department of Cellular and Molecular Physiology, Yale University
School of Medicine, New Haven, Connecticut 06520, Department of
Pharmacology, Medical College of Ohio, Toledo, Ohio 43699
APStracts 3:0088F, 1996.
The human ATP1AL1 gene encodes a protein which is expressed in brain,
kidney, and skin, and which is highly homologous to the recently
cloned non gastric isoforms of H,K-ATPase. We have generated
polyclonal antibodies against the protein encoded by ATP1AL1 and used
them to monitor the protein's expression and distribution in
transfection studies. The protein was retained in the ER when it was
transiently expressed alone in COS cells. In COS cells co-transfected
with ATP1AL1 plus gastric H,K-ATPase [beta]-subunit cDNAs, both
proteins reached the surface. Stably transfected lines of HEK-293
cells expressing both of these proteins demonstrate a 86Rb+-uptake
activity sensitive to both 2-methyl,8-(phenylmethoxy)imidazo(1,2
-a)pyridine 3-acetonitrile (SCH 28080) and ouabain (Ki's 131 and 42
[mu]M, respectively). Outward proton fluxes were measured in the same
cells as the spontaneous intracellular pH (pHi) recovery in cells
loaded with a pH-sensitive dye (BCECF) 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 which can be inhibited by 1 mM ouabain. Comparison
of the 86Rb+ influx and proton efflux, however, does not support
equal H+-Rb+ (K+) exchange mediated by this pump under the conditions
of pHi-monitoring experiments. Moreover, while the acid extrusion
activity mediated by the pump shows a marked pH dependence, the 86Rb+
-uptake activity present in the cells expressing the ATP1AL1-gH,K
[beta] complex can not be stimulated by acute lowering of
intracellular pH. These data suggest that the ATP1AL1 encoded protein
is the catalytic [alpha]-subunit of a human K+(Rb+)-dependent ATPase.
The possible implications of the discrepancy between 86Rb+ -uptake
and pHi -monitoring data are discussed.
Received 17 August 1995; accepted in final form 19 April 1996.
APS Manuscript Number F275-5.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 19 May 96