The effect of β-subunit assembly on function and localization of the colonic H+,K+-ATPase α-subunit

Abstract

Background. Previous experiments from our laboratory have demonstrated that HKα2 coimmunoprecipitated with β1-Na +,K+-ATPase. Although HKα2 is expressed abundantly in the apical membrane of distal colon, the demonstration that β1 localizes to this same membrane in distal colon has not been demonstrated previously. Methods. Immunolocalization was performed in distal colon using a polyclonal antibody against HKα2 and a monoclonal antibody against β1. Results. The results demonstrate that HKα2 localizes to the apical membrane. Two pools of β1-Na+,K+-ATPase were detected. The first localized to the apical membrane. The second pool was detected in the basolateral membrane when distal colon sections were deglycosylated with glycosidase F. Therefore, our results demonstrate that β1 localizes to the apical membrane with HKα2, and supports the view that β1 is the physiologic β-subunit for HKα2. We tested, therefore, the efficiency of the two β-subunits expressed in distal colon (β1 and β3) to support the activity of HKα2. Human embryonic kidney HEK-293 cells were transiently cotransfected with HKα2 plus β1 or HKα2 plus β3. Subsequently, 86Rb+-uptake and plasma membrane localization were evaluated. The results demonstrate that both HKα2/β1 and HKα2/ β3 support 86Rb+-uptake. However, 86Rb+-uptake measured in the cells cotransfected with HKα2 plus β1 exceeded that measured in cells expressing HKα2/β3. Fluorescence microscopy using enhanced green fluorescent protein cloned at the amino-terminus of HKα2 demonstrated protein migration to the plasma membrane in cells cotransfected with EGFP-HKα2 plus β1. In contrast, in cells cotransfected with EGFP-HKα2 plus β3, the vast majority of the protein remained confined to intracellular compartments. The significantly higher 86Rb +-uptake corresponded to additional localization of HKα2 to the plasma membrane when coexpressed with β1 compared to β3. Conclusion. Taken together, these and previous results from our laboratory indicate that β1-Na+,K+-ATPase is likely to represent the most physiologic and efficient subunit for HKα2 assembly in distal colon.