The effect of beta-subunit assembly on function and localization of the colonic H+,K+-ATPase alpha-subunit.
BACKGROUND
Previous experiments from our laboratory have demonstrated that HKalpha(2) coimmunoprecipitated with beta(1)-Na(+),K(+)-ATPase. Although HKalpha(2) is expressed abundantly in the apical membrane of distal colon, the demonstration that beta(1) 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 HKalpha(2) and a monoclonal antibody against beta(1).


RESULTS
The results demonstrate that HKalpha(2) localizes to the apical membrane. Two pools of beta(1)-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 beta(1) localizes to the apical membrane with HKalpha(2), and supports the view that beta(1) is the physiologic beta-subunit for HKalpha(2). We tested, therefore, the efficiency of the two beta-subunits expressed in distal colon (beta(1) and beta(3)) to support the activity of HKalpha(2). Human embryonic kidney HEK-293 cells were transiently cotransfected with HKalpha(2) plus beta(1) or HKalpha(2) plus beta(3). Subsequently, (86)Rb(+)-uptake and plasma membrane localization were evaluated. The results demonstrate that both HKalpha(2)/beta(1) and HKalpha(2)/beta(3) support (86)Rb(+)-uptake. However, (86)Rb(+)-uptake measured in the cells cotransfected with HKalpha(2) plus beta(1) exceeded that measured in cells expressing HKalpha(2)/beta(3). Fluorescence microscopy using enhanced green fluorescent protein cloned at the amino-terminus of HKalpha(2) demonstrated protein migration to the plasma membrane in cells cotransfected with EGFP-HKalpha(2) plus beta(1). In contrast, in cells cotransfected with EGFP-HKalpha(2) plus beta(3), the vast majority of the protein remained confined to intracellular compartments. The significantly higher (86)Rb(+)-uptake corresponded to additional localization of HKalpha(2) to the plasma membrane when coexpressed with beta(1) compared to beta(3).


CONCLUSIONS
Taken together, these and previous results from our laboratory indicate that beta(1)-Na(+),K(+)-ATPase is likely to represent the most physiologic and efficient subunit for HKalpha(2) assembly in distal colon.
Li
Jian
J
Department of Internal Medicine, Wake Forest University Medical School, Winston-Salem, North Carolina 27157, USA.
Codina
Juan
J
Petroske
Elizabeth
E
Werle
Mike J
MJ
Willingham
Mark C
MC
DuBose
Thomas D
TD
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D016764:Cell Polarity / Q000502:physiology; D003106:Colon / Q000166:cytology / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007679:Kidney Medulla / Q000166:cytology / Q000201:enzymology; D008856:Microscopy, Fluorescence; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012414:Rubidium Radioisotopes; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
C503551:ATP1B1 protein, human; C503552:Atp1b1 protein, mouse; C503554:Atp1b1 protein, rat; D021122:Protein Subunits; D012414:Rubidium Radioisotopes; C498531:ATP1A2 protein, human; C498532:Atp1a2 protein, rat; D000254:Sodium-Potassium-Exchanging ATPase