Background: The present experiments were designed to study the importance of the carboxy-terminus of colonic H(+), K(+)-ATPase alpha-subunit (HKalpha(2)), for both function as well as integrity of assembly with beta1-Na(+), K(+)-ATPase.
Methods: For this purpose, a mutation of 84 amino acids in the carboxy-terminus was created (DeltaHKalpha(2)) and HEK-293 cells were used as expression systems for functional studies using (86)Rb(+)-uptake, coimmunoprecipitation using specific antibodies and fluorescence microscopy using green fluorescent protein.
Results: The results demonstrate that comparable levels of expression of HKalpha(2) and DeltaHKalpha(2) mRNA were observed when cells were cotransfected with beta1 subunit. However, the abundance of expression of full length HKalpha(2) protein exceeded that of the truncated protein DeltaHKalpha(2). Ouabain-sensitive (86)Rb(+)-uptake was present only in cells cotransfected with HKalpha(2)/beta(1), indicating that the mutation was incapable of sustaining functionality. Coimmunoprecipitation experiments demonstrated that HKalpha(2) protein was immunoprecipitated more abundantly than DeltaHKalpha(2) when coexpressed with beta1. The use of sucrose gradients and green fluorescence protein immunofluorescence demonstrated that while the DeltaHKalpha(2)/beta(1) complex was confined to the endoplasmic reticulum, the HKalpha(2)/beta(1) complex translocated to the plasma membrane.
Conclusion: Taken together, our results are consistent with the view that the carboxy-terminus of HKalpha(2) facilitates the proper folding of the HKalpha(2)/beta(1) complex allowing translocation of the heterodimer to the plasma membrane where potassium uptake occurs. Otherwise, the alpha/beta complex is destined for degradation.