The carboxy terminus of the colonic H+,K+-ATPase α-subunit is required for stable β subunit assembly and function

The carboxy terminus of the colonic H+,K+-ATPase α-subunit is required for stable β subunit assembly and function.BackgroundThe present experiments were designed to study the importance of the carboxy-terminus of colonic H+,K+-ATPase α-subunit (HKα2), for both function as well as integrity of assembly with β1-Na+,K+-ATPase.MethodsFor this purpose, a mutation of 84 amino acids in the carboxy-terminus was created (ΔHKα2) and HEK-293 cells were used as expression systems for functional studies using 86Rb+-uptake, coimmunoprecipitation using specific antibodies and fluorescence microscopy using green fluorescent protein.ResultsThe results demonstrate that comparable levels of expression of HKα2 and ΔHKα2 mRNA were observed when cells were cotransfected with β1 subunit. However, the abundance of expression of full length HKα2 protein exceeded that of the truncated protein ΔHKα2. Ouabain-sensitive 86Rb+-uptake was present only in cells cotransfected with HKα2/β1, indicating that the mutation was incapable of sustaining functionality. Coimmunoprecipitation experiments demonstrated that HKα2 protein was immunoprecipitated more abundantly than ΔHKα2 when coexpressed with β1. The use of sucrose gradients and green fluorescence protein immunofluorescence demonstrated that while the ΔHKα2/β1 complex was confined to the endoplasmic reticulum, the HKα2/β1 complex translocated to the plasma membrane.ConclusionTaken together, our results are consistent with the view that the carboxy-terminus of HKα2 facilitates the proper folding of the HKα2/β1 complex allowing translocation of the heterodimer to the plasma membrane where potassium uptake occurs. Otherwise, the α/β complex is destined for degradation