Ion Channels – Membrane Transport – Integrative Physiology
Kidney International (2004) 65, 1301–1310; doi:10.1111/j.1523-1755.2004.00507.x
The carboxy terminus of the colonic H+,K+-ATPase 
-subunit is required for stable 
subunit assembly and function
JIAN LI, JUAN CODINA, ELIZABETH PETROSKE, MIKE J WERLE and THOMAS D DUBOSE JR.
Department of Internal Medicine, Section on Nephrology, Wake Forest University School of Medicine, Winston-Salem, North Carolina; Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas
Correspondence: Thomas D. DuBose, Jr., M.D., Department of Internal Medicine, Wake Forest University School of Medicine Medical Center Blvd., Winston-Salem, NC 27157. E-mail: tdubose@wfubmc.edu
Received 8 September 2003; Revised 27 October 2003; Accepted 11 November 2003.
Abstract
The carboxy terminus of the colonic H+,K+-ATPase 
-subunit is required for stable 
subunit assembly and function.
Background
The present experiments were designed to study the importance of the carboxy-terminus of colonic H+,K+-ATPase 
-subunit (HK2), for both function as well as integrity of assembly with 
1-Na+,K+-ATPase.
Methods
For this purpose, a mutation of 84 amino acids in the carboxy-terminus was created (
HK2) 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.
Results
The results demonstrate that comparable levels of expression of HK2 and HK2 mRNA were observed when cells were cotransfected with 1 subunit. However, the abundance of expression of full length HK2 protein exceeded that of the truncated protein HK2. Ouabain-sensitive 86Rb+-uptake was present only in cells cotransfected with HK2/1, indicating that the mutation was incapable of sustaining functionality. Coimmunoprecipitation experiments demonstrated that HK2 protein was immunoprecipitated more abundantly than HK2 when coexpressed with 1. The use of sucrose gradients and green fluorescence protein immunofluorescence demonstrated that while the HK2/1 complex was confined to the endoplasmic reticulum, the HK2/1 complex translocated to the plasma membrane.
Conclusion
Taken together, our results are consistent with the view that the carboxy-terminus of HK2 facilitates the proper folding of the HK2/1 complex allowing translocation of the heterodimer to the plasma membrane where potassium uptake occurs. Otherwise, the / complex is destined for degradation.
Keywords:
colonic H+,K+-ATPase, carboxy terminus, potassium transport, urinary acidification, Na+,K+-ATPase
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