A carboxy-terminus motif of HKα2 is necessary for assembly and function

Background. The present experiments were designed to study the importance of the carboxy-terminus of HKalpha(2), for both function and integrity of assembly with beta(1)-Na+,K+-ATPase. Methods. For this purpose, stop codons were created, by polymerase chain reaction (PCR), at different positions in the carboxy-terminus of HKalpha2. Subsequently, chimeras between HKalpha(2) and the carboxy-terminus of alpha(1)-Na+,K+-ATPase or with the carboxy-terminus of the gastric H+,K+-ATPase were created. Human embryonic kidney HEK-293 cells were used as expression systems for functional studies using Rb-86(+) uptake and alpha/beta assembly using specific antibodies. Results. The results demonstrate that the entire carboxy-terminus of HKalpha(2) is required for optimal protection of the alpha/beta complex from degradation and for functionality as evidenced by Rb-86(+) uptake. The results also demonstrate that there was flexibility in the sequence of the carboxy-terminus. The last two tyrosines (Y1035Y1036) of HKalpha(2) could be mutated to alanines and the carboxy-terminus of HKalpha(2) could be replaced by the carboxy-terminus of alpha(1)- Na+,K+-ATPase while preserving transport activity. Conclusion. The entire carboxy-terminus of HKalpha(2) is required for stable assembly with beta(1)-Na+,K+-ATPase and functionality.