A carboxy-terminus motif of HKalpha2 is necessary for assembly and function.
BACKGROUND
The present experiments were designed to study the importance of the carboxy-terminus of HKalpha2, for both function and integrity of assembly with beta1-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 HKalpha2 and the carboxy-terminus of alpha1-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 86Rb+ uptake and alpha/beta assembly using specific antibodies.


RESULTS
The results demonstrate that the entire carboxy-terminus of HKalpha2 is required for optimal protection of the alpha/beta complex from degradation and for functionality as evidenced by 86Rb+ uptake. The results also demonstrate that there was flexibility in the sequence of the carboxy-terminus. The last two tyrosines (Y1035Y1036) of HKalpha2 could be mutated to alanines and the carboxy-terminus of HKalpha2 could be replaced by the carboxy-terminus of alpha1-Na+,K+-ATPase while preserving transport activity.


CONCLUSIONS
The entire carboxy-terminus of HKalpha2 is required for stable assembly with beta1-Na+,K+-ATPase and functionality.
Codina
Juan
J
Sections of Nephrology and Molecular Medicine, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
Li
Jian
J
Dubose
Thomas D
TD
D000595:Amino Acid Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D000939:Epitopes / Q000235:genetics; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012414:Rubidium Radioisotopes; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection
C503551:ATP1B1 protein, human; D000939:Epitopes; D021122:Protein Subunits; D016271:Proto-Oncogene Proteins c-myc; D012414:Rubidium Radioisotopes; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase