The isoform-specific region of the Na,K-ATpase catalytic subunit: Role in enzyme kinetics and regulation by protein kinase C

Marie Josée Duran, Sandrine V. Pierre, Deborah L. Carr, Thomas A. Pressley

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

Comparisons of the primary structures of the Na,K-ATPase α-isoforms reveal the existence of regions of structural divergence, suggesting that they are involved in unique functions. One of these regions is the isoform-specific region (ISR), located near the ATP binding site in the major cytoplasmic loop. To evaluate its importance, we constructed mutants of the rodent wild-type α1 and α3 isoforms in which the ISR was replaced with irrelevant sequences, i.e., the analogous region from the rat gastric H,K-ATPase catalytic subunit or a region from the human c-myc oncogene. Opossum kidney (OK) cells were transfected with wild-type rat α1, α3, or their corresponding chimeras and selected in ouabain. Introduction of either mutant produced ouabain-resistant colonies, consistent with functional expression of the chimeric protein and indicating that the ISR is not essential for overall Na,K-ATPase function. The introduced chimeras were then characterized enzymatically by measuring the relative rate of K+ and Li+ deocclusions. Results showed that exchanges of both α1 and α3 ISRs significantly modified the sensitivity for the enzyme to either K+ or Li+. Subsequent treatment of the cells with phorbol esters revealed an altered Na,K-ATPase transport in response to protein kinase C activation for the α1 chimeras. No changes were observed for the α3 isoform, suggesting that it is not sensitive to PKC regulation. These results demonstrated that the ISR plays an important role in ion deocclusion and in the response to PKC (only for the α1 isoform).

Original languageEnglish
Pages (from-to)16174-16183
Number of pages10
JournalBiochemistry
Volume43
Issue number51
DOIs
StatePublished - Dec 28 2004

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