Li X, Alvarez B, Casey JR, Reithmeier RA, Fliegel L

Department of Biochemistry and Physiology, Canadian Institute of Health Research Membrane Protein Group, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.

We examined the ability of carbonic anhydrase II to bind to and affect the transport efficiency of the NHE1 isoform of the mammalian Na(+)/H(+) exchanger. The C-terminal region of NHE1 was expressed in Escherichia coli fused with an N-terminal glutathionine S-transferase or with a C-terminal polyhistidine tag. Using a microtiter plate binding assay we showed that the C-terminal region of NHE1 binds carbonic anhydrase II (CAII) and binding was stimulated by low pH and blocked by antibodies against the C-terminal of NHE1. The binding to NHE1 was confirmed by demonstrating protein-protein interaction using affinity blotting with CAII and immobilized NHE1 fusion proteins. CAII co-immunoprecipitated with NHE1 from CHO cells suggesting the proteins form a complex in vivo. In cells expressing CAII and NHE1, the H(+) transport rate was almost 2-fold greater than in cells expressing NHE1 alone. The CAII inhibitor acetazolamide significantly decreased the H(+) transport rate of NHE1 and transfection with a dominant negative CAII inhibited NHE1 activity. Phosphorylation of the C-terminal of NHE1 greatly increased the binding of CAII. Our study suggests that NHE1 transport efficiency is influenced by CAII, likely through a direct interaction at the C-terminal region. Regulation of NHE1 activity by phosphorylation could involve modulation of CAII binding.

Mesh Terms:
Animals, Binding Sites, Blotting, Western, CHO Cells, Carbonic Anhydrase II, Cricetinae, Dose-Response Relationship, Drug, Electrophoresis, Polyacrylamide Gel, Histidine, Hydrogen-Ion Concentration, Phosphorylation, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins, Sodium-Hydrogen Antiporter, Time Factors, Transfection

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