Carbonic anhydrase II binds to and enhances activity of the Na+/H+ exchanger.
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.
Li
Xiuju
X
Department of Biochemistry and Physiology, Canadian Institute of Health Research Membrane Protein Group, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Alvarez
Bernardo
B
Casey
Joseph R
JR
Reithmeier
Reinhart A F
RA
Fliegel
Larry
L
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D024402:Carbonic Anhydrase II / Q000737:chemistry* / Q000378:metabolism*; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D006639:Histidine; D024402:Carbonic Anhydrase II