Relief of autoinhibition of the electrogenic Na-HCO_3 cotransporter NBCel-B: role of IRBIT vs. amino-terminal truncation

摘要

Two maneuvers known to stimulate electrogenic sodium bicarbonate cotransporter 1 (NBCel) activity are 1) deletion from the cytosolic amino-terminus (Nt) of NBCel -C of an 87-amino acid sequence that contains an autoinhibitory domain (AID); and 2) binding of the protein IRBIT to elements within the same 87-amino acid module in a different variant, NBCel-B. Helpful to understanding the relationship between these two phenomena would be an appreciation of the relative magnitude of stimulation caused by each maneuver for the same NBCel variant. In the present study, we performed two-electrode voltage-clamp on Xenopus oocytes expressing human NBCel-B constructs, with and without human IRBIT constructs. We find that removal of the AID stimulates NBCel-B to the same extent as coexpression of wild-type IRBIT. The potency of wild-type IRBIT apparently is reduced by the action of endogenous oocyte protein phosphatases: a mutant IRBIT that cannot be influenced by the action of protein phosphatase-1 stimulates NBCel-B to an extent 50% greater than can be achieved by removal of the NBCel-B AID. Thus the stimulatory effect of IRBIT cannot be explained solely by masking of autoinhibitory determinants within the AID. Finally, we find that an NBCel-B construct that lacks amino acid residues 2-16 of the Nt is fully autoinhibited, but cannot be stimulated by IRBIT, indicating that autoinhibitory and IRBIT-binding determinants within the cytosolic Nt are not identical.