Relief of autoinhibition of the electrogenic Na-HCO(3) [corrected] cotransporter NBCe1-B: role of IRBIT vs.amino-terminal truncation.
Two maneuvers known to stimulate electrogenic sodium bicarbonate cotransporter 1 (NBCe1) activity are 1) deletion from the cytosolic amino-terminus (Nt) of NBCe1-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, NBCe1-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 NBCe1 variant. In the present study, we performed two-electrode voltage-clamp on Xenopus oocytes expressing human NBCe1-B constructs, with and without human IRBIT constructs. We find that removal of the AID stimulates NBCe1-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 NBCe1-B to an extent 50% greater than can be achieved by removal of the NBCe1-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 NBCe1-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.
Dept. of Physiology and Biophysics, Case Western Reserve Univ. School of Medicine, Cleveland, OH 44106-4970, USA.
Seong-Ki
SK
Lee
Walter F
WF
Boron
Mark D
MD
Parker
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes; D018408:Patch-Clamp Techniques; D054645:Protein Phosphatase 1 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D027982:Sodium-Bicarbonate Symporters / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
C470184:CLECL1 protein, human; D037181:Lectins, C-Type; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; D049452:Green Fluorescent Proteins; D054645:Protein Phosphatase 1