Functional reassembly of NBCe1‐A from co‐expressed cytosolic and transmembrane domains

Abstract

The renal electrogenic sodium bicarbonate cotransporter NBCe1‐A is composed of three major domains: a ~45 kDa cytosolic amino‐terminal domain (Nt), a ~60 kDa transmembrane domain (TMD), and a ~10 kDa cytosolic carboxy‐terminal domain (Ct). Expressed in isolation in Xenopus oocytes, neither the Nt nor an NBCe1 construct that lacks the Nt (TMD/Ct) mediates detectable electrogenic NBC activity. However, oocytes co‐expressing Nt and TMD/Ct from two separate cRNAs exhibit substantial NBCe1 activity. Furthermore, in oocytes that are expressing TMD/Ct from cRNA, NBCe1 activity can be elicited by injection of recombinant Nt protein. The reconstitution of NBCe1 activity by Nt and TMD/Ct does not require the NBCe1‐Ct, as evidenced by reconstitution of NBCe1‐like activity in oocytes that are co‐expressing Nt and the chimeric TMD/Ct construct “NBCe1‐TMD/AE1‐Ct” in which the Ct of NBCe1 is replaced by the Ct of AE1. These data are consistent with the hypotheses that the Nt and the intracellular face of the TMD of NBCe1 contain reciprocal binding sites and that binding of the Nt to the TMD is required to produce functional NBCe1. Control of Nt occupancy at its binding site on the TMD could represent a novel means by which NBCe1 activity, and thereby HCO3− reabsorption, is regulated.