TM1 Residues Face the Ion Translocation Pathway of kNBC1 (NBCe1‐A)

Abstract

All members of the SLC4 transporter family that have been shown to mediate bicarbonate (carbonate) transport have an identical 8-residue motif in the junction of extracellular loop 1 and transmembrane segment 1 (TM1). A spontaneous S427L mutation in TM1 of NBC1 (NBCe1), encoded by SLC4A4, impairs the function of the cotransporter resulting in proximal renal tubular acidosis and ocular abnormalities. Whether residues in TM1 are part of the ion conducting pathway is unknown and was addressed in the present study. We utilized SCAM methodology (substituted cysteine accessibility method) and subsequent exposure to MTS (methanethiosulfonate) reagents. Each residue in TM1 was individually replaced with cysteine on the wt-kNBC1 (NBCe1-A) backbone. The flux through the cotransporter was measured using BCECF to determine intracellular pH changes and intracellular buffer capacity in HEK293 cells expressing each mutant. The flux through S427C, T438C, F443C, G445C, and G448C mutants was decreased by ~50%. The flux mediated by a T442C mutant (located within the 8-residue consensus motif) was normal, and was blocked by MTSES, MTSET or MTSEA. L426C, S427C, L430C, and L434C flux was stimulated 10-25% by MTSEA. Several mutants (N439C, I441C, G444C, L446C, and L447C) were not expressed normally on the plasma membrane suggesting that these residues are required for targeting/protein folding. Our results indicate that residues within TM1 line the ion translocation pathway of NBC1. Supported by the NIH.