Topology Determination of the Transmembrane Domain of the Electrogenic Sodium Bicarbonate Cotransporter NBCe1‐A

NBCe1‐A electrogenically cotransports sodium and bicarbonate across the basolateral membrane of proximal tubule cells. The illusive transport mechanism of NBCe1‐A requires structural analysis of the transport protein. NBCe1 has been proposed to traverse the lipid bilayer at least 10 times based on in vitro glycosylation assay. To further determine the topology of NBCe1‐A in vivo , we performed cysteine scanning mutagenesis coupled with sulfhydryl specific chemistry. Fifty six cysteine residues were individually introduced throughout a modified NBCe1‐A protein with five cytoplasmic endogenous cysteines mutated to serine. Location of introduced cysteine residues were determined by whole cell labeling with two cysteine specific reagents: membrane permeant biotin maleimide (BM) and membrane impermeant MTS‐TAMRA. An introduced cysteine labeled with BM indicates its surface location, whereas lack of labeling indicates the residue resides in the lipid bilayer or in a BM inaccessible conformation. Introduced cysteines labeled with MTS‐TAMRA indicate they are exclusively located on the extracellular surface of the protein. Residues D449, A450, T451 and D452 in proposed extracellular loop 1, N503 in loop 2, E714, T718 and R722 in loop 4 were labeled both by BM and MTS‐TAMRA, demonstrating the first half of the transmembrane domain contains 8 transmembrane segments, highly homologous to AE1. The majority of introduced cysteines in the remaining transmembrane domain were not labeled by BM, suggesting that folding of this region is highly compacted unlike AE1. Based on these results, we propose a new topology model for the transmembrane domain of NBCe1‐A. Supported by NIH