Previous studies from our laboratory have shown that the oligomeric structure of NBCe1‐A is predominantly a dimer. We also demonstrated that oligomerization of NBCe1‐A is absolutely dependent on S‐S bond formation. Whether NBCe1‐A monomers are functional is currently unknown. To address this question, we took advantage of our recent finding that the NBCe1‐AT442C mutant functions normally but can be completely inhibited by the MTS reagents MTSEA, MTSES, and MTSET. In contrast these agents have no effect of wt‐NBCe1‐A. The following concatameric constructs of wt‐NBCe1‐A and NBCe1‐AT442C were studied: wt‐NBCe1‐A‐linker‐wt‐NBCe1‐A; wt‐NBCe1‐A‐linker‐NBCe1‐AT442C; NBCe1‐AT442C‐linker‐wt‐NBCe1‐A; and NBCe1‐AT442C‐linker‐NBCe1‐AT442C. The length of the linker varied inversely with the baseline concatamer function, and concatamers with the shortest linker (Thr‐Gly) functioning optimally. Flux through the four concatamers was measured in HEK‐293T cells using BCECF and quantitating the cell buffer capacity. Non‐denaturing gel electrophoresis demonstrated that each of the four concatameric constructs is monomeric. The function of the four concatamers was similar to wt‐NBCe1‐A and NBCe1‐AT442C. In the presence of the MTS reagents MTSES and MTSET, the function of both mixed‐concatamers was decreased to 40–53% of normal. The function of the wt‐NBCe1‐A homo‐concatamer was unaffected, whereas both reagents blocked the function of the mutant NBCe1‐AT442C homo‐concatamer. Our results demonstrate that the NBCe1‐A monomer is functional.Supported by the NIH

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