Coupling Modes and Stoichiometry of Cl−/HCO3− Exchange by slc26a3 and slc26a6

The SLC26 transporters are a family of mostly luminal Cl− and HCO3− transporters. The transport mechanism and the Cl−/HCO3− stoichiometry are not known for any member of the family. To address these questions, we simultaneously measured the HCO3− and Cl− fluxes and the current or membrane potential of slc26a3 and slc26a6 expressed in Xenopus laevis oocytes and the current of the transporters expressed in human embryonic kidney 293 cells. slc26a3 mediates a coupled 2Cl−/1HCO3− exchanger. The membrane potential modulated the apparent affinity for extracellular Cl− of Cl−/HCO3− exchange by slc26a3. Interestingly, the replacement of Cl− with NO3− or SCN− uncoupled the transport, with large NO3− and SCN− currents and low HCO3− transport. An apparent uncoupled current was also developed during the incubation of slc26a3-expressing oocytes in HCO3−-buffered Cl−-free media. These findings were used to develop a turnover cycle for Cl− and HCO3− transport by slc26a3. Cl− and HCO3− flux measurements revealed that slc26a6 mediates a 1Cl−/2HCO3− exchange. Accordingly, holding the membrane potential at 40 and −100 mV accelerated and inhibited, respectively, Cl−-mediated HCO3− influx, and holding the membrane potential at −100 mV increased HCO3−-mediated Cl− influx. These findings indicate that slc26a6 functions as a coupled 1Cl−/2HCO3− exchanger. The significance of isoform-specific Cl− and HCO3− transport stoichiometry by slc26a3 and slc26a6 is discussed in the context of diseases of epithelial Cl− absorption and HCO3− secretion

Intracellular Cl− as a signaling ion that potently regulates Na+/HCO3− transporters

Cl(-) is a major anion in mammalian cells involved in transport processes that determines the intracellular activity of many ions and plasma membrane potential. Surprisingly, a role of intracellular Cl(-) (Cl(-) in) as a signaling ion has not been previously evaluated. Here we report that Cl(-) in functions as a regulator of cellular Na(+) and HCO3 (-) concentrations and transepithelial transport through modulating the activity of several electrogenic Na(+)-HCO3 (-) transporters. We describe the molecular mechanism(s) of this regulation by physiological Cl(-) in concentrations highlighting the role of GXXXP motifs in Cl(-) sensing. Regulation of the ubiquitous Na(+)-HCO3(-) co-transport (NBC)e1-B is mediated by two GXXXP-containing sites; regulation of NBCe2-C is dependent on a single GXXXP motif; and regulation of NBCe1-A depends on a cryptic GXXXP motif. In the basal state NBCe1-B is inhibited by high Cl(-) in interacting at a low affinity GXXXP-containing site. IP3 receptor binding protein released with IP3 (IRBIT) activation of NBCe1-B unmasks a second high affinity Cl(-) in interacting GXXXP-dependent site. By contrast, NBCe2-C, which does not interact with IRBIT, has a single high affinity N-terminal GXXP-containing Cl(-) in interacting site. NBCe1-A is unaffected by Cl(-) in between 5 and 140 mM. However, deletion of NBCe1-A residues 29-41 unmasks a cryptic GXXXP-containing site homologous with the NBCe1-B low affinity site that is involved in inhibition of NBCe1-A by Cl(-) in. These findings reveal a cellular Cl(-) in sensing mechanism that plays an important role in the regulation of Na(+) and HCO3 (-) transport, with critical implications for the role of Cl(-) in cellular ion homeostasis and epithelial fluid and electrolyte secretion