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.