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- cotransport (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.