A Carboxy‐Terminal Domain in KCC2 Confers Constitutive K + ‐Cl − Cotransport

Abstract

The neuronal K+-Cl− cotransporter KCC2 plays a crucial role in determining intracellular chloride activity and thus the neuronal response to GABA. Of the four KCCs, KCC2 is unique in mediating constitutive K+-Cl− cotransport under isotonic conditions; the other three KCCs are exclusively swelling-activated. Two generations of chimeric KCC4-KCC2 cDNAs initially localized this characteristic to within a KCC2-specific C-terminal expansion, encompassing two predicted PEST domains. Substitution of this region in KCC2 with KCC4 resulted in a chimeric KCC that was devoid of isotonic activity, with intact swelling-activated transport. Further chimeras demonstrated that a domain just distal to the PEST domains confers isotonic transport; sequential mutagenesis of KCC4 revealed that residues 1021-1035 of KCC2 are sufficient. Swelling-activated K+-Cl− cotransport is abrogated by calyculin A, whereas isotonic transport mediated by KCC chimeras and KCC2 is completely resistant to this serine-threonine phosphatase inhibitor. In summary, a 15-residue C-terminal domain in KCC2 is both necessary and sufficient for constitutive K+-Cl− cotransport. This is to our knowledge the first identification of a discrete cytoplasmic domain that modulates the volume sensitivity of a given class of ion transporter. Furthermore, unlike swelling-activated transport, constitutive K+-Cl− cotransport mediated by KCC2 is completely independent of serine-threonine phosphatase activity, suggesting that these two modes of transport are activated by distinct mechanisms.