M935K mutation impairs phosphorylation of threonines in the cytoplasmic tail of Kcc1. (A) Alignment of human and mouse KCC1 and KCC3 showing sites of conserved methionine in relation to known regulatory threonines. (B) ⁸⁶Rb influx in HEK293 cells expressing wild-type Kcc1 (WT) or various Kcc1 mutants in isotonic and hypotonic conditions. *P < .05 compared with WT; ^#P < .05 compared with M935K. (C) Western blot of anti-HA immunoprecipitates probed with anti-phosphothreonine (pThr) or anti-HA antibodies. D, dimer; M, monomer. (D) Western blot of anti-HA immunoprecipitates from HEK293 cells expressing wild-type or the homologous Kcc3 mutant M1000K demonstrating impaired phospho-threonine. (E) A schematic illustrating the regulation of KCC1 and the inhibitory effect of M935K mutation on nearby threonines leading to constitutive activation of K-Cl cotransport.