The M935K mutation increases KCC1 cotransporter activity. (A) ⁸⁶Rb efflux over time for red blood cells in isotonic conditions in the presence of oubain and bumetanide. (B) ⁸⁶Rb efflux assays demonstrating increased efflux from Kcc1^M935K/M935K red cells in isotonic (iso) but not hypotonic (hypo) conditions and is reversible by the phosphatase inhibitor, calyculin A (CalA). Mean ± SD representative of 2 independent experiments from 3 mice of each genotype. (C) Western blot (anti-HA) from HEK293 cells transfected with tetracycline-inducible vector expressing wild-type (WT) or M935K mutant hemagglutinin (HA)-tagged Kcc1. (D) ⁸⁶Rb influx over 2 minutes in HEK293 cells expressing WT or M935K Kcc1. Results are the mean ± SD of 3 independent experiments of cells grown in duplicate relative to the non–tetracycline-induced cells. (E) Size of HEK293 cells expressing WT or M935K Kcc1 measured by forward scatter. Results are the mean ± SD of 3 independent experiments of cells grown in tetracycline. (F) ⁸⁶Rb influx over 2 minutes in HEK293 cells expressing WT or M1000K HA-tagged Kcc3 in isotonic conditions. Results are the mean ± SD of 2 independent experiments of cells grown in duplicate. Student t test: *P < .05; **P < .01; ***P < .001.