K-Cl cotransport in red blood cells from patients with KCC3 isoform mutants

Red blood cells (RBCs) possess the K-Cl cotransport (KCC) isoforms 1, 3, and 4. Mutations within a given isoform may affect overall KCC activity. In a double-blind study, we analyzed, with Rb as a K congener, K fluxes (total flux, ouabain-sensitive Na+/K+ pump, and bumetanide-sensitive Na-K-2Cl cotransport, Cl-dependent, and ouabain- and bumetanide-insensitive KCC with or without stimulation by N-ethylmaleimide (NEM) and staurosporine or Mg removal, and basal channel-mediated fluxes, osmotic fragility, and ions and water in the RBCs of 8 controls, and of 8 patients with hereditary motor and sensory neuropathy with agenesis of corpus callosum (HMSN-ACC) with defined KCC3 mutations (813FsX813 and Phe529FsX532) involving the truncations of 338 and 619 C-terminal amino acids, respectively. Water and ion content and, with one exception, mean osmotic fragility, as well as K fluxes without stimulating agents, were similar in controls and HMSN-ACC RBCs. However, the NEM-stimulated KCC was reduced 5-fold (p < 0.0005) in HMSN-ACC vs control RBCs, as a result of a lower Vmax (p < 0.05) rather than a lower Km (p = 0.109), accompanied by corresponding differences in Cl activation. Low intracellular Mg activated KCC in 6 out of 7 controls vs 1 out of 6 HMSN-ACC RBCs, suggesting that regulation is compromised. The lack of differences in staurosporine-activated KCC indicates different action mechanisms. Thus, in HMSN-ACC patients with KCC3 mutants, RBC KCC activity, although indistinguishable from that of the control group, responded differently to biochemical stressors, such as thiol alkylation or Mg removal, thereby indirectly indicating an important contribution of KCC3 to overall KCC function and regulation.