Characterization of Glial Cell K-Cl Cotransport

Background: The molecular mechanism of K-Cl cotransport (KCC) consists of at least 4 isoforms, KCC 1, 2, 3, and 4 which, in multiple combinations, exist in most cells, including erythrocytes and neuronal cells. Methods: We utilized reverse-transcriptase-polymerase chain reaction (RT-PCR) and ion flux studies to characterize KCC activity in an immortalized in vitro cell model for fibrous astrocytes, the rat C6 glioblastoma cell. Isoform-specific sets of oligonucleotide primers were synthesized for NKCC1, KCC1, KCC2, KCC3, KCC4, and also for NKCC1 and actin. K-Cl cotransport activity was determined by measuring either the furosemide-sensitive, or the Cl ^- -dependent bumetanide-insensitive Rb ⁺ (a K ⁺ congener) influx in the presence of the Na/K pump inhibitor ouabain. Rb ⁺ influx was measured at a fixed external Cl concentrations, [Cl ^- ] _e , as a function of varying external Rb concentrations, [Rb ⁺ ] _e , and at a fixed [Rb ⁺ ] _e as a function of varying [Cl ^- ] _e , and with equimolar Cl replacement by anions of the chaotropic series. Results: RT-PCR of C6 glioblastoma (C6) cells identified mRNA for three KCC isoforms (1, 3, and 4). NKCC1 mRNA was also detected. The apparent K _m for KCC-mediated Rb ⁺ influx was 15 mM [Rb ⁺ ] _e , and V _max 12.5 nmol Rb ⁺ * mg protein ^-1 * minute ^-1 . The calculated apparent K _m for external Cl ^- was 13 mM and V _max 14.4 nmol Rb ⁺ * mg protein ^-1 * minute ^-1 . The anion selectivity sequence of the furosemide-sensitive Rb ⁺ influx was Cl ^- >>Br-=NO ₃ ^- >I ^- =SCN ^- >>Sfm ^- (sulfamate). Established activators of K-Cl cotransport, hyposmotic shock and N -ethylmaleimide (NEM) pretreatment, stimulated furosemide-sensitive Rb ⁺ influx. A ñ50% NEM-induced loss of intracellular K ⁺ was prevented by furosemide. Conclusion: We have identified by RT-PCR the presence of three distinct KCC isoforms (1, 3, and 4) in rat C6 glioblastoma cells, and functionally characterized the anion selectivity and kinetics of their collective sodium-independent cation-chloride cotransport activity.