Characterization of Glial Cell K-Cl Cotransport

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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 Km for KCC-mediated Rb+ influx was 15 mM [Rb+]e, and Vmax 12.5 nmol Rb+ * mg protein-1 * minute-1. The calculated apparent Km for external Cl- was 13 mM and Vmax 14.4 nmol Rb+ * mg protein-1 * minute-1. The anion selectivity sequence of the furosemide-sensitive Rb+ influx was Cl->>Br-=NO3->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.