K-Cl cotransport: Immunohistochemical and ion flux studies in human embryonic kidney (HEK293) cells transfected with full-length and C-terminal-domain-truncated KCC1 cDNAs

Coupled K and CI movements are mediated by several isoforms of the K-CI cot ransporter (COT) encoded by the KCC genes. The ubiquitous KCC1 isoform, imp ortant for cell volume and ion homeostasis, has 12 transmembrane domains (T mds), and cytoplasmic N- and C- terminal domains (Ntd and Ctd). This study investigates the cellular localization of KCC1 by confocal microscopy, acti vation of K-CI COT by various non-osmotic and osmotic interventions with ne t unidirectional K and Rb fluxes at 37 degreesC, and the effect of Ctd dele tion on K-CI COT regulation. Human embryonic kidney (HEK293) cells were tra nsfected with full-length (fl) rabbit (rb)KCC1 and -CtdKCC1 cDNAs obtained after truncation at nucleotide 2011. Normal cells exposed to polyclonal ant i-Ctd antibodies against Ctd epitopes within a 77 amino acid sequence (a.a. 943-1020) revealed granular membrane and cytoplasmic immunostaining, presum ably endogenous KCC1. Additional diffuse membrane and cytoplasmic immunoflu orescence in flKCC1-transfected cells was absent in -CtdKCC1 -transfected c ells. Monoclonal antibodies against a c-myc epitope at the protein Ntd show ed both membrane and cytosolic fluoresence. Basal and N-ethylmaleimide (NEM )-stimulated Rb influxes through K-CI GOT, calculated as Cl-dependent Rb fl uxes, were 2-3-fold higher in flKCC1-transfected than in normal cells. NEM stimulation of K-Cl COT was highest in flKCC1-transfected cells, significan tly lower in stably and abrogated in transiently -CtdKCC1-transfected cells . Furosemide, calyculin and genistein inhibited basal and NEM-stimulated K- Cl COT in normal and transfected cells. Staurosporine and hydroxylamine wer e ineffective stimulators. No effect of pH(o) changes (16.3-8.4) was observ ed in basal or NEM-stimulated K-Cl GOT, in both normal and transfected cell s. However, inhibition by NEM occurred at pH(o) 8.4. Furthermore, in a Cl-i ndependent manner, NEM lowered cell K content by > 30% and hypotonicity (21 0-70mOsM) stimulated furosemide-sensitive Rb influx and K loss. Thus, in cu ltured normal and KCC1-transfected cells, K-C1 COT shows significant differ ences from erythrocytes, and NEM and cell swelling open furosemide-sensitiv e and Cl-independent K/Rb channels. Failure of K-CI COT in cells transfecte d with Ctd-truncated KCC1 to respond to NEM suggests a role of the Ctd for signal transduction. Copyright (C) 2001 S. Karger AG, Basel.