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 Cl movements are mediated by several isoforms of the K-Cl cotransporter (COT) encoded by the KCC genes. The ubiquitous KCC1 isoform, important for cell volume and ion homeostasis, has 12 transmembrane domains (Tmds), and cytoplasmic N- and C-terminal domains (Ntd and Ctd). This study investigates the cellular localization of KCC1 by confocal microscopy, activation of K-Cl COT by various non-osmotic and osmotic interventions with net unidirectional K and Rb fluxes at 37( degrees )C, and the effect of Ctd deletion on K-Cl COT regulation. Human embryonic kidney (HEK293) cells were transfected with full-length (fl) rabbit (rb)KCC1 and - CtdKCC1 cDNAs obtained after truncation at nucleotide 2011. Normal cells exposed to polyclonal anti-Ctd antibodies against Ctd epitopes within a 77 amino acid sequence (a.a.943-1020) revealed granular membrane and cytoplasmic immunostaining, presumably endogenous KCC1. Additional diffuse membrane and cytoplasmic immunofluorescence in flKCC1-transfected cells was absent in -CtdKCC1-transfected cells. Monoclonal antibodies against a c-myc epitope at the protein Ntd showed both membrane and cytosolic fluorescence. Basal and N-ethylmaleimide (NEM)-stimulated Rb influxes through K-Cl COT, calculated as Cl-dependent Rb fluxes, were 2-3-fold higher in flKCC1-transfected than in normal cells. NEM stimulation of K-Cl COT was highest in flKCC1-transfected cells, significantly 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 were ineffective stimulators. No effect of pH(0) changes (6.3-8.4) was observed in basal or NEM-stimulated K-Cl COT, in both normal and transfected cells. However, inhibition by NEM occurred at pH(0) 8.4. Furthermore, in a Cl-independent manner, NEM lowered cell K content by >30% and hypotonicity (210-70mOsM) stimulated furosemide-sensitive Rb influx and K loss. Thus, in cultured normal and KCC1-transfected cells, K-Cl COT shows significant differences from erythrocytes, and NEM and cell swelling open furosemide-sensitive and Cl-independent K/Rb channels. Failure of K-Cl COT in cells transfected with Ctd-truncated KCC1 to respond to NEM suggests a role of the Ctd for signal transduction.
Lauf
P K
PK
Department of Physiology and Biophysics, Wright State University School of Medicine, Dayton, OH 45435, USA. peter.lauf@wright.edu
Zhang
J
J
Gagnon
K B
KB
Delpire
E
E
Fyffe
R E
RE
Adragna
N C
NC
D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D018076:DNA, Complementary; D005033:Ethylmaleimide / Q000494:pharmacology; D005665:Furosemide / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D017136:Ion Transport; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009994:Osmolar Concentration; D010080:Oxazoles / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D012413:Rubidium / Q000378:metabolism; D027981:Symporters; D014162:Transfection; D000096922:K Cl- Cotransporters
D002352:Carrier Proteins; D002712:Chlorides; D018076:DNA, Complementary; D005033:Ethylmaleimide; D005665:Furosemide; D019833:Genistein; D010080:Oxazoles; D011188:Potassium; D012413:Rubidium; D027981:Symporters; D000096922:K Cl- Cotransporters