Ion channels, Ca2+ signaling, and reporter gene expression in antigen-specific mouse T cells.
Whole cell recordings were performed in parallel with measurements of intracellular Ca2+ ([Ca2+]i) and gene expression using the murine T cell hybridoma, B3Z, a cell line stably infected with a lacZ reporter gene, driven by the minimal IL-2 promoter (NF-AT, nuclear factor of activated T cells). The physiologic roles of ion channels in B3Z cells were investigated by correlating the pharmacology of channel block with [Ca2+]i, and expression of lacZ. In B3Z cells and activated human T cells, the major component of voltage-gated K+ (K(V)) current had biophysical and pharmacologic properties associated with type n channels encoded by Kv1.3; a minor K(V) component was charybdotoxin (CTX) resistant. Ca2+-activated K+ (K(Ca)) current was sensitive to CTX, but not to margatoxin (MgTX). Inwardly rectifying K+ (K(IR)) current was blocked completely by 200 microM of Ba2+. Outwardly rectifying Cl- currents were induced by cell swelling. An inwardly rectifying Ca2+ current (I(CRAC)) was activated by dialyzing the cell with 10 mM EGTA and 10 microM IP3. CTX reduced thapsigargin-stimulated [Ca2+]i signaling and gene expression by approximately 25%. Although the thapsigargin-stimulated [Ca2+]i signal was resistant to complete inhibition by K+ channel blockers, it was very sensitive to the K+ diffusion potential and Cl- removal, suggesting that drug-resistant K+ channels and perhaps Cl- channels can maintain a sufficiently negative membrane potential to drive Ca2+ influx. Neither [Ca2+]i signaling nor gene expression induced by stimulation of the CD3-epsilon subunit of the TCR was inhibited by ion channel blockers used in this study. We conclude that several channel types can contribute to maintenance of Vm, Ca2+ signals, and gene expression.
Kerschbaum
H H
HH
Department of Physiology and Biophysics, University of California, Irvine 92717, USA.
Negulescu
P A
PA
Cahalan
M D
MD
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology; D002478:Cells, Cultured; D002586:Cesium / Q000494:pharmacology; D018999:Charybdotoxin / Q000494:pharmacology; D018118:Chloride Channels; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D007473:Ion Channels / Q000502:physiology*; D051379:Mice; D015221:Potassium Channels / Q000502:physiology; D013601:T-Lymphocytes / Q000378:metabolism*
D015220:Calcium Channels; D018118:Chloride Channels; D007473:Ion Channels; D015221:Potassium Channels; D018999:Charybdotoxin; D002586:Cesium; D002118:Calcium