Kerschbaum H H, Negulescu P A, Cahalan M D
Department of Physiology and Biophysics, University of California, Irvine 92717, USA.
J Immunol. 1997 Aug 15;159(4):1628-38.
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.
采用小鼠T细胞杂交瘤B3Z进行全细胞记录，并同时测量细胞内Ca2+（[Ca2+]i）和基因表达。B3Z细胞系稳定感染了由最小IL-2启动子（活化T细胞核因子，NF-AT）驱动的lacZ报告基因。通过将通道阻断药理学与[Ca2+]i及lacZ表达相关联，研究了B3Z细胞中离子通道的生理作用。在B3Z细胞和活化的人T细胞中，电压门控K+（K(V)）电流的主要成分具有与Kv1.3编码的n型通道相关的生物物理和药理学特性；一个较小的K(V)成分对蝎毒素（CTX）具有抗性。Ca2+激活的K+（K(Ca)）电流对CTX敏感，但对玛格毒素（MgTX）不敏感。内向整流K+（K(IR)）电流被200μM的Ba2+完全阻断。细胞肿胀可诱导外向整流Cl-电流。通过用10 mM乙二醇双四乙酸（EGTA）和10μM肌醇-1,4,5-三磷酸（IP3）透析细胞，可激活内向整流Ca2+电流（I(CRAC)）。CTX使毒胡萝卜素刺激的[Ca2+]i信号传导和基因表达降低约25%。尽管毒胡萝卜素刺激的[Ca2+]i信号对K+通道阻滞剂的完全抑制具有抗性，但它对K+扩散电位和Cl-去除非常敏感，这表明耐药性K+通道以及可能的Cl-通道可以维持足够负的膜电位以驱动Ca2+内流。本研究中使用的离子通道阻滞剂均未抑制由T细胞受体（TCR）的CD3-ε亚基刺激诱导的[Ca2+]i信号传导或基因表达。我们得出结论，几种通道类型可有助于维持膜电位（Vm）、Ca2+信号和基因表达。
