Forscher P, Oxford G S
J Gen Physiol. 1985 May;85(5):743-63. doi: 10.1085/jgp.85.5.743.
Modulation of voltage-dependent Ca channels by norepinephrine (NE) was studied in chick dorsal root ganglion cells using the whole-cell configuration of the patch-clamp technique. Cells dialyzed with K+ and 2-10 mM EGTA exhibited Ca action potentials that were reversibly decreased in duration and amplitude by NE. Ca channel currents were isolated from other channel contributions by using: (a) tetrodotoxin (TTX) to block gNa, (b) internal K channel impermeant ions (Cs or Na/N-methylglucamine mixtures) as K substitutes, (c) external tetraethylammonium (TEA) to block K channels, (d) internal EGTA to reduce possible current contribution from Ca-activated channels. A marked decline (rundown) of Ca conductance was observed during continual dialysis, which obscured reversible NE effects. The addition of 2-5 mM MgATP to the intracellular solutions greatly retarded Ca channel rundown and permitted a clear assessment of modulatory drug effects. The inclusion of an intracellular creatine phosphate/creatine phosphokinase nucleotide regeneration system further stabilized Ca channels, which permitted recording of Ca currents for up to 3 h. NE reversibly decreased both steady state Ca currents and Ca tail currents in Cs/EGTA/MgATP-dialyzed cells. A possible role of several putative intracellular second messengers in NE receptor-Ca channel coupling was investigated. Cyclic AMP or cyclic GMP added to the intracellular solutions at concentrations several orders of magnitude higher than the Kd for activation of cyclic nucleotide-dependent protein kinases did not block or mask the expression of the NE-mediated decrease in gCa. Addition of internal EGTA to a final concentration of 10 mM also did not affect the expression of the NE response. These results suggest that neither cyclic AMP nor cyclic GMP nor Ca is acting as a second messenger coupling the NE receptor to the down-modulated Ca channel population.
采用膜片钳技术的全细胞模式，研究了去甲肾上腺素（NE）对鸡背根神经节细胞电压依赖性钙通道的调节作用。用K⁺和2 - 10 mM乙二醇双四乙酸（EGTA）透析的细胞表现出钙动作电位，NE可使其持续时间和幅度可逆性降低。通过以下方法从其他通道电流中分离出钙通道电流：（a）用河豚毒素（TTX）阻断钠通道（gNa）；（b）用内部钾通道不通透的离子（Cs或Na/甲基葡糖胺混合物）替代钾；（c）用外部四乙铵（TEA）阻断钾通道；（d）用内部EGTA减少钙激活通道可能产生的电流贡献。在持续透析过程中观察到钙电导显著下降（衰减），这掩盖了NE的可逆性作用。向细胞内溶液中添加2 - 5 mM的MgATP可大大延缓钙通道的衰减，并能清晰评估调节药物的作用。加入细胞内磷酸肌酸/肌酸磷酸激酶核苷酸再生系统可进一步稳定钙通道，从而能够记录长达3小时的钙电流。在Cs/EGTA/MgATP透析的细胞中，NE可使稳态钙电流和钙尾电流可逆性降低。研究了几种假定的细胞内第二信使在NE受体 - 钙通道偶联中的可能作用。以比激活环核苷酸依赖性蛋白激酶的解离常数（Kd）高几个数量级的浓度向细胞内溶液中添加环磷酸腺苷（cAMP）或环磷酸鸟苷（cGMP），并未阻断或掩盖NE介导的钙电导降低的表达。将内部EGTA最终浓度调至10 mM也不影响NE反应的表达。这些结果表明，cAMP、cGMP和Ca均未作为将NE受体与下调的钙通道群体偶联的第二信使。
