Valeyev A Y, Hackman J C, Holohean A M, Wood P M, Katz J L, Davidoff R A
Neurophysiology and Spinal Cord Pharmacology Laboratories, Veterans Affairs Medical Center, Miami, Florida 33101, USA.
J Neurophysiol. 1999 Jul;82(1):10-5. doi: 10.1152/jn.1999.82.1.10.
Whole cell and cell-attached patch-clamp techniques characterized the neurosteroid anesthetic alphaxalone's (5alpha-pregnane-3alpha-ol-11,20-dione) effects on GABAA receptors and on Cl- currents in cultured embryonic (5- to 8-wk old) human dorsal root ganglion neurons. Alphaxalone applied by pressure pulses from closely positioned micropipettes failed to potentiate the inward Cl- currents produced by application of GABA. In the absence of GABA, alphaxalone (0.1-5.0 microM) directly evoked inward currents in all dorsal root ganglion neurons voltage-clamped at negative membrane potentials. The amplitude of the current was directly proportional to the concentration of alphaxalone (Hill coefficient 1.3 +/- 0.15). The alphaxalone-induced whole cell current was carried largely by Cl- ions. Its reversal potential was close to the theoretical Cl- equilibrium potential, changing with a shift in the external Cl- concentration as predicted by the Nernst equation for Cl- ions. And because the alphaxalone-current was not suppressed by the competitive GABAA receptor antagonist bicuculline or by the channel blockers picrotoxin and t-butylbicyclophosphorothionate (TBPS; all at 100 microM), it did not appear to result from activation of GABAA receptors. In contrast to GABA-currents in the same neurons, the whole cell current-voltage curves produced in the presence of alphaxalone demonstrated strong inward rectification with nearly symmetrical bath and pipette Cl- concentrations. Fluctuation analysis of the membrane current variance produced by 1.0 microM alphaxalone showed that the power density spectra were best fitted to double Lorentzian functions. The elementary conductance for alphaxalone-activated Cl- channels determined by the relationship between mean amplitude of whole cell current and variance was 30 pS. Single-channel currents in cell-attached patches when the pipette solution contained 10 microM alphaxalone revealed a single conductance state with a chord conductance of approximately 29 pS. No subconductance states were seen. The current-voltage determinations for the single-channels activated by alphaxalone demonstrated a linear relationship. Mean open and shut times of single alphaxalone-activated channels were described by two exponential decay functions. Taken together, the results indicate that in embryonic human DRG neurons, micromolar concentrations of alphaxalone directly activate Cl- channels whose electrophysiological and pharmacological properties are distinct from those of Cl- channels associated with GABAA receptors.
全细胞和细胞贴附式膜片钳技术表征了神经甾体麻醉剂α-香螺甾酮（5α-孕烷-3α-醇-11,20-二酮）对培养的胚胎（5至8周龄）人背根神经节神经元中GABAA受体和Cl-电流的影响。通过紧邻的微吸管施加压力脉冲来施加α-香螺甾酮，未能增强应用GABA所产生的内向Cl-电流。在不存在GABA的情况下，α-香螺甾酮（0.1 - 5.0微摩尔）在所有钳制于负膜电位的背根神经节神经元中直接诱发内向电流。电流幅度与α-香螺甾酮浓度成正比（希尔系数1.3±0.15）。α-香螺甾酮诱导的全细胞电流主要由Cl-离子携带。其反转电位接近理论Cl-平衡电位，随着外部Cl-浓度的变化而改变，正如Cl-离子的能斯特方程所预测的那样。并且由于α-香螺甾酮电流不受竞争性GABAA受体拮抗剂荷包牡丹碱或通道阻滞剂印防己毒素和叔丁基双环磷硫酰亚胺（TBPS；均为100微摩尔）的抑制，它似乎不是由GABAA受体的激活所导致。与同一神经元中的GABA电流相反，在存在α-香螺甾酮的情况下产生的全细胞电流-电压曲线在浴槽和吸管Cl-浓度几乎对称时表现出强烈的内向整流。对1.0微摩尔α-香螺甾酮产生的膜电流方差进行波动分析表明，功率密度谱最适合双洛伦兹函数。由全细胞电流的平均幅度与方差之间的关系所确定的α-香螺甾酮激活的Cl-通道的基本电导为30皮安。当吸管溶液含有10微摩尔α-香螺甾酮时，细胞贴附式膜片中的单通道电流显示出单一电导状态，弦电导约为29皮安。未观察到亚电导状态。由α-香螺甾酮激活的单通道的电流-电压测定显示出线性关系。单个α-香螺甾酮激活通道的平均开放和关闭时间由两个指数衰减函数描述。综上所述，结果表明在胚胎人背根神经节神经元中，微摩尔浓度的α-香螺甾酮直接激活Cl-通道，其电生理和药理特性与与GABAA受体相关的Cl-通道不同。
