Whole cell voltage-clamp studies performed in isolated adult neurosecretory cells identified as dorsal unpaired median (DUM) neurons of the terminal abdominal ganglion of the cockroach Periplaneta americana have allowed us to reveal a complex voltage-dependent outward current regulating the pacemaker activity. 2. The global outward current remaining after tetrodotoxin treatment was activated by depolarization above -50 mV, showing steep voltage dependence and outward rectification. 3. We used tail current analysis to determine the ionic selectivity of this outward current. The reversal potentials for two extracellular potassium concentrations (-92.7 and -65.4 mV for 3.1 and 10 mM, respectively) is consistent with the expected equilibrium potential for potassium ions. 4. Both peak and sustained components of the global outward K+ current were reduced by external application of 20 mM tetraethylammonium chloride, 10 nM iberiotoxin, 1 nM charybdotoxin (CTX) and 1 mM cadmium chloride. Subtraction of current recorded in CTX solution from that in control solution revealed an unusual biphasic Ca(2+)-dependent K+ current. The fast transient current resistant to 5 mM 4-aminopyridine (4-AP) is distinguished by its dependence on holding potential and time course from the late sustained current. 5. In addition, two other components of CTX-resistant outward K+ current could be separated by sensitivity to 4-AP, time course, and voltage dependence. Beside a calcium-independent delayed outwardly rectifying current, a 4-AP-sensitive fast transient current resembling the A-current has been also identified. It activates at negative potential (about -65 mV) and unlike the A-current of other neurons, it inactivates rapidly with complex inactivation kinetics. A-like current is half-inactivated at -63.5 mV and half-activated at -35.6 mV. 6. Our findings demonstrate for the first time in DUM neuron cell bodies the existence of multiple potassium currents underlying the spontaneous electrical activity. Their identification and characterization represent a fundamental step in further understanding the pacemaker properties of these insect neurosecretory cells.
摘要
在美洲大蠊终腹神经节中被鉴定为背侧无对中（DUM）神经元的分离成年神经分泌细胞上进行的全细胞电压钳研究，使我们得以揭示一种调节起搏活动的复杂电压依赖性外向电流。2. 河豚毒素处理后剩余的整体外向电流在高于 -50 mV 的去极化作用下被激活，呈现出陡峭的电压依赖性和外向整流特性。3. 我们使用尾电流分析来确定这种外向电流的离子选择性。两种细胞外钾浓度（3.1 mM 和 10 mM 时分别为 -92.7 mV 和 -65.4 mV）下的反转电位与钾离子预期的平衡电位一致。4. 整体外向钾电流的峰值和持续成分都因外部施加 20 mM 氯化四乙铵、10 nM 埃博霉素、1 nM 卡律蝎毒素（CTX）和 1 mM 氯化镉而降低。从对照溶液记录的电流中减去在 CTX 溶液中记录的电流，揭示出一种不寻常的双相钙依赖性钾电流。对 5 mM 4 - 氨基吡啶（4 - AP）有抗性的快速瞬态电流，其对保持电位和时间进程的依赖性与晚期持续电流不同。5. 此外，CTX 抗性外向钾电流的另外两个成分可以通过对 4 - AP 的敏感性、时间进程和电压依赖性来分离。除了一种钙依赖性延迟外向整流电流外，还鉴定出一种类似于 A 电流的 4 - AP 敏感快速瞬态电流。它在负电位（约 -65 mV）时激活，与其他神经元的 A 电流不同，它以复杂的失活动力学迅速失活。类 A 电流在 -63.5 mV 时半失活，在 -35.6 mV 时半激活。6. 我们的研究结果首次在 DUM 神经元胞体中证明了自发电活动背后存在多种钾电流。它们的鉴定和表征是进一步理解这些昆虫神经分泌细胞起搏特性的基本步骤。
