Single-channel properties of voltage-dependent calcium channels were investigated in rat melanotropes in short-term primary culture. Unitary currents were resolved using the cell-attached configuration. 2. Depolarizations higher than -50 mV activated a population of 8.1-pS calcium channels [low-voltage activated (LVA)]. The LVA channel ensembles displayed a monoexponential time course of inactivation and a sigmoidal time course of activation fitted best by an m2h Hodgkin-Huxley-type model. Microscopic kinetic analysis suggested that at least one open state, two closed states, and one inactivated state are involved in channel gating. 3. At potentials positive to -20 mV a second class of calcium channels was activated with a conductance of 24.7 pS [high-voltage activated (HVA)]. HVA channels display different gating modes. Gating with high open probability (mode 2) and low open probability (mode 1) as well as blank traces (mode 0) are observed. The HVA channels were heterogeneous with respect to their inactivation properties. Ensembles that decayed entirely during a 300-ms test pulse as well as nondecaying ensembles were observed. Both HVA channel subtypes displayed sigmoidal activation, which was fitted by an m2 model. Microscopic kinetic analysis suggested that at least one open state and two closed states are involved in mode two gating of both HVA channel subtypes. 4. Depolarizing prepulses did not recruit or facilitate calcium channel activity in response to a test pulse, but inactivating HVA channel activity was strongly reduced. Depolarizing prepulses (+50 mV) did not affect the probability of opening of the noninactivating HVA channel. 5. The voltage dependence and kinetics of the LVA as well as both HVA channels are in good agreement with previously published data on the properties of the various calcium current components derived from whole-cell recordings of rat melanotropes. The data suggest that a T-type as well as two L-type channels (an inactivating and noninactivating channel) underlie the calcium current in these cells.
摘要
在短期原代培养的大鼠促黑素细胞中研究了电压依赖性钙通道的单通道特性。使用细胞贴附式配置解析了单位电流。2. 高于 -50 mV 的去极化激活了一群 8.1 皮秒的钙通道 [低电压激活 (LVA)]。LVA 通道群体显示出单指数失活时间进程和由 m2h 霍奇金 - 赫胥黎型模型最佳拟合的 S 型激活时间进程。微观动力学分析表明，通道门控至少涉及一个开放状态、两个关闭状态和一个失活状态。3. 在高于 -20 mV 的电位下，激活了第二类钙通道，其电导为 24.7 皮秒 [高电压激活 (HVA)]。HVA 通道表现出不同的门控模式。观察到高开放概率 (模式 2)、低开放概率 (模式 1) 以及无信号迹线 (模式 0) 的门控。HVA 通道在失活特性方面是异质的。观察到在 300 毫秒测试脉冲期间完全衰减的群体以及不衰减的群体。两种 HVA 通道亚型均显示出 S 型激活，由 m2 模型拟合。微观动力学分析表明，两种 HVA 通道亚型的模式 2 门控至少涉及一个开放状态和两个关闭状态。4. 去极化预脉冲不会募集或促进响应测试脉冲的钙通道活性，但会强烈降低失活的 HVA 通道活性。去极化预脉冲 (+50 mV) 不影响非失活 HVA 通道的开放概率。5. LVA 以及两种 HVA 通道的电压依赖性和动力学与先前发表的关于从大鼠促黑素细胞全细胞记录中获得的各种钙电流成分特性的数据高度一致。数据表明，这些细胞中的钙电流由一个 T 型以及两个 L 型通道 (一个失活通道和一个非失活通道) 构成。
