Voltage- and calcium-dependent inactivation of calcium channels in Lymnaea neurons.
Voltage- and calcium-dependent inactivation of calcium channels in Lymnaea neurons.
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DOI：
10.1085/jgp.114.4.535
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发表时间：
1999-10
期刊：
影响因子：
--
通讯作者：
Byerly L
中科院分区：
文献类型：
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作者：
Gera S;Byerly L
采用膜片钳技术研究了淡水蜗牛（lynaea stagnation）神经元中Ca2+通道失活。在存在高浓度的细胞内Ca2+缓冲液（5 mM EGTA）时，这些Ca2+通道的失活完全依赖于电压；它不受渗透二价离子的身份或细胞外Ca2+内流量的影响，也不受较高水平的细胞内Ca2+缓冲的影响。在这些条件下测量的失活，尽管独立于Ca2+内流，但具有钟形电压依赖性，这通常被认为是Ca2+依赖性失活的标志。当细胞内Ca2+缓冲液浓度降至0.1 mM EGTA时，淋巴神经元确实发生Ca2+依赖性失活。然而，Ca2+依赖性失活的幅度不随Ca2+内流线性增加，但饱和相对少量的Ca2+内流。从负电位失活的恢复是双指数的，并且在细胞内存在不同浓度的EGTA时具有相同的时间常数。然而，由于细胞内EGTA的减少，缓慢组分的振幅选择性地增强，从而减慢了整体恢复速度。5mm EGTA完全抑制Ca2+依赖性失活的能力表明Ca2+结合位点距离通道蛋白本身有一定距离。没有证据表明丝氨酸/苏氨酸磷酸化在Ca2+通道失活中的作用。细胞松弛素B （Cytochalasin B）是一种微丝干扰物，被发现可以大大增加Ca2+通道失活的数量，但肌动蛋白丝参与细胞松弛素B对Ca2+通道失活的影响，无法使用其他药理化合物验证。因此，这些神经元中Ca2+依赖性失活的机制仍然未知，但似乎与哺乳动物l型Ca2+通道的机制不同。
Ca2+ channel inactivation in the neurons of the freshwater snail, Lymnaea stagnalis, was studied using patch-clamp techniques. In the presence of a high concentration of intracellular Ca2+ buffer (5 mM EGTA), the inactivation of these Ca2+ channels is entirely voltage dependent; it is not influenced by the identity of the permeant divalent ions or the amount of extracellular Ca2+ influx, or reduced by higher levels of intracellular Ca2+ buffering. Inactivation measured under these conditions, despite being independent of Ca2+ influx, has a bell-shaped voltage dependence, which has often been considered a hallmark of Ca2+-dependent inactivation. Ca2+-dependent inactivation does occur in Lymnaea neurons, when the concentration of the intracellular Ca2+ buffer is lowered to 0.1 mM EGTA. However, the magnitude of Ca2+-dependent inactivation does not increase linearly with Ca2+ influx, but saturates for relatively small amounts of Ca2+ influx. Recovery from inactivation at negative potentials is biexponential and has the same time constants in the presence of different intracellular concentrations of EGTA. However, the amplitude of the slow component is selectively enhanced by a decrease in intracellular EGTA, thus slowing the overall rate of recovery. The ability of 5 mM EGTA to completely suppress Ca2+-dependent inactivation suggests that the Ca2+ binding site is at some distance from the channel protein itself. No evidence was found of a role for serine/threonine phosphorylation in Ca2+ channel inactivation. Cytochalasin B, a microfilament disrupter, was found to greatly enhance the amount of Ca2+ channel inactivation, but the involvement of actin filaments in this effect of cytochalasin B on Ca2+ channel inactivation could not be verified using other pharmacological compounds. Thus, the mechanism of Ca2+-dependent inactivation in these neurons remains unknown, but appears to differ from those proposed for mammalian L-type Ca2+ channels.