Ward S M, Kenyon J L
Department of Physiology & Cell Biology/MS 352, University of Nevada School of Medicine, Reno, NV, 89557, USA.
Cell Calcium. 2000 Oct;28(4):233-46. doi: 10.1054/ceca.2000.0151.
In order to learn about the endogenous Ca2+-buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca2+ release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca2+-activated Cl- currents (I(ClCa)) in protocols that manipulated Ca2+-buffering. I(ClCa)was activated by Ca2+ influx via voltage-gated Ca2+ channels or by Ca2+ release via RyRs activated by 10 mM caffeine. I(ClCa)was measured in neurons at 20 degrees C and 35 degrees C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20 degrees C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+ buffers. The amplitude of I(ClCa)activated by Ca2+ influx or Ca2+ at 20 degrees C was similar in the amphotericin neurons and neurons dialyzed with an 'unbuffered' pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca2+ binding molecules. Thus, endogenous mobile Ca2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20 degrees C to 35 degrees C increased the amplitude and the rate of deactivation of I(ClCa)consistent with an increased rate of Ca2+ buffering by fixed endogenous Ca2+-buffers. Dialysis with 2 mM EGTA/0.1 microM free Ca2+ reduced the amplitude and increased the rate of deactivation of I(ClCa)activated by Ca2+ influx and abolished I(ClCa)activated by Ca2+ release. Dialysis with 2 mM BAPTA/0.1 microM free Ca2+ abolished I(ClCa)activated by Ca2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 microM free Ca2+ caused the persistent activation of I(ClCa). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+ channels and the Ca2+-activated Cl- channels are separated by 50-400 nm and that the RyRs are more than 600 nm from the plasma membrane.
为了了解鸡背根神经节（DRG）神经元细胞质中的内源性Ca2+缓冲情况以及ryanodine受体Ca2+释放通道（RyRs）与质膜之间的距离，我们在操纵Ca2+缓冲的实验方案中监测了Ca2+激活的Cl-电流（I(ClCa)）的幅度和时间进程。I(ClCa)通过电压门控Ca2+通道的Ca2+内流或由10 mM咖啡因激活的RyRs的Ca2+释放来激活。使用保留内源性Ca2+缓冲的两性霉素穿孔膜片钳技术，在20℃和35℃下测量神经元中的I(ClCa)，或者在20℃下用设计用于替代内源性Ca2+缓冲剂的移液管溶液透析的神经元中测量I(ClCa)。在两性霉素神经元和用含有10 mM柠檬酸盐和3 mM ATP作为唯一Ca2+结合分子的“无缓冲”移液管溶液透析的神经元中，20℃时由Ca2+内流或Ca2+激活的I(ClCa)幅度相似。因此，内源性可移动Ca2+缓冲剂在鸡DRG神经元中相对不重要。将神经元从20℃加热到35℃会增加I(ClCa)的幅度和失活速率，这与固定的内源性Ca2+缓冲剂增加Ca2+缓冲速率一致。用2 mM乙二醇双四乙酸（EGTA）/0.1 microM游离Ca2+透析会降低由Ca2+内流激活的I(ClCa)的幅度并增加其失活速率，同时消除由Ca2+释放激活的I(ClCa)。用2 mM 1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）/0.1 microM游离Ca2+透析会消除由Ca2+内流或释放激活的I(ClCa)。用42 mM羟乙基乙二胺三乙酸（HEEDTA）/0.5 microM游离Ca2+透析会导致I(ClCa)持续激活。使用Ca2+扩散模型进行的计算表明，电压门控Ca2+通道和Ca2+激活的Cl-通道之间的距离为50 - 400 nm，并且RyRs与质膜的距离超过600 nm。
