In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: modulation by calcium and GTP gamma S.
In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: modulation by calcium and GTP gamma S.
复制标题
轴膜周围髓磷脂和白质网格蛋白包被的囊泡中离子通道的体外分析：钙和 GTP γ S 的调节。
DOI：
10.1007/bf00968722
复制
发表时间：
1994
影响因子：
4.4
通讯作者：
Sapirstein,VS
中科院分区：
文献类型：
--
作者：
Cherksey,B;Durrie,R;Braun,PE;Sapirstein,VS
本研究分析了牛轴膜周围髓鞘和白质来源的笼状蛋白包被小泡的K+通道活性。通过膜泡与跨膜片钳吸管形成的磷脂双层膜的融合来评估通道活性。在轴突周围观察到自发的K+通道，波幅分别为25-30、45-55和80-100ps，平均开放时间为1-2ms。6-甲基二氢吡喃-2-酮增加了轴膜-髓鞘50pS通道的开放概率。外膜-髓磷脂K+通道活性受钙离子调节。当Ca~(2+)加到双层膜的一侧时，活性几乎或没有变化。加入10μM总钙后，K+通道活性变化不大。然而，在80μM时，总钙通道的所有K+通道活动均被抑制，同时100pS的氯离子−通道被激活。轴突周围髓鞘的K+通道活动也受G蛋白的调节。将γS加入双分子膜的膜上，可抑制在所有保持电位下存在的45-50pS通道的活性。内吞被囊泡，部分通过G蛋白介导的事件形成；白质被覆小泡被G蛋白和K+通道活性分析。以前的研究表明，这些小泡来自轴突周围区域，富含α亚基G0、Gsα和Giα以及低分子量G蛋白ras。与用GTPγS处理的轴膜周围髓鞘一样，囊泡膜只有50pS通道。该通道在所有保持电位下都是激活的，开放时间为1-6毫秒。在与囊泡膜融合的双层膜中加入γS，在低电压下可抑制该通道的活动，但在保持电位大于或等于45 mV时，可诱导该通道处于过度激活状态。6-甲基-二氢吡喃-2-酮(20μM)也能激活囊泡50pS K+通道。
This study reports the analysis of K+channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K+channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K+channel activity was regulated by Ca2+. Little or no change in activity was observed when Ca2+was added to thecisside of the bilayer. Addition of 10 μM total Ca2+also resulted in little change in K+channel activity. However, at 80 μM total Ca2+all K+channel activity was suppressed along with the activation of a 100 pS Cl−channel. The K+channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPγS to thetransside of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K+channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the α subunits of G0, Gsα, and Giαand the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPγS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPγS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K+channel was also activated by the 6-methyl-dihydropyron-2-one (20 μM).