Dept. of Ophthalmology and Visual Sciences, Univ. of Nebraska Medical Center, Omaha, NE 68198-5840, USA.
J Neurophysiol. 2012 Jun;107(12):3468-78. doi: 10.1152/jn.00779.2011. Epub 2012 Mar 21.
Synaptic communication requires proper coupling between voltage-gated Ca(2+) (Ca(V)) channels and synaptic vesicles. In photoreceptors, L-type Ca(V) channels are clustered close to synaptic ribbon release sites. Although clustered, Ca(V) channels move continuously within a confined domain slightly larger than the base of the ribbon. We hypothesized that expanding Ca(V) channel confinement domains should increase the number of channel openings needed to trigger vesicle release. Using single-particle tracking techniques, we measured the expansion of Ca(V) channel confinement domains caused by depletion of membrane cholesterol with cholesterol oxidase or methyl-β-cyclodextrin. With paired whole cell recordings from cones and horizontal cells, we then determined the number of Ca(V) channel openings contributing to cone Ca(V) currents (I(Ca)) and the number of vesicle fusion events contributing to horizontal cell excitatory postsynaptic currents (EPSCs) following cholesterol depletion. Expansion of Ca(V) channel confinement domains reduced the peak efficiency of release, decreasing the number of vesicle fusion events accompanying opening of each Ca(V) channel. Cholesterol depletion also inhibited exocytotic capacitance increases evoked by brief depolarizing steps. Changes in efficiency were not due to changes in I(Ca) amplitude or glutamate receptor properties. Replenishing cholesterol restored Ca(V) channel domain size and release efficiency to control levels. These results indicate that cholesterol is important for organizing the cone active zone. Furthermore, the finding that cholesterol depletion impairs coupling between channel opening and vesicle release by allowing Ca(V) channels to move further from release sites shows that changes in presynaptic Ca(V) channel mobility can be a mechanism for adjusting synaptic strength.
突触通讯需要电压门控 Ca(2+)（Ca(V)）通道和突触小泡之间的适当偶联。在光感受器中，L 型 Ca(V)通道聚集在突触带释放位点附近。尽管聚集在一起，Ca(V)通道在一个稍大于带底部的受限域内连续移动。我们假设扩大 Ca(V)通道限制域应该会增加触发囊泡释放所需的通道打开数量。使用单颗粒跟踪技术，我们测量了膜胆固醇耗竭用胆固醇氧化酶或甲基-β-环糊精引起的 Ca(V)通道限制域的扩张。然后，我们通过从锥体和水平细胞进行配对的全细胞记录，确定了导致锥体 Ca(V)电流（I(Ca)）的 Ca(V)通道打开数量和导致水平细胞兴奋性突触后电流（EPSC）的囊泡融合事件数量在胆固醇耗竭后。Ca(V)通道限制域的扩展降低了释放的峰值效率，减少了每个 Ca(V)通道打开时伴随的囊泡融合事件数量。胆固醇耗竭还抑制了短暂去极化步骤引起的胞吐电容增加。效率的变化不是由于 I(Ca)幅度或谷氨酸受体特性的变化引起的。补充胆固醇将 Ca(V)通道域大小和释放效率恢复到对照水平。这些结果表明胆固醇对于组织锥体活跃区很重要。此外，胆固醇耗竭通过允许 Ca(V)通道从释放位点进一步移动而损害通道打开和囊泡释放之间的偶联的发现表明，突触前 Ca(V)通道迁移率的变化可以是调节突触强度的一种机制。
