Department of Molecular and Cellular Physiology, and Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94304-5453, USA.
Proc Natl Acad Sci U S A. 2012 Jul 17;109(29):11830-5. doi: 10.1073/pnas.1209318109. Epub 2012 Jul 2.
Tight coupling of Ca(2+) channels to the presynaptic active zone is critical for fast synchronous neurotransmitter release. RIMs are multidomain proteins that tether Ca(2+) channels to active zones, dock and prime synaptic vesicles for release, and mediate presynaptic plasticity. Here, we use conditional knockout mice targeting all RIM isoforms expressed by the Rims1 and Rims2 genes to examine the contributions and mechanism of action of different RIMs in neurotransmitter release. We show that acute single deletions of each Rims gene decreased release and impaired vesicle priming but did not alter the extracellular Ca(2+)-responsiveness of release (which for Rims gene mutants is a measure of presynaptic Ca(2+) influx). Moreover, single deletions did not affect the synchronization of release (which depends on the close proximity of Ca(2+) channels to release sites). In contrast, deletion of both Rims genes severely impaired the Ca(2+) responsiveness and synchronization of release. RIM proteins may act on Ca(2+) channels in two modes: They tether Ca(2+) channels to active zones, and they directly modulate Ca(2+)-channel inactivation. The first mechanism is essential for localizing presynaptic Ca(2+) influx to nerve terminals, but the role of the second mechanism remains unknown. Strikingly, we find that although the RIM2 C(2)B domain by itself significantly decreased Ca(2+)-channel inactivation in transfected HEK293 cells, it did not rescue any aspect of the RIM knockout phenotype in cultured neurons. Thus, RIMs primarily act in release as physical Ca(2+)-channel tethers and not as Ca(2+)-channel modulators. Different RIM proteins compensate for each other in recruiting Ca(2+) channels to active zones, but contribute independently and incrementally to vesicle priming.
钙通道与突触前活跃区的紧密偶联对于快速同步神经递质释放至关重要。RIM 是一种多结构域蛋白，可将钙通道固定在活跃区，对接并引发突触囊泡释放，并介导突触前可塑性。在这里，我们使用靶向由 Rims1 和 Rims2 基因表达的所有 RIM 同工型的条件性基因敲除小鼠，研究不同 RIM 在神经递质释放中的作用和作用机制。我们发现，每个 Rims 基因的急性单一缺失都会降低释放并损害囊泡引发，但不会改变释放的细胞外 Ca(2+)-反应性（对于 Rims 基因突变体，这是测量突触前 Ca(2+)流入的指标）。此外，单一缺失不会影响释放的同步性（这取决于 Ca(2+)通道与释放位点的接近程度）。相比之下，删除两个 Rims 基因严重损害了 Ca(2+)的反应性和释放的同步性。RIM 蛋白可能以两种模式作用于钙通道：它们将钙通道固定在活跃区，并且它们直接调节钙通道失活。第一种机制对于将局部的突触前 Ca(2+)内流定位到神经末梢是必不可少的，但第二种机制的作用仍然未知。引人注目的是，我们发现，尽管单独的 RIM2 C(2)B 结构域显著降低了转染的 HEK293 细胞中的钙通道失活，但它不能挽救培养神经元中任何 RIM 缺失表型的任何方面。因此，RIM 主要作为物理钙通道系绳而不是钙通道调节剂在释放中起作用。不同的 RIM 蛋白在将钙通道募集到活跃区方面相互补偿，但独立地和增量地对囊泡引发做出贡献。
