Ford Kevin J, Davis Graeme W
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94158.
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94158
J Neurosci. 2014 Oct 29;34(44):14517-25. doi: 10.1523/JNEUROSCI.2203-14.2014.
The strength and dynamics of synaptic transmission are determined, in part, by the presynaptic action potential (AP) waveform at the nerve terminal. The ion channels that shape the synaptic AP waveform remain essentially unknown for all but a few large synapses amenable to electrophysiological interrogation. The Drosophila neuromuscular junction (NMJ) is a powerful system for studying synaptic biology, but it is not amenable to presynaptic electrophysiology. Here, we demonstrate that Archaerhodopsin can be used to quantitatively image AP waveforms at the Drosophila NMJ without disrupting baseline synaptic transmission or neuromuscular development. It is established that Shaker mutations cause a dramatic increase in neurotransmitter release, suggesting that Shaker is predominantly responsible for AP repolarization. Here we demonstrate that this effect is caused by a concomitant loss of both Shaker and slowpoke (slo) channel activity because of the low extracellular calcium concentrations (0.2-0.5 mM) used typically to assess synaptic transmission in Shaker. In contrast, at physiological extracellular calcium (1.5 mM), the role of Shaker during AP repolarization is limited. We then provide evidence that calcium influx through synaptic CaV2.1 channels and subsequent recruitment of Slo channel activity is important, in concert with Shaker, to ensure proper AP repolarization. Finally, we show that Slo assumes a dominant repolarizing role during repetitive nerve stimulation. During repetitive stimulation, Slo effectively compensates for Shaker channel inactivation, stabilizing AP repolarization and limiting neurotransmitter release. Thus, we have defined an essential role for Slo channels during synaptic AP repolarization and have revised our understanding of Shaker channels at this model synapse.
突触传递的强度和动力学部分取决于神经末梢处的突触前动作电位（AP）波形。除了少数几个适合进行电生理研究的大突触外，塑造突触AP波形的离子通道基本上仍不为人所知。果蝇神经肌肉接头（NMJ）是研究突触生物学的一个强大系统，但它不适合进行突触前电生理研究。在这里，我们证明古紫质可用于定量成像果蝇NMJ处的AP波形，而不会破坏基线突触传递或神经肌肉发育。已经确定，Shaker突变会导致神经递质释放显著增加，这表明Shaker主要负责AP复极化。在这里我们证明，这种效应是由于通常用于评估Shaker中突触传递的低细胞外钙浓度（0.2 - 0.5 mM）导致Shaker和慢poke（slo）通道活性同时丧失所致。相比之下，在生理细胞外钙浓度（1.5 mM）下，Shaker在AP复极化过程中的作用有限。然后我们提供证据表明，通过突触CaV2.1通道的钙内流以及随后Slo通道活性的募集与Shaker协同作用，对于确保适当的AP复极化很重要。最后，我们表明在重复神经刺激期间，Slo发挥主要的复极化作用。在重复刺激期间，Slo有效地补偿了Shaker通道的失活，稳定了AP复极化并限制了神经递质释放。因此，我们确定了Slo通道在突触AP复极化过程中的重要作用，并修正了我们对这个模型突触处Shaker通道的理解。
