title: Archaerhodopsin voltage imaging: synaptic calcium and BK channels stabilize action potential repolarization at the Drosophila neuromuscular junction.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94158.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Ford
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94158 Graeme.Davis@ucsf.edu.
authors.forename: Graeme W
authors.initials: GW
authors.lastname: Davis
mesh_terms: D000200:Action Potentials / Q000502:physiology*; D000818:Animals; D019843:Archaeal Proteins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D004330:Drosophila; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D009469:Neuromuscular Junction / Q000502:physiology*; D017494:Proton Pumps
chemical_list: D019843:Archaeal Proteins; D002118:Calcium; D015220:Calcium Channels; D051036:Large-Conductance Calcium-Activated Potassium Channels; C055476:archaerhodopsin protein, Archaea; D017494:Proton Pumps
keywords: BK channel; Kv1 channel; NMJ; presynaptic; short term plasticity; voltage imaging