Ca2+ from one or two channels controls fusion of a single vesicle at the frog neuromuscular junction.
Neurotransmitter release is triggered by the cooperative action of approximately five Ca2+ ions entering the presynaptic terminal through Ca2+ channels. Depending on the organization of the active zone (AZ), influx through one or many channels may be needed to cause fusion of a vesicle. Using a combination of experiments and modeling, we examined the number of channels that contribute Ca2+ for fusion of a single vesicle in a frog neuromuscular AZ. We compared Ca2+ influx to neurotransmitter release by measuring presynaptic action potential-evoked (AP-evoked) Ca2+ transients simultaneously with postsynaptic potentials. Ca2+ influx was manipulated by changing extracellular [Ca2+] (Ca(ext)) to alter the flux per channel or by reducing the number of open Ca2+ channels with omega-conotoxin GVIA (omega-CTX). When Ca(ext) was reduced, the exponent of the power relationship relating release to Ca2+ influx was 4.16 +/- 0.62 (SD; n = 4), consistent with a biochemical cooperativity of approximately 5. In contrast, reducing influx with omega-CTX yielded a power relationship of 1.7 +/- 0.44 (n = 5) for Ca(ext) of 1.8 mM and 2.12 +/- 0.44 for Ca(ext) of 0.45 mM (n = 5). Using geometrically realistic Monte Carlo simulations, we tracked Ca2+ ions as they entered through each channel and diffused in the terminal. Experimental and modeling data were consistent with two to six channel openings per AZ per AP; the Ca2+ that causes fusion of a single vesicle originates from one or two channels. Channel cooperativity depends mainly on the physical relationship between channels and vesicles and is insensitive to changes in the non-geometrical parameters of our model.
Shahrezaei
Vahid
V
Department of Physics, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
Cao
Alex
A
Delaney
Kerry R
KR
D000200:Action Potentials / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000378:metabolism / Q000502:physiology*; D008561:Membrane Fusion / Q000502:physiology*; D009469:Neuromuscular Junction / Q000378:metabolism / Q000502:physiology*; D017729:Presynaptic Terminals / Q000378:metabolism / Q000502:physiology; D011894:Rana pipiens; D013572:Synaptic Vesicles / Q000378:metabolism / Q000502:physiology*
D015220:Calcium Channels; D002118:Calcium