Abstract
The exogenous Ca2+ chelator EGTA (ethylene glycol tetraacetic acid) has been widely used to probe the coupling distance between Ca2+ channels and vesicular Ca2+ sensors for neurotransmitter release. Because of its slow forward rate for binding, EGTA is thought to not capture calcium ions in very proximity to a channel, whereas it does capture calcium ions at the remote distance. However, in this study, our reaction diffusion simulations (RDSs) of Ca2+ combined with a release calculation using vesicular sensor models indicate that a high concentration of EGTA decreases Ca2+ and vesicular release in the nanodomain of single channels. We found that a key determinant of the effect of EGTA on neurotransmitter release is the saturation of the vesicular sensor. When the sensor is saturated, the reduction in the Ca2+ concentration by EGTA is masked. By contrast, when the sensor is in a linear range, even a small reduction in Ca2+ by EGTA can decrease vesicular release. In proximity to a channel, the vesicular sensor is often saturated for a long voltage step, but not for a brief Ca2+ influx typically evoked by an action potential. Therefore, when EGTA is used as a diagnostic tool to probe the coupling distance, care must be taken regarding the presynaptic Ca2+ entry duration as well as the property of the vesicular Ca2+ sensor.
Highlights
The release of neurotransmitter is triggered by an increase in the intracellular Ca2+ concentration ([Ca2+]i) in the presynaptic nerve terminal (Katz, 1969)
[Ca2+]i = iCa/(4π · F · DCa · r) where iCa is the amplitude of the constant Ca2+ current of a single voltage-gated calcium channels (VGCCs), F is the Faraday constant, and DCa is the diffusion coefficient of free Ca2+ (Neher, 1986)
Because several Ca2+ buffers are present in the presynaptic terminal, a calcium ion often binds to a buffer molecule before it reaches vesicular Ca2+ sensors; Ca2+ buffers dampen the [Ca2+]i
Summary
The release of neurotransmitter is triggered by an increase in the intracellular Ca2+ concentration ([Ca2+]i) in the presynaptic nerve terminal (Katz, 1969). The probability of vesicular release, i.e., the output from the presynaptic nerve terminal, is determined by the combination of Ca2+ influx, the properties of Ca2+ buffers and Ca2+ sensors, and the distance between VGCCs and Ca2+ sensors This coupling distance between VGCCs and Ca2+ sensors is an important determinant of the [Ca2+]i sensed by the Ca2+ sensor, because the spatial concentration gradient of Ca2+ formed around the open channel is steep. This gradient was first postulated using a mathematical model of Ca2+ diffusion, referred to as the “Ca2+ microdomain” (Chad and Eckert, 1984; Fogelson and Zucker, 1985), and later experimentally observed in the squid giant presynaptic terminal (Llinás et al, 1992) and the frog neuromuscular junction (DiGregorio et al, 1999)
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