Probing the Endogenous Ca²⁺Buffers at the Presynaptic Terminals of the Crayfish Neuromuscular Junction

Abstract

Ca2+ indicators of varying affinity and mobility were pressure injected into the presynaptic axon of the inhibitor of the crayfish neuromuscular junction (NMJ). Fluorescence transients recorded at a 2-kHz resolution were used to probe physiological parameters governing the decay of fluorescence transients within 100 ms after an action potential (early decay). Blocking Ca2+ extrusion or Ca2+ sequestration processes did not significantly alter early decay, arguing against a role for either mechanism. Fluorescence transients recorded with low mobility or fixed indicators exhibited early decay similar to that recorded with indicators of comparable affinity but high mobility, suggesting that early decay was not due to the rate of Ca2+-indicator diffusion. The extent of early decay correlated closely with the affinity, but not mobility, of the Ca2+ sensitive dyes tested. These results implicate intrinsic buffers with slow Ca2+ binding kinetics as the most likely determinants of early decay. However, computer simulations showed that intrinsic buffers with a slow binding rate are unlikely to be the only ones present in the system because the slow kinetics would be unable to buffer incoming Ca2+ during an action potential and would result in momentary indicator saturation. In fact, experimental data show that the peak amplitude of an action potential activated Ca+ transient is about 20% of the maximal fluorescence intensity activated by prolonged Ca2+ influx. We conclude that endogenous buffering at the crayfish NMJ includes both fast and slow components, the former being fast enough to compete with fast Ca2+ indicators, and the latter dictating the early decay.

Modeling of quantal neurotransmitter release kinetics in the presence of fixed and mobile calcium buffers

“…Endogenous Ca 2+ buffers play a principal role in evoked quantal release (Sala and Hernandez-Cruz 1990;Nowycky and Pinter 1993;Lin et al 2005). As already stated, after the arrival of the action potential and entry of Ca 2+ through the voltage-gated channels, more than 95% of the Ca 2+ ions are immediately bound to the buffers within a distance of 10-50 nm from the point of Ca 2+ entry (Augustine and Neher 1992a;Neher 1995).…”

“…No active transport of Ca 2+ across the membrane is postulated, as according to the data of Lin et al (2005)blocking Ca 2+ extrusion or Ca 2+ sequestration processes did not significantly alter the kinetics of Ca 2+ concentration changes, arguing against a role for either mechanism. This made it possible to consider the diffusion process in the active zone to be a homogeneous diffusion due to the spherical symmetry of the region.…”

A General Model of Synaptic Transmission and Short-Term Plasticity

“…In another model (Matveev et al, 2002), we allowed Ca 2+ to act on one target to facilitate release with two components that arose from Ca 2+ diffusion characteristics. But this model required immobile buffers with fast kinetics that are inconsistent with data on presynaptic buffer properties (Lin et al, 2005; V. Matveev and J.-W. Lin, personal communication) and required a high local tortuosity severely restricting Ca 2+ diffusion and an unrealistic fura-2 immobilization.…”

“…In contrast to approaches in which model parameters were relatively unconstrained, we began with extensive data that impose stringent constraints on endogenous buffers and pumps (Lin et al, 2005; V. Matveev and J.-W. Lin, personal communication) and chose unvarying parameters consistent with those constraints. We used a realistic model of synaptotagmin, separating Ca 2+ binding to C2A and C2B domains and choosing parameters similar to those used by others and agreeing with kinetic biochemical data (Bollmann et al, 2000; Chapman, 2008; Schneggenburger and Neher, 2000).…”

“…Analysis of presynaptic Ca 2+ transients reveals a slowly binding buffer, B s , and a fast equilibrating buffer, B f (Lin et al, 2005; V. Matveev and J.-W. Lin, personal communication). Our simulations used values for total amounts ( B s total and B f total ), on rates ( k s on and k f on ), and affinities ( K D, s and K D, f ) that are in the middle of ranges inferred experimentally (Table 1).…”