Rapid calcium-dependent exocytosis underlies neurotransmitter release from nerve terminals. Despite the fundamental importance of this process, neither the relationship between presynaptic intracellular calcium ion concentration ([Ca2+]i) and rate of exocytosis, nor the maximal rate of secretion is known quantitatively. To provide this information, we have used flash photolysis of caged Ca2+ to elevate [Ca2+]i rapidly and uniformly in synaptic terminals, while measuring membrane capacitance as an index of exocytosis and monitoring [Ca2+]i with a Ca(2+)-indicator dye. When [Ca2+]i was abruptly increased to > 10 microM, capacitance rose at a rate that increased steeply with [Ca2+]i. The steepness suggested that at least four calcium ions must bind to activate synaptic vesicle fusion. Half-saturation was at 194 microM, and the maximal rate constant was 2,000-3,000 s-1. A given synaptic vesicle can exocytose with high probability within a few hundred microseconds, if [Ca2+]i rises above 100 microM. These properties provide for the extremely rapid signalling required for neuronal communication.

The kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2+ were studied by capacitance (Cm) measurements with millisecond time resolution. After elevation of the internal Ca2+ concentration ([Ca2+]i), Cm rises rapidly with one or more exponentials. The time constant of the fastest component decreases for higher [Ca2+]i (range 3-600 microM) over three orders of magnitude before it saturates at approximately 1 ms. The corresponding maximal rates of secretion can be as fast as 100,000 fF/s or 40,000 vesicles/s. There is a Ca(2+)-dependent delay before Cm rises, which may reflect the kinetics of multiple Ca2+ ions binding to the secretory apparatus. The initial rise in Cm is described by models containing a sequence of two to four single Ca(2+)-binding steps followed by a rate-limiting exocytosis step. The predicted Ca2+ dissociation constant (Kd) of a single Ca(2+)-binding site is between 7 and 21 microM. At [Ca2+]i > 30 microM clear indications of a fast endocytotic process complicate the analysis of the secretory response.

Thermodynamic, structural, and magnetic criteria, the properties of the charge distributions, and low-energy ionization processes are theoretically analyzed to learn about the role of π-electron delocalization in recently synthesized stable singlet carbenes (Arduengo et al. J. Am. Chem. Soc. 1991, 113, 361) and silylenes (Denk et al. J. Am. Chem. Soc. 1994, 116, 2691) of the imidazol-2-ylidene type and also in related model systems. The different approaches show consistently that cyclic electron delocalization does indeed occur in the CC unsaturated imidazol-2-ylidene systems, in particular with respect to the corresponding C−C saturated imidazolin-2-ylidenes. However, the conclusion regarding the degree of conjugation and aromaticity depends on the criteria used, being quite small according to the “atoms-in-molecules” charge analysis but more significant according to the energetic and the magnetic properties. According to all criteria, the aromatic character of imidazol-2-ylidenes is less pronounced compared to benzene or the imidazolium cation. π-Electron resonance is found to be less extensive in the silylenes compared to their carbene analogs.

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