Mobilization of ACh quanta in snake motor nerve terminals is depressed by elevated [Ca2+]i

	Mobilization of ACh quanta in snake motor nerve terminals is depressed by elevated [Ca²⁺]_i
Research field:	Other
Authors:	Parsons RL, Calupca MA, Merriam LA
Address of presenting author:	Department of Anatomy and Neurobiology University of Vermont College of Medicine C427 Given Building Burlington, Vermont 05405 USA
E-mail:	rparsons@zoo.uvm.edu
Phone:	802 656-2230
Fax:	802 656-8704
Text of abstract	Introduction During repetitive presynaptic stimulation at the neuromuscular junction, synaptic vesicles are mobilized from a reserve pool to the readily releasable pool and join with recycled, refilled synaptic vesicles to replace those vesicles lost from release sites during exocytosis (Van der Kloot & Molgo, 1994). Vesicle mobilization and recycling allow the nerve terminal to maintain the required high quantum content of endplate currents with prolonged stimulation. Mechanisms regulating vesicle mobilization are not well defined. The present study investigated, in vesamicol-treated snake twitch motoneuron terminals, whether sustained elevation of the intracellular calcium concentration ([Ca²⁺]_i) affected mobilization of quanta from the reserve pool to the readily releasable pool during prolonged K⁺-stimulation. Methods Experiments were done in vitro on isolated, vesamicol (5 mM)-treated custocutaneous nerve-muscle preparations dissected from garter snakes following rapid decapitation. Vesamicol blocks the vesicular ACh transporter (Parsons et al., 1993). In vesamicol (5 mM), MEPCs represent currents generated by ACh from preformed quanta and not from recycling vesicles. We determined MEPC frequencies, recorded from endplates voltage clamped to -150 mV, as a function of time in vesamicol-treated, nerve-muscle preparations challenged with elevated K⁺ (35 mM) (Parsons et al., 1999). Results MEPC frequency increased markedly during the initial period of K⁺ stimulation, but then declined progressively. The number of quanta released during exposure to 35 K⁺ was estimated by integrating the MEPC frequency versus time relationship. With control conditions (external solution: 1 mM Ca²⁺ and 4.2 mM Mg²⁺) ~500,000 quanta were released with a time constant of 15 minutes. With external Ca²⁺ raised to 3.6 mM and Mg²⁺ omitted, ~216,000 quanta were released with a time constant of 6 minutes. Positive FM1-43 staining of motor nerve terminals still occurred at times when MEPC frequency was essentially eliminated indicating marked endocytosis continued (Betz & Bewick, 1992). In the absence of vesamicol, after 34 min in 35 K⁺ with 3.6 Ca²⁺, the MEPC frequency was 242 ± 14 sec^-¹ indicating exocytosis continued. Conclusions We suggest that a sustained elevation of [Ca²⁺]_i depressed mobilization of quanta from the reserve pool to the readily releasable pool during prolonged K⁺ stimulation. References Betz, W. & Bewick, G.S. 1992. Science 255, 48-54. Parsons, R.L., Calupca, M.A., Merriam, L.A. & Prior, C. 1999. J. Neurophysiol. 81, 2696-2700. Parsons, S.M., Prior, C. & Marshall, I.G. 1993. Int. Rev. Neurobiol. 35, 279-390. Van der Kloot, W. & Molgo, J. 1994. Physiol. Rev. 74, 899-991.
Keywords:	Presynaptic mechanisms, Neurotransmitter mobilization, Motor nerve terminals, Neuromuscular junction

Created 2000-04-27