Importance of Full-Collapse Vesicle Exocytosis for Synaptic Fatigue-Resistance at Rat Fast and Slow Muscle Neuromuscular Junctions.

Neurotransmitter release during trains of activity usually involves two vesicle pools (readily releasable pool, or RRP, and reserve pool, or RP) and two exocytosis mechanisms ("full-collapse" and "kiss-and-run"). However, synaptic terminals are adapted to differing patterns of use and the relationship of these factors to enabling terminals to adapt to differing transmitter release demands is not clear. We have therefore tested their contribution to a terminal's ability to maintain release, or synaptic fatiguability in motor terminals innervating fast-twitch (fatiguable), and postural slow-twitch (fatigue-resistant) muscles. We used electrophysiological recording of neurotransmission and fluorescent dye markers of vesicle recycling to compare the effects of kinase inhibitors of varying myosin light chain kinase (MLCK) selectivity (staurosporine, wortmannin, LY294002 & ML-9) on vesicle pools, exocytosis mechanisms, and sustained neurotransmitter release, using postural-type activity train (20 Hz for 10 min) in these muscles. In both muscles, a small, rapidly depleted vesicle pool (the RRP) was inhibitor insensitive, continuing to release FM1-43, which is a marker of full-collapse exocytosis. MLCK-inhibiting kinases blocked all remaining FM1-43 loss from labelled vesicles. However, FM2-10 release only slowed, indicating continuing kiss-and-run exocytosis. Despite this, kinase inhibitors did not affect transmitter release fatiguability under normal conditions. However, augmenting release in high Ca2+ entirely blocked the synaptic fatigue-resistance of terminals in slow-twitch muscles. Thus, full-collapse exocytosis from most vesicles (the RP) is not essential for maintaining release during a single prolonged train. However, it becomes critical in fatigue-resistant terminals during high vesicle demand.

Site-directed mutagenesis studies on the Drosophila octopamine/tyramine receptor.

The cloned Drosophila octopamine/tyramine receptor can be coupled to second messenger pathways in an agonist-specific fashion by the endogenously occurring biogenic amines, octopamine and tyramine, when expressed in Chinese hamster ovary cells. We have mutated to alanine a range of receptor amino acids that could potentially form hydrogen bonds with the beta-hydroxyl group of octopamine based on homologies with alpha- and beta-adrenergic receptor subtypes. After stable expression of the mutant receptors in CHO cells we have compared the ability of octopamine and tyramine to displace [(3)H]yohimbine binding to membrane fractions from the mutant cell lines with their ability to modulate adenylyl cyclase activity in intact cells. The results suggest that none of the mutated amino acids residues, at least in isolation, are likely to be involved in interactions with the beta-hydroxyl group of the octopamine side chain. It is possible that amino acids not mutated in the present study are somehow involved in this interaction. Alternatively, it is also possible that the beta-hydroxyl group of the octopamine side chain is capable of interacting with more than one of the amino acids mutated in the present study.