Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis.

Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC50 ~ 15 µM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC50 = 479 µM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37-fold improvement in potency over dynasore for liposome-stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36-fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin-dependent endocytosis of transferrin in multiple cell types (IC50 of 5.7 and 5.8 µM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin-independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity-dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non-specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin-mediated endocytosis.

Isoflurane inhibits transmitter release and the presynaptic action potential.

BACKGROUND: Isoflurane inhibits the excitatory postsynaptic current (EPSC) at many synapses. Accumulated evidence suggests the involvement of a presynaptic mechanism. However, the extent of the presynaptic contribution has not been quantitatively studied. Furthermore, the mechanism underlying the presynaptic contribution remains unclear. METHODS: To estimate the presynaptic contribution, the authors compared the effects of isoflurane on the presynaptic capacitance jump, which is proportional to vesicle release, and the postsynaptic glutamate receptor-mediated EPSC at a calyx-type synapse in rat brainstem. The authors determined whether isoflurane affects the waveform of the action potential recorded from nerve terminals. By studying the relation between the EPSC and the presynaptic action potential at the same synapse, the authors determined whether isoflurane inhibits the EPSC by decreasing the presynaptic action potential. RESULTS: Isoflurane at 0.35-1.05 mM reduced the EPSC and the presynaptic capacitance jump to a similar degree without affecting the miniature EPSC (an indicator of quantal size), suggesting that isoflurane inhibits the EPSC predominantly by reducing glutamate release. Isoflurane reduced the presynaptic action potential by approximately 3-8%. The EPSC was proportional to the presynaptic action potential amplitude raised to a power of 10.2. Based on this relation, inhibition of the presynaptic action potential contributed to 62-78% of isoflurane-induced inhibition of the EPSC. CONCLUSIONS: Isoflurane inhibits the EPSC predominantly by inhibition of transmitter release. Isoflurane reduces the presynaptic action potential amplitude, which may contribute significantly to its inhibitory effect on the EPSC.

Capacitance measurements at the calyx of Held in the medial nucleus of the trapezoid body.

We have recently applied Lindau-Neher's capacitance measurement technique to study vesicle trafficking at the calyx-type synapse in the rat medial nucleus of the trapezoid body (MNTB) in slice conditions. This application made the MNTB synapse an excellent model for the study of exocytosis and endocytosis at conventional active zones. However, the application was only made at calyces that are presumably equivalent to a single-compartment circuit because their passive current transients decayed mono-exponentially. Here, we determined whether the application could be extended to majority of calyces whose passive current transients decayed bi-exponentially. By comparison of calyces with mono- or bi-exponential decay in their passive current transients, we found similar properties in respect to: (1) the capacitance jump induced by trains of action-potential equivalent stimuli, which reflects exocytosis; (2) the size of a releasable vesicle pool; (3) the time course of the decay after the capacitance jump, which reflects endocytosis; and (4) the transient capacitance artifact observed in the presence of Cd(2+) that blocks exocytosis. These similar properties were also obtained from modeling calyces as a single- or two-compartment circuit. Thus, capacitance measurements may be extended to the majority of calyces, which may facilitate the study of rapid vesicle trafficking at conventional active zones.

Single and multiple vesicle fusion induce different rates of endocytosis at a central synapse.

During synaptic transmission, neurotransmitter-laden vesicles fuse with the presynaptic membrane and discharge their contents into the synaptic cleft. After fusion, the vesicular membrane is retrieved by endocytosis for reuse. This recycling mechanism ensures a constant supply of releasable vesicles at the nerve terminal. The kinetics of endocytosis have been measured mostly after intense or non-physiological stimulation. Here we use capacitance measurements to resolve the fusion and retrieval of single and multiple vesicles following mild physiological stimulation at a mammalian central synapse. The time constant of endocytosis after single vesicle fusion was 56 ms; after a single action potential or trains at < or = 2 Hz it was about 115 ms, but increased gradually to tens of seconds as the frequency and the number of action potentials increased. These results indicate that an increase in the rate of exocytosis at the active zone induces a decrease in the rate of endocytosis. Existing models, including inhibition of endocytosis by Ca(2+), could not account for these results our results suggest that an accumulation of unretrieved vesicles at the plasma membrane slows endocytosis. These findings may resolve the debate about the dependence of endocytosis kinetics on the stimulation frequency, and suggest a potential role of regulation of endocytosis in short-term synaptic depression.