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Chad Grabner, Steven H DeVries; Fast fusion kinetics of primed vesicles at a mammalian cone photoreceptor synapse. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4526.
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© ARVO (1962-2015); The Authors (2016-present)
Cone photoreceptor ribbon synapses signal the end of a light step by releasing a burst of transmitter into the synaptic cleft. The timing and synchronicity of release determine the peak cleft glutamate concentration and hence influence the amplitude of the postsynaptic response; however, postsynaptic responses are subject to non-linearities. Membrane capacitance measurements provide a direct readout of vesicle fusion, and such measurements have not been obtained at the mammalian cone synapse.
Recordings were obtained from ground squirrel (Ictidomys tridecemlineatus) retina. Membrane capacitance (Cm) was measured in whole-cell voltage clamp using the ‘sine+dc’ routine and a HEKA EPC-10 amplifier. Cells were held at -70 mV, excepted during stimulation. Patch electrodes contained a CsCl based solution with 10 mM EGTA. Slices were bathed in a bicarbonate buffered saline (5% CO2) supplemented with TBOA (200 µM) and CsCl (5 mM).
Release kinetics were examined by varying stimulus duration over a range of 1-30 ms and stepped to -10 mV. The change in Cm at 1 and 30 ms differed by only 2-fold (11.4 ± 1.7 vs. 22.6 ± 1.8 fF; n = 5 cells), and the plot of Cm over pulse duration was well-fit by a double exponential with time constants of 0.9 ms (16 fF ~290 vesicles) and 10 ms (7 fF ~127 vesicles). From a separate set of cells the relationship between Cm and membrane voltage levels was explored with a family of steps between -50 and 30 mV, given for 1 or 30 ms. Release reached a maximum at -10 mV for both short and long steps, the release function was bell-shaped, which suggests the profile of Ca2+ entry greatly influences the apparent vesicle pool size. HEPES (15 mM), which blocks inhibitory proton feedback onto cone voltage-dependent Ca2+ channels, was able to shift release to the first kinetic phase.
The combined size of the two fast pools equals or slightly exceeds the size of the vesicle pool that is estimated to be membrane docked at a cone’s ~20 ribbons. The bulk of transmitter is released at a very high rate upon depolarization, but there also seems to be a slower component that may be a consequence of the retarding effect of proton feedback.
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