channel mobility : effects of 1 cholesterol depletion on release at the cone ribbon synapse

18 Synaptic communication requires proper coupling between voltage-gated calcium (CaV) 19 channels and synaptic vesicles. In photoreceptors, L-type CaV channels are clustered close to 20 synaptic ribbon release sites. Although clustered, CaV channels move continuously within a 21 confined domain slightly larger than the base of the ribbon. We hypothesized that expanding 22 CaV channel confinement domains should increase the number of channel openings needed to 23 trigger vesicle release. Using single particle tracking techniques, we measured the expansion 24 of CaV channel confinement domains caused by depletion of membrane cholesterol with 25 cholesterol oxidase or methyl-β-cycylodextrin. With paired whole cell recordings from cones 26 and horizontal cells, we then determined the number of CaV channel openings contributing to 27 cone CaV currents (ICa) and the number of vesicle fusion events contributing to horizontal cell 28 excitatory post-synaptic currents (EPSCs) following cholesterol depletion. Expansion of CaV 29 channel confinement domains reduced the peak efficiency of release, decreasing the number 30 of vesicle fusion events accompanying opening of each CaV channel. Cholesterol depletion 31 also inhibited exocytotic capacitance increases evoked by brief depolarizing steps. Changes in 32 efficiency were not due to changes in ICa amplitude or glutamate receptor properties. 33 Replenishing cholesterol restored CaV channel domain size and release efficiency to control 34 levels. These results indicate that cholesterol is important for organizing the cone active zone. 35 Furthermore, the finding that cholesterol depletion impairs coupling between channel opening 36 and vesicle release by allowing CaV channels to move further from release sites shows that 37 changes in presynaptic CaV channel mobility can be a mechanism for adjusting synaptic 38 strength. 39