Figure 5. About half of the phorbol ester potentiation of evoked and spontaneous EPSC depends on PKC phosphorylation of Munc18-1.

(A and B) Evoked EPSC traces (top) and spontaneous EPSCs (middle) are shown both before (left) and after (right) application of 1 µM PDBu to the slice. The bottom panels show time plots of evoked EPSC amplitudes and their potentiation by PDBu. Data for a M18^WT rescued synapse (A) and for a M18^SA rescued synapse (B) are shown. (C) Average time courses of normalized EPSC amplitudes during PDBu potentiation for synapses rescued with M18^SA (red symbols) and M18^WT (black, n = 7 and n = 6 cells, respectively). (D and E) Quantifications of the average and individual values for EPSC potentiation (D, Figure 5—source data 1A) and for the baseline EPSC amplitudes (E, Figure 5—source data 1B) in synapses rescued with M18^SA (red) and with M18^WT (black). Note that ∼half of the potentiation of evoked EPSC amplitudes depended on an intact Munc18-1 phosphorylation site. (F) Average time courses of normalized spontaneous EPSC frequency before and after PDBu application, both for synapses rescued with Munc18^SA (red symbols) and with M18^WT (black; n = 4 and 6, respectively). (G) Quantification of absolute mEPSC frequencies before and after PDBu application, for synapses rescued with M18^WT and M18^SA. For the M18^SA data, an outlier data point with an unusually high baseline frequency (4 Hz; pink symbols) was removed when calculating the average absolute mEPSC frequencies (see Figure5—source data 1C). (H) Average relative potentiation of mEPSC frequency under both conditions. Note that about half of the potentiation of spontaneous release depends on the PKC phosphorylation of Munc18-1 (see Figure5—source data 1D).

DOI: http://dx.doi.org/10.7554/eLife.01715.017

Figure 5—figure supplement 1. Model of Munc18-1 PKC phosphorylation and de-phosphorylation and its effect on presynaptic plasticity.

Following elevation of residual Ca²⁺, conventional PKC will be activated, and phosphorylate Munc18-1. Munc18-1 is de-phosphorylated during the decay of PTP by a phosphatase (PP). We assume that Munc18-1 is bound to the partially formed SNARE complex of docked and readily releasable vesicles, and that PKC phosphorylation of Munc18-1 represents a switch towards a higher release probability of readily releasable vesicles. Note that phorbol esters, which are DAG analogues, activate a wider range of presynaptic C1 domain proteins including PKC and Munc13-1. See also ‘Discussion’.

DOI: http://dx.doi.org/10.7554/eLife.01715.019