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Changes in membrane cholesterol content can alter protein kinase activity, however, it is not known whether kinases regulating transmitter release are sensitive to membrane cholesterol content. Here we have used the cholesterol extracting agent methyl-beta-cyclodextrin to measure the effects of acute cholesterol reduction on transmitter release from cultured cerebellar neurons. Cholesterol depletion increased the frequency of spontaneous transmitter release without altering the amplitude and time course of mEPSCs. Evoked transmitter release was decreased by cholesterol extraction and the paired pulse ratio was also decreased. Alterations in synaptic transmission were not associated with failure of action potential generation or changes in presynaptic Ca(2+) signaling. Both the increase in mEPSC frequency and the change in paired pulse ratio were blocked by the broad spectrum protein kinase inhibitor staurosporine. The increase in mEPSC frequency was also sensitive to selective inhibitors of PKC and PKA. Our results therefore demonstrate that the activity of presynaptic protein kinases that regulate spontaneous and evoked neurotransmitter release is sensitive to changes of membrane cholesterol content.

Cholesterol-Dependent Kinase Activity Regulates Transmitter Release from Cerebellar Synapses