The Role of Certain Phospho-Proteins in Regulating the Mode of Synaptic Vesicle Exocytosis

Abstract

The presynaptic release of neurotransmitter requires the exocytosis of synaptic vesicles (SVs). There are three pools of such vesicles: the readily releasable pool (RRP), the reserve pool (RP) and the resting pool (RtP). Rat brain cerebral cortical synaptosomes were utilised in the presence of 5 mM extracellular Ca2+, and various chemical stimuli have been established to release the RRP (1 mM 4-aminopyridine, 4AP5C) or the RRP and the RP (raised potassium [30 mM, HK5C] or ionomycin [5 µM, ION5C]) of glutamate (Glu) containing SVs.
Exocytosis of SVs involves the formation of a fusion pore between the vesicular membrane and the plasma membrane (PM) at the active zone (AZ), and once this is formed Glu can immediately exit the lumen of the vesicle and enter the extracellular space. The vesicle itself undergoes one of two processes: (i) full fusion (FF) whereby the fusion pore expands, and the vesicular membrane fully inserts into the PM with the vesicle being subsequently recycled by clathrin dependent endocytosis at the peri-active zone; (ii) kiss-and-run (KR) whereby the fusion pore closes < 0.5 s after opening. The contribution of each of these distinct pathways in normal transmission is still a matter of debate and in particular not much is known about the biochemical mechanisms underlying KR mode.
Reported herein, the role of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) in regulating the exocytosis of the SV pools and their mode was investigated. The kinase PKA was studied using a specific inhibitor, KT5720 and a specific activator, Sp-5,6-DCl-cBIMPS (cBIMPS). It was observed that PKA inhibition did not interfere with the exocytosis of the RRP or RP but switched the RRP SVs to an FF mode for the dynamin dependent KR mechanism but not for the NMII dependent mechanism. Conversely, PKA activation had no effect on the Glu release from the SV pools but switched some of the RP SVs to a KR mechanism. Pre-treatment with the drug MiTMAB – which prevents dynamin binding to lipid membranes – did not perturb the dynamin dependent KR mode suggesting that the dynamin is already associated with the membrane(s) prior to the application of the stimulus or the drug. This may indicate that only a proportion of the total terminal dynamin contributes to the KR mechanism, which is associated with the PM.
The role of cAMP itself was studied by activating adenylate cyclase (AC) with forskolin or inhibiting this enzyme with 9-cyclopentyl-adenine monomethanesulfonate (9-cp-ade). The inhibitor 9-cp-ade had no effect on any stimulated release of the pools of SVs nor did it affect the mode of exocytosis. Activation of AC had two distinct effects: (i) the release of the RP of SVs was inhibited; (ii) the 4AP5C evoked RRP SVs which underwent FF mode were switched to KR mode. Forskolin increased change in intracellular Ca2+ with 4AP5C stimulation, which may have caused the RRP to switch to KR mode with 4AP5C; however, change in intracellular Ca2+ decreased with HK5C stimulation, which may have caused the loss of RP release. The forskolin induced inhibition of RP SV release was due to AC activation since this was prevented when synaptosomes were pre-treated with 9-cp-ade, but the cAMP was not acting on PKA as direct PKA activation did not inhibit the RP SV release. The cAMP produced was working on exchange proteins directly activated by cAMP (EPAC), evidenced as specific inhibitor of EPACs ESI-09 prevented the action of forskolin such that the RP of SVs could be released by HK5C.
The role of actin cytoskeleton in the release of the pools of SVs and the mode of exocytosis was studied. Intriguingly, stabilising microfilaments with jasplakinolide did not perturb the release of the RRP or RP with any of the stimuli and neither did it affect the mode of exocytosis. Clearly, the first round of SV exocytosis does not need active reformation of actin microfilaments as microfilament stabilisation of does not affect SV release dynamics. However, disassembly of microfilaments with latrunculin A did have major effects: (i) it inhibited the release of the RP of SVs induced by either ION5C or HK5C; (ii) it switched the mode of exocytosis of the RRP to FF mode. Both the dynamin dependent and the NMII dependent KR mode of the RRP have been shown to require an intact actin cytoskeleton.
These results again emphasise that SVs can undergo both KR and FF mode and that these processes are regulated by PKA, EPAC and the cytoskeleton. Future experiments will aim to find relationships between these enzymes and other enzymes such as PKC and see if they are related to the microfilaments. Such knowledge may also help to ascertain whether there are specific phosphorylation sites on dynamin and NMII that control KR mode. Results with MiTMAB appear to highlight that for dynamin the phosphorylation change may only occur on a very minor population of the total dynamin.