| Abstract No.: | 203 |
| Country: | Canada |
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| Title: | PHOSPHORYLATION AND PRESYNAPTIC STRENGTH |
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| Authors/Affiliations: | 1 Milton P. Charlton* and Lorelei B. Silverman-Gavrila
1 University of Toronto, Department of Physiology, ON, Canada
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| Content: | Transmitter release at high probability phasic synapses of crayfish neuromuscular junctions depresses by over 50% in 60 minutes when stimulated at 0.2 Hz. This low-frequency depression (LFD) is regulated by several protein kinases and phosphatases (Silverman-Gavrila et al., 2005 J Neurosci. 25(12):3168-80). Inhibition of presynaptic protein phosphatase 2B (calcineurin) by intracellular presynaptic injection of an impermeant calcineurin autoinhibitory peptide inhibited LFD and resulted in facilitation of transmitter release but high-frequency (20 Hz) depression was not affected. Since this inhibitor had no effect when injected into the postsynaptic cell, only presynaptic calcineurin activity
is necessary for LFD. Calcineurin is activated by Ca2+ and by calmodulin. Activation of calcineurin near Ca2+ channels where a large, brief Ca2+ signal is available might not be affected by a slow Ca2+ buffer. Indeed, buffering of residual presynaptic Ca2+ by EGTA-AM did not inhibit LFD. Therefore, calcineurin activation may be restricted to areas close to the active zone. Calcineurin can also be constitutively activated by proteolysis of the autoinhibitory domain by the Ca2+-dependent protease calpain. We found calpain immunoreactivity at crayfish neuromuscular junctions. Pharmacological inhibition of calpain with inhibitor-I or PD145305 both blocked LFD, while the inactive negative control compound PD150606 was ineffective. Therefore it is likely that calcineurin activated by presynaptic calpain may regulate LFD.
To examine changes in phosphoproteins during LFD we removed motor axons and nerve terminals after induction of LFD or treatment with various drugs that affect kinase and phosphatase activity. Proteins were extracted, separated by PAGE and stained with phospho-specific ratio stains (ProQ-Diamond/SYPRO Ruby) to identify bands for analysis by mass spectrometry. Phosphorylation of actin and tubulin was decreased during LFD; this could affect their polymerization. Actin, phosphoactin and tubulin were found in presynaptic terminals by immunocytochemistry. During LFD there was loss of phosphoactin and tubulin immunoreactivity in presynaptic boutons. The involvement of cytoskeletal elements in LFD was tested pharmacologically. The actin depolymerising drugs cytochalasin and latrunculin and microtubule stabilizer taxol inhibited LFD. Therefore, dephosphorylation of presynaptic actin and tubulin and subsequent changes in the cytoskeleton may regulate LFD. LFD is unlike long term depression (LTD) found in mammalian synapses because the latter requires postsynaptic calcineurin activity. Thus the simpler invertebrate synapse discloses a novel phosphorylation dependent presynaptic depression mechanism.
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