title: Myosin light chain kinase is not a regulator of synaptic vesicle trafficking during repetitive exocytosis in cultured hippocampal neurons.
abstract: The mechanism by which synaptic vesicles (SVs) are recruited to the release site is poorly understood. One candidate mechanism for trafficking of SVs is the myosin-actin motor system. Myosin activity is modulated by myosin light chain kinase (MLCK), which in turn is activated by calmodulin. Ca(2+) signaling in presynaptic terminals, therefore, may serve to regulate SV mobility along actin filaments via MLCK. Previous studies in different types of synapses have supported such a hypothesis. Here, we further investigated the role of MLCK in neurotransmitter release at glutamatergic synapses in cultured hippocampal neurons by examining the effects of two MLCK inhibitors, 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine.HCl (ML-7) and wortmannin. Bath application of ML-7 enhanced short-term depression of EPSCs to repetitive stimulation, whereas it reduced presynaptic release probability. However, ML-7 also inhibited action potential amplitude and voltage-gated Ca(2+) channel currents. These effects were not mimicked by wortmannin, suggesting that ML-7 was not specific to MLCK in hippocampal neurons. When SV exocytosis was directly triggered by a Ca(2+) ionophore, calcimycin, to bypass voltage-gated Ca(2+) channels, ML-7 had no effect on neurotransmitter release. Furthermore, when SV exocytosis elicited by electrical field stimulation was monitored by styryl dye, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide], the unloading kinetics of the dye was not altered in the presence of wortmannin. These data indicate that MLCK is not a major regulator of presynaptic SV trafficking during repetitive exocytosis at hippocampal synapses.
authors.affiliation: Medical Research Council Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, London WC1E 6BT, United Kingdom.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Tokuoka
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Goda
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D001369:Axons / Q000378:metabolism; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D004307:Dose-Response Relationship, Radiation; D004558:Electric Stimulation / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D006624:Hippocampus / Q000166:cytology*; D007150:Immunohistochemistry / Q000379:methods; D009219:Myosin-Light-Chain Kinase / Q000502:physiology*; D016900:Neurofilament Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D051381:Rats; D013572:Synaptic Vesicles / Q000187:drug effects / Q000502:physiology*
chemical_list: D004791:Enzyme Inhibitors; C073804:FM1 43; D016900:Neurofilament Proteins; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; C051774:neurofilament protein H; D009219:Myosin-Light-Chain Kinase; D002118:Calcium