Myosin light chain kinase is not a regulator of synaptic vesicle trafficking during repetitive exocytosis in cultured hippocampal neurons.
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.
Tokuoka
Hirofumi
H
Medical Research Council Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, London WC1E 6BT, United Kingdom.
Goda
Yukiko
Y
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*
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