Myosin Light Chain Kinase Is Not a Regulator of Synaptic Vesicle Trafficking During Repetitive Exocytosis in Cultured Hippocampal Neurons

J Neurosci. 2006 Nov 8;26(45):11606-14. doi: 10.1523/JNEUROSCI.3400-06.2006.

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials / drug effects
  • Action Potentials / physiology
  • Action Potentials / radiation effects
  • Analysis of Variance
  • Animals
  • Animals, Newborn
  • Axons / metabolism
  • Calcium / metabolism
  • Calcium / pharmacology
  • Cells, Cultured
  • Dose-Response Relationship, Radiation
  • Electric Stimulation / methods
  • Enzyme Inhibitors / pharmacology
  • Excitatory Postsynaptic Potentials / drug effects
  • Excitatory Postsynaptic Potentials / physiology
  • Excitatory Postsynaptic Potentials / radiation effects
  • Exocytosis / drug effects
  • Exocytosis / physiology*
  • Exocytosis / radiation effects
  • Hippocampus / cytology*
  • Immunohistochemistry / methods
  • Myosin-Light-Chain Kinase / physiology*
  • Neurofilament Proteins / metabolism
  • Neurons / cytology
  • Neurons / drug effects
  • Neurons / physiology*
  • Patch-Clamp Techniques / methods
  • Pyridinium Compounds / metabolism
  • Quaternary Ammonium Compounds / metabolism
  • Rats
  • Synaptic Vesicles / drug effects
  • Synaptic Vesicles / physiology*

Substances

  • Enzyme Inhibitors
  • FM1 43
  • Neurofilament Proteins
  • Pyridinium Compounds
  • Quaternary Ammonium Compounds
  • neurofilament protein H
  • Myosin-Light-Chain Kinase
  • Calcium