Houeland Gry, Nakhost Arash, Sossin Wayne S, Castellucci Vincent F
Department of Physiology, University of Montreal, Montreal, Quebec, Canada.
J Neurophysiol. 2007 Jan;97(1):134-43. doi: 10.1152/jn.00122.2006. Epub 2006 Sep 13.
Activation of phosphokinase C (PKC) can increase transmitter release at sensory-motor neuron synapses in Aplysia, but the target of PKC phosphorylation has not been determined. One putative target of PKC at synapses is the synaptosomal-associated protein of 25 kDa (SNAP-25), a member of the SNARE protein complex implicated in synaptic vesicle docking and fusion. To determine whether PKC regulated transmitter release through phosphorylation of SNAP-25, we cloned Aplysia SNAP-25 and expressed enhanced green fluorescent protein (EGFP)-coupled SNAP-25 constructs mutated at the PKC phosphorylation site Ser198 in Aplysia sensory neurons. We found several distinct effects of expression of EGFP-SNAP-25 constructs. First, the rates of synaptic depression were slowed when cells contained SNAP-25 with phosphomimetic residues Glu or Asp. Second, PDBu-mediated increases in transmitter release at naïve synapses were blocked in cells expressing nonphosphorylated-state SNAP-25. Finally, expression of EGFP-coupled SNAP-25 but not uncoupled SNAP-25 inhibited 5-HT-mediated reversal of depression and the ability of EGFP-coupled SNAP-25 to inhibit the reversal of depression was affected by changes at Ser198. These results suggest SNAP-25 and phosphorylation of SNAP-25 by PKC can regulate transmitter release at Aplysia sensory-motor neuron synapses by a number of distinct processes.
蛋白激酶C（PKC）的激活可增加海兔感觉运动神经元突触处的神经递质释放，但PKC磷酸化的靶点尚未确定。突触处PKC的一个假定靶点是25 kDa的突触体相关蛋白（SNAP - 25），它是SNARE蛋白复合体的成员，与突触小泡的对接和融合有关。为了确定PKC是否通过SNAP - 25的磷酸化来调节神经递质释放，我们克隆了海兔SNAP - 25，并在海兔感觉神经元中表达了在PKC磷酸化位点Ser198处突变的增强型绿色荧光蛋白（EGFP）偶联的SNAP - 25构建体。我们发现EGFP - SNAP - 25构建体的表达有几种不同的效应。首先，当细胞含有带有模拟磷酸化残基Glu或Asp的SNAP - 25时，突触抑制的速率减慢。其次，在表达非磷酸化状态SNAP - 25的细胞中，PDBu介导的幼稚突触处神经递质释放的增加被阻断。最后，EGFP偶联的SNAP - 25而非未偶联的SNAP - 25的表达抑制了5 - HT介导的抑郁逆转，并且EGFP偶联的SNAP - 25抑制抑郁逆转的能力受Ser198处变化的影响。这些结果表明，SNAP - 25以及PKC对SNAP - 25的磷酸化可通过多种不同过程调节海兔感觉运动神经元突触处的神经递质释放。
