The Ca(V)2α1 EF-hand F helix tyrosine, a highly conserved locus for GPCR inhibition of Ca(V)2 channels

The sensory neuron of Aplysia californica participates in several forms of presynaptic plasticity including homosynaptic depression, heterosynaptic depression, facilitation and the reversal of depression. The calcium channel triggering neurotransmitter release at most synapses is Ca(V)2, consisting of the pore forming α1 subunit (Ca(V)2α1), and auxiliary Ca(V)β, and Ca(V)α2δ subunits. To determine the role of the Ca(V)2 channel in presynaptic plasticity in Aplysia, we cloned Aplysia Ca(V)2α1, Ca(V)β, and Ca(V)α2δ and over-expressed the proteins in Aplysia sensory neurons (SN). We show expression of exogenous Ca(V)2α1 in the neurites of cultured Aplysia SN. One proposed mechanism for heterosynaptic depression in Aplysia is through inhibition of Ca(V)2. Here, we demonstrate that heterosynaptic depression of the Ca(V)2 calcium current is inhibited when a channel with a Y-F mutation at the conserved Src phosphorylation site is expressed, showing the strong conservation of this mechanism over evolution. We also show that the Y-F mutation reduces heterosynaptic inhibition of neurotransmitter release, highlighting the physiological importance of this mechanism for the regulation of synaptic efficacy. These results also demonstrate our ability to replace endogenous Ca(V)2 channels with recombinant channels allowing future examination of the structure function relationship of Ca(V)2 in the regulation of transmitter release in this system.