The CaV2\u03b11 EF-hand F helix tyrosine, a highly conserved locus for GPCR inhibition of CaV2 channels

Tyler W Dunn,Wayne S Sossin,Philippe Séguéla,Xiaotang Fan,Ariel R Ase

doi:10.1038/s41598-018-21586-5

Tyler W Dunn, Wayne S Sossin + Show 3 more

Open Access

https://doi.org/10.1038/s41598-018-21586-5

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Abstract

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 CaV2, consisting of the pore forming α1 subunit (CaV2α1), and auxiliary CaVβ, and CaVα2δ subunits. To determine the role of the CaV2 channel in presynaptic plasticity in Aplysia, we cloned Aplysia CaV2α1, CaVβ, and CaVα2δ and over-expressed the proteins in Aplysia sensory neurons (SN). We show expression of exogenous CaV2α1 in the neurites of cultured Aplysia SN. One proposed mechanism for heterosynaptic depression in Aplysia is through inhibition of CaV2. Here, we demonstrate that heterosynaptic depression of the CaV2 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 CaV2 channels with recombinant channels allowing future examination of the structure function relationship of CaV2 in the regulation of transmitter release in this system.

Highlights

Conserved, found in most CaV2 sequences, and point mutation of the Y1747 ortholog to phenylalanine in the Aplysia CaV2α1 (Y1501F) reduces the inhibition of the calcium transient with dopamine and 5HT1A activation measured with fluorescence imaging
CaV2α1 Y1501F expression in presynaptic sensory neurons reduced the heterosynaptic depression at sensory to motor neuron synapses with 5HT1A activation
We have cloned the pore forming subunit of the CaV2 calcium channel from Aplysia californica (CaV2 alpha 1 subunit- CaV2α1) using primers designed from searching the Aplysia transcriptosome for sequences with homology to the cloned Lymnaea CaV2 channel

Summary

Introduction

We demonstrate that heterosynaptic depression of the CaV2 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 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 demonstrate our ability to replace endogenous CaV2 channels with recombinant channels allowing future examination of the structure function relationship of CaV2 in the regulation of transmitter release in this system. CaV2α1 Y1501F expression in presynaptic sensory neurons reduced the heterosynaptic depression at sensory to motor neuron synapses with 5HT1A activation This indicates that the VI-inhibition of the CaV2 current through Src kinase phosphorylation of the F-helix EF-hand of CaV2α1 is a physiologically important and highly conserved mechanism of CaV2 regulation. The block of the inhibition with Y1501F expression appears to be complete at 48 h, indicating near complete substitution of the endogenous alpha 1 subunits with recombinant subunits at this time point

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