Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene targeted mice

Abstract

The voltage-gated sodium channel NaV1.7 plays a critical role in pain pathways. Besides action potential propagation, NaV1.7 regulates neurotransmitter release, integrates depolarizing inputs over long periods and regulates transcription. In order to better understand these functions, we generated an epitope-tagged NaV1.7 mouse that showed normal pain behavior. Analysis of NaV1.7 complexes affinity-purified under native conditions by mass spectrometry revealed 267 NaV1.7 associated proteins including known interactors, such as the sodium channel {beta}3 subunit (Scn3b) and collapsin response mediator protein (Crmp2), and novel interactors. Selected novel NaV1.7 protein interactors membrane-trafficking protein synapototagmin-2 (Syt2), G protein-regulated inducer of neurite outgrowth 1 (Gprin1), L-type amino acid transporter 1 (Lat1) and transmembrane P24 trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated using co-immunoprecipitation and functional assays. The information provided with this physiologically normal epitope-tagged mouse should provide useful insights into the pain mechanisms associated with NaV1.7 channel function.