Nav1.8 N-terminal-peptide Blocks p11-assisted Membrane Translocation In Primary Sensory Neurons

NaV1.8 is a predominant contributor of the tetrodotoxin-resistant (TTX-r) sodium-current in nociceptive dorsal root ganglion (DRG) neurons. Several neuropathy models have shown increased NaV1.8 immunoreactivity in the peripheral nerve fibers that exhibit increased TTX-r current. Thus, it is postulated that increased membrane translocation of NaV1.8 may be involved in the pathogenesis of neuropathic pain. The N-terminal (127 amino acids) cytoplasmic domain of NaV1.8 possesses a specific affinity to p11 annexin II subunit. This molecular association between Nav1.8 and p11 was demonstrated to promote translocation of NaV1.8 to cell membrane. OBJECTIVE: We have hypothesized that a targeted disruption of p11-assisted NaV1.8 intracellular translocation provides a potential therapeutic treatment for peripheral neuropathies. To reach this goal, we designed and evaluated a synthetic peptide competing the p11-Nav1.8 association. METHODS: We constructed a lentivirus-based expression vector carrying 3XFLAG-NaV1.8 N-terminal [1-127] fusion peptide with IRES-GFP. The specific interaction between Nav1.8 [1-127] peptide and p11 was examined by immunoprecipitation pull-down experiment using lentivirus-transduced rat DRG neurons. The degree of Nav1.8 membrane translocation was examined by immunocytology of DRG neurons treated with Nav [1-127] peptide. RESULTS: Using the fluorescence-activated cell sorting assay (FACS), GFP-positive lentivirus-transduced cells were assessed to be 80% of DRG cell culture. Western blot analysis of anti-3XFLAG antibody immunoprecipitate revealed the presence of both Nav [1-127] peptide and p11 suggesting their molecular interaction. Immunocytological analysis using anti-NaV1.8 antibody showed that the membrane distribution of endogenous NaV1.8 was decreased in GFP+ and IB4+ DRG neurons. CONCLUSIONS: These data indicate that the p11-assisted NaV1.8 membrane translocation may be effectively reduced with the NaV1.8 N-terminal synthetic peptide. This novel strategy of targeting the p11-NaV1.8 binding may potentially be useful in alleviating neuropathic pain symptoms. Supported in part by NIH C06RR14529 and R21NS049137.