The recently discovered tetrodotoxin (TTX)-resistant and sensory neuron specific SNS (alternatively called the PN3 or NaV1.8) voltage gated sodium channel plays a critical role in neuropathic pain. Protein kinase A(PKA)-mediated phosphorylation of the SNS channel enhances the current possibly contributing to ectopic action potential firing and neuropathic pain. However, how phosphorylation of the SNS channel results in the modulation of channel function is not known. This study takes a multidisciplinary approach to study the detailed mechanisms responsible for the phosphorylationmediated alteration of the SNS channel function. Specifically, we will test the hypothesis that PKAinduced modulation of the SNS channel results from a charge-charge interaction between the LI-II loop and the loop-receptor on the cytoplasmic domain) of the channel protein. The study consists of 2-electrode voltage clamp characterization of SNS channels expressed in Xenopus oocytes, inside-out macropatch current and single channel study of channels expressed in HEK293 cells, and a 2-hybrid and co-immunoprecipitation study of protein-protein interaction between the LI-II loop and its putative loop receptor. Site-directed mutagenesis is used extensively to identifity the specific Ser residue(s) mediating the PKA-action. The electrostatic interaction hypothesis is tested through maniputations of the ionic strength and the pH. The conceptual innovation lies in our capitalizing on the observation that PKA-mediated phosphorylation results in a potentiation of current for SNS but an inhibition for BIIA channels leading to our novel hypothesis. The technical innovation lies in our ability to integrate state-of-art molecular manipulation of the sodium channel protein and biochemical approaches with a rigorous biophysical analysis of the channel function using first-latency and conditional probability techniques. A detailed molecular-level understanding of SNS channel modulation is critical to gain a deeper understanding of neuropathic pain and for the development of novel therapeutic strategies for a disease process with major medical and economic implications but with few effective treatments at present.
