Project Summary: Mechanisms and specificity of sodium channel trafficking: Developing a novel analgesic strategy. The burden of pain is significant and current pain treatments are often ineffective and addictive. Alternatives are urgently needed. Voltage-gated sodium channel NaV1.7 is preferentially expressed in pain- sensing neurons. Mutations in NaV1.7 can cause disorders ranging from intense pain (gain-of-function) to complete painlessness (loss-of-function) in humans, suggesting that its inhibition could provide analgesia without CNS side-effects or addictive potential. However, ongoing efforts to develop inhibitors of NaV1.7 conductance at the cell membrane have not yet resulted in new therapies. We propose an alternative strategy for inhibition of NaV1.7 function; reducing the number of channels at the cell surface by modulating their trafficking to and from the cell membrane. Achieving this goal would require identifying and modulating mechanisms that specifically mediate NaV1.7 trafficking. This project will investigate whether NaV1.7 is trafficked by specific mechanisms. Whether NaVs are trafficked by dedicated mechanisms or together with other axonal proteins with different functions is a fundamental question. NaV1.7 and NaV1.8 are functionally related, as they both contribute to neuronal depolarization and promote pain. In contrast, voltage-gated potassium (KV) channels oppose neuronal excitation and suppress pain. This proposal will test the hypothesis that ion channels with different physiological functions are trafficked separately from each other according to their functions. Previous attempts to observe sodium channel trafficking using fluorescent protein tags have failed because the substantial pool of sodium channels in the cytoplasm and at the cell membrane conceal the weak signal of individual vesicles carrying few channels. To overcome this, we developed Optical Pulse-chase Axonal Long-distance (OPAL) imaging, which utilizes functional human NaV channels tagged with self-labeling proteins (HaloTag and SNAPTag) and microfluidic chambers to selectively label channels that are being actively trafficked in axons. This method allows live visualization of sodium channel vesicular sorting, axonal transport, and endocytosis in distal sensory axons for the first time. In the proposed experiments, we will examine two major aspects of axonal trafficking in turn: Aim 1 will investigate anterograde trafficking to distal terminals and Aim 2 will interrogate endocytosis and retrograde trafficking. In each Aim, we will 1) Determine whether NaVs are sorted into specific vesicles by live co- localization imaging with tagged vesicle markers, 2) Determine whether different but functionally related NaV isoforms are trafficked together, and 3) Determine whether functionally opposite NaV and KV channels are trafficked together or separately. Together, these experiments will explain the logic of axonal vesicular transport and potentially provide...