Depolarizing Na V and hyperpolarizing K V channels are co-trafficked in sensory neurons.

Neuronal excitability relies on coordinated action of functionally distinct ion channels. Voltage-gated sodium (Na V ) and potassium (K V ) channels have distinct but complementary roles in firing action potentials: Na V channels provide depolarizing current while K V channels provide hyperpolarizing current. Mutations and dysfunction of multiple Na V and K V channels underlie disorders of excitability, including pain and epilepsy. Modulating ion channel trafficking may offer a potential therapeutic strategy for these diseases. A fundamental question, however, is whether these channels with distinct functional roles are transported independently or packaged together in the same vesicles in sensory axons. We have used Optical Pulse-Chase Axonal Long-distance (OPAL) imaging to investigate trafficking of Na V and K V channels and other axonal proteins from distinct functional classes in live rodent sensory neurons (from male and female rats). We show that, similar to Na V 1.7 channels, Na V 1.8 and K V 7.2 channels are transported in Rab6a-positive vesicles, and that each of the Na V channel isoforms expressed in healthy, mature sensory neurons - Na V 1.6, Na V 1.7, Na V 1.8, and Na V 1.9 - are co-transported in the same vesicles. Further, we show that multiple axonal membrane proteins with different physiological functions - Na V 1.7, K V 7.2, and TNFR1 - are co-transported in the same vesicles. However, vesicular packaging of axonal membrane proteins is not indiscriminate, since another axonal membrane protein - NCX2 - is transported in separate vesicles. These results shed new light on the development and organization of sensory neuron membranes, revealing complex sorting of axonal proteins with diverse physiological functions into specific transport vesicles. Significance Statement Normal neuronal excitability is dependent on precise regulation of membrane proteins including Na V and K V channels, and imbalance in the level of these channels at the plasma membrane could lead to excitability disorders. Ion channel trafficking could potentially be targeted therapeutically, which would require better understanding of the mechanisms underlying trafficking of functionally diverse channels. Optical Pulse-chase Axonal Long-distance (OPAL) imaging in live neurons permitted examination of the specificity of ion channel trafficking, revealing co-packaging of axonal proteins with opposing physiological functions into the same transport vesicles. This suggests that additional trafficking mechanisms are necessary to regulate levels of surface channels and reveals an important consideration for therapeutic strategies that target ion channel trafficking for the treatment of excitability disorders.