Depolarizing Na_V and Hyperpolarizing K_V Channels Are Co-Trafficked in Sensory Neurons

Abstract

Neuronal excitability relies on coordinated action of functionally distinction 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 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) is cotransported 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 cotransported 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.

The fates of internalized NaV1.7 channels in sensory neurons: Retrograde cotransport with other ion channels, axon-specific recycling, and degradation

“…This is perhaps unsurprising given that late endosomes arise from homotypic fusion of many early endosomes ( 44 ), which may be expected to lead to the mixing of cargos. This integrated transport is consistent with our report that multiple ion channels and axonal membrane proteins are trafficked together in the same vesicles in the anterograde direction ( 28 ). However, it is also possible that certain axonal proteins may be handled in a more specific manner via earlier endocytic or recycling processes, as has been demonstrated for opioid receptors ( 45 ).…”

“…We next sought to determine whether the Rab7 endosomes that transport Na V 1.7 away from distal axons carry only Na V 1.7 or also contain other axonal membrane proteins. To test this, we engineered multiple axonal ion channels with extracellular Halo tags or SNAP tags: SNAP-Na V 1.7, Halo-Na V 1.8, Halo-K V 7.2 ( 28 ) as well as an extracellularly tagged Halo-TrkA. SNAP tag is an enzymatic tag that functions similarly to Halo tag but has a different specific cognate ligand, enabling simultaneous use with Halo tag.…”

“…Instead, most previous studies of Na V channel trafficking relied on reporter proteins fused to channel fragments, fixed immunocytochemistry, and biochemical methods ( 3 , 32 , 33 , 34 ). However, we have developed novel tagged channels and imaging methods, which have allowed the observation of full-length sodium channel trafficking in live neurons ( 15 , 28 ). In the present study, we build upon these advances to make the first real-time observations of Na V channel retrograde trafficking.…”

“…The construction of plasmids encoding Halo-Na V 1.7, SNAP-Na V 1.7, Halo-Na V 1.8, and Halo-K V 7.2 was described previously ( 28 ). The codon-optimized Halo-Na V 1.7 construct followed the protocol for the construction of the noncodon-optimized Halo-Na V 1.7 ( 15 ) and was described previously ( 28 ).…”

“…The construction of plasmids encoding Halo-Na V 1.7, SNAP-Na V 1.7, Halo-Na V 1.8, and Halo-K V 7.2 was described previously ( 28 ). The codon-optimized Halo-Na V 1.7 construct followed the protocol for the construction of the noncodon-optimized Halo-Na V 1.7 ( 15 ) and was described previously ( 28 ). Briefly, the final construct topology is in order from the N terminus: 1 to 30 amino acids β4 signal peptide, 3× myc tag (EQKLISEEDL), Halo-tag enzyme (297 amino acids) (Promega), 3× hemagglutinin tag (YPYDVPDYA), 21 amino-acid transmembrane segment (β4 163–183), 7 amino-acid linker (SGLRSAT), hNa V 1.7-R. SNAP-Na V 1.7 was derived from the noncodon-optimized Halo-Na V 1.7 construct ( 15 ) by replacing the Halo tag with the 182 amino acid SNAP f (New England BioLabs) using megamutagenesis.…”