Depolarizing NaV and hyperpolarizing KV channels are co-trafficked in sensory neurons.

Neuronal excitability relies on coordinated action of functionally distinct ion channels. Voltage-gated sodium (NaV) and potassium (KV) channels have distinct but complementary roles in firing action potentials: NaV channels provide depolarizing current while KV channels provide hyperpolarizing current. Mutations and dysfunction of multiple NaV and KV 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 NaV and KV channels and other axonal proteins from distinct functional classes in live rodent sensory neurons (from male and female rats). We show that, similar to NaV1.7 channels, NaV1.8 and KV7.2 channels are transported in Rab6a-positive vesicles, and that each of the NaV channel isoforms expressed in healthy, mature sensory neurons - NaV1.6, NaV1.7, NaV1.8, and NaV1.9 - are co-transported in the same vesicles. Further, we show that multiple axonal membrane proteins with different physiological functions - NaV1.7, KV7.2, 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 StatementNormal neuronal excitability is dependent on precise regulation of membrane proteins including NaV and KV 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.

Sodium Channels in Human Pain Disorders: Genetics and Pharmacogenomics.

Acute pain is adaptive, but chronic pain is a global challenge. Many chronic pain syndromes are peripheral in origin and reflect hyperactivity of peripheral pain-signaling neurons. Current treatments are ineffective or only partially effective and in some cases can be addictive, underscoring the need for better therapies. Molecular genetic studies have now linked multiple human pain disorders to voltage-gated sodium channels, including disorders characterized by insensitivity or reduced sensitivity to pain and others characterized by exaggerated pain in response to normally innocuous stimuli. Here, we review recent developments that have enhanced our understanding of pathophysiological mechanisms in human pain and advances in targeting sodium channels in peripheral neurons for the treatment of pain using novel and existing sodium channel blockers.

The AMPK Activator A769662 Blocks Voltage-Gated Sodium Channels: Discovery of a Novel Pharmacophore with Potential Utility for Analgesic Development.

Voltage-gated sodium channels (VGSC) regulate neuronal excitability by governing action potential (AP) generation and propagation. Recent studies have revealed that AMP-activated protein kinase (AMPK) activators decrease sensory neuron excitability, potentially by preventing sodium (Na+) channel phosphorylation by kinases such as ERK or via modulation of translation regulation pathways. The direct positive allosteric modulator A769662 displays substantially greater efficacy than other AMPK activators in decreasing sensory neuron excitability suggesting additional mechanisms of action. Here, we show that A769662 acutely inhibits AP firing stimulated by ramp current injection in rat trigeminal ganglion (TG) neurons. PT1, a structurally dissimilar AMPK activator that reduces nerve growth factor (NGF) -induced hyperexcitability, has no influence on AP firing in TG neurons upon acute application. In voltage-clamp recordings, application of A769662 reduces VGSC current amplitudes. These findings, based on acute A769662 application, suggest a direct channel blocking effect. Indeed, A769662 dose-dependently blocks VGSC in rat TG neurons and in Nav1.7-transfected cells with an IC50 of ~ 10 µM. A769662 neither displayed use-dependent inhibition nor interacted with the local anesthetic (LA) binding site. Popliteal fossa administration of A769662 decreased noxious thermal responses with a peak effect at 5 mins demonstrating an analgesic effect. These data indicate that in addition to AMPK activation, A769662 acts as a direct blocker/modulator of VGSCs, a potential mechanism enhancing the analgesic property of this compound.