Cindy Barbosa

Aberrant epilepsy-associated mutant Nav1.6 sodium channel activity can be targeted with cannabidiol.

Mutations in brain isoforms of voltage-gated sodium channels have been identified in patients with distinct epileptic phenotypes. Clinically, these patients often do not respond well to classic anti-epileptics and many remain refractory to treatment. Exogenous as well as endogenous cannabinoids have been shown to target voltage-gated sodium channels and cannabidiol has recently received attention for its potential efficacy in the treatment of childhood epilepsies. In this study, we further investigated the ability of cannabinoids to modulate sodium currents from wild-type and epilepsy-associated mutant voltage-gated sodium channels. We first determined the biophysical consequences of epilepsy-associated missense mutations in both Nav1.1 (arginine 1648 to histidine and asparagine 1788 to lysine) and Nav1.6 (asparagine 1768 to aspartic acid and leucine 1331 to valine) by obtaining whole-cell patch clamp recordings in human embryonic kidney 293T cells with 200 μM Navβ4 peptide in the pipette solution to induce resurgent sodium currents. Resurgent sodium current is an atypical near threshold current predicted to increase neuronal excitability and has been implicated in multiple disorders of excitability. We found that both mutations in Nav1.6 dramatically increased resurgent currents while mutations in Nav1.1 did not. We then examined the effects of anandamide and cannabidiol on peak transient and resurgent currents from wild-type and mutant channels. Interestingly, we found that cannabidiol can preferentially target resurgent sodium currents over peak transient currents generated by wild-type Nav1.6 as well as the aberrant resurgent and persistent current generated by Nav1.6 mutant channels. To further validate our findings, we examined the effects of cannabidiol on endogenous sodium currents from striatal neurons, and similarly we found an inhibition of resurgent and persistent current by cannabidiol. Moreover, current clamp recordings show that cannabidiol reduces overall action potential firing of striatal neurons. These findings suggest that cannabidiol could be exerting its anticonvulsant effects, at least in part, through its actions on voltage-gated sodium channels, and resurgent current may be a promising therapeutic target for the treatment of epilepsy syndromes.

Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: The role of TRPC channels†

Transient Receptor Potential Canonical (TRPC) channels are implicated in modulating neurite outgrowth. The expression pattern of TRPCs changes significantly during brain development, suggesting that fine‐tuning TRPC expression may be important for orchestrating neuritogenesis. To study how alterations in the TRPC expression pattern affect neurite outgrowth, we used nerve growth factor (NGF)‐differentiated rat pheochromocytoma 12 (PC12) cells, a model system for neuritogenesis. In PC12 cells, NGF markedly up‐regulated TRPC1 and TRPC6 expression, but down‐regulated TRPC5 expression while promoting neurite outgrowth. Overexpression of TRPC1 augmented, whereas TRPC5 overexpression decelerated NGF‐induced neurite outgrowth. Conversely, shRNA‐mediated knockdown of TRPC1 decreased, whereas shRNA‐mediated knockdown of TRPC5 increased NGF‐induced neurite extension. Endogenous TRPC1 attenuated the anti‐neuritogenic effect of overexpressed TRPC5 in part by forming the heteromeric TRPC1–TRPC5 channels. Previous reports suggested that TRPC6 may facilitate neurite outgrowth. However, we found that TRPC6 overexpression slowed down neuritogenesis, whereas dominant negative TRPC6 (DN‐TRPC6) facilitated neurite outgrowth in NGF‐differentiated PC12 cells. Consistent with these findings, hyperforin, a neurite outgrowth promoting factor, decreased TRPC6 expression in NGF‐differentiated PC12 cells. Using pharmacological and molecular biological approaches, we determined that NGF up‐regulated TRPC1 and TRPC6 expression via a p75NTR–IKK2‐dependent pathway that did not involve TrkA receptor signaling in PC12 cells. Similarly, NGF up‐regulated TRPC1 and TRPC6 via an IKK2 dependent pathway in primary cultured hippocampal neurons. Thus, our data suggest that a balance of TRPC1, TRPC5, and TRPC6 expression determines neurite extension rate in neural cells, with TRPC6 emerging as an NGF‐dependent “molecular damper” maintaining a submaximal velocity of neurite extension. J. Cell. Physiol. 227: 1408–1419, 2012.

Navβ4 Regulates Fast Resurgent Sodium Currents and Excitability in Sensory Neurons

BackgroundIncreased electrical activity in peripheral sensory neurons including dorsal root ganglia (DRG) and trigeminal ganglia neurons is an important mechanism underlying pain. Voltage gated sodium channels (VGSC) contribute to the excitability of sensory neurons and are essential for the upstroke of action potentials. A unique type of VGSC current, resurgent current (INaR), generates an inward current at repolarizing voltages through an alternate mechanism of inactivation referred to as open-channel block. INaRs are proposed to enable high frequency firing and increased INaRs in sensory neurons are associated with pain pathologies. While Nav1.6 has been identified as the main carrier of fast INaR, our understanding of the mechanisms that contribute to INaR generation is limited. Specifically, the open-channel blocker in sensory neurons has not been identified. Previous studies suggest Navβ4 subunit mediates INaR in central nervous system neurons. The goal of this study was to determine whether Navβ4 regulates INaR in DRG sensory neurons.ResultsOur immunocytochemistry studies show that Navβ4 expression is highly correlated with Nav1.6 expression predominantly in medium-large diameter rat DRG neurons. Navβ4 knockdown decreased endogenous fast INaR in medium-large diameter neurons as measured with whole-cell voltage clamp. Using a reduced expression system in DRG neurons, we isolated recombinant human Nav1.6 sodium currents in rat DRG neurons and found that overexpression of Navβ4 enhanced Nav1.6 INaR generation. By contrast neither overexpression of Navβ2 nor overexpression of a Navβ4-mutant, predicted to be an inactive form of Navβ4, enhanced Nav1.6 INaR generation. DRG neurons transfected with wild-type Navβ4 exhibited increased excitability with increases in both spontaneous activity and evoked activity. Thus, Navβ4 overexpression enhanced INaR and excitability, whereas knockdown or expression of mutant Navβ4 decreased INaR generation.ConclusionINaRs are associated with inherited and acquired pain disorders. However, our ability to selectively target and study this current has been hindered due to limited understanding of how it is generated in sensory neurons. This study identified Navβ4 as an important regulator of INaR and excitability in sensory neurons. As such, Navβ4 is a potential target for the manipulation of pain sensations.

Substituted N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamides: potent anticonvulsants that affect frequency (use) dependence and slow inactivation of sodium channels.

We prepared 13 derivatives of N-(biphenyl-4′-yl)methyl (R)-2-acetamido-3-methoxypropionamide that differed in type and placement of a R-substituent in the terminal aryl unit. We demonstrated that the R-substituent impacted the compound’s whole animal and cellular pharmacological activities. In rodents, select compounds exhibited excellent anticonvulsant activities and protective indices (PI = TD50/ED50) that compared favorably with clinical antiseizure drugs. Compounds with a polar, aprotic R-substituent potently promoted Na+ channel slow inactivation and displayed frequency (use) inhibition of Na+ currents at low micromolar concentrations. The possible advantage of affecting these two pathways to decrease neurological hyperexcitability is discussed.