Non-technical summary Abnormal pain sensitivity associated with inherited and acquired pain disorders occurs through increased excitability of peripheral sensory neurons in part due to changes in the properties of voltage-gated sodium channels (Navs). Resurgent sodium currents (I NaR ) are atypical currents believed to be associated with increased excitability of neurons and may have implications in pain. Mutations in Nav1.7 (peripheral Nav isoform) associated with two genetic pain disorders, inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD), enhance Nav1.7 function via distinct mechanisms. We show that changes in Nav1.7 function due to mutations associated with PEPD, but not IEM, are important in I NaR generation, suggesting that I NaR may play a role in pain associated with PEPD. This knowledge provides us with a better understanding of the mechanism of I NaR generation and may lead to the development of specialized treatment for pain disorders associated with I NaR .Abstract Inherited erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD) are inherited pain syndromes predominantly caused by missense mutations in the peripheral neuronal voltage-gated sodium channel (Nav) isoform Nav1.7. While both IEM and PEPD mutations increase neuronal excitability, IEM mutations primarily enhance activation and PEPD mutations impair inactivation. In addition, one PEPD mutation, Nav1.7-I1461T, has been shown to increase resurgent sodium currents in dorsal root ganglion (DRG) neurons. Because resurgent currents have been implicated in increased neuronal excitability, we asked whether (1) additional PEPD mutations located within the putative inactivation gate and docking sites and (2) IEM mutations might also increase these unusual currents. Resurgent currents are generated following open-channel block at positive potentials by an endogenous blocking particle and subsequent expulsion of this blocker upon repolarization to moderately negative potentials. Here we used a mimetic of the putative blocking particle, the Navβ4 peptide, to determine if enhanced resurgent currents are induced by three distinct PEPD mutations and two IEM mutations in stably transfected HEK293 cells. We demonstrate that (1) Nav1.7-mediated resurgent currents are observed in HEK293 cells with the Navβ4 peptide in the recording pipette, (2) while the PEPD mutants M1627K, T1464I and V1299F exhibit enhanced resurgent current amplitudes compared to wild-type, the IEM mutants I848T and L858H do not, and (3) there is a strong correlation between the decay time constant of open-channel fast inactivation and resurgent current amplitude. These data suggest that resurgent currents may play a role in the neuronal hyperexcitability associated with PEPD, but not IEM, mutations.

The present results indicate that ethanol increases postsynaptic GABA(A) receptor sensitivity, enhances action potential-independent GABA release onto VTA-DA neurons, and that this latter effect is independent of GABA(B) auto-receptor inhibition of GABA release.

Activation of ventral tegmental area (VTA)-dopaminergic (DA) neurons by ethanol has been implicated in the rewarding and reinforcing actions of ethanol. GABAergic transmission is thought to play an important role in regulating the activity of DA neurons. We have reported previously that ethanol enhances GABA release onto VTA-DA neurons in a brain slice preparation. Because intraterminal Ca 2ϩ levels regulate neurotransmitter release, we investigated the roles of Ca 2ϩ -dependent mechanisms in ethanol-induced enhancement of GABA release. Acute ethanol enhanced miniature inhibitory postsynaptic current (mIPSC) frequency in the presence of the nonspecific voltage-gated Ca 2ϩ channel inhibitor, cadmium chloride, even though basal mIPSC frequency was reduced by cadmium. Conversely, the inositol-1,4,5-triphosphate receptor inhibitor, 2-aminoethoxydiphenylborane, and the sarco/endoplasmic reticulum Ca 2ϩ ATPase pump inhibitor, cyclopiazonic acid, eliminated the ethanol enhancement of mIPSC frequency. Recent studies suggest that the G protein-coupled receptor, 5-hydroxytryptamine (5-HT) 2C , may modulate GABA release in the VTA. Thus, we also investigated the role of 5-HT 2C receptors in ethanol enhancement of GABAergic transmission. Application of 5-HT and the 5-HT 2C receptor agonist, Ro-60-0175 [(␣S)-6-chloro-5-fluoro-␣-methyl-1H-indole-1-ethanamine fumarate], alone enhanced mIPSC frequency of which the latter was abolished by the 5-HT 2C receptor antagonist, SB200646 [N-(1-methyl-5-indoyl)-N-(3-pyridyl)urea hydrochloride], and substantially diminished by cyclopiazonic acid. Furthermore, SB200646 abolished the ethanol-induced increase in mIPSC frequency and had no effect on basal mIPSC frequency. These observations suggest that an increase in Ca 2ϩ release from intracellular stores via 5-HT 2C receptor activation is involved in the ethanol-induced enhancement of GABA release onto VTA-DA neurons.

Chronic and neuropathic pain constitute significant health problems affecting millions of individuals each year. Pain sensations typically originate in sensory neurons of the peripheral nervous system which relay information to the central nervous system (CNS). Pathological pain sensations can arise as result of changes in excitability of these peripheral sensory neurons. Voltage-gated sodium channels are key determinants regulating action potential generation and propagation; thus, changes in sodium channel function can have profound effects on neuronal excitability and pain signaling. At present, most of the clinically available sodium channel blockers used to treat pain are non-selective across sodium channel isoforms and can contribute to cardio-toxicity, motor impairments, and CNS side effects. Numerous strides have been made over the last decade in an effort to develop more selective and efficacious sodium channel blockers to treat pain. The purpose of this review is to highlight some of the more recent developments put forth by research universities and pharmaceutical companies alike in the pursuit of developing more targeted sodium channel therapies for the treatment of a variety of neuropathic pain conditions.

Paroxysmal extreme pain disorder (PEPD) and inherited erythromelalgia (IEM) are inherited pain syndromes arising from different sets of gain-of-function mutations in the sensory neuronal sodium channel isoform Nav1.7. Mutations associated with PEPD, but not IEM, result in destabilized inactivation of Nav1.7 and enhanced resurgent sodium currents. Resurgent currents arise after relief of ultra-fast open-channel block mediated by an endogenous blocking particle and are thought to influence neuronal excitability. As such, enhancement of resurgent currents may constitute a pathological mechanism contributing to sensory neuron hyperexcitability and pain hypersensitivity associated with PEPD. Furthermore, pain associated with PEPD, but not IEM, is alleviated by the sodium channel inhibitor carbamazepine. We speculated that selective attenuation of PEPD-enhanced resurgent currents might contribute to this therapeutic effect. Here we examined whether carbamazepine and two other sodium channel inhibitors, riluzole and anandamide, exhibit differential inhibition of resurgent currents. To gain further insight into the potential mechanism(s) of resurgent currents, we examined whether these inhibitors produced correlative changes in other properties of sodium channel inactivation. Using stably transfected human embryonic kidney 293 cells expressing wild-type Nav1.7 and the PEPD mutants T1464I and M1627K, we examined the effects of the three drugs on Nav␤4 peptide-mediated resurgent currents. We observed a correlation between resurgent current inhibition and a drug-mediated increase in the rate of inactivation and inhibition of persistent sodium currents. Furthermore, although carbamazepine did not selectively target resurgent currents, anandamide strongly inhibited resurgent currents with minimal effects on the peak transient current amplitude, demonstrating that resurgent currents can be selectively targeted.

Chronic ethanol exposure may induce neuroadaptive responses in N-methyl-D-aspartate (NMDA) receptors, which are thought to underlie a variety of alcohol-related brain disorders. Here, we demonstrate that hyperexcitability triggered by withdrawal from chronic ethanol exposure is associated with increases in both synaptic NMDA receptor expression and activation. Withdrawal from chronic ethanol exposure (75 mM ethanol, 5-9 days) elicited robust and prolonged epileptiform activity in CA1 pyramidal neurons from hippocampal explants, which was absolutely dependent upon NMDA receptor activation but independent of chronic inhibition of protein kinase A (PKA). Analysis of Sr 2ϩ -supported asynchronous NMDA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) was employed to assess changes in NMDA neurotransmission. After chronic exposure, ethanol withdrawal was associated with an increase in mEPSC amplitude 3.38-fold over that after withdrawal from acute ethanol exposure. Analysis of paired evoked ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid EPSCs and spontaneous mEPSCs indicated that withdrawal after chronic exposure was also associated with a selective increase in action potential evoked but not spontaneous transmitter release probability. Immunoblot analysis revealed significant increases in total NR1, NR2A, and NR2B subunit expression after chronic exposure and unaffected by PKA-inhibition manner. Confocal imaging studies indicate that increased NR1 subunit expression was associated with increased density of NR1 expression on dendrites in parallel with a selective increase in the size of NR1 puncta on dendritic spines. Therefore, neuroadaptation to chronic ethanol exposure in NMDA synaptic transmission is responsible for aberrant network excitability after withdrawal and results from changes in both postsynaptic function as well as presynaptic release.

Activation of the dopaminergic (DA) neurons of the ventral tegmental area (VTA) by ethanol has been implicated in its rewarding and reinforcing effects. We previously demonstrated that ethanol enhances GABA release onto VTA-DA neurons via activation of 5-HT 2C receptors and subsequent release of calcium from intracellular stores. Here we demonstrate that excitation of VTA-DA neurons by ethanol is limited by an ethanol-enhancement in GABA release.In this study, we performed whole-cell voltage clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs) and cell-attached recordings of action potential firing from VTA-DA neurons in midbrain slices from young Long Evans rats. Acute exposure to ethanol (75 mM) transiently enhanced the firing rate of VTA-DA neurons as well as the frequency of mIPSCs. Simultaneous blockade of both GABA A and GABA B receptors (Picrotoxin (75 μM) and SCH50911 (20 μM)) disinhibited VTA-DA firing rate whereas a GABA A agonist (muscimol, 1 μM) strongly inhibited firing rate. In the presence of picrotoxin, ethanol enhanced VTA-DA firing rate more than in the absence of picrotoxin. Additionally, a sub-maximal concentration of muscimol together with ethanol inhibited VTA-DA firing rate more than muscimol alone. DAMGO (3 μM) inhibited mIPSC frequency but did not block the ethanol-enhancement in mIPSC frequency. DAMGO (1 and 3 μM) had no effect on VTA-DA firing rate. Naltrexone (60 μM) had no effect on basal or ethanol-enhancement of mIPSC frequency. Additionally, naltrexone (20 and 60 μM) did not block the ethanol-enhancement in VTA-DA firing rate. Overall, the present results indicate that the ethanol enhancement in GABA release onto VTA-DA neurons limits the stimulatory effect of ethanol on VTA-DA neuron activity and may have implications for the rewarding properties of ethanol.Correspondence: Richard Morrisett, Ph.D., The University of Texas at Austin, PHAR-Pharmacology, 1 University Station, A1915, Austin, TX 78712-0125, 512-471-1018, 512-475-6088 (fax), ramorris@mail.utexas.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2012 January 13. Activation of dopaminergic (DA) neurons of the ventral tegmental area (VTA) by ethanol and other drugs of abuse is believed to be a critical component in the development and expression of drug dependence and addiction (Gatto et al., 1994;Robbins and Everitt, 1996;Wise, 1996;Koob et al., 1998;Schultz, 2002;Appel, 2004). The VTA is situated in the midbrain and sends projections to the nucleus accumbens (NAc), p...

Voltage-gated sodium (Na) channel inhibitors are used clinically as analgesics and local anesthetics. However, the absence of Na channel isoform selectivity of current treatment options can result in adverse cardiac and central nervous system side effects, limiting their therapeutic utility. Human hereditary gain- or loss-of-pain disorders have demonstrated an essential role of Na1.7 sodium channels in the sensation of pain, thus making this channel an attractive target for new pain therapies. We previously identified a novel, state-dependent human Na1.7 selective inhibitor (PF-05089771, IC = 11 nM) that interacts with the voltage-sensor domain (VSD) of domain IV. We further characterized the state-dependent interaction of PF-05089771 by systematically varying the voltage, frequency, and duration of conditioning prepulses to provide access to closed, open, and fast- or slow-inactivated states. The current study demonstrates that PF-05089771 exhibits a slow onset of block that is depolarization and concentration dependent, with a similarly slow recovery from block. Furthermore, the onset of block by PF-05089771 develops with similar rates using protocols that bias channels into predominantly fast- or slow-inactivated states, suggesting that channel inhibition is less dependent on the availability of a particular inactivated state than the relative time that the channel is depolarized. Taken together, the inhibitory profile of PF-05089771 suggests that a conformational change in the domain IV VSD after depolarization is necessary and sufficient to reveal a high-affinity binding site with which PF-05089771 interacts, stabilizing the channel in a nonconducting conformation from which recovery is slow.