Cox, J.J. et al. An SCN9A channelopathy causes congenital inability to experience pain. Nature 444, 894-898
Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrooke's Hospital, Cambridge CB2 0XY, UK. Nature
(Impact Factor: 41.46).
01/2007; 444(7121):894-8. DOI: 10.1038/nature05413
The complete inability to sense pain in an otherwise healthy individual is a very rare phenotype. In three consanguineous families from northern Pakistan, we mapped the condition as an autosomal-recessive trait to chromosome 2q24.3. This region contains the gene SCN9A, encoding the alpha-subunit of the voltage-gated sodium channel, Na(v)1.7, which is strongly expressed in nociceptive neurons. Sequence analysis of SCN9A in affected individuals revealed three distinct homozygous nonsense mutations (S459X, I767X and W897X). We show that these mutations cause loss of function of Na(v)1.7 by co-expression of wild-type or mutant human Na(v)1.7 with sodium channel beta(1) and beta(2) subunits in HEK293 cells. In cells expressing mutant Na(v)1.7, the currents were no greater than background. Our data suggest that SCN9A is an essential and non-redundant requirement for nociception in humans. These findings should stimulate the search for novel analgesics that selectively target this sodium channel subunit.
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- "In recent years, Na V 1.7 has emerged as a validated pain target based on several human genetic studies. Gain-of-function mutations in the SNC9A gene encoding the pore-forming α-subunit of Na V 1.7, have been shown to cause painful inherited neuropathies (Cheng et al., 2011; Dabby et al., 2010; Estacion et al., 2008; Theile et al., 2011; Yang et al., 2004) whereas loss-of-function mutations in SCN9A result in a congenital indifference to all forms of pain (Cox et al., 2006). This suggests that subtype-selective inhibitors of Na V 1.7 are likely to be useful analgesics for treating a broad range of pain conditions (England and Rawson, 2010). "
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ABSTRACT: Many spider venom-peptides are known to modulate the activity of the voltage-gated sodium channel NaV1.7, which has emerged as a promising analgesic target. A class of spider venom-peptides (NaSpTx1) in particular have been found to potently inhibit NaV1.7 (nanomolar IC50), and shown to produce analgesic effects in animals. However, one member of this family, μ TRTX Hhn2b (Hhn2b), does not inhibit mammalian NaV channels expressed in dorsal root ganglia at concentrations up to 100 μM. This peptide is classified as a NaSpTx1 member by virtue of its cysteine spacing and sequence conservation over functionally important residues. Here, we have performed detailed structural and functional analyses of Hhn2b leading us to identify two non-pharmacophore residues that contribute to hNaV1.7 inhibition by non-overlapping mechanisms. These findings allowed us to produce a double mutant of Hhn2b that shows nanomolar inhibition of hNaV1.7. Traditional structure/function analysis did not provide sufficient resolution to identify the mechanism underlying the observed gain of function. However, by solving the high-resolution structure of both the wild type and mutant peptides using advanced multidimensional NMR experiments, we were able to uncover a previously unknown network of interactions that stabilize the pharmacophore region of this class of venom-peptides. We further monitored the lipid binding properties of the peptides and identified that one of the key amino acid substitutions also selectively modulates the binding of the peptide to anionic lipids. These results will further aid the development of peptide-based analgesics for the treatment of chronic pain.
Available from: Fernanda Cardoso
- "Remarkably, individuals with loss-of-function mutations in the gene encoding the pore-forming a subunit of human Na V 1.7 (hNa V 1.7) have a complete insensitivity to pain (Cox et al., 2006), with no other sensory deficits except a lack of smell (anosmia) due to the role of hNa V 1.7 in olfaction (Weiss et al., 2011; Rupasinghe et al., 2012). Thus, hNa V 1.7 has become an exciting, genetically validated target for treating pain disorders (Liu and Wood, 2011; King and Vetter, 2014; Minett et al., 2014). "
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ABSTRACT: Spider venoms are a rich source of ion channel modulators with therapeutic potential. Given the analgesic potential of subtype-selective inhibitors of voltage-gated sodium (NaV) channels, we screened spider venoms for inhibitors of human NaV1.7 using a high-throughput fluorescent assay. Here, we describe the discovery of a novel NaV1.7 inhibitor, μ-TRTX-Tp1a (Tp1a), isolated from the venom of the Peruvian green-velvet tarantula Thrixopelma pruriens. Recombinant and synthetic forms of this 33-residue peptide preferentially inhibited hNav1.7 > hNav1.6 > hNav1.2 > hNav1.1 > hNav1.3 channels in fluorescent assays. NaV1.7 inhibition was diminished (IC50 11.5 nM), and the association rate decreased, for the C-terminal acid form of Tp1a compared to the native amidated form (IC50 2.1 nM), suggesting that the peptide C-terminus contributes to its interaction with hNaV1.7. Tp1a had no effect on human voltage-gated calcium channels or nicotinic acetylcholine receptors at 5 μM. Unlike most spider toxins that modulate NaV channels, Tp1a inhibited hNav1.7 without significantly altering the voltage-dependence of activation or inactivation. Tp1a proved to be analgesic by reversing spontaneous pain induced in mice by intraplantar injection in OD1, a scorpion toxin that potentiates hNav1.7. The structure of Tp1a as determined using NMR spectroscopy revealed a classical inhibitor cystine knot motif (ICK). The molecular surface of Tp1a presents a hydrophobic patch surrounded by positively charged residues, with subtle differences to other ICK spider toxins that might contribute to its different pharmacological profile. Tp1a may help guide the development of more selective and potent hNaV1.7 inhibitors for treatment of chronic pain.
The American Society for Pharmacology and Experimental Therapeutics.
Available from: Julie Kaae Klint
- "as an analgesic target. Gain-of-function mutations in the SNC9A gene encoding hNaV1.7 cause painful inherited neuropathies (Yang et al., 2004; Fertleman et al., 2006; Estacion et al., 2008; Cheng et al., 2011; Theile et al., 2011), whereas loss-offunction mutations result in congenital indifference to all forms of pain (Cox et al., 2006). Moreover, single nucleotide polymorphisms in SCN9A are associated with differences in pain sensitivity (Reimann et al., 2010; Duan et al., 2013; Reeder et al., 2013). "
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ABSTRACT: Chronic pain is a serious worldwide health issue, with current analgesics having limited efficacy and dose-limiting side effects. Humans with loss-of-function mutations in the voltage-gated sodium channel NaV 1.7 (hNaV 1.7) are indifferent to pain, making hNaV 1.7 a promising target for analgesic development. Since spider venoms are replete with NaV channel modulators, we examined their potential as a source of hNaV 1.7 inhibitors.
We developed a high-throughput fluorescent-based assay to screen spider venoms against hNaV 1.7 and isolate 'hit' peptides. To examine the binding site of these peptides, we constructed a panel of chimeric channels in which the S3b-S4 paddle motif from each voltage sensor domain of hNaV 1.7 was transplanted into the homotetrameric KV 2.1 channel.
We screened 205 spider venoms and found that 40% contain at least one inhibitor of hNaV 1.7. By deconvoluting 'hit' venoms, we discovered seven novel members of the NaSpTx family 1. One of these peptides, Hd1a (peptide μ-TRTX-Hd1a from venom of the spider Haplopelma doriae), inhibited hNaV 1.7 with a high level of selectivity over all other subtypes, except hNaV 1.1. We showed that Hd1a is a gating modifier that inhibits hNaV 1.7 by interacting with the S3b-S4 paddle motif in channel domain II. The structure of Hd1a, determined using heteronuclear NMR, contains an inhibitor cystine knot motif that is likely to confer high levels of chemical, thermal and biological stability.
Our data indicate that spider venoms are a rich natural source of hNaV 1.7 inhibitors that might be useful leads for the development of novel analgesics.
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