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J Physiol
2010 May 15;588Pt 10:1695-707. doi: 10.1113/jphysiol.2010.187112.
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An in vivo tethered toxin approach for the cell-autonomous inactivation of voltage-gated sodium channel currents in nociceptors.
Stürzebecher AS
,
Hu J
,
Smith ES
,
Frahm S
,
Santos-Torres J
,
Kampfrath B
,
Auer S
,
Lewin GR
,
Ibañez-Tallon I
.
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Understanding information flow in sensory pathways requires cell-selective approaches to manipulate the activity of defined neurones. Primary afferent nociceptors, which detect painful stimuli, are enriched in specific voltage-gated sodium channel (VGSC) subtypes. Toxins derived from venomous animals can be used to dissect the contributions of particular ion currents to cell physiology. Here we have used a transgenic approach to target a membrane-tethered isoform of the conotoxin MrVIa (t-MrVIa) only to nociceptive neurones in mice. T-MrVIa transgenic mice show a 44 +/- 7% reduction of tetrodotoxin-resistant (TTX-R) VGSC current densities. This inhibition is permanent, reversible and does not result in functional upregulation of TTX-sensitive (TTX-S) VGSCs, voltage-gated calcium channels (VGCCs) or transient receptor potential (TRP) channels present in nociceptive neurones. As a consequence of the reduction of TTX-R VGSC currents, t-MrVIa transgenic mice display decreased inflammatory mechanical hypersensitivity, cold pain insensitivity and reduced firing of cutaneous C-fibres sensitive to noxious cold temperatures. These data validate the use of genetically encoded t-toxins as a powerful tool to manipulate VGSCs in specific cell types within the mammalian nervous system. This novel genetic methodology can be used for circuit mapping and has the key advantage that it enables the dissection of the contribution of specific ionic currents to neuronal function and to behaviour.
Figure 1. Generation of BAC transgenic mice encoding membrane-tethered MrVIa toxinA, diagram showing t-MrVIa (green ribbon) including a FLAG epitope for immunodetection, a flexible poly (asparagine-glycine) linker (grey) and a GPI tether to the cell membrane. B, schematic overview of the BAC modification by two-step homologous recombination. The modified shuttle vector (SV) contains the t-toxin cassette (green), flanked by a secretion signal (sec) and a polyA (pA), inserted between two recombination boxes (A and B in red) corresponding to the sequences flanking the initiator methionine of the Scn10a gene encoding the Nav1.8 VGSC subunit. Recombination (red lines) with the Scn10a BAC results in the modified BAC and the free shuttle vector. Scn10a gene promoter (black arrow), Scn10a exons (grey boxes), selection markers (dark green boxes), NcoI restriction sites for Southern blot (N), PI-SceI restriction site for BAC linearisation (P). C, Southern blot of transgenic founder lines. Lines 2 and 27 showed the highest relative ratio of the transgene band (∼2.3 kb) in comparison with the endogenous Scn10a allele (∼1.5 kb) indicative of high BAC copy number.
Figure 2. Cell-autonomous inhibition of sodium currents in nociceptors of Tg-t-MrVIa miceA, co-expression of Scn10a and t-MrVIa transcripts in DRGs of Tg-t-MrVIa mice detected by RT-PCR analysis (Sk, skin; Bs, brain stem; Co, cortex. B, in situ hybridisation on spinal cord sections of mouse embryos (E15) show strong t-MrVIa signal in DRGs but not in spinal cord (Sc) of Tg-t-MrVIa mice. Vc, vertebral column. Scale bar: 100 μm. C, t-MrVIa transcripts were detected by in situ hybridisation in nociceptors (arrows) but not mechanoreceptors (arrowheads) of Tg-t-MrVIa mice (2–4 weeks old). Scale bar: 50 μm. D–H, voltage-gated currents were evoked by step depolarisations from −50 mV to +40 mV preceded by a hyperpolarizing prepulse from −60 to −120 mV to prevent inactivation. D and E, representative traces and current–voltage relationships of inward currents indicate a significant reduction of sodium currents in nociceptors of Tg-t-MrVIa mice in comparison to wild-type littermates. F, current–voltage relations of outward currents are not affected in nociceptors of Tg-t-MrVIa mice. G and H, representative traces and current–voltage relations of inward currents, evoked by the same protocol shown in D, indicate no differences in mechanoreceptors of Tg-t-MrVIa mice and wild-type littermates. I, peak VGSC current densities are significantly reduced in nociceptors of Tg-t-MrVIa mice (243.5 ± 37.2 pA pF−1) with respect to wild-type mice (413.7 ± 37.2 pA pF−1) (n= 32 per group) and unchanged in mechanoreceptors (n= 9 per group). P < 0.05 two-way ANOVA in E and H; P > 0.05 two-way ANOVA in F; P < 0.05 t test in I. J and K, representative traces (J) and current–voltage relations (K) of voltage-gated calcium currents, evoked by step depolarisations from −90 mV to +40 mV, indicate no differences in nociceptors of Tg-t-MrVIa mice and wild-type littermates (n= 10 per group). L, representative action potentials used to identify nociceptors and mechanoreceptors.
Figure 3. t-MrVIa acts at the cell membrane and specifically blocks TTX-R currents with no compensatory upregulation of other VGSCsA, t-MrVIa (FLAG: red) colocalizes with the membrane marker WGA (blue) in DRG neurones co-electroporated with t-MrVIa and cytoplasmic EGFP (upper panel). PI-PLC treatment eliminates FLAG immunoreactivity showing specific cleavage of the GPI-anchored toxin from the membrane (lower panel). Scale bars: 5 μm. B, mammalian cells transfected with t-MrVIa show expression of the toxin in the membrane fraction by FLAG immunoprecipitation. C, bar graph indicating the quantification of VGSC peak currents in nociceptors of wild-type (Wt) and Tg-t-MrVIa mice (n= 32 cells per group) before and after TTX and PI-PLC treatment (n= 12 cells). The current densities of total VGSC and TTX-R currents are significantly reduced in Tg-t-MrVIa mice compared to wild-type mice, and significantly recover from t-toxin inhibition after PI-PLC treatment. TTX-S currents are not significantly affected in Tg-t-MrVIa mice. D, current–voltage relationships of TTX-R currents indicate a significant inhibition of sodium currents in nociceptors of Tg-t-MrVIa mice in comparison to Wt littermates (P < 0.05 two-way-ANOVA), and no significant inhibition, with respect to Wt, in Tg-t-MrVIa mice after PI-PLC treatment (P > 0.05 two-way ANOVA). E, peak current measurements in nociceptors treated with TTX and live-labelled with isolectin B4 indicate that IB4+ve nociceptors display more TTX-R than IB4–ve nociceptors in Wt and Tg mice and that t-MrVIa inhibition is significant in both subpopulations but more pronounced in IB4+ve nociceptors (n= 10–22 cells per group). F, the mean amplitude of action potentials is significantly reduced in IB4+ve nociceptors (Wt: 89.2 ± 2.3 mV, Tg-t-MrVIa: 74.3 ± 3.9 mV) but not in IB4–ve nociceptors and mechanoreceptors (t test). (n= 9–20 per group.) G, immunodetection of substance P (red) and isolectin B4 (green) in dorsal spinal cord indicate no differences in afferent innervation between Wt and Tg-t-MrVIa adult mice. Scale bar: 50 μm, t test in C and E.
Figure 4. Tg-t-MrVIa adult mice show reduced inflammatory hyperalgesia and insensitivity to cold painA, no differences in inflammatory thermal hyperalgesia induced by intraplantar injection of carrageenan were observed between wild-type (Wt) and Tg-t-MrVIa mice (n= 10 mice). B, induced inflammation causes mechanical hyperalgesia in Wt mice (peak at 4 h) and significantly reduced hyperalgesia in Tg-t-MrVIa (n= 10 mice). Dashed line indicates baseline pain response. C, nocifensive cold responses scored during 90 s on a 0°C-cooled plate showed significantly reduced responses to noxious cold in Tg-t-MrVIa mice (n= 10 mice). D, temperature preference tests quantifying the time mice spent in either of two plates kept at 30°C and 10°C (n= 8 mice). E, histogram indicating the firing frequency of cold-sensitive C-fibres with a threshold >10°C over a cooling gradient from 30 to 0°C. The firing frequency did not differ between Wt and Tg-t-MrVIa mice (fibres: n= 8–12 fibres per group). F, histogram indicating the firing frequency of cold-sensitive C-fibres with a threshold < 10°C responding to noxious cold stimuli over a cooling gradient from 30 to 0°C. The firing frequency of cooling units with a threshold below 10°C is significantly reduced in Tg-t-MrVIa compared to Wt mice (fibres: n= 4–8 fibres per group). The lower trace corresponds to the cold ramp stimuli used in both cases. P > 0.05 two-way ANOVA in A and E; P < 0.05 two-way ANOVA in B and F; P < 0.05 t test in C.
Akopian,
The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways.
1999, Pubmed
Akopian,
The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways.
1999,
Pubmed
Amaya,
The voltage-gated sodium channel Na(v)1.9 is an effector of peripheral inflammatory pain hypersensitivity.
2006,
Pubmed
Auer,
Silencing neurotransmission with membrane-tethered toxins.
2010,
Pubmed
Baker,
Tethered-toxin debut gets cold reception.
2010,
Pubmed
Baker,
Protein kinase C mediates up-regulation of tetrodotoxin-resistant, persistent Na+ current in rat and mouse sensory neurones.
2005,
Pubmed
Bautista,
The menthol receptor TRPM8 is the principal detector of environmental cold.
2007,
Pubmed
Benn,
Developmental expression of the TTX-resistant voltage-gated sodium channels Nav1.8 (SNS) and Nav1.9 (SNS2) in primary sensory neurons.
2001,
Pubmed
Brock,
Tetrodotoxin-resistant impulses in single nociceptor nerve terminals in guinea-pig cornea.
1998,
Pubmed
Cummins,
The roles of sodium channels in nociception: Implications for mechanisms of pain.
2007,
Pubmed
Cummins,
A novel persistent tetrodotoxin-resistant sodium current in SNS-null and wild-type small primary sensory neurons.
1999,
Pubmed
Daly,
Structures of muO-conotoxins from Conus marmoreus. I nhibitors of tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels in mammalian sensory neurons.
2004,
Pubmed
Dib-Hajj,
NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and down-regulated after axotomy.
1998,
Pubmed
Dong,
Small interfering RNA-mediated selective knockdown of Na(V)1.8 tetrodotoxin-resistant sodium channel reverses mechanical allodynia in neuropathic rats.
2007,
Pubmed
Gong,
Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication.
2002,
Pubmed
Hargreaves,
A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia.
1988,
Pubmed
Holford,
Manipulating neuronal circuits with endogenous and recombinant cell-surface tethered modulators.
2009,
Pubmed
Hu,
Mechanosensitive currents in the neurites of cultured mouse sensory neurones.
2006,
Pubmed
Ibañez-Tallon,
Tethering naturally occurring peptide toxins for cell-autonomous modulation of ion channels and receptors in vivo.
2004,
Pubmed
,
Xenbase
Ibañez-Tallon,
Novel modulation of neuronal nicotinic acetylcholine receptors by association with the endogenous prototoxin lynx1.
2002,
Pubmed
,
Xenbase
Jarvis,
A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat.
2007,
Pubmed
Lawson,
Phenotype and function of somatic primary afferent nociceptive neurones with C-, Adelta- or Aalpha/beta-fibres.
2002,
Pubmed
Lewin,
Mechanosensation and pain.
2004,
Pubmed
Luo,
Genetic dissection of neural circuits.
2008,
Pubmed
Matsutomi,
Multiple types of Na(+) currents mediate action potential electrogenesis in small neurons of mouse dorsal root ganglia.
2006,
Pubmed
Milenkovic,
Speed and temperature dependences of mechanotransduction in afferent fibers recorded from the mouse saphenous nerve.
2008,
Pubmed
Müller,
The bHLH factor Olig3 coordinates the specification of dorsal neurons in the spinal cord.
2005,
Pubmed
Nassar,
Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain.
2004,
Pubmed
Renganathan,
Na(v)1.5 underlies the 'third TTX-R sodium current' in rat small DRG neurons.
2002,
Pubmed
Rush,
A single sodium channel mutation produces hyper- or hypoexcitability in different types of neurons.
2006,
Pubmed
Sangameswaran,
A novel tetrodotoxin-sensitive, voltage-gated sodium channel expressed in rat and human dorsal root ganglia.
1997,
Pubmed
,
Xenbase
Snape,
Excitability parameters and sensitivity to anemone toxin ATX-II in rat small diameter primary sensory neurones discriminated by Griffonia simplicifolia isolectin IB4.
2010,
Pubmed
Stucky,
Isolectin B(4)-positive and -negative nociceptors are functionally distinct.
1999,
Pubmed
Terlau,
Conus venoms: a rich source of novel ion channel-targeted peptides.
2004,
Pubmed
Thompson,
Treatment challenges and complications with ziconotide monotherapy in established pump patients.
2006,
Pubmed
Zimmermann,
Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures.
2007,
Pubmed
