Damage-sensing nociceptors in the skin provide an indispensable protective function thanks to their specialized ability to detect and transmit hot temperatures that would block or inflict irreversible damage in other mammalian neurons. Here we show that the exceptional capacity of skin C-fiber nociceptors to encode noxiously hot temperatures depends on two tetrodotoxin (TTX)-resistant sodium channel α-subunits: NaV1.8 and NaV1.9. We demonstrate that NaV1.9, which is commonly considered an amplifier of subthreshold depolarizations at 20°C, undergoes a large gain of function when temperatures rise to the pain threshold. We also show that this gain of function renders NaV1.9 capable of generating action potentials with a clear inflection point and positive overshoot. In the skin, heat-resistant nociceptors appear as two distinct types with unique and possibly specialized features: one is blocked by TTX and relies on NaV1.9, and the second type is insensitive to TTX and composed of both NaV1.8 and NaV1.9. Independent of rapidly gated TTX-sensitive NaV channels that form the action potential at pain threshold, NaV1.8 is required in all heat-resistant nociceptors to encode temperatures higher than ∼46°C, whereas NaV1.9 is crucial for shaping the action potential upstroke and keeping the NaV1.8 voltage threshold within reach.

Department of Cellular Neurophysiology Institute of Physiology Academy of Sciences of the Czech Republic Prague Czech Republic

Department of Mathematics and Computer Science Bethel University St Paul MN

Department of Physiology and Pathophysiology Friedrich Alexander Universität Erlangen Nürnberg Erlangen Germany

Klinik für Anästhesiologie am Universitätsklinikum Erlangen Friedrich Alexander Universität Erlangen Nürnberg Erlangen Germany

Klinik für Anästhesiologie und Intensivmedizin Medizinische Hochschule Hannover Hannover Germany

Zobrazit více v PubMed

Akopian A.N., Souslova V., England S., Okuse K., Ogata N., Ure J., Smith A., Kerr B.J., McMahon S.B., Boyce S., et al. . 1999. The tetrodotoxin-resistant sodium channel SNS has a specialized function in pain pathways. Nat. Neurosci. 2:541–548. 10.1038/9195 PubMed DOI

Alle H., Roth A., and Geiger J.R.. 2009. Energy-efficient action potentials in hippocampal mossy fibers. Science. 325:1405–1408. 10.1126/science.1174331 PubMed DOI

Amaya F., Wang H., Costigan M., Allchorne A.J., Hatcher J.P., Egerton J., Stean T., Morisset V., Grose D., Gunthorpe M.J., et al. . 2006. The voltage-gated sodium channel Na(v)1.9 is an effector of peripheral inflammatory pain hypersensitivity. J. Neurosci. 26:12852–12860. 10.1523/JNEUROSCI.4015-06.2006 PubMed DOI PMC

Baker M.D. 2005. Protein kinase C mediates up-regulation of tetrodotoxin-resistant, persistent Na+ current in rat and mouse sensory neurones. J. Physiol. 567:851–867. 10.1113/jphysiol.2005.089771 PubMed DOI PMC

Bessou P., and Perl E.R.. 1969. Response of cutaneous sensory units with unmyelinated fibers to noxious stimuli. J. Neurophysiol. 32:1025–1043. 10.1152/jn.1969.32.6.1025 PubMed DOI

Blair N.T., and Bean B.P.. 2002. Roles of tetrodotoxin (TTX)-sensitive Na+ current, TTX-resistant Na+ current, and Ca2+ current in the action potentials of nociceptive sensory neurons. J. Neurosci. 22:10277–10290. 10.1523/JNEUROSCI.22-23-10277.2002 PubMed DOI PMC

Bretag A.H. 1969. Synthetic interstitial fluid for isolated mammalian tissue. Life Sci. 8:319–329. 10.1016/0024-3205(69)90283-5 PubMed DOI

Caterina M.J., Leffler A., Malmberg A.B., Martin W.J., Trafton J., Petersen-Zeitz K.R., Koltzenburg M., Basbaum A.I., and Julius D.. 2000. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science. 288:306–313. 10.1126/science.288.5464.306 PubMed DOI

Coste B., Osorio N., Padilla F., Crest M., and Delmas P.. 2004. Gating and modulation of presumptive NaV1.9 channels in enteric and spinal sensory neurons. Mol. Cell. Neurosci. 26:123–134. 10.1016/j.mcn.2004.01.015 PubMed DOI

Coste B., Crest M., and Delmas P.. 2007. Pharmacological dissection and distribution of NaN/Nav1.9, T-type Ca2+ currents, and mechanically activated cation currents in different populations of DRG neurons. J. Gen. Physiol. 129:57–77. 10.1085/jgp.200609665 PubMed DOI PMC

Cox J.J., Reimann F., Nicholas A.K., Thornton G., Roberts E., Springell K., Karbani G., Jafri H., Mannan J., Raashid Y., et al. . 2006. An SCN9A channelopathy causes congenital inability to experience pain. Nature. 444:894–898. 10.1038/nature05413 PubMed DOI PMC

Cummins T.R., Dib-Hajj S.D., Black J.A., Akopian A.N., Wood J.N., and Waxman S.G.. 1999. A novel persistent tetrodotoxin-resistant sodium current in SNS-null and wild-type small primary sensory neurons. J. Neurosci. 19:RC43 10.1523/JNEUROSCI.19-24-j0001.1999 PubMed DOI PMC

Dib-Hajj S.D., Tyrrell L., Black J.A., and Waxman S.G.. 1998. NaN, a novel voltage-gated Na channel, is expressed preferentially in peripheral sensory neurons and down-regulated after axotomy. Proc. Natl. Acad. Sci. USA. 95:8963–8968. 10.1073/pnas.95.15.8963 PubMed DOI PMC

Dib-Hajj S., Black J.A., Cummins T.R., and Waxman S.G.. 2002. NaN/Nav1.9: A sodium channel with unique properties. Trends Neurosci. 25:253–259. 10.1016/S0166-2236(02)02150-1 PubMed DOI

Dib-Hajj S.D., Black J.A., and Waxman S.G.. 2015. NaV1.9: A sodium channel linked to human pain. Nat. Rev. Neurosci. 16:511–519. 10.1038/nrn3977 PubMed DOI

Dittert I., Benedikt J., Vyklický L., Zimmermann K., Reeh P.W., and Vlachová V.. 2006. Improved superfusion technique for rapid cooling or heating of cultured cells under patch-clamp conditions. J. Neurosci. Methods. 151:178–185. 10.1016/j.jneumeth.2005.07.005 PubMed DOI

Enyedi P., and Czirják G.. 2010. Molecular background of leak K+ currents: Two-pore domain potassium channels. Physiol. Rev. 90:559–605. 10.1152/physrev.00029.2009 PubMed DOI

Fang X., Djouhri L., McMullan S., Berry C., Waxman S.G., Okuse K., and Lawson S.N.. 2006. Intense isolectin-B4 binding in rat dorsal root ganglion neurons distinguishes C-fiber nociceptors with broad action potentials and high Nav1.9 expression. J. Neurosci. 26:7281–7292. 10.1523/JNEUROSCI.1072-06.2006 PubMed DOI PMC

Fujii T., and Ibata Y.. 1982. Effects of heating on electrical activities of guinea pig olfactory cortical slices. Pflugers Arch. 392:257–260. 10.1007/BF00584306 PubMed DOI

Griffin J.D., and Boulant J.A.. 1995. Temperature effects on membrane potential and input resistance in rat hypothalamic neurones. J. Physiol. 488:407–418. 10.1113/jphysiol.1995.sp020975 PubMed DOI PMC

Hampl M., Eberhardt E., O’Reilly A.O., and Lampert A.. 2016. Sodium channel slow inactivation interferes with open channel block. Sci. Rep. 6:25974 10.1038/srep25974 PubMed DOI PMC

Hodgkin A.L., and Katz B.. 1949. The effect of temperature on the electrical activity of the giant axon of the squid. J. Physiol. 109:240–249. 10.1113/jphysiol.1949.sp004388 PubMed DOI PMC

Huth T., Rittger A., Saftig P., and Alzheimer C.. 2011. β-Site APP-cleaving enzyme 1 (BACE1) cleaves cerebellar Na+ channel β4-subunit and promotes Purkinje cell firing by slowing the decay of resurgent Na+ current. Pflugers Arch. 461:355–371. 10.1007/s00424-010-0913-2 PubMed DOI

Jo S., and Bean B.P.. 2011. Inhibition of neuronal voltage-gated sodium channels by brilliant blue G. Mol. Pharmacol. 80:247–257. 10.1124/mol.110.070276 PubMed DOI PMC

Leipold E., Hanson-Kahn A., Frick M., Gong P., Bernstein J.A., Voigt M., Katona I., Oliver Goral R., Altmüller J., Nürnberg P., et al. . 2015. Cold-aggravated pain in humans caused by a hyperactive NaV1.9 channel mutant. Nat. Commun. 6:10049 10.1038/ncomms10049 PubMed DOI PMC

Leo S., D’Hooge R., and Meert T.. 2010. Exploring the role of nociceptor-specific sodium channels in pain transmission using Nav1.8 and Nav1.9 knockout mice. Behav. Brain Res. 208:149–157. 10.1016/j.bbr.2009.11.023 PubMed DOI

Liu C.J., Dib-Hajj S.D., Black J.A., Greenwood J., Lian Z., and Waxman S.G.. 2001. Direct interaction with contactin targets voltage-gated sodium channel Na(v)1.9/NaN to the cell membrane. J. Biol. Chem. 276:46553–46561. 10.1074/jbc.M108699200 PubMed DOI

Lolignier S., Bonnet C., Gaudioso C., Noël J., Ruel J., Amsalem M., Ferrier J., Rodat-Despoix L., Bouvier V., Aissouni Y., et al. . 2015. The Nav1.9 channel is a key determinant of cold pain sensation and cold allodynia. Cell Reports. 11:1067–1078. 10.1016/j.celrep.2015.04.027 PubMed DOI

Lyfenko A., Vlachová V., Vyklický L., Dittert I., Kress M., and Reeh P.W.. 2002. The effects of excessive heat on heat-activated membrane currents in cultured dorsal root ganglia neurons from neonatal rat. Pain. 95:207–214. 10.1016/S0304-3959(01)00401-8 PubMed DOI

Matsutomi T., Nakamoto C., Zheng T., Kakimura J., and Ogata N.. 2006. Multiple types of Na(+) currents mediate action potential electrogenesis in small neurons of mouse dorsal root ganglia. Pflugers Arch. 453:83–96. 10.1007/s00424-006-0104-3 PubMed DOI

McCoy E.S., Taylor-Blake B., Street S.E., Pribisko A.L., Zheng J., and Zylka M.J.. 2013. Peptidergic CGRPα primary sensory neurons encode heat and itch and tonically suppress sensitivity to cold. Neuron. 78:138–151. 10.1016/j.neuron.2013.01.030 PubMed DOI PMC

Minett M.S., Eijkelkamp N., and Wood J.N.. 2014. Significant determinants of mouse pain behaviour. PLoS One. 9:e104458 10.1371/journal.pone.0104458 PubMed DOI PMC

Nevo E. 2011. Evolution under environmental stress at macro- and microscales. Genome Biol. Evol. 3:1039–1052. 10.1093/gbe/evr052 PubMed DOI PMC

Ostman J.A., Nassar M.A., Wood J.N., and Baker M.D.. 2008. GTP up-regulated persistent Na+ current and enhanced nociceptor excitability require NaV1.9. J. Physiol. 586:1077–1087. 10.1113/jphysiol.2007.147942 PubMed DOI PMC

Priest B.T., Murphy B.A., Lindia J.A., Diaz C., Abbadie C., Ritter A.M., Liberator P., Iyer L.M., Kash S.F., Kohler M.G., et al. . 2005. Contribution of the tetrodotoxin-resistant voltage-gated sodium channel NaV1.9 to sensory transmission and nociceptive behavior. Proc. Natl. Acad. Sci. USA. 102:9382–9387. 10.1073/pnas.0501549102 PubMed DOI PMC

Renganathan M., Cummins T.R., and Waxman S.G.. 2001. Contribution of Na(v)1.8 sodium channels to action potential electrogenesis in DRG neurons. J. Neurophysiol. 86:629–640. 10.1152/jn.2001.86.2.629 PubMed DOI

Rogers M., Zidar N., Kikelj D., and Kirby R.W.. 2016. Characterization of endogenous sodium channels in the ND7-23 neuroblastoma cell line: Implications for use as a heterologous ion channel expression system suitable for automated patch clamp screening. Assay Drug Dev. Technol. 14:109–130. 10.1089/adt.2016.704 PubMed DOI PMC

Rugiero F., Mistry M., Sage D., Black J.A., Waxman S.G., Crest M., Clerc N., Delmas P., and Gola M.. 2003. Selective expression of a persistent tetrodotoxin-resistant Na+ current and NaV1.9 subunit in myenteric sensory neurons. J. Neurosci. 23:2715–2725. 10.1523/JNEUROSCI.23-07-02715.2003 PubMed DOI PMC

Rush A.M., and Waxman S.G.. 2004. PGE2 increases the tetrodotoxin-resistant Nav1.9 sodium current in mouse DRG neurons via G-proteins. Brain Res. 1023:264–271. 10.1016/j.brainres.2004.07.042 PubMed DOI

Rush A.M., Craner M.J., Kageyama T., Dib-Hajj S.D., Waxman S.G., and Ranscht B.. 2005. Contactin regulates the current density and axonal expression of tetrodotoxin-resistant but not tetrodotoxin-sensitive sodium channels in DRG neurons. Eur. J. Neurosci. 22:39–49. 10.1111/j.1460-9568.2005.04186.x PubMed DOI

Rush A.M., Cummins T.R., and Waxman S.G.. 2007. Multiple sodium channels and their roles in electrogenesis within dorsal root ganglion neurons. J. Physiol. 579:1–14. 10.1113/jphysiol.2006.121483 PubMed DOI PMC

Shen K.F., and Schwartzkroin P.A.. 1988. Effects of temperature alterations on population and cellular activities in hippocampal slices from mature and immature rabbit. Brain Res. 475:305–316. 10.1016/0006-8993(88)90619-1 PubMed DOI

Smith E.S., Omerbašić D., Lechner S.G., Anirudhan G., Lapatsina L., and Lewin G.R.. 2011. The molecular basis of acid insensitivity in the African naked mole-rat. Science. 334:1557–1560. 10.1126/science.1213760 PubMed DOI

St Pierre M., Reeh P.W., and Zimmermann K.. 2009. Differential effects of TRPV channel block on polymodal activation of rat cutaneous nociceptors in vitro. Exp. Brain Res. 196:31–44. 10.1007/s00221-009-1808-3 PubMed DOI

Stucky C.L., and Lewin G.R.. 1999. Isolectin B(4)-positive and -negative nociceptors are functionally distinct. J. Neurosci. 19:6497–6505. 10.1523/JNEUROSCI.19-15-06497.1999 PubMed DOI PMC

Tate S., Benn S., Hick C., Trezise D., John V., Mannion R.J., Costigan M., Plumpton C., Grose D., Gladwell Z., et al. . 1998. Two sodium channels contribute to the TTX-R sodium current in primary sensory neurons. Nat. Neurosci. 1:653–655. 10.1038/3652 PubMed DOI

Usoskin D., Furlan A., Islam S., Abdo H., Lönnerberg P., Lou D., Hjerling-Leffler J., Haeggström J., Kharchenko O., Kharchenko P.V., et al. . 2015. Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing. Nat. Neurosci. 18:145–153. 10.1038/nn.3881 PubMed DOI

Vandewauw I., De Clercq K., Mulier M., Held K., Pinto S., Van Ranst N., Segal A., Voet T., Vennekens R., Zimmermann K., et al. . 2018. A TRP channel trio mediates acute noxious heat sensing. Nature. 555:662–666. 10.1038/nature26137 PubMed DOI

Vanoye C.G., Kunic J.D., Ehring G.R., and George A.L. Jr. 2013. Mechanism of sodium channel NaV1.9 potentiation by G-protein signaling. J. Gen. Physiol. 141:193–202. 10.1085/jgp.201210919 PubMed DOI PMC

Vetter I., Hein A., Sattler S., Hessler S., Touska F., Bressan E., Parra A., Hager U., Leffler A., Boukalova S., et al. . 2013. Amplified cold transduction in native nociceptors by M-channel inhibition. J. Neurosci. 33:16627–16641. 10.1523/JNEUROSCI.1473-13.2013 PubMed DOI PMC

Volgushev M., Vidyasagar T.R., Chistiakova M., Yousef T., and Eysel U.T.. 2000. Membrane properties and spike generation in rat visual cortical cells during reversible cooling. J. Physiol. 522:59–76. 10.1111/j.1469-7793.2000.0059m.x PubMed DOI PMC

Vyklický L., Vlachová V., Vitásková Z., Dittert I., Kabát M., and Orkand R.K.. 1999. Temperature coefficient of membrane currents induced by noxious heat in sensory neurones in the rat. J. Physiol. 517:181–192. 10.1111/j.1469-7793.1999.0181z.x PubMed DOI PMC

Waxman S.G., and Zamponi G.W.. 2014. Regulating excitability of peripheral afferents: Emerging ion channel targets. Nat. Neurosci. 17:153–163. 10.1038/nn.3602 PubMed DOI

Winter Z., Gruschwitz P., Eger S., Touska F., and Zimmermann K.. 2017. Cold temperature encoding by cutaneous TRPA1 and TRPM8-carrying fibers in the mouse. Front. Mol. Neurosci. 10:209 10.3389/fnmol.2017.00209 PubMed DOI PMC

Zakon H.H. 2012. Adaptive evolution of voltage-gated sodium channels: The first 800 million years. Proc. Natl. Acad. Sci. USA. 109(Suppl 1):10619–10625. 10.1073/pnas.1201884109 PubMed DOI PMC

Zhang M.M., Wilson M.J., Azam L., Gajewiak J., Rivier J.E., Bulaj G., Olivera B.M., and Yoshikami D.. 2013. Co-expression of Na(V)β subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking μ-conotoxins. Br. J. Pharmacol. 168:1597–1610. 10.1111/bph.12051 PubMed DOI PMC

Zimmermann K., Leffler A., Fischer M.M., Messlinger K., Nau C., and Reeh P.W.. 2005. The TRPV1/2/3 activator 2-aminoethoxydiphenyl borate sensitizes native nociceptive neurons to heat in wildtype but not TRPV1 deficient mice. Neuroscience. 135:1277–1284. 10.1016/j.neuroscience.2005.07.018 PubMed DOI

Zimmermann K., Leffler A., Babes A., Cendan C.M., Carr R.W., Kobayashi J., Nau C., Wood J.N., and Reeh P.W.. 2007. Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures. Nature. 447:855–858. 10.1038/nature05880 PubMed DOI

Zimmermann K., Hein A., Hager U., Kaczmarek J.S., Turnquist B.P., Clapham D.E., and Reeh P.W.. 2009. Phenotyping sensory nerve endings in vitro in the mouse. Nat. Protoc. 4:174–196. 10.1038/nprot.2008.223 PubMed DOI PMC

Zimmermann K., Lennerz J.K., Hein A., Link A.S., Kaczmarek J.S., Delling M., Uysal S., Pfeifer J.D., Riccio A., and Clapham D.E.. 2011. Transient receptor potential cation channel, subfamily C, member 5 (TRPC5) is a cold-transducer in the peripheral nervous system. Proc. Natl. Acad. Sci. USA. 108:18114–18119. 10.1073/pnas.1115387108 PubMed DOI PMC