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Phosphorylation of a chronic pain mutation in the voltage-gated sodium channel Nav1.7 increases voltage sensitivity

2020; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1074/jbc.ra120.014288

ISSN

1083-351X

Autores

Clara M. Kerth, Petra Hautvast, Jannis Körner, Angelika Lampert, Jannis Meents,

Tópico(s)

Cardiac electrophysiology and arrhythmias

Resumo

Mutations in voltage-gated sodium channels (Navs) can cause alterations in pain sensation, such as chronic pain diseases like inherited erythromelalgia. The mutation causing inherited erythromelalgia, Nav1.7 p.I848T, is known to induce a hyperpolarized shift in the voltage dependence of activation in Nav1.7. So far, however, the mechanism to explain this increase in voltage sensitivity remains unknown. In the present study, we show that phosphorylation of the newly introduced Thr residue explains the functional change. We expressed wildtype human Nav1.7, the I848T mutant, or other mutations in HEK293T cells and performed whole-cell patch-clamp electrophysiology. As the insertion of a Thr residue potentially creates a novel phosphorylation site for Ser/Thr kinases and because Nav1.7 had been shown in Xenopus oocytes to be affected by protein kinases C and A, we used different nonselective and selective kinase inhibitors and activators to test the effect of phosphorylation on Nav1.7 in a human system. We identify protein kinase C, but not protein kinase A, to be responsible for the phosphorylation of T848 and thereby for the shift in voltage sensitivity. Introducing a negatively charged amino acid instead of the putative phosphorylation site mimics the effect on voltage gating to a lesser extent. 3D modeling using the published cryo-EM structure of human Nav1.7 showed that introduction of this negatively charged site seems to alter the interaction of this residue with the surrounding amino acids and thus to influence channel function. These results could provide new opportunities for the development of novel treatment options for patients with chronic pain. Mutations in voltage-gated sodium channels (Navs) can cause alterations in pain sensation, such as chronic pain diseases like inherited erythromelalgia. The mutation causing inherited erythromelalgia, Nav1.7 p.I848T, is known to induce a hyperpolarized shift in the voltage dependence of activation in Nav1.7. So far, however, the mechanism to explain this increase in voltage sensitivity remains unknown. In the present study, we show that phosphorylation of the newly introduced Thr residue explains the functional change. We expressed wildtype human Nav1.7, the I848T mutant, or other mutations in HEK293T cells and performed whole-cell patch-clamp electrophysiology. As the insertion of a Thr residue potentially creates a novel phosphorylation site for Ser/Thr kinases and because Nav1.7 had been shown in Xenopus oocytes to be affected by protein kinases C and A, we used different nonselective and selective kinase inhibitors and activators to test the effect of phosphorylation on Nav1.7 in a human system. We identify protein kinase C, but not protein kinase A, to be responsible for the phosphorylation of T848 and thereby for the shift in voltage sensitivity. Introducing a negatively charged amino acid instead of the putative phosphorylation site mimics the effect on voltage gating to a lesser extent. 3D modeling using the published cryo-EM structure of human Nav1.7 showed that introduction of this negatively charged site seems to alter the interaction of this residue with the surrounding amino acids and thus to influence channel function. These results could provide new opportunities for the development of novel treatment options for patients with chronic pain. Voltage-gated sodium channels (Navs) are important for the proper functioning of our body, especially in the nervous system and muscle tissue including cardiac muscle cells. Human Navs have four pore-forming domains (DI–IV), each of them equipped with six transmembrane segments (S1–S6). S1–S4 of each domain contain the voltage sensor, whereas S5/S6 form the channel pore (1Ahern C.A. Payandeh J. Bosmans F. Chanda B. The hitchhiker's guide to the voltage-gated sodium channel galaxy.J. Gen. Physiol. 2016; 147: 1-24Crossref PubMed Scopus (143) Google Scholar, 2Dib-Hajj S.D. Yang Y. Black J.A. Waxman S.G. The Na(V)1.7 sodium channel: from molecule to man.Nat. Rev. Neurosci. 2013; 14: 49-62Crossref PubMed Scopus (344) Google Scholar). Nine human Nav isoforms (Nav1.1–1.9) have been described (3Meents J.E. Lampert A. Studying sodium channel gating in heterologous expression systems.in: Advanced Patch-Clamp Analysis for Neuroscientists, Neuromethods. 113. Humana Press, New York, NY2016: 37-65Google Scholar), expressed throughout different tissues (1Ahern C.A. Payandeh J. Bosmans F. Chanda B. The hitchhiker's guide to the voltage-gated sodium channel galaxy.J. Gen. Physiol. 2016; 147: 1-24Crossref PubMed Scopus (143) Google Scholar, 4Catterall W.A. Goldin A.L. Waxman S.G. International Union of Pharmacology. XLVII. Nomenclature and structure-function relationships of voltage-gated sodium channels.Pharmacol. Rev. 2005; 57: 397-409Crossref PubMed Scopus (963) Google Scholar, 5Körner J. Lampert A. Sodium channels.in: Fritzsch B. The Senses - A Comprehensive Reference. 2nd Ed. Academic press, Cambridge, MA2020: 120-141Crossref Google Scholar). Next to the pore-forming α-subunit of Nav channels, there are four β-subunits (SCNB1b–4b; β1–4), which are believed to modify the gating and expression of Navs (1Ahern C.A. Payandeh J. Bosmans F. Chanda B. The hitchhiker's guide to the voltage-gated sodium channel galaxy.J. Gen. Physiol. 2016; 147: 1-24Crossref PubMed Scopus (143) Google Scholar, 5Körner J. Lampert A. Sodium channels.in: Fritzsch B. The Senses - A Comprehensive Reference. 2nd Ed. Academic press, Cambridge, MA2020: 120-141Crossref Google Scholar). Among the nine Nav isoforms, Nav1.7 to 1.9 are relevant for the perception of pain as they are mainly located in peripheral sensory neurons of the dorsal root ganglion, sympathetic ganglion, and in olfactory sensory neurons (6Meents J.E. Bressan E. Sontag S. Foerster A. Hautvast P. Rosseler C. Hampl M. Schuler H. Goetzke R. Chi Le T.K. Kleggetveit I.P. Le Cann K. Kerth C. Rush A.M. Rogers M. et al.The role of Nav1.7 in human nociceptors: insights from human iPS cell-derived sensory neurons of erythromelalgia patients.Pain. 2019; 160: 1327-1341Crossref PubMed Scopus (26) Google Scholar, 7Dib-Hajj S.D. Geha P. Waxman S.G. Sodium channels in pain disorders: pathophysiology and prospects for treatment.Pain. 2017; 158 Suppl 1: S97-S107Crossref PubMed Scopus (36) Google Scholar, 8Wang J. Ou S.W. Wang Y.J. Distribution and function of voltage-gated sodium channels in the nervous system.Channels (Austin). 2017; 11: 534-554Crossref PubMed Scopus (36) Google Scholar, 9Waxman S.G. Merkies I.S.J. Gerrits M.M. Dib-Hajj S.D. Lauria G. Cox J.J. Wood J.N. Woods C.G. Drenth J.P.H. Faber C.G. Sodium channel genes in pain-related disorders: phenotype-genotype associations and recommendations for clinical use.Lancet Neurol. 2014; 13: 1152-1160Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). In a previous study, we have shown that human Nav1.7 is especially important for defining the threshold of the action potential in nociceptors and plays an important role in the action potential upstroke (6Meents J.E. Bressan E. Sontag S. Foerster A. Hautvast P. Rosseler C. Hampl M. Schuler H. Goetzke R. Chi Le T.K. Kleggetveit I.P. Le Cann K. Kerth C. Rush A.M. Rogers M. et al.The role of Nav1.7 in human nociceptors: insights from human iPS cell-derived sensory neurons of erythromelalgia patients.Pain. 2019; 160: 1327-1341Crossref PubMed Scopus (26) Google Scholar). Considering this role of Nav1.7, one might deduct that mutations in Nav1.7 can lead to changes in neuronal excitability and thereby to altered pain perception. Accordingly, several mutations in Nav1.7 are known to cause a variety of chronic pain syndromes, such as inherited erythromelalgia (IEM) or paroxysmal extreme pain disorder, and seem to play a role in small fiber neuropathy (7Dib-Hajj S.D. Geha P. Waxman S.G. Sodium channels in pain disorders: pathophysiology and prospects for treatment.Pain. 2017; 158 Suppl 1: S97-S107Crossref PubMed Scopus (36) Google Scholar, 10Brouwer B.A. Merkies I.S. Gerrits M.M. Waxman S.G. Hoeijmakers J.G. Faber C.G. Painful neuropathies: the emerging role of sodium channelopathies.J. Peripher. Nerv. Syst. 2014; 19: 53-65Crossref PubMed Scopus (66) Google Scholar). Patients with IEM have sudden pain attacks and synchronous erythema of the distal extremities, often triggered by exercise or exposure to higher temperatures (11Dib-Hajj S.D. Cummins T.R. Black J.A. Waxman S.G. From genes to pain: Na v 1.7 and human pain disorders.Trends Neurosci. 2007; 30: 555-563Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 12Novella S.P. Hisama F.M. Dib-Hajj S.D. Waxman S.G. A case of inherited erythromelalgia.Nat. Clin. Pract. Neurol. 2007; 3: 229-234Crossref PubMed Scopus (28) Google Scholar). Unfortunately, neither common pain treatments nor nonselective sodium channel blockers, such as lidocaine, provide satisfactory pain relief (7Dib-Hajj S.D. Geha P. Waxman S.G. Sodium channels in pain disorders: pathophysiology and prospects for treatment.Pain. 2017; 158 Suppl 1: S97-S107Crossref PubMed Scopus (36) Google Scholar, 13Davis M.D. Rooke T. Erythromelalgia.Curr. Treat. Options Cardiovasc. Med. 2006; 8: 153-165Crossref PubMed Scopus (20) Google Scholar, 14Tang Z. Chen Z. Tang B. Jiang H. Primary erythromelalgia: a review.Orphanet J. Rare Dis. 2015; 10: 127Crossref PubMed Scopus (49) Google Scholar). One selective sodium channel blocker (PF-0508977) was described to mildly relieve pain in patients with IEM, but the effects were very short lasting (15Cao L. McDonnell A. Nitzsche A. Alexandrou A. Saintot P.P. Loucif A.J. Brown A.R. Young G. Mis M. Randall A. Waxman S.G. Stanley P. Kirby S. Tarabar S. Gutteridge A. et al.Pharmacological reversal of a pain phenotype in iPSC-derived sensory neurons and patients with inherited erythromelalgia.Sci. Transl. Med. 2016; 8: 335ra356Crossref Scopus (105) Google Scholar). Different gain-of-function mutations known to cause IEM are located in the SCN9A gene, which encodes for Nav1.7 (16Wu M.T. Huang P.Y. Yen C.T. Chen C.C. Lee M.J. A novel SCN9A mutation responsible for primary erythromelalgia and is resistant to the treatment of sodium channel blockers.PLoS One. 2013; 8e55212Crossref PubMed Scopus (46) Google Scholar, 17Cummins T.R. Dib-Hajj S.D. Waxman S.G. Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy.J. Neurosci. 2004; 24: 8232-8236Crossref PubMed Scopus (293) Google Scholar, 18Lampert A. O'Reilly A.O. Dib-Hajj S.D. Tyrrell L. Wallace B.A. Waxman S.G. A pore-blocking hydrophobic motif at the cytoplasmic aperture of the closed-state Nav1.7 channel is disrupted by the erythromelalgia-associated F1449V mutation.J. Biol. Chem. 2008; 283: 24118-24127Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 19Han C. Dib-Hajj S.D. Lin Z. Li Y. Eastman E.M. Tyrrell L. Cao X. Yang Y. Waxman S.G. Early- and late-onset inherited erythromelalgia: genotype-phenotype correlation.Brain. 2009; 132: 1711-1722Crossref PubMed Scopus (93) Google Scholar, 20Cheng X. Dib-Hajj S.D. Tyrrell L. Waxman S.G. Mutation I136V alters electrophysiological properties of the Na(v)1.7 channel in a family with onset of erythromelalgia in the second decade.Mol. Pain. 2008; 4: 1Crossref PubMed Scopus (67) Google Scholar, 21Dib-Hajj S.D. Rush A.M. Cummins T.R. Hisama F.M. Novella S. Tyrrell L. Marshall L. Waxman S.G. Gain-of-function mutation in Nav1.7 in familial erythromelalgia induces bursting of sensory neurons.Brain. 2005; 128: 1847-1854Crossref PubMed Scopus (337) Google Scholar, 22Drenth J.P. te Morsche R.H. Guillet G. Taieb A. Kirby R.L. Jansen J.B. SCN9A mutations define primary erythermalgia as a neuropathic disorder of voltage gated sodium channels.J. Invest. Dermatol. 2005; 124: 1333-1338Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). One of the earliest known of such mutations is the gain-of-function mutation Nav1.7 p.I848T, which is located in the intracellular S4–S5 linker region of domain II (Fig. 1A) (16Wu M.T. Huang P.Y. Yen C.T. Chen C.C. Lee M.J. A novel SCN9A mutation responsible for primary erythromelalgia and is resistant to the treatment of sodium channel blockers.PLoS One. 2013; 8e55212Crossref PubMed Scopus (46) Google Scholar, 17Cummins T.R. Dib-Hajj S.D. Waxman S.G. Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy.J. Neurosci. 2004; 24: 8232-8236Crossref PubMed Scopus (293) Google Scholar) and which leads to an early onset of IEM (11Dib-Hajj S.D. Cummins T.R. Black J.A. Waxman S.G. From genes to pain: Na v 1.7 and human pain disorders.Trends Neurosci. 2007; 30: 555-563Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 17Cummins T.R. Dib-Hajj S.D. Waxman S.G. Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy.J. Neurosci. 2004; 24: 8232-8236Crossref PubMed Scopus (293) Google Scholar, 19Han C. Dib-Hajj S.D. Lin Z. Li Y. Eastman E.M. Tyrrell L. Cao X. Yang Y. Waxman S.G. Early- and late-onset inherited erythromelalgia: genotype-phenotype correlation.Brain. 2009; 132: 1711-1722Crossref PubMed Scopus (93) Google Scholar). Throughout all domains, the S4–S5 linker regions are known to contain a variety of disease- (and IEM-) causing mutations in Navs (14Tang Z. Chen Z. Tang B. Jiang H. Primary erythromelalgia: a review.Orphanet J. Rare Dis. 2015; 10: 127Crossref PubMed Scopus (49) Google Scholar, 16Wu M.T. Huang P.Y. Yen C.T. Chen C.C. Lee M.J. A novel SCN9A mutation responsible for primary erythromelalgia and is resistant to the treatment of sodium channel blockers.PLoS One. 2013; 8e55212Crossref PubMed Scopus (46) Google Scholar, 23Ahn H.S. Dib-Hajj S.D. Cox J.J. Tyrrell L. Elmslie F.V. Clarke A.A. Drenth J.P. Woods C.G. Waxman S.G. A new Nav1.7 sodium channel mutation I234T in a child with severe pain.Eur. J. Pain. 2010; 14: 944-950Crossref PubMed Scopus (36) Google Scholar, 24Hoeijmakers J.G. Han C. Merkies I.S. Macala L.J. Lauria G. Gerrits M.M. Dib-Hajj S.D. Faber C.G. Waxman S.G. Small nerve fibres, small hands and small feet: a new syndrome of pain, dysautonomia and acromesomelia in a kindred with a novel NaV1.7 mutation.Brain. 2012; 135: 345-358Crossref PubMed Scopus (60) Google Scholar, 25Eberhardt M. Nakajima J. Klinger A.B. Neacsu C. Huhne K. O'Reilly A.O. Kist A.M. Lampe A.K. Fischer K. Gibson J. Nau C. Winterpacht A. Lampert A. Inherited pain: sodium channel Nav1.7 A1632T mutation causes erythromelalgia due to a shift of fast inactivation.J. Biol. Chem. 2014; 289: 1971-1980Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Cheng X. Dib-Hajj S.D. Tyrrell L. Wright D.A. Fischer T.Z. Waxman S.G. Mutations at opposite ends of the DIII/S4-S5 linker of sodium channel Na V 1.7 produce distinct pain disorders.Mol. Pain. 2010; 6: 24Crossref PubMed Scopus (30) Google Scholar, 27Lampert A. Dib-Hajj S.D. Tyrrell L. Waxman S.G. Size matters: erythromelalgia mutation S241T in Nav1.7 alters channel gating.J. Biol. Chem. 2006; 281: 36029-36035Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 28Zhang L.L. Lin Z.M. Ma Z.H. Xu Z. Yang Y.L. Yang Y. Mutation hotspots of SCN9A in primary erythermalgia.Br. J. Dermatol. 2007; 156: 767-769Crossref PubMed Scopus (15) Google Scholar). The functional importance of the S4–S5 linker regions for Nav channel gating has recently been shown using cryo-EM of the ancestral bacterial Nav channel (29Wisedchaisri G. Tonggu L. McCord E. Gamal El-Din T.M. Wang L. Zheng N. Catterall W.A. Resting-state structure and gating mechanism of a voltage-gated sodium channel.Cell. 2019; 178: 993-1003.e1012Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Of interest, the authors identified three amino acid residues that are key for the opening and closing of the channel gate: I119, L123, V126 (29Wisedchaisri G. Tonggu L. McCord E. Gamal El-Din T.M. Wang L. Zheng N. Catterall W.A. Resting-state structure and gating mechanism of a voltage-gated sodium channel.Cell. 2019; 178: 993-1003.e1012Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). These data might suggest that any changes to these crucial amino acids could affect Nav gating and nociceptor excitability. The human Nav1.7 I848 residue corresponds to the crucial amino acid L123 of the bacterial Nav channel. This could explain why this mutation causes changes in the gating of Nav1.7. In fact, we and others have shown that the Nav1.7/I848T mutation induces significant changes in action potential firing behavior of human induced pluripotent stem cell–derived nociceptors, such as a decreased firing threshold, a decreased time to peak, and an enhanced upstroke of the action potential (6Meents J.E. Bressan E. Sontag S. Foerster A. Hautvast P. Rosseler C. Hampl M. Schuler H. Goetzke R. Chi Le T.K. Kleggetveit I.P. Le Cann K. Kerth C. Rush A.M. Rogers M. et al.The role of Nav1.7 in human nociceptors: insights from human iPS cell-derived sensory neurons of erythromelalgia patients.Pain. 2019; 160: 1327-1341Crossref PubMed Scopus (26) Google Scholar, 15Cao L. McDonnell A. Nitzsche A. Alexandrou A. Saintot P.P. Loucif A.J. Brown A.R. Young G. Mis M. Randall A. Waxman S.G. Stanley P. Kirby S. Tarabar S. Gutteridge A. et al.Pharmacological reversal of a pain phenotype in iPSC-derived sensory neurons and patients with inherited erythromelalgia.Sci. Transl. Med. 2016; 8: 335ra356Crossref Scopus (105) Google Scholar). Phosphorylation can modify and regulate the function or expression of Nav channels (2Dib-Hajj S.D. Yang Y. Black J.A. Waxman S.G. The Na(V)1.7 sodium channel: from molecule to man.Nat. Rev. Neurosci. 2013; 14: 49-62Crossref PubMed Scopus (344) Google Scholar, 30Vijayaragavan K.B.,M. Chahine M. Modulation of Nav1.7 and Nav1.8 peripheral nerve sodium channels by protein kinase A and protein kinase C.J. Neurophysiol. 2004; 91: 1156-1569Crossref Scopus (97) Google Scholar, 31Chatelier A. Dahllund L. Eriksson A. Krupp J. Chahine M. Biophysical properties of human Na v1.7 splice variants and their regulation by protein kinase A.J. Neurophysiol. 2008; 99: 2241-2250Crossref PubMed Scopus (39) Google Scholar, 32Scheuer T. Regulation of sodium channel activity by phosphorylation.Semin. Cell Dev. Biol. 2011; 22: 160-165Crossref PubMed Scopus (55) Google Scholar, 33Laedermann C.J. Abriel H. Decosterd I. Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.Front. Pharmacol. 2015; 6: 263Crossref PubMed Scopus (37) Google Scholar, 34Lorenzini M. Burel S. Lesage A. Wagner E. Charrière C. Chevillard P.-M. Evrard B. Maloney D. Ruff K.M. Pappu R.V. Wagner S. Nerbonne J.M. Silva J.R. Townsend R.R. Maier L.S. et al.Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation reveals multisite regulation of surface expression and gating.bioRxiv. 2020; ([preprint])https://doi.org/10.1101/2020.04.29.067835Crossref PubMed Scopus (0) Google Scholar). It was shown in Xenopus laevis oocytes that phosphorylation by protein kinase C (PKC) and protein kinase A (PKA) can increase or decrease the peak currents of Nav1.7 and Nav1.8 and more importantly induce shifts in voltage dependence of activation (30Vijayaragavan K.B.,M. Chahine M. Modulation of Nav1.7 and Nav1.8 peripheral nerve sodium channels by protein kinase A and protein kinase C.J. Neurophysiol. 2004; 91: 1156-1569Crossref Scopus (97) Google Scholar). In this light, it seems possible that phosphorylation might be responsible for the changes of the Nav1.7/I848T mutation, as this mutant provides a potential novel phosphorylation site. In this study, we show in whole-cell voltage-clamp experiments that phosphorylation of the Nav1.7/I848T mutant is responsible for the hyperpolarized shift in the voltage dependence of activation. Moreover, by introducing a negative charge to mimic the phosphorylated I848T residue, we confirm a possible phosphorylation. To try to close the gap between the newly discovered phosphorylation of the I848T mutant and its effect on channel gating, we performed mutagenesis to investigate potential molecular interactions of the phosphorylated side group and the channel pore. We show that this negatively charged side chain could interfere with noncovalent π-interactions of the surrounding phenylalanines and could thereby lead to changes in channel gating. In the following sections, the mutant Nav1.7 channel will be termed I848T, whereas the individual mutated amino acid residue will be labeled T848. The gain-of-function mutation Nav1.7/I848T (Fig. 1A) is known to cause IEM. Earlier studies have shown that the I848T mutation causes a hyperpolarizing shift in the voltage dependence of activation of Nav1.7 (16Wu M.T. Huang P.Y. Yen C.T. Chen C.C. Lee M.J. A novel SCN9A mutation responsible for primary erythromelalgia and is resistant to the treatment of sodium channel blockers.PLoS One. 2013; 8e55212Crossref PubMed Scopus (46) Google Scholar), which seems to lead to increased pain in the patient (6Meents J.E. Bressan E. Sontag S. Foerster A. Hautvast P. Rosseler C. Hampl M. Schuler H. Goetzke R. Chi Le T.K. Kleggetveit I.P. Le Cann K. Kerth C. Rush A.M. Rogers M. et al.The role of Nav1.7 in human nociceptors: insights from human iPS cell-derived sensory neurons of erythromelalgia patients.Pain. 2019; 160: 1327-1341Crossref PubMed Scopus (26) Google Scholar, 15Cao L. McDonnell A. Nitzsche A. Alexandrou A. Saintot P.P. Loucif A.J. Brown A.R. Young G. Mis M. Randall A. Waxman S.G. Stanley P. Kirby S. Tarabar S. Gutteridge A. et al.Pharmacological reversal of a pain phenotype in iPSC-derived sensory neurons and patients with inherited erythromelalgia.Sci. Transl. Med. 2016; 8: 335ra356Crossref Scopus (105) Google Scholar, 17Cummins T.R. Dib-Hajj S.D. Waxman S.G. Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy.J. Neurosci. 2004; 24: 8232-8236Crossref PubMed Scopus (293) Google Scholar). Here, we first attempted to confirm these earlier results in a heterologous expression system, using human embryonic kidney cell line HEK293T cells transiently transfected with either wildtype Nav1.7 or its I848T mutant form. First, we found that the I848T mutant produces significantly smaller currents than the wildtype channel (Fig. 1, B–C, Table 1), which has not been reported so far. An earlier study of Cummins et al. (17Cummins T.R. Dib-Hajj S.D. Waxman S.G. Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy.J. Neurosci. 2004; 24: 8232-8236Crossref PubMed Scopus (293) Google Scholar) cotransfecting Nav1.7 with the β1- and β2-subunits did not show the decrease in current density. However, when we cotransfected both β-subunits with the I848T mutant or the wildtype channel, we still observed a significant decrease in current density for the I848T mutant (Fig. S1C and Table S1). Furthermore, we identified a hyperpolarized shift in the voltage dependence of activation of −9.77 ± 1.53 mV (Fig. 1, D–E, Table 1), which is comparable with earlier results reported in heterologous systems (7Dib-Hajj S.D. Geha P. Waxman S.G. Sodium channels in pain disorders: pathophysiology and prospects for treatment.Pain. 2017; 158 Suppl 1: S97-S107Crossref PubMed Scopus (36) Google Scholar, 16Wu M.T. Huang P.Y. Yen C.T. Chen C.C. Lee M.J. A novel SCN9A mutation responsible for primary erythromelalgia and is resistant to the treatment of sodium channel blockers.PLoS One. 2013; 8e55212Crossref PubMed Scopus (46) Google Scholar, 17Cummins T.R. Dib-Hajj S.D. Waxman S.G. Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy.J. Neurosci. 2004; 24: 8232-8236Crossref PubMed Scopus (293) Google Scholar) as well as to previous results in iPS-derived nociceptors of patients with IEM (6Meents J.E. Bressan E. Sontag S. Foerster A. Hautvast P. Rosseler C. Hampl M. Schuler H. Goetzke R. Chi Le T.K. Kleggetveit I.P. Le Cann K. Kerth C. Rush A.M. Rogers M. et al.The role of Nav1.7 in human nociceptors: insights from human iPS cell-derived sensory neurons of erythromelalgia patients.Pain. 2019; 160: 1327-1341Crossref PubMed Scopus (26) Google Scholar). As expected, we did not find any difference in the voltage dependence of steady-state fast inactivation between the wildtype and I848T (Fig. 1F).Table 1Voltage-clamp parameters of the different Nav1.7 mutantsGenotypeActivationFast inactivationCurrent densitynV1/2 (mV)Slope (V/s)Gmax (pS)nV1/2 (mV)Slope (V/s)(pA/pF)WT31−18.3 ± 5.918.91 ± 1.2476.62 ± 53.1330−84.21 ± 6.826.67 ± 1.42−416.5 ± 291.5I848T30−28.06 ± 5.428.99 ± 1.4841.01 ± 29.5132−86.32 ± 5.116.48 ± 1.36−252.8 ± 167.6I848E20−24.1 ± 6.9111.09 ± 1.777.79 ± 4.5915−84.05 ± 6.66.99 ± 1.93−53.29 ± 47.09F1432L WT18−20.02 ± 4.039.14 ± 0.83110.0 ± 91.6120−83.82 ± 4.076.70 ± 1.55−340.4 ± 229F1432L IT14−40.76 ± 7.919.9 ± 2.0315.89 ± 13.429−86.02 ± 4.695.59 ± 1.11−91.68 ± 53.48F832L WT14−18.24 ± 5.429.24 ± 1.14124.7 ± 86.0911−84.06 ± 7.236.58 ± 1.12−445.6 ± 315.4F832L IT14−25.7 ± 5.138.94 ± 1.2672.34 ± 50.2611−82.89 ± 4.936.13 ± 0.74−406.9 ± 246.9F1320L WT9−15.94 ± 5.78.64 ± 1.27124.6 ± 93.529−85.97 ± 4.035.9 ± 1.18−550.4 ± 427F1320L IT14−25.70 ± 6.29.82 ± 1.2681.79 ± 46.0819−84.37 ± 4.76.38 ± 1.12−278.2 ± 80.83F1436L WT15−19.20 ± 3.628.94 ± 1.3769.16 ± 49.759−73.96 ± 3.515.59 ± 1.39−194.3 ± 107.6F1436L IT17−29.91 ± 2.59.21 ± 1.2334.21 ± 27.2613−74.40 ± 4.145.93 ± 0.79−111.0 ± 64.26All data are presented as mean ± SD. Open table in a new tab All data are presented as mean ± SD. With these experiments, we were able to confirm the gain-of-function nature of the I848T mutation (6Meents J.E. Bressan E. Sontag S. Foerster A. Hautvast P. Rosseler C. Hampl M. Schuler H. Goetzke R. Chi Le T.K. Kleggetveit I.P. Le Cann K. Kerth C. Rush A.M. Rogers M. et al.The role of Nav1.7 in human nociceptors: insights from human iPS cell-derived sensory neurons of erythromelalgia patients.Pain. 2019; 160: 1327-1341Crossref PubMed Scopus (26) Google Scholar, 16Wu M.T. Huang P.Y. Yen C.T. Chen C.C. Lee M.J. A novel SCN9A mutation responsible for primary erythromelalgia and is resistant to the treatment of sodium channel blockers.PLoS One. 2013; 8e55212Crossref PubMed Scopus (46) Google Scholar, 17Cummins T.R. Dib-Hajj S.D. Waxman S.G. Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy.J. Neurosci. 2004; 24: 8232-8236Crossref PubMed Scopus (293) Google Scholar). However, it is not clear which mechanism underlies the mutation-induced hyperpolarized shift of activation of Nav1.7. The I848T substitution creates a novel potential phosphorylation site for threonine kinases since it replaces an isoleucine with threonine, the typical substrate for this class of kinases. Owing to its intracellular location, a phosphorylation at this position seems reasonable (Fig. 1A). This implies that the hyperpolarized activation curve of the I848T mutant could be induced by phosphorylation. We used staurosporine, a nonspecific protein kinase inhibitor (23Ahn H.S. Dib-Hajj S.D. Cox J.J. Tyrrell L. Elmslie F.V. Clarke A.A. Drenth J.P. Woods C.G. Waxman S.G. A new Nav1.7 sodium channel mutation I234T in a child with severe pain.Eur. J. Pain. 2010; 14: 944-950Crossref PubMed Scopus (36) Google Scholar), to reduce phosphorylation and thereby potentially counteract the disease-causing effect of the I848T mutant. Indeed, staurosporine led to a significant reduction of the hyperpolarized shift in voltage dependence of activation for the I848T mutant (Fig. 2, A–B and Table 2). The activation curve of the staurosporine-treated I848T mutant displayed a shallower slope than the other curves (Fig. 2A and Table 2), which could suggest a mixed population of phosphorylated and unphosphorylated channels. However, increasing the concentration of staurosporine had no further effect on voltage dependence or on the slope factor (Table 2). It is therefore possible that nonspecific kinase inhibitors are not effective enough in abolishing phosphorylation of the I848T mutant. Considering that staurosporine had no effect on the voltage dependence of activation for the wildtype channel (Fig. 2 and Table 2), it is interesting to note that protein kinase–mediated phosphorylation does not seem to play an important role for the gating of the unmutated Nav1.7.Table 2Voltage-clamp parameters for Nav1.7 wildtype and I848T mutant treated with different kinase inhibitors and activatorsGenotypeActivationFast inactivationCurrent densitynV1/2 (mV)Slope (V/s)Gmax (pS)nV1/2 (mV)Slope (V/s)(pA/pF)WT31−18.3 ± 5.918.91 ± 1.2476.62 ± 53.1330−84.21 ± 6.826.67 ± 1.42−416.5 ± 291.5I848T (IT)30−28.06 ± 5.428.99 ± 1.4841.01 ± 29.5132−86.32 ± 5.116.48 ± 1.36−252.8 ± 167.6WT+ 500 nM staurosporine29−16.26 ± 6.768.8 ± 1.1262.35 ± 51.5424−88.74 ± 5.316.79 ± 1.24−310.6 ± 207.3IT + 500 nM staurosporine25−22.92 ± 3.8910.4 ± 1.6437.41 ± 28.1822−86.4 ± 5.966.57 ± 0.99−217.7 ± 139.1IT + 1 μM staurosporine19−24.74 ± 4.7710.25 ± 0.92WT + 200 nM calphostin C24−16.57 ± 6.358.15 ± 1.25102.3 ± 93.1325−83.68 ± 6.087.88 ± 1.39−399.6 ± 420.4IT + 200 nM calphostin C25−20.90 ± 4.979.55 ± 1.4157.77 ± 54.3521−81.05 ± 5.728.59 ± 1.9−216.1 ± 215.5WT + 1 μM PMA24−18.54 ± 3.618.44 ± 1.3486.69 ± 63.8128−85.02 ± 2.36.58 ± 1.13−425.6 ± 288.9IT + 1 μM PMA31−25.84 ± 49.77 ± 1.3357.84 ± 39.1327−83.47 ± 5.236.29 ± 1.06−265.9 ± 161.2WT + 10 μM H-8925−16.87 ± 4.969.21 ± 1.3263.74 ± 39.821−85.91 ± 5.046.72 ± 0.83−327.4 ± 169.6IT + 10 μM H-8925−25.39 ± 4.4910.02 ± 1.535.04 ± 7.5625−86.55 ± 4.436.54 ± 0.8−178.6 ± 111.2All data are presented as mean ± SD. Staurosporine is a nonspecific kinase inhibitor. Calphostin C is a selective protein kinase C inhibitor. PMA (phorbol 12-myristate 13-acetate) is a protein kinase C activator. H-89 is a selective protein kinase A inhibitor.

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