FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3039444660> ?p ?o ?g. }

• W3039444660 endingPage "11865" @default.
• W3039444660 startingPage "11845" @default.
• W3039444660 abstract "Nav1.6 is the primary voltage-gated sodium channel isoform expressed in mature axon initial segments and nodes, making it critical for initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may have profound effects on input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism regulating ion channel function. Because Nav1.6 and the multifunctional Ca2+/CaM-dependent protein kinase II (CaMKII) are independently linked to excitability disorders, we sought to investigate modulation of Nav1.6 function by CaMKII signaling. We show that inhibition of CaMKII, a Ser/Thr protein kinase associated with excitability, synaptic plasticity, and excitability disorders, with the CaMKII-specific peptide inhibitor CN21 reduces transient and persistent currents in Nav1.6-expressing Purkinje neurons by 87%. Using whole-cell voltage clamp of Nav1.6, we show that CaMKII inhibition in ND7/23 and HEK293 cells significantly reduces transient and persistent currents by 72% and produces a 5.8-mV depolarizing shift in the voltage dependence of activation. Immobilized peptide arrays and nanoflow LC-electrospray ionization/MS of Nav1.6 reveal potential sites of CaMKII phosphorylation, specifically Ser-561 and Ser-641/Thr-642 within the first intracellular loop of the channel. Using site-directed mutagenesis to test multiple potential sites of phosphorylation, we show that Ala substitutions of Ser-561 and Ser-641/Thr-642 recapitulate the depolarizing shift in activation and reduction in current density. Computational simulations to model effects of CaMKII inhibition on Nav1.6 function demonstrate dramatic reductions in spontaneous and evoked action potentials in a Purkinje cell model, suggesting that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate neuronal excitability. Nav1.6 is the primary voltage-gated sodium channel isoform expressed in mature axon initial segments and nodes, making it critical for initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may have profound effects on input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism regulating ion channel function. Because Nav1.6 and the multifunctional Ca2+/CaM-dependent protein kinase II (CaMKII) are independently linked to excitability disorders, we sought to investigate modulation of Nav1.6 function by CaMKII signaling. We show that inhibition of CaMKII, a Ser/Thr protein kinase associated with excitability, synaptic plasticity, and excitability disorders, with the CaMKII-specific peptide inhibitor CN21 reduces transient and persistent currents in Nav1.6-expressing Purkinje neurons by 87%. Using whole-cell voltage clamp of Nav1.6, we show that CaMKII inhibition in ND7/23 and HEK293 cells significantly reduces transient and persistent currents by 72% and produces a 5.8-mV depolarizing shift in the voltage dependence of activation. Immobilized peptide arrays and nanoflow LC-electrospray ionization/MS of Nav1.6 reveal potential sites of CaMKII phosphorylation, specifically Ser-561 and Ser-641/Thr-642 within the first intracellular loop of the channel. Using site-directed mutagenesis to test multiple potential sites of phosphorylation, we show that Ala substitutions of Ser-561 and Ser-641/Thr-642 recapitulate the depolarizing shift in activation and reduction in current density. Computational simulations to model effects of CaMKII inhibition on Nav1.6 function demonstrate dramatic reductions in spontaneous and evoked action potentials in a Purkinje cell model, suggesting that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate neuronal excitability. The voltage-gated sodium channel (Nav) family is a class of ion channels that is critical for generating and propagating action potentials (AP). Navs are large heteromultimeric protein complexes that are formed by α subunits, which are the functional pore-forming subunits of the channel, and one or more auxiliary β subunits. Sodium channel α subunits are encoded by 10 genes that control expression in various excitable cells (1Catterall W.A. Voltage-gated sodium channels at 60: structure, function and pathophysiology.J. Physiol. 2012; 590 (22473783): 2577-258910.1113/jphysiol.2011.224204Crossref PubMed Scopus (392) Google Scholar). In the brain, the isoform Nav1.6 is critical for regulating and maintaining neuronal excitability (2O'Brien J.E. Meisler M.H. Sodium channel SCN8A (Nav1.6): properties and de novo mutations in epileptic encephalopathy and intellectual disability.Front. Genet. 2013; 4 (24194747): 21310.3389/fgene.2013.00213Crossref PubMed Google Scholar). Nav1.6 is unique both in its expression pattern and biophysical properties. In adult neurons, Nav1.6 is predominantly expressed at the axon initial segment (AIS), the proximal unmyelinated region of the neuron where APs are initiated (3Hu W. Tian C. Li T. Yang M. Hou H. Shu Y. Distinct contributions of Nav1.6 and Nav1.2 in action potential initiation and backpropagation.Nat. Neurosci. 2009; 12 (19633666): 996-100210.1038/nn.2359Crossref PubMed Scopus (380) Google Scholar), and at nodes of Ranvier, where APs are propagated down the axon (3Hu W. Tian C. Li T. Yang M. Hou H. Shu Y. Distinct contributions of Nav1.6 and Nav1.2 in action potential initiation and backpropagation.Nat. Neurosci. 2009; 12 (19633666): 996-100210.1038/nn.2359Crossref PubMed Scopus (380) Google Scholar, 4Kole M.H. Stuart G.J. Signal processing in the axon initial segment.Neuron. 2012; 73 (22284179): 235-24710.1016/j.neuron.2012.01.007Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). This provides Nav1.6 with significant control of AP initiation as well as propagation of neuronal impulses (3Hu W. Tian C. Li T. Yang M. Hou H. Shu Y. Distinct contributions of Nav1.6 and Nav1.2 in action potential initiation and backpropagation.Nat. Neurosci. 2009; 12 (19633666): 996-100210.1038/nn.2359Crossref PubMed Scopus (380) Google Scholar, 5Wang X. Zhang X.G. Zhou T.T. Li N. Jang C.Y. Xiao Z.C. Ma Q.H. Li S. Elevated neuronal excitability due to modulation of the voltage-gated sodium channel Nav1.6 by Aβ1–42.Front. Neurosci. 2016; 10 (27013956): 9410.3389/fnins.2016.00094Crossref PubMed Scopus (16) Google Scholar). Furthermore, Nav1.6 displays unique biophysical properties that enable the channel to exert powerful tuning capabilities of these impulses. For example, Nav1.6 can generate large persistent currents (6Patel R.R. Barbosa C. Brustovetsky T. Brustovetsky N. Cummins T.R. Aberrant epilepsy-associated mutant Nav1.6 sodium channel activity can be targeted with cannabidiol.Brain. 2016; 139 (27267376): 2164-218110.1093/brain/aww129Crossref PubMed Google Scholar, 7Bant J.S. Raman I.M. Control of transient, resurgent, and persistent current by open-channel block by Na channel β4 in cultured cerebellar granule neurons.Proc. Natl. Acad. Sci. U. S. A. 2010; 107 (20566860): 12357-1236210.1073/pnas.1005633107Crossref PubMed Scopus (86) Google Scholar, 8Pan Y. Cummins T.R. Distinct functional alterations in SCN8A epilepsy mutant channels.J. Physiol. 2020; 598 (31715021): 381-40110.1113/JP278952Crossref PubMed Scopus (12) Google Scholar), which is an atypical form of sodium current activity that may profoundly impact the physiology of excitable cells (9Kole M.H. Stuart G.J. Is action potential threshold lowest in the axon?.Nat. Neurosci. 2008; 11 (18836442): 1253-125510.1038/nn.2203Crossref PubMed Scopus (98) Google Scholar, 10Osorio N. Cathala L. Meisler M.H. Crest M. Magistretti J. Delmas P. Persistent Nav1.6 current at axon initial segments tunes spike timing of cerebellar granule cells.J. Physiol. 2010; 588 (20173079): 651-67010.1113/jphysiol.2010.183798Crossref PubMed Google Scholar). Changes in Nav1.6 expression and function are associated with excitability disorders including neuropathic pain (11Xie W. Strong J.A. Zhang J.M. Local knockdown of the NaV1.6 sodium channel reduces pain behaviors, sensory neuron excitability, and sympathetic sprouting in rat models of neuropathic pain.Neuroscience. 2015; 291 (25686526): 317-33010.1016/j.neuroscience.2015.02.010Crossref PubMed Scopus (53) Google Scholar, 12Chen L. Huang J. Zhao P. Persson A.K. Dib-Hajj F.B. Cheng X. Tan A. Waxman S.G. Dib-Hajj S.D. Conditional knockout of NaV1.6 in adult mice ameliorates neuropathic pain.Sci. Rep. 2018; 8 (29497094): 384510.1038/s41598-018-22216-wCrossref PubMed Scopus (26) Google Scholar, 13Laedermann C.J. Abriel H. Decosterd I. Post-translational modifications of voltage-gated sodium channels in chronic pain syndromes.Front. Pharmacol. 2015; 6 (26594175): 26310.3389/fphar.2015.00263Crossref PubMed Scopus (35) Google Scholar), epilepsies (2O'Brien J.E. Meisler M.H. Sodium channel SCN8A (Nav1.6): properties and de novo mutations in epileptic encephalopathy and intellectual disability.Front. Genet. 2013; 4 (24194747): 21310.3389/fgene.2013.00213Crossref PubMed Google Scholar, 6Patel R.R. Barbosa C. Brustovetsky T. Brustovetsky N. Cummins T.R. Aberrant epilepsy-associated mutant Nav1.6 sodium channel activity can be targeted with cannabidiol.Brain. 2016; 139 (27267376): 2164-218110.1093/brain/aww129Crossref PubMed Google Scholar, 14Blumenfeld H. Lampert A. Klein J.P. Mission J. Chen M.C. Rivera M. Dib-Hajj S. Brennan A.R. Hains B.C. Waxman S.G. Role of hippocampal sodium channel Nav1.6 in kindling epileptogenesis.Epilepsia. 2009; 50 (18637833): 44-5510.1111/j.1528-1167.2008.01710.xCrossref PubMed Scopus (94) Google Scholar, 15Hargus N.J. Nigam A. Bertram 3rd, E.H. Patel M.K. Evidence for a role of Nav1.6 in facilitating increases in neuronal hyperexcitability during epileptogenesis.J. Neurophysiol. 2013; 110 (23741036): 1144-115710.1152/jn.00383.2013Crossref PubMed Scopus (0) Google Scholar, 16Ottolini M. Barker B.S. Gaykema R.P. Meisler M.H. Patel M.K. Aberrant sodium channel currents and hyperexcitability of medial entorhinal cortex neurons in a mouse model of SCN8A encephalopathy.J. Neurosci. 2017; 37 (28676574): 7643-765510.1523/JNEUROSCI.2709-16.2017Crossref PubMed Scopus (18) Google Scholar), autism-spectrum disorders (17Butler K.M. da Silva C. Shafir Y. Weisfeld-Adams J.D. Alexander J.J. Hegde M. Escayg A. De novo and inherited SCN8A epilepsy mutations detected by gene panel analysis.Epilepsy Res. 2017; 129 (27875746): 17-2510.1016/j.eplepsyres.2016.11.002Crossref PubMed Google Scholar, 18Alshammari M.A. Khan M.R. Alasmari F. Alshehri A.O. Ali R. Boudjelal M. Alhosaini K.A. Niazy A.A. Alshammari T.K. Changes in the fluorescence tracking of NaV1.6 protein expression in a BTBR T+Itpr3tf/J autistic mouse model.Neural Plast. 2019; 2019 (31933626): 1-1210.1155/2019/4893103Crossref Scopus (0) Google Scholar), ischemia (19Zhu H. Lin W. Zhao Y. Wang Z. Lao W. Kuang P. Zhou H. Transient upregulation of Nav1.6 expression in the genu of corpus callosum following middle cerebral artery occlusion in the rats.Brain Res. Bull. 2017; 132 (28434994): 20-2710.1016/j.brainresbull.2017.04.008Crossref PubMed Scopus (2) Google Scholar), and stress-induced changes in signal transduction (20Wittmack E.K. Rush A.M. Hudmon A. Waxman S.G. Dib-Hajj S.D. Voltage-gated sodium channel Nav1.6 is modulated by p38 mitogen-activated protein kinase.J. Neurosci. 2005; 25 (16014723): 6621-663010.1523/JNEUROSCI.0541-05.2005Crossref PubMed Scopus (86) Google Scholar, 21Wu J.-X. Tong L. Hu L. Xia C.-M. Li M. Chen Q.-H. Chen F.-X. Du D.-S. Upregulation of Nav1.6 expression in the rostral ventrolateral medulla of stress-induced hypertensive rats.Hypertens. Res. 2018; 41 (30287879): 1013-102210.1038/s41440-018-0105-6Crossref PubMed Scopus (3) Google Scholar). Notably, many of these changes are significantly influenced by intracellular mediators, including protein-protein interactions and post-translational modifications. Phosphorylation is a powerful mechanism that can rapidly modulate Nav function and expression. The most widely studied phosphorylation-mediated modulation of neuronal Navs has focused on the roles of protein kinases A and C (22Numann R. Catterall W.A. Scheuer T. Functional modulation of brain sodium channels by protein kinase C phosphorylation.Science. 1991; 254 (1656525): 115-11810.1126/science.1656525Crossref PubMed Google Scholar, 23Vijayaragavan K. Boutjdir 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 (14657190): 1556-156910.1152/jn.00676.2003Crossref PubMed Scopus (0) Google Scholar, 24Tan Z.Y. Priest B.T. Krajewski J.L. Knopp K.L. Nisenbaum E.S. Cummins T.R. Protein kinase C enhances human sodium channel hNav1.7 resurgent currents via a serine residue in the domain III-IV linker.FEBS Lett. 2014; 588 (25240195): 3964-396910.1016/j.febslet.2014.09.011Crossref PubMed Google Scholar, 25Li M. West J.W. Lai Y. Scheuer T. Catterall W.A. Functional modulation of brain sodium channels by cAMP-dependent phosphorylation.Neuron. 1992; 8 (1319185): 1151-115910.1016/0896-6273(92)90135-ZAbstract Full Text PDF PubMed Google Scholar, 26Cantrell A.R. Catterall W.A. Neuromodulation of Na+ channels: an unexpected form of cellular plasticity.Nat. Rev. Neurosci. 2001; 2 (11389473): 397-40710.1038/35077553Crossref PubMed Scopus (282) Google Scholar) and p38 mitogen-activated protein kinase (20Wittmack E.K. Rush A.M. Hudmon A. Waxman S.G. Dib-Hajj S.D. Voltage-gated sodium channel Nav1.6 is modulated by p38 mitogen-activated protein kinase.J. Neurosci. 2005; 25 (16014723): 6621-663010.1523/JNEUROSCI.0541-05.2005Crossref PubMed Scopus (86) Google Scholar, 27Hudmon A. Choi J.-S. Tyrrell L. Black J.A. Rush A.M. Waxman S.G. Dib-Hajj S.D. Phosphorylation of sodium channel Nav1.8 by p38 mitogen-activated protein kinase increases current density in dorsal root ganglion neurons.J. Neurosci. 2008; 28 (18354022): 3190-320110.1523/JNEUROSCI.4403-07.2008Crossref PubMed Scopus (133) Google Scholar) in excitatory dysregulation. Dysfunction in αCaMKII activity is also significantly linked to excitatory disorders and is highly co-localized with Navs in neurons (28Hund T.J. Koval O.M. Li J. Wright P.J. Qian L. Snyder J.S. Gudmundsson H. Kline C.F. Davidson N.P. Cardona N. Rasband M.N. Anderson M.E. Mohler P.J. A β(IV)-spectrin/CaMKII signaling complex is essential for membrane excitability in mice.J. Clin. Invest. 2010; 120 (20877009): 3508-351910.1172/JCI43621Crossref PubMed Scopus (188) Google Scholar, 29Liu X.B. Murray K.D. Neuronal excitability and calcium/calmodulin-dependent protein kinase type II: location, location, location.Epilepsia. 2012; 53 (22612808): 45-5210.1111/j.1528-1167.2012.03474.xCrossref PubMed Scopus (63) Google Scholar, 30Grubb M.S. Shu Y. Kuba H. Rasband M.N. Wimmer V.C. Bender K.J. Short- and long-term plasticity at the axon initial segment.J. Neurosci. 2011; 31 (22072655): 16049-1605510.1523/JNEUROSCI.4064-11.2011Crossref PubMed Scopus (91) Google Scholar, 31Gasser A. Ho T.S. Cheng X. Chang K.J. Waxman S.G. Rasband M.N. Dib-Hajj S.D. An ankyrinG-binding motif is necessary and sufficient for targeting Nav1.6 sodium channels to axon initial segments and nodes of Ranvier.J. Neurosci. 2012; 32 (22623668): 7232-724310.1523/JNEUROSCI.5434-11.2012Crossref PubMed Scopus (79) Google Scholar, 32Küry S. van Woerden G.M. Besnard T. Proietti Onori M. Latypova X. Towne M.C. Cho M.T. Prescott T.E. Ploeg M.A. Sanders S. Stessman H.A.F. Pujol A. Distel B. Robak L.A. Bernstein J.A. et al.De novo mutations in protein kinase genes CAMK2A and CAMK2B cause intellectual disability.Am. J. Hum. Genet. 2017; 101 (29100089): 768-78810.1016/j.ajhg.2017.10.003Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). CaMKII, therefore, may be contributing to pro-excitatory changes within the brain by modulating the activity of neuronal Navs. However, CaMKII phosphorylation and modulation of Nav1.6, the major isoform critical for AP initiation and propagation, has not been investigated. CaMKII is a multifunctional serine/threonine protein kinase highly concentrated in the brain and is critical for neuronal excitability (33Bayer K.U. Schulman H. CaM kinase: still inspiring at 40.Neuron. 2019; 103 (31394063): 380-39410.1016/j.neuron.2019.05.033Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). There are four genes that encode mammalian subunits, with the α subunit predominating in excitatory neurons of the forebrain and Purkinje neurons of the cerebellum (34Zalcman G. Federman N. Romano A. CaMKII isoforms in learning and memory: localization and function.Front. Mol. Neurosci. 2018; 11 (30564099): 44510.3389/fnmol.2018.00445Crossref PubMed Scopus (34) Google Scholar, 35Wang X. Zhang C. Szábo G. Sun Q.-Q. Distribution of CaMKIIα expression in the brain in vivo, studied by CaMKIIα-GFP mice.Brain Res. 2013; 1518 (23632380): 9-2510.1016/j.brainres.2013.04.042Crossref PubMed Scopus (65) Google Scholar). During excitatory events, CaMKII is transiently activated upon calcium influx and may retain persistent activity (36Colbran R.J. Smith M.K. Schworer C.M. Fong Y.L. Soderling T.R. Regulatory domain of calcium/calmodulin-dependent protein kinase II: mechanism of inhibition and regulation by phosphorylation.J. Biol. Chem. 1989; 264 (2538462): 4800-4804Abstract Full Text PDF PubMed Google Scholar, 37Miller S.G. Kennedy M.B. Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch.Cell. 1986; 44 (3006921): 861-87010.1016/0092-8674(86)90008-5Abstract Full Text PDF PubMed Scopus (611) Google Scholar, 38Fong Y.L. Taylor W.L. Means A.R. Soderling T.R. Studies of the regulatory mechanism of Ca2+/calmodulin-dependent protein kinase II: mutation of threonine 286 to alanine and aspartate.J. Biol. Chem. 1989; 264 (2550458): 16759-16763Abstract Full Text PDF PubMed Google Scholar), providing CaMKII with a unique capability to decode these events and form molecular memory of its activation (39Hudmon A. Schulman H. Neuronal Ca2+/calmodulin-dependent protein kinase II: the role of structure and autoregulation in cellular function.Annu. Rev. Biochem. 2002; 71 (12045104): 473-51010.1146/annurev.biochem.71.110601.135410Crossref PubMed Scopus (490) Google Scholar, 40Lisman J. Yasuda R. Raghavachari S. Mechanisms of CaMKII action in long-term potentiation.Nat. Rev. Neurosci. 2012; 13 (22334212): 169-18210.1038/nrn3192Crossref PubMed Scopus (601) Google Scholar, 41Coultrap S.J. Bayer K.U. CaMKII regulation in information processing and storage.Trends Neurosci. 2012; 35 (22717267): 607-61810.1016/j.tins.2012.05.003Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 42Stratton M. Lee I.H. Bhattacharyya M. Christensen S.M. Chao L.H. Schulman H. Groves J.T. Kuriyan J. Activation-triggered subunit exchange between CaMKII holoenzymes facilitates the spread of kinase activity.Elife. 2014; 3 (24473075): e0161010.7554/eLife.01610PubMed Google Scholar). Fluctuations in CaMKII activity are often associated with neuronal diseases, including epilepsy and autism-spectrum disorders (32Küry S. van Woerden G.M. Besnard T. Proietti Onori M. Latypova X. Towne M.C. Cho M.T. Prescott T.E. Ploeg M.A. Sanders S. Stessman H.A.F. Pujol A. Distel B. Robak L.A. Bernstein J.A. et al.De novo mutations in protein kinase genes CAMK2A and CAMK2B cause intellectual disability.Am. J. Hum. Genet. 2017; 101 (29100089): 768-78810.1016/j.ajhg.2017.10.003Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), ischemia (43Coultrap S.J. Vest R.S. Ashpole N.M. Hudmon A. Bayer K.U. CaMKII in cerebral ischemia.Acta Pharmacol. Sin. 2011; 32 (21685929): 861-87210.1038/aps.2011.68Crossref PubMed Scopus (66) Google Scholar, 44Ashpole N.M. Chawla A.R. Martin M.P. Brustovetsky T. Brustovetsky N. Hudmon A. Loss of calcium/calmodulin-dependent protein kinase II activity in cortical astrocytes decreases glutamate uptake and induces neurotoxic release of ATP.J. Biol. Chem. 2013; 288 (23543737): 14599-1461110.1074/jbc.M113.466235Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), and pain (45Yu H. Pan B. Weyer A. Wu H.E. Meng J. Fischer G. Vilceanu D. Light A.R. Stucky C. Rice F.L. Hudmon A. Hogan Q. CaMKII controls whether touch is painful.J. Neurosci. 2015; 35 (26490852): 14086-1410210.1523/JNEUROSCI.1969-15.2015Crossref PubMed Scopus (13) Google Scholar, 46Chen Y. Yang C. Luo F. Wang Z. The role of CAMKIIα in neuropathic pain.J. Pain. 2009; 10: S3910.1016/j.jpain.2009.01.167Abstract Full Text Full Text PDF Google Scholar, 47Qian Y. Xia T. Cui Y. Chu S. Ma Z. Gu X. The role of CaMKII in neuropathic pain and fear memory in chronic constriction injury in rats.Int. J. Neurosci. 2019; 129 (30118368): 146-15410.1080/00207454.2018.1512986Crossref PubMed Scopus (2) Google Scholar). Previous studies suggest that coupling between CaMKII and Navs contributes to regulation of neuronal excitability (28Hund T.J. Koval O.M. Li J. Wright P.J. Qian L. Snyder J.S. Gudmundsson H. Kline C.F. Davidson N.P. Cardona N. Rasband M.N. Anderson M.E. Mohler P.J. A β(IV)-spectrin/CaMKII signaling complex is essential for membrane excitability in mice.J. Clin. Invest. 2010; 120 (20877009): 3508-351910.1172/JCI43621Crossref PubMed Scopus (188) Google Scholar, 30Grubb M.S. Shu Y. Kuba H. Rasband M.N. Wimmer V.C. Bender K.J. Short- and long-term plasticity at the axon initial segment.J. Neurosci. 2011; 31 (22072655): 16049-1605510.1523/JNEUROSCI.4064-11.2011Crossref PubMed Scopus (91) Google Scholar). To this end, CaMKII inhibition has been shown to reduce excitability of small and medium trigeminal ganglion neurons in addition to decreasing sodium current amplitude (48Liang R. Liu X. Wei L. Wang W. Zheng P. Yan X. Zhao Y. Liu L. Cao X. The modulation of the excitability of primary sensory neurons by Ca2+-CaM-CaMKII pathway.Neurol. Sci. 2012; 33 (22205399): 1083-109310.1007/s10072-011-0907-7Crossref PubMed Scopus (10) Google Scholar). Moreover, CaMKII inhibition also reduces sodium current amplitude in cerebellar granule cells (49Carlier E. Dargent B. De Waard M. Couraud F. Na+ channel regulation by calmodulin kinase II in rat cerebellar granule cells.Biochem. Biophys. Res. Commun. 2000; 274 (10913349): 394-39910.1006/bbrc.2000.3145Crossref PubMed Scopus (0) Google Scholar), in which Nav1.6 is the predominant isoform (10Osorio N. Cathala L. Meisler M.H. Crest M. Magistretti J. Delmas P. Persistent Nav1.6 current at axon initial segments tunes spike timing of cerebellar granule cells.J. Physiol. 2010; 588 (20173079): 651-67010.1113/jphysiol.2010.183798Crossref PubMed Google Scholar). These studies suggest that CaMKII signaling may play a major role in neuronal excitability, possibly through modulation of Nav1.6 sodium currents. In this study, we directly investigate the potential for CaMKII to phosphorylate and modulate Nav1.6. Multiple biochemical approaches, including MS and immobilized peptide arrays, were used to identify CaMKII phosphorylation sites on Nav1.6, and whole-cell voltage-clamp electrophysiology was used to examine the functional effects of this phosphorylation on channel biophysical properties. Using computational simulations, we further sought to examine how functional changes in Nav1.6 induced by CaMKII modulation may impact spontaneous and evoked AP initiation in neurons. Electrophysiological recordings of bulk sodium currents from trigeminal neurons and cerebellar granule cells show significantly decreased transient sodium currents following CaMKII inhibition with KN93 or KN62 (48Liang R. Liu X. Wei L. Wang W. Zheng P. Yan X. Zhao Y. Liu L. Cao X. The modulation of the excitability of primary sensory neurons by Ca2+-CaM-CaMKII pathway.Neurol. Sci. 2012; 33 (22205399): 1083-109310.1007/s10072-011-0907-7Crossref PubMed Scopus (10) Google Scholar, 49Carlier E. Dargent B. De Waard M. Couraud F. Na+ channel regulation by calmodulin kinase II in rat cerebellar granule cells.Biochem. Biophys. Res. Commun. 2000; 274 (10913349): 394-39910.1006/bbrc.2000.3145Crossref PubMed Scopus (0) Google Scholar). To further explore the potential for CaMKII to regulate sodium currents in neurons, we recorded from Purkinje cells incubated with the CaMKII inhibitor CN21 conjugated to the tat cell–penetrating motif (tatCN21) or its inactive analog tatCN21Ala (50Ashpole N.M. Hudmon A. Excitotoxic neuroprotection and vulnerability with CaMKII inhibition.Mol. Cell Neurosci. 2011; 46 (21316454): 720-73010.1016/j.mcn.2011.02.003Crossref PubMed Scopus (60) Google Scholar). Sodium currents from Purkinje cells treated with no peptide (left), tatCN21 (middle), or tatCN21Ala (right) were elicited with a series of depolarizing steps ranging from −100 to +15 mV for 50 ms from a holding potential of −100 mV (Fig. 1A). Application of tatCN21 to inhibit CaMKII significantly reduced transient sodium current density by 87% (Fig. 1C) in Purkinje neurons compared with no peptide or tatCN21Ala-treated cells. We also examined persistent current generation in Purkinje neurons in the context of CaMKII inhibition by using the same depolarizing step protocol as described above. Persistent current is a noninactivating, or very slowly inactivating, sodium current and was measured at the end of the current trace (Fig. 1B). These currents are known to amplify subthreshold inputs and mediate repetitive action potential firing (9Kole M.H. Stuart G.J. Is action potential threshold lowest in the axon?.Nat. Neurosci. 2008; 11 (18836442): 1253-125510.1038/nn.2203Crossref PubMed Scopus (98) Google Scholar, 51Taddese A. Bean B.P. Subthreshold sodium current from rapidly inactivating sodium channels drives spontaneous firing of tuberomammillary neurons.Neuron. 2002; 33 (11856532): 587-60010.1016/S0896-6273(02)00574-3Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 52Stuart G. Sakmann B. Amplification of EPSPs by axosomatic sodium channels in neocortical pyramidal neurons.Neuron. 1995; 15 (7576650): 1065-107610.1016/0896-6273(95)90095-0Abstract Full Text PDF PubMed Scopus (322) Google Scholar). In addition to decreasing transient sodium currents in Purkinje neurons, CaMKII inhibition with tatCN21 also reduces persistent current density (Fig. 1D). Current densities were calculated by normalizing the current amplitude at −25 mV by the cell capacitance. We used −25 mV for transient and persistent sodium current measurements to minimize clamp error artifacts during analysis. We did not determine the impact of CaMKII inhibition on Purkinje neuron sodium current voltage dependence because of issues with space-clamp errors. Because Nav1.6 is the predominant sodium current observed in Purkinje neurons (53Schaller K.L. Caldwell J.H. Expression and distribution of voltage-gated sodium channels in the cerebellum.Cerebellum. 2003; 2 (12882229): 2-910.1080/14734220309424Crossref PubMed Scopus (89) Google Scholar), these data suggest that Nav1.6 may be a substrate for CaMKII and modulated by CaMKII signaling. While Nav1.6 is the predominant sodium current in Purkinje neurons (53Schaller K.L. Caldwell J.H. Expression and distribution of voltage-gated sodium channels in the cerebellum.Cerebellum. 2003; 2 (12882229): 2-910.1080/14734220309424Crossref PubMed Scopus (89) Google Scholar), it is conceivable that the expression of another tetrodotoxin (TTX)-sensitive voltage-gated sodium channel isoform (e.g. Nav1.1) in Purkinje neurons could contaminate our recordings. The TTX metabolite 4,9-anhydro-TTX has been reported to selectively target Nav1.6 sodium currents (54Rosker C. Lohberger B. Hofer D. Steinecker B. Quasthoff S. Schreibmayer W. The TTX metabolite 4,9-anhydro-TTX is a highly specific blocker of the Nav1.6 voltage-dependent sodium channel.Am. J. Physiol. Cell Physiol. 2007; 293 (17522141): C783-C78910.1152/ajpcell.00070.2007Crossref PubMed Scopus (0) Google Scholar). However, recent evidence shows that 4,9-anhydro-TTX also blocks Nav1.1 currents at similar concentrations required to block Nav1.6 activity (55Griffith T.N. Docter T.A. Lumpkin E.A. Tetrodotoxin-sensitive sodium channels mediate action potential firing and excitability in menthol-sensitive Vglut3-lineage sensory neurons.J. Neurosci. 2019; 39 (31300524): 7086-710110.1523/JNEUROSCI.2817-18.2019Crossref PubMed Scopus (7) Google Scholar). Therefore, we turned to a heterologous expression system, where the role of CaMKII in modulating Nav1.6 sodium currents could be explored without significant contamination from other endogenous voltage-gated sodium channel isoforms. Whole-cell voltage-clamp recordings were obtained in neuronal ND7/23 cells transiently co-transfected with GFP (positive selection) and a tetrodotoxin-resistant (TTX-R) form (Y371S point mutation) of the human Nav1.6 channel that makes Nav1.6 resistant to TTX, yet does not appear to alter its biophysical properties (56Herzog R.I. Liu C. Waxman S.G. Cummins T.R. Calmodulin binds to the C terminus of sodium channels Nav1.4 and Nav1.6 and differentially modulates their functional properties.J. Neurosci. 2003; 23 (12967988): 8261-827010.1523/JNEUROSCI.23-23-08261.2003Crossref PubMed Google Scholar). In this system, TTX can be used to block endogenous currents, allowing TTX-R Nav1.6 current to be effectively studied in isolation. Space- and voltage-clamp errors are also more effectively controlled in ND7/23 cells. The CaMKII peptide inhibitor CN21 or its inactive analog, CN21Ala, was included in the patch pipette. Whereas we observed 15.9 ± 1.9% autonomously active CaMKII in our cultured ND7/23 cells (see “Experimental procedures”), we also included Ca2+ and calmodulin in the patch pipette to promote further activation of endogenous CaMKII as described previously (57Ashpole N.M. Herren A.W. Ginsburg K.S. Brogan J.D. Johnson D.E. Cummins T.R. Bers D.M. Hudmon A. Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium ch" @default.
• W3039444660 created "2020-07-10" @default.
• W3039444660 creator A5015166106 @default.
• W3039444660 creator A5046108675 @default.
• W3039444660 creator A5058785586 @default.
• W3039444660 creator A5077534501 @default.
• W3039444660 creator A5078097835 @default.
• W3039444660 date "2020-08-01" @default.
• W3039444660 modified "2023-10-16" @default.
• W3039444660 title "CaMKII enhances voltage-gated sodium channel Nav1.6 activity and neuronal excitability" @default.
• W3039444660 cites W1486939861 @default.
• W3039444660 cites W1501923953 @default.
• W3039444660 cites W1580110901 @default.
• W3039444660 cites W1582195464 @default.
• W3039444660 cites W1596672035 @default.
• W3039444660 cites W1723799090 @default.
• W3039444660 cites W1965453901 @default.
• W3039444660 cites W1968130118 @default.
• W3039444660 cites W1969213411 @default.
• W3039444660 cites W1971265072 @default.
• W3039444660 cites W1976504173 @default.
• W3039444660 cites W1977035226 @default.
• W3039444660 cites W1979325786 @default.
• W3039444660 cites W1983187084 @default.
• W3039444660 cites W1983396112 @default.
• W3039444660 cites W1984457534 @default.
• W3039444660 cites W1985245021 @default.
• W3039444660 cites W1986189584 @default.
• W3039444660 cites W1986726759 @default.
• W3039444660 cites W1987046506 @default.
• W3039444660 cites W1990116234 @default.
• W3039444660 cites W1996575144 @default.
• W3039444660 cites W1999980742 @default.
• W3039444660 cites W2002812952 @default.
• W3039444660 cites W2002934796 @default.
• W3039444660 cites W2003242423 @default.
• W3039444660 cites W2007289876 @default.
• W3039444660 cites W2010110624 @default.
• W3039444660 cites W2013115334 @default.
• W3039444660 cites W2013960132 @default.
• W3039444660 cites W2016824667 @default.
• W3039444660 cites W2024695712 @default.
• W3039444660 cites W2026357254 @default.
• W3039444660 cites W2026602510 @default.
• W3039444660 cites W2028569392 @default.
• W3039444660 cites W2029988619 @default.
• W3039444660 cites W2033374878 @default.
• W3039444660 cites W2033940966 @default.
• W3039444660 cites W2033942553 @default.
• W3039444660 cites W2035203431 @default.
• W3039444660 cites W2038050499 @default.
• W3039444660 cites W2039072874 @default.
• W3039444660 cites W2039398588 @default.
• W3039444660 cites W2039408689 @default.
• W3039444660 cites W2040010868 @default.
• W3039444660 cites W2042506557 @default.
• W3039444660 cites W2043437163 @default.
• W3039444660 cites W2045309328 @default.
• W3039444660 cites W2049779229 @default.
• W3039444660 cites W2057537356 @default.
• W3039444660 cites W2063332150 @default.
• W3039444660 cites W2064163649 @default.
• W3039444660 cites W2065385556 @default.
• W3039444660 cites W2065978440 @default.
• W3039444660 cites W2067140034 @default.
• W3039444660 cites W2071051861 @default.
• W3039444660 cites W2072402474 @default.
• W3039444660 cites W2075953074 @default.
• W3039444660 cites W2081500927 @default.
• W3039444660 cites W2086121847 @default.
• W3039444660 cites W2091039828 @default.
• W3039444660 cites W2098674708 @default.
• W3039444660 cites W2100977258 @default.
• W3039444660 cites W2105658413 @default.
• W3039444660 cites W2107738760 @default.
• W3039444660 cites W2117120818 @default.
• W3039444660 cites W2118279723 @default.
• W3039444660 cites W2118444707 @default.
• W3039444660 cites W2126486990 @default.
• W3039444660 cites W2129228410 @default.
• W3039444660 cites W2131075809 @default.
• W3039444660 cites W2135011583 @default.
• W3039444660 cites W2135576636 @default.
• W3039444660 cites W2135945594 @default.
• W3039444660 cites W2140944550 @default.
• W3039444660 cites W2143296458 @default.
• W3039444660 cites W2147001406 @default.
• W3039444660 cites W2152695330 @default.
• W3039444660 cites W2159163865 @default.
• W3039444660 cites W2163151214 @default.
• W3039444660 cites W2165722299 @default.
• W3039444660 cites W2165826527 @default.
• W3039444660 cites W2166889327 @default.
• W3039444660 cites W2169064772 @default.
• W3039444660 cites W2178010673 @default.
• W3039444660 cites W2324430644 @default.
• W3039444660 cites W2327346420 @default.
• W3039444660 cites W2359604401 @default.

• next