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PLOS ONE  2014 

Functional Expression of Rat Nav1.6 Voltage-Gated Sodium Channels in HEK293 Cells: Modulation by the Auxiliary β1 Subunit

DOI: 10.1371/journal.pone.0085188

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Abstract:

The Nav1.6 voltage-gated sodium channel α subunit isoform is abundantly expressed in the adult rat brain. To assess the functional modulation of Nav1.6 channels by the auxiliary β1 subunit we expressed the rat Nav1.6 sodium channel α subunit by stable transformation in HEK293 cells either alone or in combination with the rat β1 subunit and assessed the properties of the reconstituted channels by recording sodium currents using the whole-cell patch clamp technique. Coexpression with the β1 subunit accelerated the inactivation of sodium currents and shifted the voltage dependence of channel activation and steady-state fast inactivation by approximately 5–7 mV in the direction of depolarization. By contrast the β1 subunit had no effect on the stability of sodium currents following repeated depolarizations at high frequencies. Our results define modulatory effects of the β1 subunit on the properties of rat Nav1.6-mediated sodium currents reconstituted in HEK293 cells that differ from effects measured previously in the Xenopus oocyte expression system. We also identify differences in the kinetic and gating properties of the rat Nav1.6 channel expressed in the absence of the β1 subunit compared to the properties of the orthologous mouse and human channels expressed in this system.

References

[1]  Hille B (2001) Ion Channels of Excitable Membranes. SunderlandMA: Sinauer. 814 p.
[2]  Catterall WA (2000) From ionic currents to molecular mechanisms: structure and function of voltage-gated sodium channels. Neuron 26: 13–25.
[3]  Patino GA, Isom LL (2010) Electrophysiology and beyond: multiple roles of Na+ channel β subunits in development and disease. Neuroscience Letters 486: 53–59.
[4]  Goldin AL (2001) Resurgence of sodium channel research. Annual Review of Physiology 63: 871–894.
[5]  Yu FH, Catterall WA (2003) Overview of the voltage-gated sodium channel family. Genome Biology 4: 207.201–207.207.
[6]  Felts PA, Yokoyama S, Dib-Hajj S, Black JA, Waxman SG (1997) Sodium channel α-subunit mRNAs I, II, III, NaG, Na6 and hNE (PN1): different expression patters in developing rat nervous system. Molecular Brain Research 45: 71–82.
[7]  Whitaker WRJ, Clare JJ, Powell AJ, Chen YH, Faull RLM, et al. (2000) Distribution of voltage-gated sodium channel α-subunit and β-subunit mRNAs in human hippocampal formation, cortex, and cerebellum. Journal of Comparative Neurology 422: 123–139.
[8]  Whitaker WRJ, Faull RLM, Waldvogel HJ, Plumpton CJ, Emson PC, et al. (2001) Comparative distribution of voltage-gated sodium channel proteins in human brain. Molecular Brain Research 88: 37–53.
[9]  Auld VJ, Goldin AL, Krafte DS, Marshall J, Dunn JM, et al. (1988) A rat brain Na+ channel α subunit with novel gating properties. Neuron 1: 449–461.
[10]  Caldwell JH, Schaller KL, Lasher RS, Peles E, Levinson SR (2000) Sodium channel Nav1.6 is localized nodes of Ranvier, dendrites, and synapses. Proceedings of the National Academy of Sciences of the United States of America 97: 5616–5620.
[11]  Hu W, Tian C, Yang M, Hou H, Shu Y (2009) Distinct contributions of Nav1.6 and Nav1.2 in action potential initiation and backpropagation. Nature Neuroscience 12: 996–1002.
[12]  Burgess DL, Kohrman DC, Galt J, Plummer NW, Jones JM, et al. (1995) Mutation of a new sodium channel gene, Scn8a, in the mouse mutant ‘motor endplate disease’. Nature Genetics 10: 461–465.
[13]  Schaller KL, Caldwell JH (2003) Expression and distribution of voltage-gated sodium channels in the cerebellum. Cerebellum 2: 2–9.
[14]  Shah BS, Stevens EB, Pinnock RD, Dixon AK, Lee K (2001) Developmental expression of the novel voltage-gated sodium channel auxiliary subunit β3, in rat CNS. Journal of Physiology 534: 763–776.
[15]  Brackenbury WJ, Calhoun JD, Chen C, Miyazaki H, Nukina N, et al. (2010) Functional reciprocity between Na+ channel Nav1.6 and β1 subunits in the coordinated regulation of excitability and neurite outgrowth. Proceedings of the National Academy of Sciences of the United States of America 107: 2283–2288.
[16]  Dietrich PS, McGivern JG, Delgado SG, Koch BD, Eglen RM, et al. (1998) Functional analysis of a voltage-gated sodium channel and its splice variant from rat dorsal root ganglia. Journal of Neurochemistry 70: 2262–2272.
[17]  Tan J, Soderlund DM (2011) Independent and joint modulation of rat Nav1.6 voltage-gated sodium channels by coexpression with the auxiliary β1 and β2 subunits. Biochemical and Biophysical Research Communications 407: 788–792.
[18]  Goldin AL (2006) Expression of ion channels in Xenopus oocytes. In: Clare JJ, Trezise DJ, editors. Expression and analysis of recombinant ion channels. Weinheim: Wiley VCH Verlag GmbH & Co. KgaA. 1–25.
[19]  Thomas P, Smart TG (2005) HEK293 cell line: a vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods 51: 187–200.
[20]  Tan J, Soderlund DM (2011) Coexpression with auxiliary β subunits modulates the action of tefluthrin on rat Nav1.6 and Nav1.3 sodium channels. Pesticide Biochemistry and Physiology 101: 256–264.
[21]  He B, Soderlund DM (2011) Differential state-dependent modification of rat Nav1.6 sodium channels expressed in human embryonic kidney (HEK293) cells by the pyrethroid insecticides tefluthrin and deltamethrin. Toxicology and Applied Pharmacology 257: 377–387.
[22]  Bezanilla F, Armstrong CM (1977) Inactivation of the sodium channel. Journal of General Physiology 70: 549–566.
[23]  Burbidge SA, Dale TJ, Powell AJ, Whitaker WRJ, Xie XM, et al. (2002) Molecular cloning, distribution and functional analysis of the Nav1.6 voltage-gated sodium channel from human brain. Molecular Brain Research 103: 80–90.
[24]  Welch NC, Lin W, Juranka PF, Morris CE, Stys PK (2008) Traditional AMPA receptor antagonists partially block Nav1.6-mediated persistent current. Neuropharmacology 55: 1165–1171.
[25]  Chen Y, Yu FH, Sharp EM, Beacham D, Scheuer T, et al. (2008) Functional properties and differential modulation of Nav1.6 sodium channels. Molecular and Cellular Neuroscience 38: 607–615.
[26]  He B, Soderlund DM (2010) Human embryonic kidney (HEK293) cells express endogenous voltage-gated sodium currents and Nav1.7 sodium channels. Neuroscience Letters 469: 268–272.
[27]  Khaliq ZM, Gouwens N, Raman IM (2003) The contribution of resurgent sodium current to high-frequency firing in Purkinje neurons: an experimental and modeling study. Journal of Neuroscience 23: 4899–4912.
[28]  Raman IM, Bean BP (2001) Inactivation and recovery of sodium currents in cerebellar Purkinje neurons: evidence for two mechanisms. Biophysical Journal 80: 729–737.
[29]  Graham FL, Smiley J, Russell WC, Nairn R (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. Journal of General Virology 36: 59–77.
[30]  Shaw G, Morse S, Ararat M, Graham FL (2002) Preferential transformation of human neuronal cells by human adeonviruses and the origin of HEK 293 cells. FASEB Journal 16: 869–871.
[31]  Moran O, Nizzari M, Conti F (2000) Endogenous expression of the β1A sodium channel subunit in HEK-293 cells. FEBS Letters 473: 132–134.
[32]  Moran O, Conti F, Tammaro P (2003) Sodium channel heterologous expression in mammalian cells and the endogenous beta-1 subunits. Neuroscience Letters 336: 175–179.
[33]  Zhao J, O’Leary ME, Chahine M (2011) Regulation of Nav1.6 and Nav1.8 peripheral nerve Na+ channels by auxiliary β-subunits. Journal of Neurophysiology 106: 608–619.
[34]  Qu Y, Curtis R, Lawson D, Gilbride K, Ge P, et al. (2001) Differential modulation of sodium channel gating and persistent sodium currents of the β1, β2, and β3 subunits. Molecular and Cellular Neuroscience 18: 570–580.
[35]  Cummins TR, F A, Renganathan M, Herzog RI, Dib-Hajj SD, et al. (2001) Nav1.3 sodium channels: rapid repriming and slow closed-state inactivation display quantitative differences after expression in a mammalian cell line and in spinal sensory neurons. Journal of Neuroscience 21: 5952–5961.
[36]  Ferrera L, Moran O (2006) β1-subunit modulates the Nav1.4 sodium channel by changing the surface charge. Experimental Brain Research 172: 139–150.
[37]  Laezza F, Lampert A, Kozel MA, Gerber BR, Rush AM, et al. (2009) FGF14 N-terminal splice variants differentially modulate Nav1.2 and Nav1.6-encoded sodium channels. Molecular and Cellular Neuroscience 42: 90–101.

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