title: Localization of sodium channel subtypes in mouse ventricular myocytes using quantitative immunocytochemistry.
abstract: Voltage-gated sodium channels are responsible for the rising phase of the action potential in cardiac muscle. Previously, both TTX-sensitive neuronal sodium channels (NaV1.1, NaV1.2, NaV1.3, NaV1.4 and NaV1.6) and the TTX-resistant cardiac sodium channel (NaV1.5) have been detected in cardiac myocytes, but relative levels of protein expression of the isoforms were not determined. Using a quantitative approach, we analyzed z-series of confocal microscopy images from individual mouse myocytes stained with either anti-NaV1.1, anti-NaV1.2, anti-NaV1.3, anti-NaV1.4, anti-NaV1.5, or anti-NaV1.6 antibodies and calculated the relative intensity of staining for these sodium channel isoforms. Our results indicate that the TTX-sensitive channels represented approximately 23% of the total channels, whereas the TTX-resistant NaV1.5 channel represented 77% of the total channel staining in mouse ventricular myocytes. These ratios are consistent with previous electrophysiological studies in mouse ventricular myocytes. NaV1.5 was located at the cell surface, with high density at the intercalated disc, but was absent from the transverse (t)-tubular system, suggesting that these channels support surface conduction and inter-myocyte transmission. Low-level cell surface staining of NaV1.4 and NaV1.6 channels suggest a minor role in surface excitation and conduction. Conversely, NaV1.1 and NaV1.3 channels are localized to the t-tubules and are likely to support t-tubular transmission of the action potential to the myocyte interior. This quantitative immunocytochemical approach for assessing sodium channel density and localization provides a more precise view of the relative importance and possible roles of these individual sodium channel protein isoforms in mouse ventricular myocytes and may be applicable to other species and cardiac tissue types.
authors: Department of Pharmacology, University of Washington, Seattle, WA 98195-7280, USA. Electronic address: ruthw@u.washington.edu.
authors: Ruth E
authors: RE
authors: Westenbroek
authors: Sebastian
authors: S
authors: Bischoff
authors: Ying
authors: Y
authors: Fu
authors: Sebastian K G
authors: SK
authors: Maier
authors: William A
authors: WA
authors: Catterall
authors: Todd
authors: T
authors: Scheuer
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D006352:Heart Ventricles / Q000378:metabolism*; D007150:Immunohistochemistry / Q000379:methods / Q000592:standards; D042541:Intracellular Space / Q000378:metabolism; D008297:Male; D051379:Mice; D032383:Myocytes, Cardiac / Q000378:metabolism*; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000378:metabolism; D020033:Protein Isoforms; D021381:Protein Transport; D015222:Sodium Channels / Q000145:classification / Q000378:metabolism*
chemical_list: D062554:NAV1.5 Voltage-Gated Sodium Channel; D020033:Protein Isoforms; D015222:Sodium Channels
keywords: Localization; Sodium channels; Ventricular myocytes
references: Arch Histol Cytol. 2004 Jun;67(2):115-34
references: 15468952
references: Prog Biophys Mol Biol. 2008 Oct-Nov;98(2-3):309-18
references: 19351521
references: J Physiol. 2010 Nov 1;588(Pt 21):4249-60
references: 20837647
references: J Neurosci. 2007 Oct 24;27(43):11543-51
references: 17959797
references: Nat Neurosci. 2006 Sep;9(9):1142-9
references: 16921370
references: FEBS Lett. 1990 Nov 26;275(1-2):195-200
references: 2175715
references: J Electrocardiol. 2002 Oct;35(4):343-9
references: 12395362
references: Cardiovasc Res. 2008 Apr 1;78(1):45-52
references: 18178574
references: Circ Res. 2006 Mar 17;98(5):667-74
references: 16484618
references: J Mol Cell Cardiol. 2001 Apr;33(4):599-613
references: 11273715
references: Am J Physiol. 1996 Aug;271(2 Pt 2):H498-506
references: 8770089
references: Biotechniques. 2000 May;28(5):884-6, 888
references: 10818693
references: Circulation. 2004 Mar 23;109(11):1421-7
references: 15007009
references: Pflugers Arch. 1985 May;404(2):190-6
references: 2409525
references: Arch Mal Coeur Vaiss. 2007 Sep;100(9):787-93
references: 18033008
references: Circulation. 1996 Dec 15;94(12):3083-6
references: 8989112
references: J Neurosci. 2007 May 30;27(22):5903-14
references: 17537961
references: J Physiol. 1999 Dec 15;521 Pt 3:587-99
references: 10601491
references: Biochem Biophys Res Commun. 2005 Sep 9;334(4):1135-40
references: 16038878
references: J Physiol. 2005 May 1;564(Pt 3):683-96
references: 15746173
references: Heart Rhythm. 2011 Dec;8(12):1923-30
references: 21767519
references: J Biol Chem. 2004 Sep 24;279(39):40748-54
references: 15272007
references: Biotechniques. 1988 Jul-Aug;6(7):632-8
references: 3273409
references: Proc Natl Acad Sci U S A. 2002 Mar 19;99(6):4073-8
references: 11891345
references: J Mol Cell Cardiol. 2013 Aug;61:133-141
references: 23702286
references: Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3289-93
references: 8159741
references: FASEB J. 2012 Jan;26(1):63-72
references: 21948246
references: Circulation. 2011 Aug 30;124(9):1001-11
references: 21824921
references: Circulation. 2001 Mar 6;103(9):1303-10
references: 11238277
references: Biochem Biophys Res Commun. 2006 Sep 29;348(3):1163-6
references: 16904633
references: J Mol Cell Cardiol. 2007 Mar;42(3):469-77
references: 17289073