Abstract
There is demand for isoform-specific ion channel inhibitors as tools to investigate the biology of endogenous ion channels and validate them as targets in drug discovery programs. There is also hope that such inhibitors may be new therapeutic agents or provide the foundation for such agents. However, in practice, it is commonly experienced that inhibitors lack sufficient specificity, fail to distinguish between members of a class of ion channel, or have other (non-ion channel) off-target effects. Due to their extraordinary specificity, antibodies offer a potentially attractive strategy for overcoming these problems. Inhibitory antibodies acting at the extracellular face of ion channels are particularly attractive because there is enhanced possibility for specificity and intracellular delivery methods are not required. Here we describe experience with such an antibody approach and methodology for generating agents based on anti-peptide polyclonal antibodies.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Nauli SM, Alenghat FJ, Luo Y, Williams E, Vassilev P, Li X, Elia AE, Lu W, Brown EM, Quinn SJ, Ingber DE, Zhou J (2003) Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat Genet 33:129–137
Li J, Sukumar P, Milligan CJ, Kumar B, Ma ZY, Munsch CM, Jiang LH, Porter KE, Beech DJ (2008) Interactions, functions, and independence of plasma membrane STIM1 and TRPC1 in vascular smooth muscle cells. Circ Res 103:e97–e104
Raz-Prag D, Grimes WN, Fariss RN, Vijayasarathy C, Campos MM, Bush RA, Diamond JS, Sieving PA (2010) Probing potassium channel function in vivo by intracellular delivery of antibodies in a rat model of retinal neurodegeneration. Proc Natl Acad Sci U S A 107:12710–12715
Chioni AM, Fraser SP, Pani F, Foran P, Wilkin GP, Diss JK, Djamgoz MB (2005) A novel polyclonal antibody specific for the Nav1.5 voltage-gated Na+ channel ‘neonatal’ splice form. J Neurosci Methods 147:88–98
Fraser SP, Diss JK, Chioni AM, Mycielska ME, Pan H, Yamaci RF, Pani F, Siwy Z, Krasowska M, Grzywna Z, Brackenbury WJ, Theodorou D, Koyuturk M, Kaya H, Battaloglu E, De Bella MT, Slade MJ, Tolhurst R, Palmieri C, Jiang J, Latchman DS, Coombes RC, Djamgoz MB (2005) Voltage-gated sodium channel expression and potentiation of human breast cancer metastasis. Clin Cancer Res 11:5381–5389
Wyatt CN, Campbell V, Brodbeck J, Brice NL, Page KM, Berrow NS, Brickley K, Terracciano CM, Naqvi RU, MacLeod KT, Dolphin AC (1997) Voltage-dependent binding and calcium channel current inhibition by an anti-alpha 1D subunit antibody in rat dorsal root ganglion neurones and guinea-pig myocytes. J Physiol 502:307–319
Xu SZ, Zeng F, Lei M, Li J, Gao B, Xiong C, Sivaprasadarao A, Beech DJ (2005) Generation of functional ion-channel tools by E3 targeting. Nat Biotechnol 23:1289–1293
Zhou BY, Ma W, Huang XY (1998) Specific antibodies to the external vestibule of voltage-gated potassium channels block current. J Gen Physiol 111:555–563
Liao YJ, Safa P, Chen YR, Sobel RA, Boyden ES, Tsien RW (2008) Anti-Ca2+ channel antibody attenuates Ca2+ currents and mimics cerebellar ataxia in vivo. Proc Natl Acad Sci U S A 105:2705–2710
Naylor J, Milligan CJ, Zeng F, Jones C, Beech DJ (2008) Production of a specific extracellular inhibitor of TRPM3 channels. Br J Pharmacol 155:567–573
Xu SZ, Beech DJ (2001) TrpC1 is a membrane-spanning subunit of store-operated Ca2+ channels in native vascular smooth muscle cells. Circ Res 88:84–87
Xu SZ, Muraki K, Zeng F, Li J, Sukumar P, Shah S, Dedman AM, Flemming PK, McHugh D, Naylor J, Cheong A, Bateson AN, Munsch CM, Porter KE, Beech DJ (2006) A sphingosine-1-phosphate-activated calcium channel controlling vascular smooth muscle cell motility. Circ Res 98:1381–1389
Xu SZ, Sukumar P, Zeng F, Li J, Jairaman A, English A, Naylor J, Ciurtin C, Majeed Y, Milligan CJ, Bahnasi YM, Al-Shawaf E, Porter KE, Jiang LH, Emery P, Sivaprasadarao A, Beech DJ (2008) TRPC channel activation by extracellular thioredoxin. Nature 45(1):69–72
Naylor J, Li J, Milligan CJ, Zeng F, Sukumar P, Hou B, Sedo A, Yuldasheva N, Majeed Y, Beri D, Jiang S, Seymour VA, McKeown L, Kumar B, Harteneck C, O’Regan D, Wheatcroft SB, Kearney MT, Jones C, Porter KE, Beech DJ (2010) Pregnenolone sulphate- and cholesterol-regulated TRPM3 channels coupled to vascular smooth muscle secretion and contraction. Circ Res 106:1507–1515
Ciurtin C, Majeed Y, Naylor J, Sukumar P, English AA, Emery P, Beech DJ (2010) TRPM3 channel stimulated by pregnenolone sulphate in synovial fibroblasts and negatively coupled to hyaluronan. BMC Musculoskelet Disord 11:111
Kumar B, Dreja K, Shah SS, Cheong A, Xu SZ, Sukumar P, Naylor J, Forte A, Cipollaro M, McHugh D, Kingston PA, Heagerty AM, Munsch CM, Bergdahl A, Hultgardh-Nilsson A, Gomez MF, Porter KE, Hellstrand P, Beech DJ (2006) Upregulated TRPC1 channel in vascular injury in vivo and its role in human neointimal hyperplasia. Circ Res 98:557–563
Bergdahl A, Gomez MF, Dreja K, Xu SZ, Adner M, Beech DJ, Broman J, Hellstrand P, Sward K (2003) Cholesterol depletion impairs vascular reactivity to endothelin-1 by reducing store-operated Ca2+ entry dependent on TRPC1. Circ Res 93:839–847
Kwan HY, Shen B, Ma X, Kwok YC, Huang Y, Man YB, Yu S, Yao X (2009) TRPC1 associates with BK(Ca) channel to form a signal complex in vascular smooth muscle cells. Circ Res 104:670–678
Rosado JA, Brownlow SL, Sage SO (2002) Endogenously expressed Trp1 is involved in store-mediated Ca2+ entry by conformational coupling in human platelets. J Biol Chem 277:42157–42163
Klionsky L, Tamir R, Holzinger B, Bi X, Talvenheimo J, Kim H, Martin F, Louis JC, Treanor JJ, Gavva NR (2006) A polyclonal antibody to the prepore loop of transient receptor potential vanilloid type 1 blocks channel activation. J Pharmacol Exp Ther 319:192–198
Gomez-Varela D, Zwick-Wallasch E, Knotgen H, Sanchez A, Hettmann T, Ossipov D, Weseloh R, Contreras-Jurado C, Rothe M, Stuhmer W, Pardo LA (2007) Monoclonal antibody blockade of the human Eag1 potassium channel function exerts antitumor activity. Cancer Res 67:7343–7349
Tradtrantip L, Zhang H, Saadoun S, Phuan PW, Lam C, Papadopoulos MC, Bennett JL, Verkman AS (2012) Anti-aquaporin-4 monoclonal antibody blocker therapy for neuromyelitis optica. Ann Neurol 71:314–322
Wei G, Meng W, Guo H, Pan W, Liu J, Peng T, Chen L, Chen CY (2011) Potent neutralization of influenza A virus by a single-domain antibody blocking M2 ion channel protein. PLoS One 6:e28309
Lo AS, Zhu Q, Marasco WA (2008) Intracellular antibodies (intrabodies) and their therapeutic potential. Handb Exp Pharmacol 181:343–373
Miller KM, De Ryck MR, Wolff CGJ, Lawson ADG, Finney HM, Baker TS (2011) Function modifying Nav1.7 antibodies. Patent number WO/2011/051350
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character if a protein. J Mol Biol 157:105–132
Kolaskar AS, Tongaonkar PS (1990) A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276:172–174
Hecquet CM, Ahmmed GU, Vogel SM, Malik AB (2008) Role of TRPM2 channel in mediating H2O2-induced Ca2+ entry and endothelial hyperpermeability. Circ Res 102:347–355
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Naylor, J., Beech, D.J. (2013). Generation of Antibodies That Are Externally Acting Isoform-Specific Inhibitors of Ion Channels. In: Gamper, N. (eds) Ion Channels. Methods in Molecular Biology, vol 998. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-351-0_19
Download citation
DOI: https://doi.org/10.1007/978-1-62703-351-0_19
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-350-3
Online ISBN: 978-1-62703-351-0
eBook Packages: Springer Protocols