Contrasting Actions of Philanthotoxin-343 and Philanthotoxin- (12) on Human Muscle Nicotinic Acetylcholine Receptors TIM J. BRIER, IAN R. MELLOR, DENIS B. TIKHONOV, IOANA NEAGOE, ZUOYI SHAO, MATT J. BRIERLEY, KRISTIAN STRØMGAARD, JERZY W. JAROSZEWSKI, POVL KROGSGAARD-LARSEN, AND PETER N. R. USHERWOOD Division of Molecular Toxicology, School of Life and Environmental Sciences, University of Nottingham, Nottingham, United Kingdom (T.J.B., I.R.M., I.N., Z.S., M.J.B. and P.N.R.U.); Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Copenhagen, Denmark (K.S., J.W.J. and P.K-L.); and Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia (D.B.T.). Received March 13, 2003; accepted June 18, 2003 This article is available online at http://molpharm.aspetjournals.org ABSTRACT Whole-cell recordings and outside-out patch recordings from TE671 cells were made to investigate antagonism of human muscle nicotinic acetylcholine receptors (nAChR) by the phil- anthotoxins, PhTX-343 and PhTX-(12). When coapplied with acetylcholine (ACh), PhTX-343 caused activation-dependent, noncompetitive inhibition (IC 50 = 17 M at -100 mV) of whole- cell currents that was strongly voltage-dependent. However, preapplication of PhTX-343 unveiled a voltage-independent antagonism that also required receptor activation, which is suggestive of desensitization enhancement. In single-channel studies, 10 M PhTX-343 significantly reduced the mean open time of channel openings evoked by 1 M ACh from 4.42  0.44 to 1.58  0.10 ms with a minor increase (1.26-fold) in mean closed time. These data indicate that PhTX-343 predom- inantly blocks the open channel gated by ACh. In contrast, PhTX-(12) caused potent (IC 50 = 0.77 M at -100 mV), acti- vation-dependent, noncompetitive inhibition of ACh-induced whole-cell currents that was only weakly voltage-dependent and suggestive of desensitization enhancement. It caused only a small decrease (7.5%) in the mean open time of channel openings induced by 1 M ACh, whereas the mean closed time was significantly increased from 200  45 ms to 586  145 ms. The different voltage-dependencies of the two modes of action of these philanthotoxins suggest two binding sites, one deep in the nAChR pore, the other near the extracellular entrance to the pore. Nicotinic acetylcholine receptors (nAChR) are distributed extensively throughout the central and peripheral nervous systems of vertebrates and invertebrates, where they medi- ate fast transmission at central synapses and neuromuscular junctions (Corringer et al., 2000; Itier and Bertrand, 2001). The human muscle cell line TE671, which is the subject of this report, expresses nAChR with sequence and subunit stoichiometry [(1) 2 1] similar to that of immature human muscle nAChR (Schoepfer et al., 1988; Lukas et al., 1999). The electrophysiological characteristics of TE671 cells, and the nAChR that they express, have been described previously (Oswald et al., 1989; Shao et al., 1998). Noncompetitive inhibitors of nAChR include antagonists of the open channel conformation of this receptor [e.g., QX-222 (Charnet et al., 1990)] and those that inhibit both closed and open channel conformations [e.g., chlorpromazine (Giraudat et al., 1986)]. These and other noncompetitive inhibitors have been extensively reviewed by Arias (1998). Philanthotoxin- 433 (PhTX-433; 4, 3, and 3 indicate the number of methylene groups between the amide/amine groups) (Eldefrawi et al., 1988; Piek and Hue, 1989) is a natural product example of a class of polyamine-containing compounds discovered in cer- tain wasp and spider venoms that noncompetitively antago- nize nAChR. In general, natural and synthetic philanthotox- ins exhibit properties that are qualitatively similar to those of polyamines, such as spermine and spermidine, but at lower concentrations (Usherwood and Blagbrough, 1991). PhTX-343 (Fig. 1), a structurally close analog of PhTX-433, is one of many synthetic analogs of the natural product (Anis et al., 1990; Bruce et al., 1990; Karst and Piek, 1991; Karst et al., 1991; Benson et al., 1992, 1993; Strømgaard et al., 1999, 2000; Bixel et al., 2000). It is a potent antagonist of ionotropic glutamate receptors mediating neuromuscular transmission in insects (Eldefrawi et al., 1988; Bruce et al., 1990), of ionotropic glutamate receptors of rat brain (Ragsdale et al., 1989; Jones et al., 1990; Brackley et al., 1993), and of recom- binant, ionotropic glutamate receptors from rat (Brackley et al., 1993; Ba ¨ hring and Mayer, 1998). The interactions of PhTX-433 and PhTX-343 with vertebrate muscle-type This work was supported by grants from the European Community BIOMED-2 (BMH4-CT97-2395), the Wellcome Trust (067496), and the Danish Medical Research Council (22-00-0372). ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; PhTX, philanthotoxin; -BgTX, -bungarotoxin; ACh, acetylcholine; MR44, N 1 -(3-{[6- ({8-[(6-aminohexyl)amino]octyl}amino)hexyl]-amino}propyl)-4-azido-2 hydroxy benzamide. 0026-895X/03/6404-954 –964$7.00 MOLECULAR PHARMACOLOGY Vol. 64, No. 4 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2523/1092601 Mol Pharmacol 64:954–964, 2003 Printed in U.S.A. 954 at ASPET Journals on March 11, 2016 molpharm.aspetjournals.org Downloaded from