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Contrasting Actions of Philanthotoxin-343 and Philanthotoxin-(12) on Human Muscle Nicotinic Acetylcholine Receptors

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.).

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 philanthotoxins, PhTX-343 and PhTX-(12). When coapplied with acetylcholine (ACh), PhTX-343 caused activation-dependent, noncompetitive inhibition (IC₅₀ = 17 µM 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 µM PhTX-343 significantly reduced the mean open time of channel openings evoked by 1 µM 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 predominantly blocks the open channel gated by ACh. In contrast, PhTX-(12) caused potent (IC₅₀ = 0.77 µM at-100 mV), activation-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 µM 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.

Address correspondence to: Dr. Ian R. Mellor, School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK. E-mail: ian.mellor{at}nottingham.ac.uk

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