Abstract
We studied desensitization of nicotinic acetylcholine (ACh) receptor channels in the clonal BC3H-1 cell line. We measured the current response to rapid perfusion of outside-out patches with 1 μM to 5 mM ACh, carbamylcholine and suberyldicholine. After binding to the receptors and opening the ion channels, all agonists induce a rapid, concentration-dependent decay of channel activity. The time constant of the current decay ranged from several seconds at low agonist concentrations to about 50 ms at saturating concentrations. The decay rate at saturating concentrations was independent of voltage. The ratio of steady-state to peak current ranged from 0.5 at low agonist concentrations to 0.02 or less at high concentrations. The rate of recovery from desensitization after removal of agonist was also measured. For ACh, the recovery time constant was 320 ms; recovery from desensitization by carbamylcholine was twice as fast. A linear kinetic scheme does not provide a complete description of these results. The data are consistent with a cyclic model, although, it is not possible to uniquely determine all of the rate constants in this scheme. The results are compared with competitive binding and single channel studies of desensitization in BC3H-1 cells.
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References
Anwyl R, Narahashi T (1980) Desensitization of the acetylcholine receptor of denervated rat soleus muscle and the effect of calcium. Br J Pharmacol 69:91–98
Brett RS, Dilger JP, Adams PR, Lancaster B (1986) A method for the rapid exchange of solutions bathing excised membrane patches. Biophys J 50:987–992
Cachelin AB, Colquhoun D (1989) Desensitization of the acetylcholine receptor of frog end-plates measured in a vaselinegap voltage clamp. J Physiol (Lond) 415:159–188
Chesnut TJ (1983) Two-component desensitization at the neuromuscular junction of the frog. J Physiol (Lond) 336:229–241
Colquhoun D, Ogden DC (1988) Activation of ion channels in the frog end-plate by high concentrations of acetylcholine. J Physiol (Lond) 395:131–159
Colquhoun D, Sakmann B (1985) Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J Physiol (Lond) 369:501–557
Connor EA, Fiekers JF, Neel DS, Parsons RL, Schnitzler RM (1984) Comparison of cholinergic activation and desensitization at snake twitch and slow muscle fibre end-plates. J Physiol (Lond) 351:657–674
Dilger JP, Brett RS (1990) Direct measurement of the concentration-and time-dependent open probability of the nicotinic acetylcholine receptor channel. Biophys J 57:723–731
Dilger JP, Brett RS, Poppers DM, Liu Y (1991) The temperature dependence of some kinetic and conductance properties of acetylcholine receptor channels. Biochim Biophys Acta 1063:253–258
Fiekers JF, Spannbauer PM, Scubon-Mulieri B, Parsons RL (1980) Voltage dependence of desensitization. J Gen Physiol 75:511–529
Franke C, Hatt H, Dudel J (1991) Steep concentration dependence and fast desensitization of nicotinic channel currents elicited by acetylcholine pulses, studied in adult vertebrate muscle. Pflügers Arch 417:509–516
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell free membrane patches. Pflügers Arch 391:85–100
Jackson MB (1988) Dependence of acetylcholine receptor channel kinetics on agonist concentration in cultured mouse muscle fibres. J Physiol (Lond) 397:555–583
Katz B, Thesleff S (1957) A study of the ‘desensitization’ produced by acetylcholine at the motor end-plate. J Physiol (Lond) 138:63–80
Liu Y (1990) A study of acetylcholine receptor channel kinetics using rapid perfusion of patches. PhD thesis, State University of New York at Stony Brook
Liu Y, Dilger J (1991) Opening rate of acetylcholine receptor channels. Biophys J 60:424–432
Maconochie DJ, Knight DE (1989) A method for making solution changes in the sub-millisecond range at the tip of a patch pipette. Pflügers Arch 414:589–596
Magazanik LG, Vyskocil F (1970) Dependence of acetylcholine desensitization on the membrane potential of frog muscle fibres and on the ionic changes in the medium. J Physiol (Lond) 210:507–518
Magleby KL, Pallotta BS (1981) A study of desensitization of acetylcholine receptors using nerve-released transmitter in the frog. J Physiol (Lond) 316:225–250
Manthey AA (1991) Voltage dependence of desensitization to carbamylcholine in frog muscle fibers reverses in low-calcium solutions. J Neurophysiol 65:980–988
Ochoa ELM, Chattopadhyay A, McNamee MG (1989) Desensitization of the nicotinic acetylcholine receptor: Molecular mechanisms and effects of modulators. Cell Mol Neurobiol 9:141–178
Ogden DC, Colquhoun D (1985) Ion channel block by acetylcholine, carbachol and suberyldicholine at the frog neuro-muscular junction. Proc R Soc Lond [Biol] 225:329–355
Papke RL, Oswald RE (1986) Effects of allosteric ligands on the gating of single channel currents in BC3H-1 cells. In: Maelicke A (ed) Nicotinic acetylcholine receptor. Springer, Berlin Heidelberg New York, pp 243–257
Sine SM, Steinbach JH (1984) Activation of a nicotinic acetylcholine receptor. Biophys J 45:175–185
Sine SM, Steinbach JH (1984) Agonists block currents through acetylcholine receptor channels. Biophys J 46:277–284
Sine SM, Steinbach JH (1986) Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by low concentrations of agonist. J Physiol (Lond) 373:129–162
Sine SM, Steinbach JH (1987) Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by high concentrations of agonist. J Physiol (Lond) 385:325–359
Sine S, Taylor P (1982) Local anesthetics and histrionicotoxin are allosteric inhibitors of the acetylcholine receptor. J Biol Chem 257:8106–8114
Sine SM, Claudio T, Sigworth FJ (1990) Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts — Single channel current kinetics reveal distinct agonist binding affinities. J Gen Physiol 96:395–437
Weber M, David-Pfeuty T, Changeux J-P (1975) Regulation of binding properties of the nicotinic receptor protein by cholinergic ligands in membrane fragments from Torpedo marmorata. Proc Natl Acad Sci USA 72:3443–3447
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Dilger, J.P., Liu, Y. Desensitization of acetylcholine receptors in BC3H-1 cells. Pflugers Arch. 420, 479–485 (1992). https://doi.org/10.1007/BF00374622
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DOI: https://doi.org/10.1007/BF00374622