Dependence of acetylcholine desensitization on the membrane potential of frog muscle fibre and on the ionic changes in the medium

I The effects of the ionophore X-537A were studied on carbamylcholine (carbachol)-induced desensitization and on tension development in relaxed potassium-depolarized frog sartorius muscles. 2 X-537A accelerated carbachol-induced desensitization in Ca2+-deficient solutions without having any effect on the conductance of the membrane in the absence of carbachol or on the extent of the carbachol-induced increase in conductance. 3 In Ca2+-deficient solution, the acceleration of desensitization by the ionophore was concentrationdependent. No effect was observed with concentrations less than 5,M and maximal acceleration was evident with 10 gM. 4 The influence of X-537A on desensitization was time-dependent. At 20 AM X-537A, there was a marked acceleration of desensitization by the end of 5 min exposure. An additional gradual acceleration occurred during a 5 to 30 min treatment. No acceleration of desensitization was evident when X-537A was simultaneously applied with carbachol to the end-plate region without prior exposure to the ionophore. 5 Desensitization also was accelerated by 30 min exposure to 20 gM X-537A in solutions containing Ca2+ or deficient in both Mg2+ and Ca2+; the rate being increased 2.8-fold in Ca2+-containing solutions, 2.9-fold in Ca2+-deficient solutions containing Mg2+, and 2.5-fold in divalent cation-deficient solutions. 6 Tension development gradually occurred in relaxed potassium-depolarized muscle preparations exposed to 20 gM X-537A. The onset of tension development occurred only after approximately 25 min of exposure both in preparations kept in Ca2+-deficient or Ca2+-containing solutions. By the end of 90 min in the ionophore, the tension developed was approximately 12% and 23% of the initial potassium contracture in those preparations maintained in the Ca2+-deficient or Ca2+-containing solutions, respectively. 7 We assume that the increase in desensitization rate following exposure to X-537A results from an elevation of the intracellular Ca2+ concentration. That muscle tension gradually increased during exposure to the ionophore supports this conclusion. The acceleration of desensitization by X-537A in the absence of external Ca2+ supports the view that the site of calcium acceleration is not on the external surface of the end-plate membrane either at or near the agonist-recognition site but rather on the inner surface.

Section: General Methodsmentioning

confidence: 99%

Effect of Ionophore X‐537a on Desensitization Rate and Tension Development in Potassium‐depolarized Muscle Fibres

DeBassio

Parsons

Schnitzler

1976

“…Early work on nicotinic AChRs of skeletal muscle ®bres has suggested that [Ca 2+ ] o is important in the control of onset and extent of desensitization (Manthey, 1966;Magazanik & Vyskocil, 1970), although it has subsequently been shown that desensitization still takes place in the absence of [Ca 2+ ] o and is reduced by an injection of EGTA into the postsynaptic ®bre (Miledi, 1980). Unlike muscle nicotinic AChRs, neuronal-type receptors of chroma n cells are highly permeable to Ca 2+ (Vernino et al, 1994) which is thus potentially capable of modulating the process of desensitization.…”

Section: Role Of Ca 2+ In Desensitization Of Nicotinic Achrsmentioning

confidence: 99%

“…In the case of nicotinic acetylcholine receptors (AChRs) early work showed that their desensitization was accelerated by high extracellular Ca 2+ levels ([Ca 2+ ] o ) (Manthey, 1966;Magazanik & Vyskocil, 1970), whereas subsequent studies have suggested that raised intracellular Ca 2+ concentration ([Ca 2+ ] i ) is actually the important factor to facilitate desensitization on muscle (Miledi, 1980;Chestnut, 1983). Progress in this ®eld has been aided by the observation that desensitization of nicotinic AChRs is a heterogeneous process comprising fast and slow phases (Feltz & Trautmann, 1982;MacOnochie & Knight, 1992), only the latter one being modulated by [Ca 2+ ] i (Cachelin & Colquhoun, 1989).…”

Section: Introductionmentioning

confidence: 99%

Imaging of intracellular calcium during desensitization of nicotinic acetylcholine receptors of rat chromaffin cells

Khiroug

Giniatullin

Sokolova

et al. 1997

1 The possible role of intracellular Ca 2+ levels ([Ca 2+ ] i ) in desensitization of nicotinic acetylcholine receptors (AChRs) was investigated in rat cultured chroma n cells by use of combined whole-cell patch clamping and confocal laser scanning microscopy with the¯uorescent dye¯uo-3. 2 On cells held at 770 mV, pressure-application of nicotine elicited inward currents with associated [Ca 2+ ] i rises mainly due to in¯ux through nicotinic AChRs. These responses were blocked by (+)-tubocurarine (10 mM) but were insensitive to a-bungarotoxin (1 mM) or Cd 2+ (0.1 mM). 3 Pressure applications of 1 mM nicotine for 2 s (conditioning pulse) evoked inward currents which faded biexponentially to a steady state level due to receptor desensitization and were accompanied by a sustained increase in [Ca 2+ ] i . Inward currents evoked by subsequent application of brief test pulses of nicotine were depressed but recovered with a time course reciprocal to the decay of the [Ca 2+ ] i transient induced by the conditioning pulse. 4 Omission of intracellular Ca 2+ chelators or use of high extracellular Ca 2+ solution (10 mM) lengthened recovery of nicotinic AChRs from desensitization while adding BAPTA or EGTA intracellularly had the opposite e ect. When the patch pipette contained¯uo-3 or no chelators, after establishing whole cell conditions the rate of recovery became progressively longer presumably due to dialysis of endogenous Ca 2+ bu ers. None of these manipulations of external or internal Ca 2+ had any e ect on onset or steady state level of desensitization. 5 High spatial resolution imaging of [Ca 2+ ] i in intact cells (in the presence of 0.1 mM Cd 2+ ) showed that its level in the immediate submembrane area decayed at the same rate as in the rest of the cell, indicating that Ca 2+ was in a strategic location to modulate (directly or indirectly) AChR desensitization. 6 The present data suggest that desensitized nicotinic AChRs are stabilized in their conformation by raised [Ca 2+ ] i and that this phenomenon retards their recovery to full activity.

“…Although desensitization is an intrinsic property of the ACh receptor, many environmental factors can alter the rate of desensitization onset, including membrane voltage (Magazanik & Vyskocil, 1970;Fiekers et al, 1980;Cachelin & Colquhoun, 1989), calcium (Manthey, 1966;Nastuk & Parsons, 1970;Fiekers et al, 1980), neurotransmitters and other drugs (Ochoa et al, 1989) and receptor phosphorylation (Huganir & Greengard, 1990).…”

Section: Introductionmentioning

confidence: 99%

Staurosporine inhibits the extent of acetylcholine receptor recovery from carbachol‐induced desensitization in snake twitch fibres

Hardwick

Coniglio

Parsons³

1991

1 The effect of the protein kinase inhibitor, staurosporine, on the extent and time course of recovery following carbachol-induced desensitization was studied in snake twitch-muscle fibres maintained in an isotonic potassium propionate solution and voltage-clamped to + 30 mV. 3 Staurosporine also produced a concentration-dependent (10nM to 0.5 pM) decrease in the amplitude of a second carbachol-induced current, following a wash period, as compared to the amplitude of the current produced by the initial carbachol application. Pretreatment with 0.5,UM K252a, another wide spectrum protein kinase inhibitor, also decreased the extent of recovery of the response to a second carbachol application following desensitization.4 Staurosporine pretreatment (0.5jUM) had no effect on either the kinetics of receptor-channel gating or the initial endplate sensitivity to agonist. This was determined by comparing the amplitude of the carbachol (540Mm)-induced currents and the amplitude and decay rate of m.e.p.cs in control and staurosporine-treated fibres. 5 Staurosporine had no effect on the time course of desensitization onset produced during the initial application of 540piM carbachol or the depth of desensitization produced by the end of a 2-3 min exposure to 540pM carbachol.6 Elevation of the external calcium concentration from 1 to 10mM during the 540pM carbachol application completely antagonized the decreased extent of recovery of m.e.p.c. amplitude produced by pretreatment with 0.5JM staurosporine. 7 We suggest that phosphorylation of a population of acetylcholine receptors is required for complete recovery from desensitization, and that staurosporine inhibits the protein kinases responsible for this phosphorylation. 8 We further propose that a transient increase in intracellular calcium, produced by an increase in calcium influx through agonist-activated endplate channels, stimulates additional protein kinase activity, which in turn, antagonizes the effect of staurosporine-treatment on recovery.