Activation of muscle nicotinic acetylcholine receptor channels by nicotinic and muscarinic agonists

Abstract

1. The dose-response parameters of recombinant mouse adult neuromuscular acetylcholine receptor channels (nAChR) activated by carbamylcholine, nicotine, muscarine and oxotremorine were measured. Rate constants for agonist association and dissociation, and channel opening and closing, were estimated from single-channel kinetic analysis. 2. The dissociation equilibrium constants were (mM): ACh (0. 16)<oxotremorine M (0.6)<carbamylcholine (0.8)<nicotine (2.6). 3. The gating equilibrium constants (opening/closing) were: ACh (45)>carbamylcholine (5.1)>oxotremorine M (0.6)>nicotine (0. 5)>muscarine (0.15). 4. Rat neuronal alpha4beta2 nAChR can be activated by all of the agonists. However, detailed kinetic analysis was impossible because the recordings lacked clusters representing the activity of a single receptor complex. Thus, the number of channels in the patch was unknown and the activation rate constants could not be determined. 5. Considering both receptor affinity and agonist efficacy, muscarine and oxotremorine are significant agonists of muscle-type nAChR. The results are discussed in terms of structure-function relationships at the nAChR transmitter binding site.

Figure 1

Molecular structures of acetylcholine, carbamylcholine, nicotine, oxotremorine and muscarine.

Figure 2

Activation properties of adult muscle type nAChR by carbamylcholine (CCh). (A) An example cluster (500 μM CCh), and open and closed intracluster interval duration histograms (mean open interval duration=0.46 ms; mean closed interval duration=0.89 ms). Interval duration histograms contain data of the whole patch from which the representative cluster is shown. The membrane potential was −100 mV; inward current is down. (B) The probability of being open within a cluster (p_o) vs agonist concentration. (C) Effective opening rate: (β′; the inverse of the slowest intracluster closed interval component) vs agonist concentration. In (B) and (C) each data point is one patch. Solid lines are fits to Equation 1 (Table 1). For comparison, data for the adult-, muscle-type nAChR activated by ACh (from Akk & Auerbach, 1996) are shown as hollow circles.

Figure 3

Single-channel kinetic analysis of CCh-activated, muscle-type nAChR. Intracluster current intervals, obtained at 100, 200, and 500 μM CCh, were simultaneously fitted by Model 1. The results of the fit are given in Table 2. Left: example single-channel currents at each concentration. Right: open and closed intracluster interval duration histograms. The solid lines are calculated from the rate constants of the fit, after incorporating a correction for missed events.

Figure 4

Adult-, muscle-type nAChR activated by nicotine. (A) An example cluster (1 mM nicotine) and intracluster open and closed interval duration histograms (mean open interval duration=0.21 ms; closed interval durations fitted using two exponentials, with time constants=0.14 and 7.9 ms). Single-channel amplitude was constant (3.1 pA) but appears to vary because of filtering. (B) The probability of being open within a cluster vs agonist concentration. (C) The effective opening vs agonist concentration. The data for the αG153S mutant receptor is shown as filled squares. In (B) and (C) each data point is one patch. Solid lines are fits by Equation 1. For comparison, data for ACh-activated nAChR (from Akk & Auerbach, 1996) are shown as hollow circles.

Figure 5

Adult, muscle-type nAChR activated by oxotremorine. (A) An example cluster (500 μM oxotremorine), and intracluster open and closed interval duration histograms (mean open interval duration=0.24 ms; mean closed interval duration=4.1 ms). (B) The probability of being open within a cluster vs agonist concentration. (C) The effective opening vs agonist concentration. In (B) and (C) each data point is one patch. Solid lines are fits by Equation 1. For comparison, data for ACh-activated nAChR (from Akk & Auerbach, 1996) are shown as hollow circles.

Figure 6

Adult-, muscle-type nAChR activated by muscarine. (A) An example cluster (100 μM muscarine), and intracluster open and closed interval duration histograms (mean open interval duration=1.47 ms; mean closed interval duration=40.4 ms). (B) The probability of being open within a cluster vs agonist concentration. (C) The effective opening vs agonist concentration. The data for the αG153S mutant receptor is shown as filled squares. In (B) and (C) each data point is one patch. Solid lines are fits by Equation 1. For comparison, data for ACh-activated nAChR (from Akk & Auerbach, 1996) are shown as hollow circles.

Figure 7

Activation of neuronal-type α₄β₂ nAChR by nicotinic and muscarinic agonists. The openings were elicited by 100 μM ACh (A), 100 μM nicotine (B), 120 μM muscarine (C) and 100 μM oxotremorine (D). The single-channel currents had similar open interval durations and conductances in the presence of these agonists (see Table 3). The means of open interval durations were 0.8 ms (ACh), 0.8 ms (nicotine), 0.8 ms (muscarine), 0.5 ms (oxotremorine). The membrane potential was −70 mV; inward current is down.