Nicotinic receptor transduction zone: invariant arginine couples to multiple electron-rich residues

Abstract

Gating of the muscle-type acetylcholine receptor (AChR) channel depends on communication between the ACh-binding site and the remote ion channel. A key region for this communication is located within the structural transition zone between the ligand-binding and pore domains. Here, stemming from β-strand 10 of the binding domain, the invariant αArg209 lodges within the hydrophobic interior of the subunit and is essential for rapid and efficient channel gating. Previous charge-reversal experiments showed that the contribution of αArg209 to channel gating depends strongly on αGlu45, also within this region. Here we determine whether the contribution of αArg209 to channel gating depends on additional anionic or electron-rich residues in this region. Also, to reconcile diverging findings in the literature, we compare the dependence of αArg209 on αGlu45 in AChRs from different species, and compare the full agonist ACh with the weak agonist choline. Our findings reveal that the contribution of αArg209 to channel gating depends on additional nearby electron-rich residues, consistent with both electrostatic and steric contributions. Furthermore, αArg209 and αGlu45 show a strong interdependence in both human and mouse AChRs, whereas the functional consequences of the mutation αE45R depend on the agonist. The emerging picture shows a multifaceted network of interdependent residues that are required for communication between the ligand-binding and pore domains.

Figure 1

(a) Structural model of the α-subunit from the Torpedo AChR (PDB code 2BG9). (b) Close-up view of the region between the ligand-binding and pore domains. Key residues are highlighted in stick representation and colored according to electronic charge (blue, positive; red, negative; and gray, neutral).

Figure 2

(a) Kinetics of activation of human and mouse AChRs and the indicated mutants. Currents elicited by 100 μM ACh are shown at a bandwidth of 10 kHz, with channel openings shown as upward deflections. Histograms of closed and open dwell times within identified clusters are shown on logarithmic time axes with PDFs generated from global kinetic fitting overlaid (see Materials and Methods; fitted rate constants are given in Table 1). (b) Energetic coupling between αArg209 and αGlu45. In each two-dimensional mutant cycle, the diagonal line indicates interresidue coupling free energy, and the horizontal and vertical lines indicate free-energy changes due to mutation. SEs of ΔΔG were computed as described in Materials and Methods. The 95% confidence limit, or twice the SE, indicates a coupling energy significantly different from zero.

Figure 3

Kinetics of activation of human and mouse AChRs and the αE45R mutant. Currents elicited by 3 mM Ch are shown at a bandwidth of 10 kHz, with channel openings shown as upward deflections. Histograms of closed and open dwell times within identified clusters of events are shown on logarithmic time axes with PDFs generated from global kinetic fitting overlaid (see Materials and Methods; fitted rate constants are given in Table 2).

Figure 4

(a) Kinetics of activation of charge-reversal mutations in human AChR. Currents elicited by 100 μM ACh are shown at a bandwidth of 10 kHz, with channel openings shown as upward deflections. Histograms of closed and open dwell times within identified clusters of events are shown on logarithmic time axes with probability density functions generated from global kinetic fitting overlaid (see Materials and Methods; fitted rate constants are given in Table 3). (b) Interresidue energetic coupling as in Fig. 2 b.

Figure 5

Kinetics of activation of hydrophobic mutations in the human AChR Currents elicited by 100 μM ACh are shown at a bandwidth of 10 kHz, with channel openings shown as upward deflections. Histograms of closed and open dwell times are shown with PDFs computed from global kinetic fitting overlaid (Table S1).

Figure 6

(a) Single-channel currents elicited by 100 μM ACh from WT and the triple-Leu-substituted AChR at a bandwidth 10 kHz. (b) Histogram of cluster mean open probability (P_open) shows a broad distribution for the triple mutant (gray bars), indicating heterogeneous kinetics, but the distribution is narrow for the WT AChR (black bars). (c) Single-channel currents elicited by 1 mM ACh from the triple-Val-substituted AChR. Clusters could not be identified even at a concentration of 1 mM ACh. (d) Open and closed time histograms were fitted by sums of exponentials using the program TAC.