Contributions of the non-alpha subunit residues (loop D) to agonist binding and channel gating in the muscle nicotinic acetylcholine receptor

Abstract

The agonist binding site of the nicotinic acetylcholine receptor has a loop-based structure, and is formed by residues located remotely to each other in terms of primary structure. Amino acid residues in sites delta57 and delta59, and the equivalent residues in the epsilon; subunit, have been identified as part of the agonist binding site and designated as loop D. The effects of point mutations in sites delta57, delta59, epsilon;55 and epsilon;57 on agonist binding and channel gating were studied. The mutated receptors were expressed transiently in HEK 293 cells and the currents were recorded using the cell-attached single-channel patch clamp technique. The results demonstrate that the mutations mainly affect channel gating with the major portion of the effect due to a reduction in the channel opening rate constant. For both the delta57/epsilon;55 and the delta59/epsilon;57 site, a mutation in the epsilon; subunit had a greater effect on channel gating than a mutation in the delta subunit. In all instances, agonist binding was affected to a lesser degree than channel gating. Previous data have placed the delta57 and delta59 residues in or near the agonist binding pocket. The data presented here suggest that these two residues (and the homologous sites in the epsilon; subunit) are not involved in specific interactions with the nicotinic agonist and that they affect the activation of the nicotinic receptor by shaping the overall structure of the agonist binding site.

Figure 1. Currents from the wild-type, and δW57F, ɛW55F and δW57F + ɛW55F mutant receptors activated by 200 μm ACh

Single-channel clusters and the first 200 ms at a higher time resolution are shown. Open events are shown downward. Membrane potential is -50 mV. Intracluster closed and open time histograms are given for the patch from which the representative cluster is shown. The continuous line in the histograms is calculated according to the activation rate constants given in Table 2 and Model 1.

Model 1

Figure 2. Currents from the wild-type, δW57F and ɛW55F mutant receptors activated by 5 mm TMA

Single-channel clusters and the first 200 ms at a higher time resolution are shown. Open events are shown downward. Membrane potential is -50 mV. Intracluster closed and open time histograms are given for the patch from which the representative cluster is shown. The continuous line in the histograms is calculated according to the activation rate constants given in Table 2 and Model 1. However, for the ɛW55F receptor, the continuous lines are fits to a single exponential with the time constants of 42 ms (closed times) and 0.5 ms (open times).

Figure 4. Currents from the δD59N, ɛG57S and δD59N + ɛG57S mutant receptors activated by 200 μm ACh

Single-channel clusters and the first 200 ms at a higher time resolution are shown. Open events are shown downward. Membrane potential is -50 mV. Intracluster closed and open time histograms are given for the patch from which the representative cluster is shown. The continuous line in the histograms is calculated according to the activation rate constants given in Table 2 and Model 1.

Figure 5. Currents from the δD59N and ɛG57S mutant receptors activated by 5 mm TMA

Single-channel clusters and the first 200 ms at a higher time resolution are shown. Open events are shown downward. Membrane potential is -50 mV. Intracluster closed and open time histograms are given for the patch from which the representative cluster is shown. The continuous line in the histograms is calculated according to the activation rate constants given in Table 2 and Model 1.

Figure 3. The effective opening rate curves for the wild-type, δW57F, ɛW55F and δW57F+ɛW55F mutant receptors

The effective opening rate curves for the wild-type receptor in the presence of ACh (open circles) or TMA (filled circles), δW57F mutant receptor in the presence of ACh (open squares), TMA (filled squares) or CCh (crossed squares), ɛW55F mutant receptor in the presence of ACh (open triangles) or TMA (filled triangles), and δW57F+ɛW55F double mutant receptor in the presence of ACh (open diamonds). The curves were fitted using eqn (1). For the δW57F receptor activated by TMA, the maximal value for effective opening rate curve was constrained at 2258 s⁻¹. For the ɛW55F receptor activated by TMA, the maximal value for an effective opening rate curve was constrained at 609 s⁻¹. The results of the fit are given in Table 1.

Figure 6. The effective opening rate curves for the wild-type, δD59N, ɛG57S and δD59N+ɛG57S mutant receptors

The effective opening rate curves for the wild-type receptor in the presence of ACh (open circles) or TMA (filled circles), δD59N mutant receptor in the presence of ACh (open squares) or TMA (filled squares), ɛG57S mutant receptor in the presence of ACh (open triangles) or TMA (filled triangles), and δD59N + ɛG57S double mutant receptor in the presence of ACh (open diamonds). The curves were fitted using eqn (1). For the ɛG57S receptor activated by TMA, the maximal value for effective opening rate curve was constrained at 2116 s⁻¹. The results of the fit are given in Table 1.