Aromatics at the murine nicotinic receptor agonist binding site: mutational analysis of the alphaY93 and alphaW149 residues

Abstract

1. Two aromatic residues of the muscle nicotinic receptor putative agonist binding site, a tyrosine in position alpha93 and a tryptophan in position alpha149, were mutated to phenylalanine and the effects of the mutations on receptor properties were investigated using single-channel patch clamp. 2. The alphaY93F mutation reduced the receptor affinity by approximately 4-fold and the channel opening rate constant by 48-fold. The alphaW149F mutation reduced the receptor affinity by approximately 12-fold and the channel opening rate constant by 93-fold. 3. The kinetic properties of hybrid receptors that contained one wild-type and one mutated alpha subunit were also examined. Only one type of hybrid receptor activity was detected. The hybrid receptors had a channel opening rate constant intermediate to those of the wild-type and mutant receptors. It was concluded that the ligand binding sites in the mutated muscle nicotinic receptor contributed equally to channel gating. In the case of the alphaW149F mutation, the presence of the mutation in one of the binding sites had no effect on the binding properties of the other, non-mutated, site. 4. The mutant channel opening and closing rate constants were also estimated in the presence of tetramethylammonium. The data suggested significant interaction between the acetyl group of acetylcholine and the alphaY93 residue.

Model 1

Model 2

Figure 1. Sample single-channel clusters and open and closed time histograms for the wild-type, hybrid and mutant αY93F receptors

The receptors were activated by 500 μm ACh. Pipette potential was +50 mV. Inward current is shown downward. A, wild-type receptor (WT). B, hybrid receptor (H). C, mutant receptor (MT). For the mutant receptor (C), only a portion of a cluster is shown. Note also that a different time scale has been used for the mutant receptor cluster. Continuous lines in histograms were calculated according to the rate constants shown in Table 2.

Figure 2. Concentration-response properties of wild-type, hybrid and mutant αY93F receptors

A, cluster open probability (P_o) versus[ACh]. B, effective opening rate (β') versus[ACh]. Effective opening rate was measured as the inverse of the duration of the longest intracluster closed time component. The data for the wild-type receptor are from Akk & Auerbach (1996). Each point represents data from one patch. The number of events per patch was 739-40 198 (αY93F hybrid) and 2520-22 227 (αY93F mutant). Continuous lines are fits to eqn (1). The results from fits are shown in Table 1.

Figure 4. Concentration-response properties of the wild-type, hybrid and mutant αW149F receptors

A, cluster open probability (P_o) versus[ACh]. B, effective opening rate (β') versus[ACh]. Effective opening rate was measured as the inverse of the duration of the longest intracluster closed time component. The data for the wild-type receptor are from Akk & Auerbach (1996). Each point represents data from one patch. The number of events per patch was 1584-11 552 (αW149F hybrid) and 910-17 562 (αW149F mutant). Continuous lines are fits to eqn (1). The results from fits are shown in Table 1.

Figure 3. Sample single-channel clusters and open and closed time histograms for the hybrid and mutant αW149F receptors

The receptors were activated by 1 mm ACh. Pipette potential was +50 mV. Inward current is shown downward. Continuous lines in histograms were calculated according to the rate constants shown in Table 2.

Figure 5. The open probability (P_o) of clusters

Intracluster P_o is plotted for one patch expressing wild-type, hybrid and mutant αY93F (A) or αW149F (B) receptors. The receptors were activated by 500 μm (A) or 2 mm (B) ACh. The clusters with the highest P_o belong to wild-type receptors, ones with the intermediate P_o to hybrid receptors, and ones with the lowest P_o to mutant receptors. Note that there is no overlap in the ranges of P_o for the three classes of clusters. The total number of clusters was 63 in A, and 90 in B.

Figure 6. Mutant and hybrid αY93F and αW149F receptors activated by 10 mm TMA

Single-channel activity and open and closed time histograms from the αY93F mutant (A) and hybrid (B) receptors, and αW149F mutant (C) and hybrid (D) receptors. For the αY93F mutant receptor, no clusters were detected at 10 mm TMA; an episode of 1200 ms in duration is shown. For the rest, a representative single-channel cluster is shown. The values within open and closed time histograms are results of fits to a single exponential. E, an effective opening rate (β') versus[TMA] for the αY93F and αW149F hybrid receptors. Each point represents data from one patch. The number of events per patch was 459-7378 (αY93F) and 598-4518 (αW149F). The continuous lines are fits to eqn (1). The concentration-response parameters are shown in Table 4.