Catch-and-Hold Activation of Muscle Acetylcholine Receptors Having Transmitter Binding Site Mutations

Abstract

Agonists turn on receptors because their target sites have a higher affinity in the active versus resting conformation of the protein. We used single-channel electrophysiology to measure the lower-affinity (LA) and higher-affinity (HA) equilibrium dissociation constants for acetylcholine in adult-type muscle mouse nicotinic receptors (AChRs) having mutations of agonist binding site amino acids. For a series of agonists and for all mutations of αY93, αG147, αW149, αY190, αY198, εW55, and δW57, the change in LA binding energy was approximately half that in HA binding energy. The results were analyzed as a linear free energy relationship between LA and HA agonist binding, the slope of which (κ) gives the fraction of the overall binding chemical potential where the LA complex is established. The linear correlation between LA and HA binding energies suggests that the overall binding process is by an integrated mechanism (catch-and-hold). For the agonist and the above mutations, κ ∼ 0.5, but side-chain substitutions of two residues had a slope that was significantly higher (0.90; αG153) or lower (0.25; εP121). The results suggest that backbone rearrangements in loop B, loop C, and the non-α surface participate in both LA binding and the LA ↔ HA affinity switch. It appears that all of the intermediate steps in AChR activation comprise a single, energetically coupled process.

Correlating Structural and Energetic Changes in Glycine Receptor Activation

“…5). The ␣1 Ϫ26Ј -␣1 19Ј coupling energy is well within the range for a significant interaction between residues in pLGICs during activation (39,40,44) and between ligand binding residues and ligand (45). We propose that this interaction represents one point of energy transfer along what is likely to be a broad reaction coordinate (broad corrugated activation barrier) as proposed by Auerbach for the related nAChR (40,46).…”

Dose–Response Analysis When There Is a Correlation between Affinity and Efficacy

“…M has been estimated for different agonists of AChR, and for all of these the extra energy of binding to the active receptor happens to be almost equal to the energy of binding to the resting receptor (M % 1.92). M has been measured both for agonists (Jadey and Auerbach, 2012) and for dozens of mutations of aromatic binding site residues (Purohit et al, 2014), and is approximately the same. Like E 0 , M appears to be a fundamental constant of the receptor.…”

“…In the preparation that I consider here-single-channel currents from adult-type mouse skeletal muscle nicotinic AChRs expressed in human embryonic kidney (HEK) cells-these confounding issues were either not present or were incorporated into the analyses so that the activation equilibrium constants were estimated accurately. Figure 2 shows K d and E 2 values for eight different agonists of endplate AChRs plotted on a log-log scale (Jadey et al, 2011;Jadey and Auerbach, 2012;Purohit et al, 2014). Remarkably, there is a linear relationship that holds over a ∼25,000-fold range in affinity.…”

“…4, which happens to look like the letter M. For each agonist, log J d is larger by a constant factor than log K d , with the factor in AChRs being M 5 1.92. (In a previous report, the factor was called k 5 1/M [Purohit et al, 2014].) In AChR, establishment of the low-affinity complex is a stopover about halfway (energetically) between the apo and high-affinity conformations of the agonist binding site.…”

Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

“…1). In adulttype AChRs, for the agonists and mutations used in this study, the HA and LA equilibrium dissociation constants are correlated and have the relationship G HA ∼2G LA (22). Combining this with Eq.…”