The Role of the M3 Helix in AChR Gating and PAM Action

Abstract

We are examining the M3 transmembrane helix of mouse muscle AChRs with regard to C↔O gating and the effects of positive allosteric modulators (PAMs). The relative ground and transition state gating energies were estimated from single-channel currents of AChRs having M3 mutations. Diliganded gating rate and equilibrium constants were measured for ∼100 mutations in 5 subunits (α, β, δ, e and γ; 20 mM choline or 500 μM ACh, −100 mV, HEK cells). In general: i) the effects of the mutations were small (≤1 kcal/mol), ii) the effects were similar among subunits, iii) the mutations in different subunits were independent, iv) the mutations have no effect on the energy from the agonist affinity change (≤∼0.3 kcal/mol) and v) most phi values were low (∼0.3). Crystal structures of AChR homologues in detergents suggest that the low phi positions in M3 are interaction sites for PAMs. We investigated the actions of PNU-120596 and IVM (1-1000nM). In WT AChRs (adult and fetal) these ligands increase substantially the frequency and lifetime of constitutive openings in a manner consistent with action at a low-phi position such as in M3. Such potentiation was also apparent at low [agonist] (<1μM ACh or choline), but at higher [agonist] gating kinetics became complex and the potentiation was less clear. The PAMs also increased substantially the frequency and lifetime of constitutive openings in some of the M3 mutants (to W).