Interactome Maps of the Acetylcholine Receptor Gate Region

Abstract

Opening of the AChR pore is regulated by a hydrophobic ‘gate’ formed by amino acid residues of the 5 M2 helices, between positions 9’-13’. In AChRs, local interactions (<∼12A) between side chains influence the C vs. O energy difference. Except for interactions with the agonist itself, the gating energy landscape appears to be essentially the same with or without ligands. We have measured local energy couplings (Ω) and gating transition pathways (Φ) at the gate, using free energies obtained from single channels in the absence of agonists. The salient results were: i) the inter-subunit interaction was strongest between immediate neighboring subunits (e.g. β-δ vs β-e) at each gate position, ii) the interaction energy was a complex function of the charge and size of the amino acid side chains, iii) the inter-subunit interactions at 9’ L were asymmetric in so far as Ω for α-β and α- e were ∼4 times greater than α- δ or β-δ; those at 13’ V were symmetric, iv) Ω of the complement pairs at different levels (e.g. 13’α-9’e and 13’β-9’δ) were greater than the of non-complement pairs (e.g. 9’α-13’δ), v) some gate mutations resulted in malleable energy landscape, manifest as a Hammond effect at 12’, and 13’ and an anti-Hammond effect at 9’. Interactions of M2 with residues in M3 and M1 are currently under investigation. An interactome map of the gate region may provide insight into the rearrangements that occur here in AChRs.