Basic residues at position 11 of α-conotoxin LvIA influence subtype selectivity between α3β2 and α3β4 nicotinic receptors via an electrostatic mechanism

Understanding the determinants of α-conotoxin (α-CTX) selectivity for different nicotinic acetylcholine receptor (nAChR) subtypes is a requisite for the design of tool compounds to study nAChRs. However, selectivity optimization of these small, disulfide rich peptides is difficult not only because of an absence of α-CTX/nAChR co-structures, but also because it is challenging to predict how a mutation to an α-CTX will alter its potency and selectivity. As a prototypical system to investigate selectivity, we employed the α-CTX LvIA that is 18-fold selective for the α3β2 nAChR over the closely related α3β4 nAChR subtype that is a target for nicotine addiction. Using two-electrode voltage clamp electrophysiology, we identified LvIA[D11R] that is 2-fold selective for the α3β4 nAChR, reversing its subtype preference. This effect is specific to the charge and not shape of LvIA[D11R], as substitution with citrulline retains selectivity for the α3β2 nAChR. Furthermore, LvIA[D11K] shows a stronger reversal, with 4-fold selectivity for the α3β4 nAChR. Motivated by these findings, using site-directed mutagenesis it was found that β2[K79A], but not β2[K78A], largely restores the antagonism of basic mutants at position 11. Finally, to understand the structural basis of this effect we used AlphaFold2 to generate models of LvIA in complex with both nAChR subtypes. Both models confirm the plausibility of an electrostatic mechanism to explain the data and also reproduce a broad range of potency and selectivity structure-activity relationships for LvIA mutants, as measured using free-energy perturbation simulations. Our work highlights how electrostatic interactions can drive α-CTX selectivity and may prove useful as a strategy for optimizing the selectivity of LvIA and other ⍺-CTXs.