Key Structural Determinants in the Agonist Binding Loops of Human β2 and β4 Nicotinic Acetylcholine Receptor Subunits Contribute to α3β4 Subtype Selectivity of α-Conotoxins

Abstract

Edited by Paul Fraser␣-Conotoxins represent a large group of pharmacologically active peptides that antagonize nicotinic acetylcholine receptors (nAChRs). The ␣3␤4 nAChR, a predominant subtype in the peripheral nervous system, has been implicated in various pathophysiological conditions. As many ␣-conotoxins have multiple pharmacological targets, compounds specifically targeting individual nAChR subtypes are needed. In this study, we performed mutational analyses to evaluate the key structural components of human ␤2 and ␤4 nAChR subunits that determine ␣-conotoxin selectivity for ␣3␤4 nAChR. ␣-Conotoxin RegIIA was used to evaluate the impact of non-conserved human ␤2 and ␤4 residues on peptide affinity. Two mutations, ␣3␤2[T59K] and ␣3␤2[S113R], strongly enhanced RegIIA affinity compared with wild-type ␣3␤2, as seen by substantially increased inhibitory potency and slower off-rate kinetics. Opposite point mutations in ␣3␤4 had the contrary effect, emphasizing the importance of loop D residue 59 and loop E residue 113 as determinants for RegIIA affinity. Molecular dynamics simulation revealed the side chains of ␤4 Lys 59 and ␤4 Arg 113 formed hydrogen bonds with RegIIA loop 2 atoms, whereas the ␤2 Thr 59 and ␤2 Ser 113 side chains were not long enough to form such interactions. Residue ␤4 Arg 113 has been identified for the first time as a crucial component facilitating antagonist binding. Another ␣-conotoxin, AuIB, exhibited low activity at human ␣3␤2 and ␣3␤4 nAChRs. Molecular dynamics simulation indicated the key interactions with the ␤ subunit are different to RegIIA. Taken together, these data elucidate the interactions with specific individual ␤ subunit residues that critically determine affinity and pharmacological activity of ␣-conotoxins RegIIA and AuIB at human nAChRs.

Fulditoxin, representing a new class of dimeric snake toxins, defines novel pharmacology at nicotinic ACh receptors

“…Pairwise alignment of mature protein sequences of human β2 and β4 subunits showed 70% homology in the extracellular domain. Homology modelling and overlay of their inter‐subunit interfaces revealed side‐chain differences between the non‐conserved residues of the β2 and β4 subunits (Cuny, Kompella, Tae, Yu, & Adams, ). These differences may account for fulditoxin's weak interaction with hα4β4 and hα3β4 subtypes in contrast to β2‐containing nAChRs.…”

“…Homology modelling and overlay of their inter-subunit interfaces revealed side-chain differences between the non-conserved residues of the β2 and β4 subunits (Cuny, Kompella, Tae, Yu, & Adams, 2016).…”

α‐Conotoxins active at α3‐containing nicotinic acetylcholine receptors and their molecular determinants for selective inhibition

“…However, AuIB was largely inactive with no activity observed at 1 μM when tested on human α3β4 (compared with an IC 50 of 0.75 μM at rat α3β4), and at 30 μM, only ~20% of ACh‐evoked current amplitude mediated by human α3β4 was inhibited, indicating that the IC 50 is >40‐fold higher for rat α3β4. At the human α3β2 subtype, AuIB exhibited a similar low inhibitory potency (Cuny et al, ).…”

“…At the rat α3β4 nAChR, residue F9 of AuIB closely interacts with residues W59 and K61 of the β4 subunit WLK (Trp‐Leu‐Lys) pocket, hypothetically via π–π and cation–π interactions. In contrast, at the human α3β4 nAChR, residue F9 of AuIB only binds outside the WLK pocket (Figure A), where although interaction is still possible with β4 residues K59 and L119, the crucial π–π interaction with W57 is absent (Cuny et al, ). At the rat homologue, the β4 W59 residue was indispensable for AuIB inhibition and K61 appeared to play an auxiliary role (Grishin et al, ).…”

“…At human α3β2, molecular modelling revealed contacts between AuIB and β2 W57, T59 and other residues. Although AuIB F9 can form the crucial π–π interaction with W57 in loop D, unlike β4 K59, the hydrophilic side chain of β2 residue T59 does not form an effective binding pocket for AuIB F9 because its interaction with the aromatic phenyl ring of AuIB F9 is energetically unfavourable (Figure B) (Cuny et al, ). In summary, AuIB at hα3β2 and hα3β4 nAChRs is stabilized ineffectively due to the lack of key interactions between AuIB F9 and β subunit agonist binding loop residues.…”