Molecular Basis for Differential Sensitivity of α-Conotoxin RegIIA at Rat and Human Neuronal Nicotinic Acetylcholine Receptors

Abstract

a-Conotoxins, as nicotinic acetylcholine receptor (nAChR) antagonists, are powerful tools for dissecting biologic processes and guiding drug development. The a3b2 and a3b4 nAChR subtypes are expressed in the central and peripheral nervous systems and play a critical role in various pathophysiological conditions ranging from nicotine addiction to the development and progression of lung cancer. Here we used the a4/7-conotoxin RegIIA, a disulfidebonded peptide from the venom of Conus regius, and its analog [N11A,N12A]RegIIA to probe the specific pharmacological properties of rat and human nAChR subtypes. nAChR subtypes were heterologously expressed in Xenopus oocytes and two-electrode voltage clamp recordings used to investigate the effects of the peptides on nAChR activity. RegIIA potently inhibited currents evoked by acetylcholine (ACh) at rat a3b2 (IC 50 5 10.7 nM), whereas a 70-fold lower potency was observed at human a3b2 nAChR (IC 50 5 704.1 nM). Conversely, there were no speciesspecific differences in sensitivity to RegIIA at the a3b4 nAChR. Receptor mutagenesis and molecular dynamics studies revealed that this difference can be attributed primarily to a single amino acid change: Glu198 on the rat a3 subunit corresponding to a proline on the human subunit. These findings reveal a novel species-and subunit-specific receptor-antagonist interaction.

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

“…␣-Conotoxin RegIIA has previously been shown to exhibit a species-specific difference in activity between human and rat ␣3␤2 nAChRs, whereas this species difference was not observed at the ␣3␤4 nAChR subtype (17). Given that both the species and subtype selectivity profiles are well described for RegIIA, it represents a suitable probe to investigate pharmacological differences between ␣3␤2 and ␣3␤4 nAChR subtypes in detail.…”

“…Site-directed Mutagenesis-Plasmid DNAs encoding human and rat ␣3, ␤2, and ␤4 nAChR subunits were subcloned into the pT7TS Xenopus expression vector (Addgene plasmid 17091) as described previously (17). Two chimeric receptors consisting of h␤2 backbone and portions of the N-terminal extracellular domain (ECD) (N-terminal T1 to loop D Glu 63 and a lesser conserved Lys 70 -His 86 segment between loops D and E) replaced with the corresponding h␤4 sequences, were generated by overlap PCR and molecular cloning.…”

“…Membrane currents from Xenopus oocytes were recorded using a single channel two-electrode voltage clamp setup (virtual ground circuit) with a GeneClamp 500B amplifier (Molecular Devices, Sunnyvale, CA) as described previously (17). Briefly, oocytes were continuously perfused with ND96 solution containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl 2 , 1 MgCl 2 , and 5 HEPES (pH 7.4) at 2 ml/min.…”

“…Furthermore, we have previously shown that the selectivity profile of RegIIA differs between nAChRs of different species, as RegIIA was equipotent at the rat (r) and human (h) ␣3␤4 subtypes but significantly less active at h␣3␤2 compared with r␣3␤2. This potency difference could be mapped to a single non-conserved Glu 198 on the r␣3 subunit (proline in human) (17).…”

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

“…Interestingly, α‐conotoxin RegIIA was significantly less potent at the human α3β2 nAChR subtype compared with rat, whereas at α3β4, no species‐specific differences in sensitivity were observed (Kompella et al, ). As the homologous receptor subtypes of rat and human are overall relatively conserved, it is believed that the binding site of RegIIA at α3β2 and α3β4 nAChRs overlaps with the agonist binding site (Kompella et al, ). Therefore, only the few residues in the ACh binding domains that differ between the species were predicted to play a role in determining selectivity.…”