± )-18-MC and (+)-catharanthine decrease drug-induced neuropathic pain.• Both drugs inhibit α9α10 at higher potency than that for α3β4 and α4β2 nAChRs.• Both drugs directly inhibit Ca V 2.2 channels but not via activation of GABA B R.• Their analgesic mechanisms may involve the inhibition of α9α10 nAChRs and/or Ca V 2.2 channels.

Snake venom neurotoxins are potent antagonists of nicotinic acetylcholine receptors (nAChRs). Here, we describe a novel member of class 3c long‐chain neurotoxin drysdalin from the venom of Drysdalia coronoides. Drysdalin lacks three of the eight conserved classical functional residues critical for nAChRs interaction. Despite such a drastic alteration of the functional site, recombinant drysdalin showed irreversible postsynaptic neurotoxicity with nanomolar potency and selectively antagonizes the rodent muscle (α1)2β1δε, and human α7 and α9α10 nAChRs, but had no significant activity at the human α3β2, α3β4, α4β2, and α4β4 nAChRs. Substitution of Leu34 and Ala37 residues with the conserved Arg had minimal impact on the potency whereas conserved Phe replacement of residue Arg30 substantially reduced or abolished inhibitory activity. In contrast, truncation of the 24‐residue long C‐terminal tail leads to complete loss in (a) activity at α9α10 nAChR; and (b) irreversibility with reduced potency at the muscle and α7 nAChRs. Overall, the non‐conserved Arg30 residue together with the uniquely long C‐terminal tail contribute to the inhibitory activity of drysdalin at the nAChRs suggesting, at least for drysdalin, functional rather than sequence conservation plays a critical role in determining the activity of the toxin.

The second of three SPRY domains (SPRY2, S1085 -V1208) located in the skeletal muscle ryanodine receptor (RyR1) is contained within regions of RyR1 that influence EC coupling and bind to imperatoxin A, a toxin probe of RyR1 channel gating. We examined the binding of the F loop (P1107-A1121) in SPRY2 to the ASI/basic region in RyR1 (T3471-G3500, containing both alternatively spliced (ASI) residues and neighboring basic amino acids). We then investigated the possible influence of this interaction on excitation contraction (EC) coupling. A peptide with the F loop sequence and an antibody to the SPRY2 domain each enhanced RyR1 activity at low concentrations and inhibited at higher concentrations. A peptide containing the ASI/basic sequence bound to SPRY2 and binding decreased ~10-fold following mutation or structural disruption of the basic residues. Binding was abolished by mutation of three critical acidic F loop residues. Together these results suggest that the ASI/basic and SPRY2 domains interact in an F loop regulatory module. Although a region that includes the SPRY2 domain influences EC coupling, as does the ASI/basic region, Ca2+ release during ligand- and depolarization-induced RyR1 activation were not altered by mutation of the three critical F loop residues following expression of mutant RyR1 in RyR1-null myotubes. Therefore the electrostatic regulatory interaction between the SPRY2 F loop residues (that bind to imperatoxin A) and the ASI/basic residues of RyR1 does not influence bi-directional DHPR-RyR1 signaling during skeletal EC coupling, possibly because the interaction is interrupted by the influence of factors present in intact muscle cells.