Abstract
Neurotoxicity following South American Crotalus rattlesnake bite is primarily caused by crotoxin, the most abundant component in their venom. Despite the central role of voltage-gated calcium channels (Ca(V)) in neurotransmission, direct targetability by crotoxin has been poorly explored. Crotoxin is a non-covalent heterodimer formed by an acidic subunit (CA) and a basic toxic phospholipase A(2) subunit (CB). Here, we chromatographically isolated the CB subunit from Crotalus vegrandis and studied its effect on Ca(V) heterologously expressed in tsA201 cells using the whole-cell patch-clamp technique. Mass spectrometry analysis identified a protein that matched with 97% sequence coverage the CBc isoform from Crotalus durissus terrificus. Isolated CB exhibited moderate phospholipase activity that was not correlated to its cytotoxic effect on cultured tsA201 cells. Using Ba(2+) as a charge carrier to prevent the enzymatic activity, we found that CB inhibited currents mediated by the N-type Ca(V)2.2 and Ca(V)1.2 L-type calcium channels, in a dose-dependent manner, with higher potency for the latter, and negligible changes in the voltage dependence of channel activation. Our results reveal a novel phospholipase-independent biological activity and a molecular target of CB providing new insights into the pathophysiology of Crotalus snakebite envenoming with potential clinical therapeutic implications.