Crotoxin B from the South American Rattlesnake Crotalus vegrandis Blocks Voltage-Gated Calcium Channels Independent of Its Intrinsic Catalytic Activity

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 A₂ 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 Ba²⁺ as a charge carrier to prevent the enzymatic activity, we found that CB inhibited currents mediated by the N-type Ca_V2.2 and Ca_V1.2 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.