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

## TL;DR

A protein from a South American rattlesnake venom blocks calcium channels in cells, causing neurotoxic effects unrelated to its enzyme activity.

## Contribution

The study identifies a novel, enzyme-independent mechanism by which a venom protein inhibits calcium channels.

## Key findings

- Isolated CB subunit inhibits N-type and L-type calcium channels in a dose-dependent manner.
- CB's cytotoxic effect is not correlated with its phospholipase activity.
- CB's inhibition of calcium channels is independent of its catalytic function.

## 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 (CaV) 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 CaV 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 CaV2.2 and CaV1.2 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.