Identification of a peptide toxin from Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels

We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spa tulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying f ractions on stretch-activated channels (SACs) in outside-out patches from a dult rat astrocytes. Although the channel gating kinetics were different be tween cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance (sim ilar to 45 pS at -100 mV) and a mild rectification were unaffected by outsi de-out formation. GsMT-4 produced a complete block of SACs in outside-out p atches and appeared specific since it had no effect on whole-cell voltage-s ensitive currents. The equilibrium dissociation constant of similar to 630 nM was calculated from the ratio of association and dissociation rate const ants. In hypotonically swollen astrocytes, GsMTx-4 produces similar to 40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produ ced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cel l size. This is the first report of a peptide toxin that specifically block s stretch-activated currents. The toxin affect on swelling-activated whole- cell currents implicates SACs in volume regulation.