Fluorescently Labeled Tarantula Toxin Reveals Voltage Dependent Adhesion to K+ Channels

Abstract

Among the most potent allosteric modulators of voltage-gated potassium channels are peptide toxins from arachnids. Guangxitoxin-1E is an exemplar of this class of ligands, having effects at nanomolar concentrations and shifting gating of Kv2.1 to a positive voltage range, beyond physiological potentials. We have synthesized biologically active variants of this toxin with unique chemically reactive groups that allow conjugation to probes that allow visualization of toxin adherence to channels on living cells. The association of toxin with Kv2.1 channels was found to be strongly voltage dependent, having different affinities for resting and activated states of voltage sensors. To compose structural hypotheses for the toxin-channel interactions that underlie allosteric effector properties, we built structural models of the toxin-channel interaction using the Rosetta-Membrane homology method and the Kv1.2-2.1 coordinates as template, to create models of Kv2.1 voltage sensing domains. Conformations of activated and resting states were generated following a sliding helix model. The toxin was then docked to these multiple voltage sensor conformations using the Rosetta Dock method to identify low energy complexes. Experimentally, measurement of toxin association and dissociation rates by voltage clamp and with fluorescently labeled toxins create constraints that define its mechanism of action. The differential adherence of toxin to resting and activated voltage sensor conformations drives allosteric modulation of channels. By measuring gating state-dependent affinities, this methodology creates a new means of assessing the potency and action of allosteric ion channel modulators. In combination with computational structure prediction and design this strategy could lead to development of voltage sensor modulators with novel state-dependence.