Allosteric Stabilization of Fully Resting Voltage Sensors by a Tarantula Toxin

Abstract

The mechanism of a tarantula toxin's action on the voltage gating of a K channel was investigated. An oxidation-resistant variant of guangxitoxin-1E (GxTX) with methionine 35 replaced by norleucine, was synthesized and found to retain biological activity. When applied to voltage-clamped CHO-K1 cells expressing rat Kv2.1, this GxTX was found to shift channel opening to more positive voltages. In response to short stimulating voltage steps, the voltage shift of conductance saturated at micromolar GxTX concentrations. Prolonged or repetitive pulses to positive potentials ejected GxTX from Kv2.1 channels, revealing the decreased affinity of GxTX for activated voltage sensors. GxTX positively shifted Kv2.1 gating currents, and prevented outward gating charge movement at negative voltages. The modulation of gating charge movement indicates that GxTX stabilizes gating charges in their most internal conformation. Single Kv2.1 channels with GxTX bound exhibited a similar unitary conductance as without tarantula toxin, but had an increased latency to first opening in response to positive voltage steps. A diminished mean open time in the presence of GxTX confirms that channel openings occur with toxin bound and suggests a mechanism for the toxin-induced decrease in peak conductance of macroscopic currents. A simple allosteric model was developed where GxTX stabilizes the earliest resting state of voltage sensors. In this model, GxTX binds activated voltage sensors with decreased affinity, and exerts only a feeble destabilizing influence on the dominant open state. The difference in binding affinity between resting and activated voltage sensors suggests potential for development of GxTX as a probe of voltage sensor conformation in living cells.