The Tarantula Toxin Guangxitoxin-1e Traps K+ Channel Voltage Sensor In A Fully Resting Conformation

Allosteric ligands modulate protein activity by altering the energy landscape of conformational change in ligand-protein complexes. Here, we investigate how specific steps in voltage-activation path of an ion channel are altered by a potent allosteric inhibitor. The K+ channel peptide inhibitor guangxitoxin-1E (GxTX) is partial inverse agonist of the voltage gated potassium channel subtype Kv2.1 from rat. At saturating concentrations of GxTX, Kv2.1 requires higher voltages to activate. The GxTX modulated conductance requires more voltage to activate. It has more sigmoidal activation kinetics, activates slower, and deactivates more rapidly. With GxTX bound, all gating charge requires more voltage to translocate, indicating that GxTX stabilizes resting voltage sensors by 20,000-fold, or 9 kBT. Remarkably, with GxTX bound, the rate of fast outward gating charge movement lost its positive voltage dependence. This suggests that GxTX can bind to channels in all conformations, but when gating charge is most intracellular, the toxin-channel complex adopts a stable conformation from which escape is time-dependent, but not voltage-sensitive. Channel kinetics were analyzed with a model that included a single conducting state for the channel, preceded by two voltage-dependent gating steps in each subunit, one independent and one concerted between subunits. When fit to the sigmoidicity and activation kinetics of Kv2.1 ionic currents, this model was predictive of the measured gating currents. However, with GxTX bound, this model was no longer able to predict gating current kinetics, indicating that a new conformational transition was rate limiting toxin-channel activation. We conclude that GxTX modulates Kv2.1 ion channels by “trapping” each voltage sensor in a conformation which is insensitive to voltage, yet where all gating charge remains on the intracellular side of the transmembrane electric field. Following escape from the GxTX-“trapped” state, gating charges can translocate outward, allowing channels to open with GxTX bound.