Exploring the Effect of Gambierol on the Gating Machinery of Kv3.1 Channels

Abstract

Gambierol is a ladder-shaped polyether toxin that acts as a gating modifier to inhibit Kv3.1 channels with nanomolar affinity. Binding determinants for gambierol have been identified at an interface between S5 and S6, located outside the permeation pathway. However, the high gambierol sensitivity of Kv3.1 channels could not be fully transplanted to the insensitive Kv2.1 channel by introducing the S5-S6 determinants. To explore whether also the voltage-sensing domain (VSD) is a determinant for gambierol sensitivity, we exchanged the complete VSD (S1-S4), parts of the VSD (the S1-S3a region and the Sb-S4 paddle), and the electromechanical coupling (L45+S6c) between Kv3.1 and Kv2.1. Our results show that the L45+S6c and the S1-S3a region did not alter the affinity of Kv3.1 channels for gambierol. In contrast, the distal part of the VSD, the S3b-S4 paddle, displayed a 100-fold decrease in affinity compared to WT Kv3.1. Since all VSD chimeras displayed similar biophysical properties and remained sensitive to well-known pore blockers, the loss in gambierol sensitivity in the S3b-S4 paddle chimera is most likely not the result of allosteric effects. Molecular-Dynamics simulations indicated that the S3b-S4 paddle motif resides in proximity of gambierol and that the structure and position of the VSD may regulate the space of the binding site between the pore domain (S5 and S6) and the gating machinery. Hence, our results suggest that the VSD, and especially the S3b-S4 paddle motif, contributes to the structure and/or the accessibility of the binding site for gambierol. (This research was supported by FWO grant G0433.12N to DJS and JT).