Observation Of Multiple Potassium Channel Closed State Structures By Voltage Clamp Spectroscopy

A primary goal of molecular physiology is to understand how proteins change their conformation. Here, we describe a spectral imaging method for measuring conformational changes of wild-type ion channels bound by a fluorescent probe containing a novel environment-sensitive red fluorophore. JP is a derivative of the aminophenoxazone fluorophore which has an enhanced bathochromic response to the polarity of its surroundings. To understand the structural changes that underlie the gating of Kv2-type voltage-gated K+ channels, we conjugated JP to variants of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to the voltage-sensor domain of Kv2 channels, and imaged fluorescence emissions from JP-GxTX conjugates in complex with Kv2.1 channels on the surface of patch-clamped cells. These voltage clamp spectroscopy measurements revealed that JP-GxTX fluorescence is dependent on the labeling site on GxTX, and Kv2 voltage activation. Using spectral images from voltage clamped cells, we have developed methods of fitting emission spectra to identify structural changes of the complex. Emission spectra of the JP-GxTX variants indicate that at least two conformations of the channel-toxin complex occur at all voltages. Depolarization redshifts the emission peaks of one variant, indicating a transition to a more polar environment during activation gating. Depolarization increases fluorescence emission from another JP-GxTX variant with little spectral change. Recent atomistic structures, of the structurally homologous peptide Protoxin-II bound to voltage-sensor domain II of Nav1.7, provide further insight into the conformational changes reported by JP-GxTX variants bound to Kv2.1. Voltage-dependent changes in fluorescence spectra reveal details of the dynamic structural changes of these voltage-gated channels.