Voltage-Clamp Fluorimetry Of Kv1.2 Channels Show Two Unique Phases Of Quenching Associated With Channel Activation

Abstract

Voltage-clamp fluorimetry can be used to visualize real-time changes in protein structure relative to a locally introduced fluorophore, and thus enhance our understanding of ion channel gating. Since the crystal structure is available for Kv1.2, it seems important to compare the voltage-dependent fluorescence reports from this channel with those already recorded from Shaker potassium channels. In the absence of introduced cysteine residues, we were unable to record voltage-dependent fluorescence signals from wild-type channels incubated with tetramethylrhodamine maleimide (TMRM). However, signals were obtained from an introduced cysteine at A291 in the Kv1.2 S3-S4 linker. Depolarization resulted in two separate components of quenching, which may underlie two different conformational changes in the protein. A slow quenching phase was observed upon depolarization from a holding potential of −120 mV and was essentially complete by −50 mV. The rate of this quenching was not significantly voltage-dependent, with time constants between 35 ms at −70 mV and 29 ms at +80 mV. The voltage-dependence of the slow component suggests that it reports on conformational changes preceding opening. A more rapid quenching component was observed upon depolarizations positive to −40 mV, with time constants from 2-10 ms. It had a similar voltage-dependence to the conductance-voltage relationship for potassium currents through Kv1.2, suggesting that it may report on channel rearrangements associated with opening. Dissociation of channel gating charge movement from pore opening by the incorporation of the ILT triple mutation in the S4 domain abolished the fast phase of fluorescence quenching at potentials up to +80 mV. This suggests that the fast quenching reports on conformational changes associated with channel opening, and that slow fluorescence quenching reflects protein rearrangements occurring earlier in the gating process.