Fast and slow voltage sensor rearrangements during activation gating in Kv1.2 channels detected using tetramethylrhodamine fluorescence

Abstract

The Kvl.2 channel, with its high, resolution crystal structure, provides an ideal model for investigating conformational changes associated with channel gating, and fluorescent probes attached at the extracellular end of S4 are a powerful way to gain, a more complete understanding of the voltage-dependent activity of these dynamic proteins. Tetramethylrhodamine-5-maleimide (TMRM) attached at. A291C reports two distinct rearrangements of the voltr age sensor domains, and a comparative fluorescence scan, of the S4 and S3-S4 linker residues in Shaker and KvI.2 shows important differences in their emission, at other homologous residues. Kvl.2 shows a rapid decrease in A291C emission, with a time constant of 1.5 ± 0.1 ms at 60 mV (n= 11) that correlates with gating currents and reports on translocation of the S4 and S3-S4 linker. However, unlike any Kv channel studied to date, this fast component: is dwarfed by a larger, slower quenching of TMRM' emission, during depolarizations between 120 and 50 mV (T = 21.4 ± 2.1 ms at 60 mV, V1/2 of -73.9 ± 1.4 mV) that is not seen in either Shaker or Kvl.5 and that comprises >60% of the total signal at all activating potentials. The slow fluorescence relaxes after repolarization in a voltagedependent manner that matches the time course of Kvl.2 ionic current, deactivation. Fluorophores placed directly in Sl and S2 at. 1187 and T219 recapitulate the time course and voltage dependence of slow quenching. The slow component is lost, when the extracellular S1-S2 linker of Kvl.2 is replaced with that of Kvl.5 or Shaker, suggesting that it arises from a continuous internal rearrangement within the voltage sensor, initiated at negative potentials but prevalent throughout the activation process, and which must be reversed for the channel to close. ©2010 Horne et al.