Characterizing Voltage-Dependent Conformational Changes in the K Channel with Fluorescence

Abstract

We examined voltage-dependent conformational changes in three specific regions of the Shaker potassium channel with site-directed fluorescent labeling: the fourth transmembrane segment (S4), the second transmembrane segment (S2), and the putative pore region. The fluorescence changes displayed distinctive properties that correlate with gating, activation, and slow inactivation of the channel. The fluorescence signals measured near the S2 and S4 segments suggest that the S2 segment may undergo voltage-sensitive conformational changes that precede those in the S4 segment. In contrast, fluorescence changes in the pore correlated with the voltage dependence and time course of ionic activation and slow inactivation. Spectroscopy indicated that the mechanism of fluorescence change involves voltage-dependent quenching of the probe in an aqueous environment by other parts of the protein.

Conformational dynamics of the Na + /K + -ATPase probed by voltage clamp fluorometry

“…The same observations, i.e., slowed kinetics on cysteine substitution and further reduction of the rate constant after TMRM attachment, were previously reported in Shaker K ϩ channel studies (14). However, the slower kinetics observed for NaK ØCys (N790C) does not impair the assignment of the observed fluorescence signals to the E 1 P-E 2 P conformational change.…”

“…The technique of sitedirected fluorescence labeling in combination with VCF has extensively been used for the detection of structural rearrangements underlying voltage sensor movements in voltage-gated cation channels (13)(14)(15). VCF implies electrophysiological measurements on integral membrane proteins in which single cysteines are introduced in putative reporter sites.…”

“…The fluorescence quantum yield of the rhodamine dye is sensitive to environmental hydrophobicity changes (13,14). The assumption that the fluorophor can discriminate between E 1 (high fluorescence) and E 2 (low fluorescence) states implies that it resides in a sheltered, hydrophobic environment in E 1 states, whereas it encounters a more exposed, quenching environment in E 2 states.…”

“…Therefore, we expressed the Na ϩ ͞K ϩ -ATPase heterologously in Xenopus oocytes and used the method of site-directed fluorescence labeling combined with voltage clamp fluorometry (VCF), which was pioneered in the laboratories of E. Y. Isacoff (13) and F. Bezanilla (14,15) on the Shaker potassium channel.…”

The conformational shifts of the voltage sensing domains between NavRh and NavAb

“…During the outward R4 transfer across the occluding Phe from Na v Ab to Na v Rh, R1-R4 on the S4 segment appear to be positioned at almost identical altitudes within the lipid bilayer, while both vertical and horizontal shifts of the S2 segment result in the R4 transfer across the occluding Phe (Supplementary information, Movie S7). This structural analysis appears to be supported by the experimental observation that the S2 segments undergo conformational changes preceding S4 in the shaker K + channel [7]. Notably, the lipid composition must be important to the electrophysiological property of voltage-gated channels as the VSDs undergo lateral rotation within the membrane bilayer, an analysis consistent with numerous lines of experimental evidence [8,9].…”