Small vertical movement of a K+ channel voltage sensor measured with luminescence energy transfer

Abstract

Voltage-gated ion channels open and close in response to voltage changes across electrically excitable cell membranes. Voltage-gated potassium (Kv) channels are homotetramers with each subunit constructed from six transmembrane segments, S1-S6 (ref. 2). The voltage-sensing domain (segments S1-S4) contains charged arginine residues on S4 that move across the membrane electric field, modulating channel open probability. Understanding the physical movements of this voltage sensor is of fundamental importance and is the subject of controversy. Recently, the crystal structure of the KvAP channel motivated an unconventional 'paddle model' of S4 charge movement, indicating that the segments S3b and S4 might move as a unit through the lipid bilayer with a large (15-20-A) transmembrane displacement. Here we show that the voltage-sensor segments do not undergo significant transmembrane translation. We tested the movement of these segments in functional Shaker K+ channels by using luminescence resonance energy transfer to measure distances between the voltage sensors and a pore-bound scorpion toxin. Our results are consistent with a 2-A vertical displacement of S4, not the large excursion predicted by the paddle model. This small movement supports an alternative model in which the protein shapes the electric field profile, focusing it across a narrow region of S4 (ref. 6).

Figure 1

Cartoon representation of the paddle model. LRET measures distances from donor labelled sites (blue circles) on the S3b-S4 paddle (structure taken from the isolated voltage-sensor). The voltage-sensing arginines are shown in red. The energy transfer acceptors (green circles) are attached to the top of the channel with a scorpion toxin. The paddle model predicts a change in distance, D_c - D_o, of 10 Å, estimated by a conservative geometric calculation assuming a 15 Å vertical translation (red arrows).

Figure 2

LRET raw data and distance calculations. Acceptor sensitized emission at two extreme voltages are shown (left) for the E333C mutant near the top of the paddle. The time constants displayed a small voltage dependence corresponding to a small movement 0.8 Å away from the toxin (top-right). The distances calculated from the two lifetime components and the weighted-average (Methods) are shown. L361C showed voltage dependent movement of 0.8 Å towards the toxin (bottom-right). Error bars for the average distance represent standard error of the mean (n = 13 for E333C, n = 8 for L361C).

Figure 3

Average distances for many Shaker sites. The S4 and S3b sites are homologous to sites on the KvAP voltage-sensing paddle. The distances for S4 change just 0.8 Å, consistent with an approximately 2 Å vertical translation. S3b moves in the direction opposite to S4, moving just 0.8 Å away from the toxin. Sites in the S3-S4 linker are clearly closer to the toxin than the transmembrane segments, as expected, and move no more than a few angstroms.

Figure 4

A model of Shaker with docked agitoxin predicts four distances for each LRET experiment. The coordinates (left) provide an opportunity to compare our measurements with a picture of Shaker that has S3 and S4 placed against the pore domain. Distances for L361C on S4 are shown measured from alpha-carbons (right).