Brevia

Membrane Insertion of a Potassium-Channel Voltage Sensor

See allHide authors and affiliations

Science  04 Mar 2005:
Vol. 307, Issue 5714, pp. 1427
DOI: 10.1126/science.1109176

This article has a correction. Please see:

Abstract

The mechanism of voltage gating in K+ channels is controversial. The paddle model posits that highly charged voltage-sensor domains move relatively freely across the lipid bilayer in response to membrane depolarization; competing models picture the charged S4 voltage-sensor helix as being shielded from lipid contact by other parts of the protein. We measured the apparent free energy of membrane insertion of a K+-channel S4 helix into the endoplasmic reticulum membrane and conclude that S4 is poised very near the threshold of efficient bilayer insertion. Our results suggest that the paddle model is not inconsistent with the high charge content of S4.

The structure of the KvAP voltage-dependent K+ channel shows two distinct features: the voltage-sensor domains and the central pore domain (1). The critical element in the sensor domains in virtually all voltagegated ion channels is the S4 helix, which contains four or more regularly spaced Arg residues interspersed with hydrophobic residues. Voltage activation has been suggested to involve paddles, each composed of a mildly hydrophobic S3 helix packed against a highly charged S4 helix, that move through the lipid bilayer in response to membrane depolarization (2). This controversial mechanism of gating is at odds with models that envisage the S4 segment as isolated from the lipid bilayer in canaliculi within the channel protein. At the heart of the controversy is the idea that the paddle model is implausible, because of the energetic cost of burying charges in a lipid bilayer (3).

We investigated the membrane insertion of the KvAP S4 helix using an experimental system (4, 5) that permits accurate measurements of the apparent free energy (ΔGapp) of translocon-mediated integration of transmembrane (TM) helices into the endoplasmic reticulum (ER) membrane (Fig. 1A). Using this system, we previously derived a base biological hydrophobicity scale and demonstrated that the contribution to ΔGapp can depend strongly on a residue's position within the helix (4).

Fig. 1.

(A) Our test protein (Escherichia coli Lep) has two N-terminal TM segments (TM1 and TM2), a cytoplasmic loop (P1), and a large luminal domain (P2). The KvAP S4 segment was inserted into the P2 domain, where it is flanked by two glycosylation acceptor sites (G1 and G2). If S4 integrates across the membrane, only G1 will be glycosylated (left); otherwise both G1 and G2 will be glycosylated (right). The apparent free energy of insertion of S4 is defined as ΔGapp = –RT ln(f1g/f2g), where R is the gas constant, T is the absolute temperature, and f1g and f2g are the fractions of singly and doubly glycosylated molecules, respectively. (B) Membrane integration of S4 and S4mut. Plasmids encoding the constructs were transcribed and translated in vitro in the absence (–) and presence (+) of dog pancreas rough microsomes (RM) (5). White dot, unglycosylated protein; one black dot, single glycosylation; two black dots, double glycosylation. (C) Scans with a single Arg (red) or Gly (green) in the test segments 1R/6L/12A and 1G/4L/14A (5). The positions of Arg and Gly are indicated on the x axis. Circles indicate the positions in S4; the repositioned Arg residues in S4mut are dashed.

The isolated S4 helix inserts to a measurable extent as a TM helix (ΔGapp = 0.5 kcal/mol) (Fig. 1B). An S4-related segment with two of the Arg residues moved one step toward the C terminus (S4mut) inserts even better (ΔGapp = 0.0 kcal/mol). Membrane integration in this system likely reflects a peptide's ability to partition across a lipid bilayer (4); thus, we conclude that the S4 helix is poised near the threshold of efficient bilayer insertion, which makes sense for a voltage-dependent switch. Earlier studies have shown that the S4 helix can associate to some extent with the ER membrane as assayed by resistance to alkaline extraction (6).

To resolve how a segment containing four Arg residues can show this kind of behavior, we scanned single Arg and Gly residues across a hydrophobic, 19-residue-long segment and measured ΔGapp for each construct. The contribution of the Arg residue to ΔGapp depended strongly on its position within the segment (Fig. 1C). Using the base biological hydrophobicity scale (4) without taking the positional variation for the Arg and Gly contributions into account yields an estimated ΔGMath of 3.9 kcal/mol for the S4 segment (5). However, including the position-dependent corrections derived from Fig. 1C reduces ΔGMath to 0.9 kcal/mol (0.4 kcal/mol for S4mut), close to the experimental value. If one or two of the Arg residues in S4 are partially shielded from direct lipid contact in the intact K+ channel structure, as has been proposed (7), ΔGapp might be further reduced.

Our results suggest that the paddle model is not inconsistent with the high Arg content of S4. They also highlight the importance of residue position within TM helices as a determinant of insertion efficiency, an aspect that current TM helix prediction schemes do not address adequately. The physical basis for the TM stability of S4 is not entirely clear, but molecular dynamics simulations suggest that the membrane-buried Arg may be accompanied by a few water molecules (8).

Supporting Online Material

http://www.sciencemag.org/cgi/content/full/1109176/DC1

Materials and Methods

References and Notes

View Abstract

Navigate This Article