PerspectiveStructural Biology

Voltage Sensor Meets Lipid Membrane

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Science  19 Nov 2004:
Vol. 306, Issue 5700, pp. 1304-1305
DOI: 10.1126/science.1105528

Electrical impulses propagate rapidly along the membranes of living cells. The molecular components that make this possible are proteins known as voltage-dependent ion channels. These channels open in response to changes in the voltage across the cell membrane, and it is precisely this voltage-dependent property that allows them to propagate electrical impulses. Thirty years ago, Armstrong and Bezanilla demonstrated that when voltage-dependent ion channels experience a change in the membrane voltage, tiny electrical charges known as gating charges move relative to the membrane electric field (1). This fundamental observation suggested that a transmembrane voltage change exerts an electric force on the gating charges, causing the pore within the channel protein to open.

Now we know that voltage-dependent potassium ion (K+) channels contain an ion-selective pore domain with a gate, and a voltage sensor domain (segments S1 to S4) attached to the pore (see the figure). The gating charges correspond to positively charged arginine residues located on the otherwise hydrophobic S4 segment. Thus, voltage-sensing results from a repositioning of the arginine residues within the membrane electric field that is associated with structural rearrangements of the voltage sensor, and these structural rearrangements are linked to the opening of the pore's gate (2, 3).

A model of the KvAP K+ channel with a voltage-sensor at the protein-lipid interface.

Accessibility and mobility data mapped onto a representation of the K+ channel show the relation of the channel's voltage sensor to the lipid membrane, water surfaces, and pore (14). The figure depicts one possible explanation for the accessibility data reported by Perozo and colleagues (14), with two exposed and two buried positive charges in an apparently open conformation of the voltage sensor. Four identical subunits (brown) of the voltage-dependent K+ channel surround a central ion-conduction pore. Each subunit has a voltage sensor comprising α-helical segments S1 to S4. Accessibility measurements suggest that S1 and S2 are closest to the pore. In contrast, S3 and S4 reside at the protein-lipid interface, with one surface (yellow) exposed to lipid, another (red) exposed to protein, and with water (blue) coating the “top.” The S4 helix contains four positively charged arginine residues (asterisks) that allow the channel to sense changes in membrane voltage.

CREDIT: KATHARINE SUTLIFF/SCIENCE

The voltage sensor's structure and the process by which the gating charges are repositioned have been subjects of intense controversy. On the basis of electrophysiological studies, a number of structural models of the voltage-dependent K+ channel have been proposed. These models share the feature of an S4 helix that is isolated from the lipid membrane by a protein wall consisting of helices S1, S2, and S3 on the channel's lipid-facing perimeter (48). They posit that a voltage change across the membrane causes a translation or rotation of the S4 helix, which would move the S4 helix and its positively-charged arginine residues within an aqueous “gating pore.” Recently, x-ray crystal structures (9), biotin-avidin accessibility studies (10), and electron microscopy (11) of KvAP, a prokaryotic voltage-dependent K+ channel, have suggested a different model. In this model (the paddle model) the voltage sensor is a highly mobile domain, and it is “inside-out” in the sense that helices S1, S2, and S3 do not isolate S4 from the membrane; instead, S4 itself is located at the protein-lipid interface. Specifically, S4 engages part of S3 to form a helix-turn-helix “paddle” that could somehow move at the protein-lipid interface. It is the location of S4 that is at the center of the controversy. Is S4 at the protein-lipid interface or is it shielded from the lipid membrane by S1, S2, and S3? The paddle model is based on a collection of data—a full-length crystal structure with obvious distortions of its voltage sensor (12), a crystal structure of the isolated voltage sensor (13), and accessibility data (10)—and thus is conceptual, not atomic, and in many respects still needs to be defined.

A recent report in Science by Perozo and his co-workers (14) presents new data on the structure of the KvAP voltage sensor. These authors studied the spin-label side-chain accessibility and mobility of KvAP K+ channels in lipid membranes using electron paramagnetic resonance (EPR) spectroscopy. This is a particularly informative technique for analyzing membrane proteins because it uses accessibility parameters determined from the spectral effects of lipid-soluble (O2) and water-soluble (NiEDDA) relaxing agents to distinguish between lipid-accessible and water-accessible surfaces (15). A spin-label side chain at a specific position on a protein can thus be classified into one of three categories: buried beneath the protein surface, on the surface exposed to aqueous solution, or on the surface exposed to lipid. Furthermore, a side-chain mobility value provides additional information; surface positions tend to have a higher mobility value than those buried inside the protein.

All voltage-dependent ion channels undergo conformational changes. Which conformation did Perozo and his colleagues analyze? The KvAP voltage sensor is held in a closed conformation when the voltage is negative (for example, −100 mV) on the inner membrane surface relative to the outside surface of the cell. The sensor moves to its open conformation upon membrane “depolarization” to 0 mV, causing the pore to open. After a few seconds at 0 mV, the pore becomes inactivated and ion conduction stops by an as yet unknown mechanism, although in all likelihood the voltage sensor remains roughly in its open conformation. This is the condition under which the EPR experiments were carried out by Perozo's group: membranes at 0 mV and the voltage sensor presumably open.

What did they find? In a spin-label scan of the voltage sensor (helical segments S1 to S4), the pattern of accessibility satisfies expectations for a protein that spans the membrane several times, that is, the water-exposed residues occur in the hydrophilic loops between transmembrane segments. This result is presented as being inconsistent with the paddle model, and the inconsistency is demonstrated through accessibility calculations made on an atomic version of a paddle model. This analysis is inappropriate because the paddle model, as originally presented, contained uncertainties (particularly with respect to the positions of S1 and S2) that precluded calculations of atomic coordinates (10).

Nevertheless, the new data convey a very telling message: Side-chain mobility and lipid accessibility both increase as measurements proceed from S1 to S4. As the authors point out, this suggests that S1 and S2 are buried in a protein environment, whereas S3 and S4 are more exposed to the lipid membrane (see the figure).

Side-chain accessibility and mobility data in the absence of distance constraints are insufficient to deduce a complex protein structure. But the authors propose a structural interpretation based on the assumption that the crystal structure of the isolated voltage sensor (13) approximates a native conformation in the membrane lipid bilayer. Given this assumption, they then ask which orientation of the crystal structure of the isolated sensor and pore best satisfy the constraints imposed by the spin-label data? Perozo and colleagues conclude that S1 and S2 must represent the surface that lies against the pore, away from the membrane, thus accounting for their buried location. They conclude that S3 and S4 helices reside on the outer perimeter of the protein against the membrane, thus accounting for their high lipid exposure and high mobility. In reality, the voltage sensor in situ may adopt a somewhat different structure than that of the isolated voltage sensor. This is especially true at its covalent attachment site (S4 of the voltage sensor to S5 of the pore) where differences are implied by the spin-label data, and at the surface formed by S1 and S2, which somehow forms an interface with the pore. But the basic conclusion seems inescapable: S3 and S4 reside at the protein-lipid interface, against the lipid membrane (see the figure).

In the crystal structures of KvAP, the carboxyl-terminal half of S3 and S4 form the helical-hairpin paddle of the voltage sensor (9). Although such an analysis was not presented in the paper, when the spin-label data are mapped onto the paddle a striking pattern constrains the orientation of the paddle in the membrane. As shown in the figure, one face is exposed entirely to lipid (yellow), whereas the other is exposed to water near the hairpin turn (blue) and to a mixture of protein and lipid (red and yellow) further “down,” as though the paddle's long axis were roughly perpendicular to the membrane plane with one face toward the channel and the other toward the membrane.

Where are the arginine residues? The authors state that four out of six are buried. Yet the answer to this question with respect to the positive charges involved in gating requires careful consideration. In the Shaker voltage-dependent K+ channel isolated from fruit fly, the amino-terminal four S4 arginine residues contribute to the gating charge, whereas the arginine residues near the carboxyl-terminal end of S4 do not (16, 17). The crystal structures of KvAP provide an explanation for this: The gating-sensitive arginines correspond precisely to those located on the voltage-sensor paddle, whereas the gating-silent arginines are found off the paddle, on the so-called S4 to S5 linker (9). Of the four paddle arginines, the spin-label data suggest that two (the third and fourth) are buried within the protein and two (the first and second) are exposed on the surface. The first of these is exposed only to lipid, whereas the second is exposed to lipid and water (see the asterisks in the figure).

The new data from Perozo and his colleagues are entirely consistent with the concepts proposed for the open conformation of the paddle model (10). Many structural aspects of the voltage-gated K+ channel are still uncertain. For instance, the new spin-label data by Perozo's group do not address the question of how the voltage sensor moves. Fluorescence measurements of the Shaker K+ channel have led to the hypothesis of small (∼2 Å) charge movements across a strong electric field that is highly focused by aqueous crevasses penetrating the protein surface (3). In contrast, avidin accessibility experiments on the KvAP channel suggest large (at least 15 Å) movements of the voltage-sensor paddle at the protein-lipid interface (10). There will no doubt be further disagreements over movements of the voltage sensor, and these will drive our understanding still further. What we need next are new structures, additional biochemical and functional analyses, accessibility data on the closed conformation of the channel, and a better chemical understanding of the protein-lipid interface. All of these are sure to come our way in the near future.

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