For the Latest Information, Tune to Channel KcsA

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Science  02 Jul 1999:
Vol. 285, Issue 5424, pp. 59-61
DOI: 10.1126/science.285.5424.59

Potassium ion (K+) channels are transmembrane proteins that regulate K+ ion flux across the cell membrane with remarkable selectivity and efficiency. Their activity underlies fundamental biological processes such as electrical signaling, osmotic balance, and signal transduction (1). Last year, MacKinnon and colleagues published the crystal structure of KcsA, the K+ channel of the bacterium Streptomyceslividans (2), ushering the field into a new era. Building on this landmark study, Roux and MacKinnon now report on page 100 a computational analysis of the electrostatic forces that stabilize K+ ions inside the central cavity of the KcsA ion channel (3). Further, Perozo et al. (4) on page 73 describe the conformational changes that take place in KcsA as it opens and closes, a process called gating that can be followed by electron paramagnetic resonance (EPR).

Although the crystal structure of the KcsA channel has yielded valuable information about permeation (that is, how the channel selectively translocates K+ ions across the membrane), it has not offered definitive answers about how the channel opens and closes (gates), arguably the most important question in ion channel physiology. Different ion channels gate in different ways: Some are activated by changes in cell membrane voltage, others by binding of ligand. KcsA is activated by changes in extracellular pH.

In their study, Perozo et al. trapped the KcsA channel in both the open and closed conformations and then analyzed the difference in the EPR signal (5). They introduced cysteine residues at select locations in each of the four identical subunits of KcsA—in transmembrane helices 1 and 2 (TM1 and TM2) and in the pore α helices (see the figure). They then labeled the helices with nitroxide spin labels, and analyzed the change in spin-label mobility and intersubunit spin-spin coupling as the channel gated in response to changes in pH. They found that TM1 and TM2 underwent conformational changes (rigid-body translations and counterclockwise rotations around the channel's central cavity) as the pore opened. Opening of the pore seemed to be directly coupled to the movement of the four TM2 helices: Their displacement increased the diameter of the permeation pathway at the point where the helices converge. The involvement of the four pore α helices in gating is still speculative. Although the amino terminus of each pore helix remains immobile during gating, there does appear to be a small movement at the carboxyl terminus. Several other proteins with transmembrane helices, including the acetylcholine receptor (6) and rhodopsin (7), are also activated by a change in conformation in the absence of any change in secondary structure.

K+ ions on the move.

Depicted are two of the four subunits of the bacterial K+ channel, KcsA. Each subunit is composed of two transmembrane helices, an outer helix (TM1) and an inner helix (TM2), and an interior fold called the pore α helix. The central cavity and the four pore α helices help to preferentially select monovalent over divalent cations and to stabilize the ion as it passes through the membrane. Movement of the four TM2 helices opens and closes the pore (a process called gating), allowing K+ ions to exit the cell cytosol. TM1 corresponds to S5 in the fruit fly K+ channel (Shaker) and TM2 to S6.

The Perozo study is a good example of the fruitful melding of structural information from KcsA (a prokaryotic ion channel) and functional information from eukaryotic channels such as the fruit fly K+ channel (Shaker), for which no high-resolution structure exists. For example, chemical modification of cysteine residues introduced into S6—a putative transmembrane segment of the Shaker channel homologous to TM2 in KcsA—showed that the gating of Shaker is accompanied by a conformational change in the carboxyl terminus of S6 akin to the displacement seen in TM2 (8). The findings in KcsA and Shaker were interpreted as evidence for the existence of a gate in the intracellular region of the pore that controls entry of K+ into the pore from the cell cytosol. This intracellular gate seems to be distinct from the narrow section of the pore that acts as a selectivity filter, allowing only K+ ions (and not Na+ions) to enter the pore. A series of ion-trapping experiments initiated by Armstrong (9) also supports this view. The extracellular region surrounding the selectivity filter appears not to be involved in opening and closing of the pore because binding of scorpion toxin to the extracellular region of the Ca2+-activated K+ channel (another type of K+ channel with a homologous pore) has almost no effect on gating (10). This fits nicely with the Perozo results, which show no movement of this region during KcsA gating. Together these observations suggest that conformational changes during gating are highly conserved in K+ channels from many different organisms. The agreement between findings in ion channels from very different organisms exemplifies the usefulness of prokaryotes for understanding eukaryote, and ultimately mammalian, ion channel biology.

Each subunit of KcsA has two transmembrane helices, whereas most of the eukaryotic channels studied so far (for example, voltage-activated channels) are far more complex. If the conformational changes observed by Perozo et al. occur in eukaryotic channels, then how are the voltage sensors in voltage-activated channels coupled to the displacement of the transmembrane helices? The answer will probably have to await analysis of a high-resolution structure of a eukaryotic voltage-activated channel. Together with what we already know about voltage-activated ion channels from functional measurements, this may lead to an unprecedented degree of understanding of a biological macromolecule.

In a complementary report, Roux and MacKinnon present an incisive computational analysis of the electrostatic profile of the KcsA pore. By solving the finite difference Poisson equation they dissect the electrostatic influence of the central cavity and the four pore α helices on the stability of the K+ ion at the pore's center (see the figure). The low electrical polarization of the membrane hydrocarbons creates a high energy barrier to the passage of ions, a key problem for the pore to overcome (11). As expected, the investigators found that the water-filled central cavity of the pore was the key stabilizing element, but the pore helices also revealed some interesting properties. The authors estimate that as much as 80% of the stabilization of K+ ions by protein arises from the amide-carbonyl dipoles of the first 13 amino acids of the pore α helices. Consequently, K+ ion stability in the pore's central cavity is critically dependent on the exact orientation of the helices with respect to the central cavity and their rearrangement during the course of gating. The most surprising but satisfying finding is that the electrostatic effects of the ion-binding site in the cavity are tuned to preferentially accommodate a monovalent cation. It will be interesting to see whether the same principles hold for other monovalent and divalent cation-selective channels.

The conformational change in the carboxyl terminus of the pore α helices observed by Perozo et al., combined with the contribution of these helices to the stability of the K+ions in the pore determined by Roux and MacKinnon, suggests a possible explanation for a long-standing puzzle about the origin of subconductance levels in different ion channels (12, 13). (Sometimes channel molecules conduct less than the maximum level of current, and this is referred to as subconductance.) On the basis of these two studies, we speculate that movements of the pore α helices may alter the stability of the ion in the central cavity, thereby affecting the amount of current passing through the channel. In Shaker channels, subconductance states are traversed as the pore opens and closes (12). It is possible that this is caused by the pore α helices occupying a transient intermediate state, creating a different affinity binding site for a K+ ion during gating. The support for this hypothesis will have to come from further experiments and computational analyses.


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