Structure and Function of the KcsA Potassium Channel

from Streptomyces lividans

Typical voltage-gated potassium channels contain four alpha-subunits in a tetrameric assembly. Each subunit is composed of six transmembrane segments, labeled S1-S6. Both the N and C-termini are located on the cytoplasmic side of the membrane. The selectivity filter on each subunit is in the membrane-embedded loop between S5 and S6. The S4 region is probably the voltage sensor. Potassium channels that activate and later inactivate in response to membrane depolarization (like the classic sodium channel) often have a "ball-and-chain„ segment at the N-terminus which occludes the inner pore during inactivation.

Until 1998, models of ion channel pores were based on hypotheses which came from biophysical and biochemical analyses. In 1998, Rod MacKinnon's group reported the x-ray crystal structure of a prokaryotic K+ channel from Streptomyces lividans (Doyle et al. (1998) Science 285, 69-77). The homologies between the KcsA protein and the channels of eukaryotic cells, especially excitable cells, has allowed confident use of this structure to elaborate many important aspects of channel activity at the molecular level.

The KcsA protein of Streptomyces lividans is a bacterial potassium channel protein that is gated by pH instead of voltage. Neverthless, the KcsA protein sequence is similar to many other types of potassium channels. KcsA channels are a passive transport pathway through which potassium leaves or enters the cell, down its electrochemical gradient. They exhibit the two characteristic features of an ion channel:1) high selectivity and 2) high throughput. These properties can be understood in terms of certain parts of the protein structure.

Sequence homologies and functional characterizations show that the KcsA channel is very similar to many voltage-gated potassium channels, such as the Shaker family. In particular, the pore loop, which exists within the membrane and is responsible for the high flux rate of K+ ions despite strong selectivity for K+ over other monovalent cations, is strongly conserved among other K+ selective ion channels. The sequence domain of the selectivity filter is shown in red (below). The blue residues line the pore cavity and the inner pore, and play a strong role in ionic permeation as well as activation gating. The functional channel is a homotetramer like most other potassium channels. Unlike voltage-gated potassium channels, this channel lacks the first 4 membrane-spanning segments termed S1-S4. However, the KcsA sequence shares very strong homology with voltage-gated ion channels in the S5, S6 helices - the outer and inner(transmembrane, TM1 and TM2) helices, respectively, in KcsA.

Overall Architecture

Structurally, the KcsA channel is a homotetramer composed of four α-subunits arranged with four-fold symmetry around the pore. The channel complex resembles a tea cup, with the mouth facing the extracellular environment and the bottom facing the cytoplasm. Each of the four subunits consists of two transmembrane α-helices (TM1 and TM2, residues 1-52 and 85-119, respectively). The two transmembrane α-helices are connected by a segment of about 30 amino acids comprising the pore region (P), which includes three main structural domains: the 'turret', the pore helix and the selectivity filter.

The inner transmembrane helix of each subunit faces the central pore while the outer helix faces the phospholipid membrane. The inner helices are tilted with respect to the membrane such that there is a relatively wide outer vestibule. The inner helices also mediate the subunit interactions which maintain the critical homotetramer structure of KcsA.

Ribbon representation of two subunits ofthe KcsA tetramer. The two trans-membrane helices, TM1 and TM2, and the pore loop, P, are shown. The two loops in the upper corners represent the ‘turret’, associated with the binding of many channel blockers. The spheres show positions seen for Rb+ and Cs+ ions in the X-ray structure. The lower two spheres represents two overlapping positions. A third, non- overlapping site,observed lower down in the central cavity, is not shown.

Top and side views of the KcsA tetramer in ribbon representation. Each subunit is in a different color.

Between the outer helix and the pore helix is the turret region (residues 53-60), forming the outer vestibule, which interacts with most channel blockers. Both the extracellular and intracellular openings (outer and inner vestibules) are lined with acidic amino acids. Their negative charge at physiological pH attracts the potassium ion to the pore opening and facilitates ion conduction through the channel.The selectivity filter (at the extracellular side of the channel) is in a short extended chain region between the pore helix and the inner trans-memb-rane helix. At this point the channel is so narrow that a K+ ion cannot enter without being fully dehydrated. The filter is comprised of the back-bone carbonyls from residues: threonine (T75), valine (V76), glycine (G77) and

tyrosine (Y78), and glycine (G79). The signature motif GYG is completely conserved in all K+ selective channels. Two of the residues of this short extended chain are characterized by unusual dihedral angles corres-ponding to the left handed α (helix) conformation of the Ramachandran

diagram. These alternate with residues in the more usual right handed a conformation, and the effect is to direct the carbonyl oxygens of 3 conse-cutive peptide bonds into the channel, while the side chains point away.

Mutagenesis studies on the Shaker potassium channel mapped onto the KcsA structure (mutation sites labeled only on one of the two subunits shown). White residues represent the binding site for charybdotoxin; the yellowresidue (Y82) is the external TEA binding site; the mustard/orange residue at the base of the selectivity filter (T74) is the internal TEA binding site. The green residues are accessible in both open and closed states; the purple residues are accessible only when the channel is open. Red residues are absolutely required for K+ selectivity.

Stick model of KcsA. The internal channel surface (minimal radius) is red.

The Pore Structure

The actual channel, which is formed by the inner helices, is lined with hydrophobic amino acids, creating a situation that does not seem to favor ion conduction. However, the inner chamber (cavity) is certainly filled with water - it is large enough to accommodate 50-60 water molecules, and this provides a polar and polarizable environment that stabilizes a cation here.

The overall length of the pore is 45 Å and the diameter varies greatly along the length. The inner helices form a narrow tunnel at the intra-cellular side of the membrane, with a length of approximately 18 Å. This tunnel leads into a wide cavity (about 10 Å wide) near the middle of the membrane. The tunnel and the cavity are wide enough that a K+ ion can move throughout this region while remaining hydrated. However, at the extracellular end, the cavity narrows into the selectivity filter which is so narrow that for a K+ ion to move through the filter, it must shed its hydration shell. The cavity is lined primarily with hydrophobic residues while the selectivity filter seems to be primarily polar due to the main chain peptide carbonyl groups of the GYG signature sequence which line this narrowest part of the pore.

Ionic Binding Within the Pore

Selectivity for the potassium ion (Pauling radius 1.33 Å) over a smaller ion such as sodium (radius, 0.95 Å) or lithium (radius, 0.60 Å), arises from the fact that the filter requires a large sphere for interaction. The energy needed to compensate for the stripping away of water molecules can only be equaled by interactions with all four subunit carbonyls, and the interactions between the ion and the carbonyls from all four subunits are maximized for the potassium ion. Since the sodium ion is too small to interact with all four subunits, it is energetically unfavorable for the ion to dehydrate and move through the channel.

Smaller cations such as Li+ and Na+ are excluded because their small dehydrated radius do not allow binding to the selectivity filter sufficient to compensate for the energetic penalty of desolvation. The larger alkali cations, Rb+ (radius 1.48Å) and Cs+ (radius 1.69 Å) do permeate the channel and their much higher electron density allows them to be imaged in the channel to reveal single ion binding sites. The selectivity filter contains two cation binding sites 7.5 Å apart. A third ion is seen in the central cavity, below the selectivity filter (MacKinnon suggests that the high electron density within this cavity may not be an ionic binding site per se, but rather a "hydrated cation cloud").

Molecular surface of KcsA and contour of the pore as a cutaway view displaying the solvent accessible surface of the K+ channel. The electrostatic potential is color coded red for negative and blue for positive. The yellow areas indicate hydrophobic side chains of several semi-conserved residues in the inner cavity. The green spheres represent K+ ion positions in the conduction pathway, identified from Rb+ and Cs+ diffraction density.

For an ion crossing the membrane through a simple pore, the electrostatic energy is maximal at the mid-point. This is quite contrary to what is seen here, where a relatively favorable state is seen as net binding, inspite of the fact that the centre of the pore is composed mainly of hydrophobic residues. Thus, the channel design provides mechanisms to counteract the destabilizing influence of the low dielectric.

One design factor is the sheer size of the central cavity, which allows a substantial number (50-60) of water molecules to be present at all times. By surrounding the cation with water, the cation can polarize the water, rather than the hydrophobic environment which has a very low dielectric constant, and the destabilizing influence of the low dielectric environment is minimized.

Additionally, like all α-helices, the pore helix has a macroscopic dipole moment, thenegative end of which points towards the center of the cavity and further reduces thedestabilizing effect of the hydrophobic cavity residues. In fact, the electric field from the pore helix dipole is greater (≥ 0.01 V/Å) than that due to the membrane potential (100 mV over the membrane thickness).

This architecture ensures a relatively low resistance pathway from the cytoplasm to the selectivity filter, allowing for a high throughput rate of permeation.

The Selectivity Filter

The side chains of the amino acids in the selectivity filter do not participate directly in cation-coordination. Rather this is accomplished by their main chain carbonyl groups. The main chain atoms of the signature sequence create a stack of sequential oxygen rings and allow many sites of proper dimensions for coordinating a K+ ion. A water molecule is apparently located between the two K+ ions within the selectivity filter, but the bound cations remain dehydrated when bound to this extremely narrow region of the permeation pathway. The inner site of the two mayactually consist of two overlapping sites, with rapid movement of an ion between them indicative of the flat energy landscape necessary for rapid motion through the filter.

Detailed (stereo) view of the K+ selectivity filter, with the nearest subunit removed. The Val (V) and Tyr (Y) side chains are directed away from the filter. Two K+ ions are shown (green), roughly 7.5 Å apart, with a single water molecule between them (red). The lower (inner) K+ is depicted as in rapid equilibrium between adjacent coordination sites.

Interaction between the side chains of the TVGY(G) (Thr-Val-Gly-Tyr-Gly) and the inner helices forms a large "sheet" of aromatic amino acids which, via hydrogen bonding between Tyr OH groups and van der Waals interactions within this sheet, ensure the pore diameter remainsconstant. A glutamate residue, behind the selectivity filter, probably hydrogen bonds to one of the peptide NH groups of the GYG sequence and also holds the filter in place.

Such features may play an important role in preventing Na+ ions from being accommodated by the selectivity filter. Unless there is a mechanism to stabilize the diameter of the selectivity filter, a Na+ ion may induce the filter to narrow, thereby solvating the Na+ more effectively and allowing its permeation.

Section of the KcsA channel perpendicular to the pore, through the selectivity filter, and viewed from the cytoplasm (from “below”).

The view highlights the network of aromatic residues surrounding thefilter and proposed to hold it at a fixed diameter. Tyrosine-78 (Y78) interacts through hydrogen bonding and van der Waals contacts with two Trp residues (W67 and W68) from the pore helix.

When an ion enters the selectivity filter, it must become dehydrated. Dehydration of acation is a very energetically expensive process and thus, the carbonyl oxygens must take the place of the water, stabilizing the dehydrated cation. K+ ions apparently fit into this filter so precisely that the energetic cost of dehydration is almost balanced by the stabilization effect of the main chain carbonyl groups. However, this strong coordination is incommensurate with rapid flux of K+ ions through the pore. Several models have been proposed which involve repulsion between the two coordinated cations within the filter, thus allowing rapid permeation. The 7.5 Å distance between cation binding sites within the filter is consistent with such a repulsive effect. Up to three potassium ions can fit into the channel at one time - two in the selectivity filter and one below it at the top of the vestibule. If only one ion is present in the selectivity filter, the interactions are too strong to allow rapid conductance through the channel.This is consistent with the fact that conductance rates are dependent on ion concentrations. Once a second potassium ion enters the filter, the ions repel one another. The repulsive force between ions equals the attractive forces between the first ion and the filter. This allows the first ion to pass into the inner chamber, where it can be hydrated by water and stabilized by the pore helix dipole moment, expelling any ion already in the chamber in to the intracellular space.

This represents strong evidence for a multi-ion (single file) conduction mechanism, as originally proposed many years ago on the basis of biophysical studies of channel conductances. The rate of conductance is therefore limited by the movement of the potassium ion through the selectivity filter (12 Å), but, in this way, it is possible for the channel to be highly specific for potassium ions and yet maintain high rates of conduction.

Summary(i) The pore is constructed of the inner helices arranged as an inverted teepee, with theselectivity filter held at its wide end. This architecture also describes the pore of cyclicnucleotide-gated channels and probably Na+ and Ca2+ channels as well.

(ii) The narrow selectivity filter is only 12 Å long, whereas the remainder of the pore is wider and has a relatively inert hydrophobic lining. These structural and chemical properties favor a high K+ throughput by minimizing the distance over which K+ interacts strongly with the channel.

(iii) A large water-filled cavity and helix dipoles help to overcome the high electrostatic energy barrier facing a cation in the low dielectric membrane center.

(iv) The K+ selectivity filter is lined by carbonyl oxygen atoms, which provide multiple closely spaced sites. The filter is constrained in an optimal geometry so that a dehydrated K+ ion fits with proper coordination but the Na+ ion is too small.

(v) Two K+ ions at close proximity in the selectivity filter repel each other. The repulsion overcomes the otherwise strong interaction between ion and protein and allows rapid conduction in the setting of high selectivity.