What do ion channels look like? How does the channel protein span the membrane? What happens to the structure of the channel when it opens and closes? Where along the length of the channel protein do drugs and transmitters bind?
Biochemical and molecular biological studies have provided a basic understanding of channel structure and function. All ion channels are large integral-membrane proteins with a core transmembrane domain that contains a central aqueous pore spanning the entire width of the membrane. The channel protein often contains carbohydrate groups attached to its external surface. The pore-forming region of many channels is made up of two or more subunits, which may be identical or different. In addition, some channels have auxiliary subunits that modify their functional properties. These subunits may be cytoplasmic or embedded in the membrane (Figure 5–9).
Ion channels are integral membrane proteins composed of several subunits.
A. Ion channels can be constructed as hetero-oligomers from distinct subunits (left), as homo-oligomers from a single type of subunit (middle), or from a single polypeptide chain organized into repeating motifs, where each motif functions as the equivalent of one subunit (right).
B. In addition to one or more α-subunits that form a central pore, some channels contain auxiliary subunits (β or γ) that modulate the gating of the pore.
The genes for all the major classes of ion channels have been cloned and sequenced. The amino acid sequence of a channel, inferred from its DNA sequence, can be used to create a structural model of the channel protein. Regions of secondary structure—the arrangement of the amino acid residues into α-helixes and β-sheets—as well as regions that are likely to correspond to membrane-spanning domains of the channel are predicted based on the structures of related proteins that have been experimentally determined using electron and X-ray diffraction analysis. This type of analysis identified the presence of four hydrophobic regions, each around 20 amino acids in length, in the amino acid sequence of a subunit of the ACh receptor channel. Each of these regions is thought to form an α-helix that spans the membrane (Figure 5–10).
The secondary structure of membrane- spanning proteins.
A. A proposed secondary structure for a subunit of the nicotinic acetylcholine (ACh) receptor channel present in skeletal muscle. Each cylinder (M1–M4) represents a putative membrane- spanning α-helix comprised of approximately 20 hydrophobic amino acid residues. The membrane segments are connected by cytoplasmic or extracellular segments (loops) of hydrophilic residues. The amino terminus (NH2) and carboxy terminus (COOH) of the protein lie on the extracellular side of the membrane.
B. The membrane-spanning regions of an ion channel protein can be identified using a hydrophobicity plot. Here a running average of hydrophobicity is plotted for the entire amino acid sequence of the α-subunit of the nicotinic ACh receptor. Each point in the plot represents an average hydrophobic index of a sequence of 19 amino acids and corresponds to the midpoint of the sequence. The amino acid sequence of the subunit is inferred from the nucleotide sequence of the cloned receptor subunit gene. Four of the hydrophobic regions (M1–M4) correspond to the membrane-spanning segments. The hydrophobic region at the far left in the plot is the signal sequence that positions the hydrophilic amino terminus of the protein on the extracellular surface of the cell during protein synthesis. The signal sequence is cleaved from the mature protein. (Reproduced, with permission, from Schofield et al. 1987.)
Additional insights into channel structure and function have been obtained by comparing the amino acid sequences of the same type of channel from different species. Regions that show a high degree of similarity (that is, have been highly conserved through evolution) are likely to be important in maintaining the structure and function of the channel. Likewise, conserved regions in different but related channels are likely to serve a common biophysical function. For example, voltage-gated channels selective for different cations have a specific membrane-spanning segment that contains positively charged amino acids (lysine or arginine) spaced at every third position along an α-helix. This motif is observed in all voltage-gated cation channels, but not in transmitter-gated channels, suggesting that this charged region is important for voltage gating (see Chapter 7).
Once a structure for a channel has been proposed, it can be tested in several ways. For example, antibodies can be raised against synthetic peptides that correspond to different hydrophilic regions in the protein sequence. Immunocytochemistry can then be used to determine whether the antibody binds to the extracellular or cytoplasmic surface of the membrane, thus defining whether a particular region of the channel is extracellular or intracellular.
The functional consequences of changes in a channel's primary amino acid sequence can be explored through a variety of techniques. One particularly versatile approach is to use genetic engineering to construct channels with parts that are derived from the genes of different species—so-called chimeric channels. This technique takes advantage of the fact that the same type of channel can have somewhat different properties in different species. For example, the bovine ACh receptor channel has a slightly greater single-channel conductance than the ACh receptor channel in electric fish. By comparing the properties of a chimeric channel to those of the two original channels, one can assess the functions of specific regions of the channel. This technique has been used to identify the membrane-spanning segment that forms the lining of the pore of the ACh receptor channel (see Chapter 9).
The roles of different amino acid residues or stretches of residues can be tested using site-directed mutagenesis, a type of genetic engineering in which specific amino acid residues are substituted or deleted. Finally, one can exploit the naturally occurring mutations in channel genes. A number of inherited and spontaneous mutations in the genes that encode ion channels in nerve or muscle produce changes in channel function that can underlie certain neurological diseases. Many of these mutations are caused by localized changes in single amino acids within channel proteins, demonstrating the importance of that region for channel function.
Ion Channels Can Be Grouped into Gene Families
The great diversity of ion channels in a multicellular organism is underscored by the recent sequencing of the human genome. Our genome contains nine genes encoding variants of voltage-gated Na+ channels, 10 genes for different Ca+ channels, over 75 genes for K+ channels, 70 genes for ligand-gated channels, and more than a dozen genes for Cl− channels. Fortunately, the evolutionary relationships between the genes that encode ion channels provide a relatively simple framework with which to categorize them.
Most of the ion channels that have been described in nerve and muscle cells fall into a few gene superfamilies. Members of each gene superfamily have similar amino acid sequences and transmembrane topology and, importantly, related functions. Each superfamily is thought to have evolved from a common ancestral gene by gene duplication and divergence. Several superfamilies can be further classified into families of genes with more closely related structure and function.
One superfamily encodes ligand-gated ion channels that are receptors for the neurotransmitters ACh, GABA, glycine, or serotonin. All of these receptors are composed of five subunits, each of which has four transmembrane α-helixes (Figure 5–11A). In addition, the extracellular domain that forms the receptor for the ligand contains a conserved loop of 13 amino acids flanked by pair of cysteine residues that form a disulfide bond. As a result, this receptor superfamily is referred to as the cys-loop receptors. Ligand-gated channels can be classified by their ion selectivity in addition to their agonist. The genes that encode glutamate receptor channels belong to a separate gene family.
Three superfamilies of ion channels.
A. Members of a large family of ligand-gated channels, such as the acetylcholine receptor channel, have five closely related subunits, each of which contains four transmembrane α-helixes (M1–M4). Each cylinder in the figure represents a single transmembrane α-helix.
B. The gap-junction channel is formed from a pair of hemichannels, one each in the pre- and postsynaptic cell membranes that join in the space between two cells. Each hemichannel is made of six identical subunits, each containing four transmembrane α-helixes. Gap-junction channels serve as conduits between the cytoplasm of the pre- and postsynaptic cells at electrical synapses (see Chapter 8).
C. The voltage-gated Na+ channel is formed from a single polypeptide chain that contains four homologous domains or repeats (motifs I–IV), each with six membrane-spanning α-helixes (S1–S6). The S5 and S6 segments are connected by an extended strand of amino acids, the P-region, which dips into and out of the external surface of the membrane to form the selectivity filter of the pore.
Gap-junction channels, which bridge the cytoplasm of two cells at electrical synapses (see Chapter 8), are encoded by a separate gene superfamily. A gap-junction channel is composed of 12 identical subunits, each of which has four membrane-spanning segments (Figure 5–11B).
The genes that encode the voltage-gated ion channels responsible for generating the action potential belong to another family. These channels are usually activated by membrane depolarization and are selective for Ca2+, Na+, or K+. All voltage-gated channels have a similar architecture, with a core motif composed of six transmembrane segments termed S1–S6 (Figure 5–11C). The S5 and S6 segments are connected by a highly conserved P-region, which loops into and out of the extracellular face of the membrane and forms the selectivity filter of the channel. Voltage-gated Na+ and Ca2+ channels are composed of a large subunit that contains four repeats of this basic motif. Voltage-gated K+ channels are composed of four separate subunits, each containing one motif. This structure is shared by other, more distantly related channel families (see Chapter 7).
The major gene family encoding the voltage-gated K+ channels is distantly related to two families of K+ channels, each with distinctive properties and structure (Figure 5–12). One family consists of the genes encoding inward-rectifying K+ channels, which are open at the resting potential and close rapidly during depolarization. Each channel subunit has only two transmembrane segments connected by a pore-forming P-region. A second family is composed of subunits with two repeated pore-forming segments. These channels may also contribute to the resting K+ conductance.
Four related families of ion channels with P-regions
A. Voltage-gated K+ channels are composed of four subunits. Each K+ channel subunit corresponds to one repeated domain of a voltage-gated Na+ channel, with six transmembrane segments and a pore-forming P-region.
B. Inward-rectifying K+ channels are composed of four subunits, each of which has only two transmembrane segments connected by a P-region.
C. A third type of K+ channel is composed of subunits that contain two repeats of the inward-rectifying K+ channel subunit, with each repeat containing a P-region. Two of these subunits are presumed to form a channel with four P-regions.
D. Glutamate receptor channels form a distinct P-region channel family. In these receptors the amino terminus is extracellular and the P-region enters and exits the cytoplasmic side of the membrane. The bacterial GluR0 subunit contains two transmembrane segments (left). In higher organisms the subunits contain a third transmembrane segment (right).
The sequencing of the genomes of a variety of species has led to the identification of additional ion channel gene families, found in organisms from bacteria to humans. Channels with related P-regions have been identified that are only very distantly related to the family of voltage-gated channels. These channels include the glutamate-gated channels, in which the P-region is inverted: It enters and leaves the internal surface of the membrane (see Figure 5–12D). Finally, the transient receptor potential (TRP) family of nonselective cation channels (named after a mutant Drosophila strain in which light evokes an abnormal transient receptor potential in photoreceptors) comprises a very large group of channels that contain a P-region. Like the voltage-gated K+ channels, TRP channels also contain six transmembrane segments but are usually gated by intracellular ligands. TRP channels are important for Ca2+ metabolism in all cells, visual signaling in insects, and pain, heat, and cold sensation in the nervous system of higher animals. Recent evidence implicates TRP channels in mechanosensation and hearing in insects and fish and in certain taste sensations in mammals.
A number of other families of channels have been identified, distinct from those considered above. These include Cl− channels that help set the resting potential of certain nerve and skeletal muscle cells, and a class of ligand-gated cation channels activated by ATP, which functions as a neurotransmitter at certain synapses. With the completion of the human genome project, it is likely that nearly all of the major classes of ion channel genes have now been identified.
However, the diversity of ion channels is even greater than the large number of ion channel genes. Because most channels in a given subfamily are composed of multiple subunits, each of which may be encoded by a family of closely related genes, combinatorial permutations of these subunits can generate a diverse array of heteromultimeric channels with different functional properties. Further diversity can be produced by alternative splicing of precursor mRNA transcribed from a single gene. Finally, the sequence of a transcript can be altered by a process termed RNA editing. As with enzyme isoforms, variants of a channel with slightly different properties may be expressed at distinct stages of development (Figure 5–13), in different cell types throughout the brain (Figure 5–14), and even in different regions within a cell. These subtle variations in structure and function presumably allow channels to perform highly specific functions.
Variants of an ion channel gene can be expressed at different points in development to fine tune channel properties.
These examples of currents through individual acetylcholine receptor channels were recorded from frog skeletal muscle at three stages of development: early (1.1 days), intermediate (2.4 days), and late (48 days). In immature muscle the single channels have a relatively small conductance and a relatively long open time. In mature muscle the channel conductance is larger and the average open time is shorter. At intermediate stages of development the population of channel variants is mixed; both brief, large-conductance channel openings and longer-lasting, small-conductance channel openings are evident. This functional difference results from a developmental switch in subunit composition caused by a change in gene expression. Both immature and mature forms of the receptor have α-, β-, and δ-subunits. However, the γ-subunit expressed early in development is replaced by the ε-subunit as the animal matures. (Reproduced, with permission, from Owens and Kullberg 1989.)
Variants of a potassium channel are expressed in different regions of the brain.
Autoradiograms show the expression patterns of mRNA transcripts of the four genes comprising the KV 3 subfamily of voltage-gated K+ channel subunits (determined by in situ hybridization). In each autoradiogram the dark areas represent high densities of expression. The brain was sectioned at the level of the posterior thalamus. The thalamic nuclei shown are VPL, ventral posterior lateral; VPM, ventral posterior medial; MD, medial dorsal; LD, lateral dorsal; VM, ventromedial; PO, posterior; and RT, reticular. Hippocampal regions CA1 and CA3; DG, dentate gyrus; and ZI, zona incerta. (Reproduced, with permission, from Weiser et al. 1994.)
The rich variety of ion channels in different types of neurons may make it possible to develop drugs that can activate or block channels in selected regions of the nervous system. Such drugs would, in principle, have maximum therapeutic effectiveness with minimum side effects.
The Closed and Open Structures of Potassium Channels Have Been Resolved by X-Ray Crystallography
Rod MacKinnon and his colleagues provided the first high-resolution X-ray crystallographic analysis of the molecular architecture of an ion-selective channel. To overcome the difficulties inherent in obtaining crystals of integral membrane proteins, they initially focused on a bacterial K+ channel, termed KcsA. These channels were useful for crystallography as they can be expressed at high levels for purification, are relatively small, and have a simple transmembrane topology that is similar to inward-rectifier K+ channels present in higher organisms, including mammals (see Figure 5–12B). The structure of the channel was further simplified using molecular engineering to truncate cytoplasmic regions that are not essential for forming the ion-selective pore.
The crystal structure determined from the modified KcsA protein provides several important insights into the mechanisms by which the channel facilitates the movement of K+ ions across the hydrophobic lipid bilayer. The channel is made up of four identical subunits arranged symmetrically around a central pore (Figure 5–15A). Each subunit has two membrane-spanning α-helixes, an inner and outer helix, that are connected by the P-loop, which forms the selectivity filter of the channel. At the extracellular end of the channel the two α-helixes tilt away from the central axis of the pore so that the structure resembles an inverted teepee.
The X-ray crystal structure of a bacterial potassium channel.
(Reproduced, with permission, from Doyle et al. 1998.)
A. Each of the four subunits of the K+ channel KcsA contributes two membrane-spanning helixes, the outer helix (blue) and the inner helix (red). The P-region (white) lies near the extracellular surface of the channel pore and consists of a short α-helix (pore helix) and a loop that forms the selectivity filter of the channel. A K+ ion is in the center of the pore. The view is looking down the channel from outside the cell.
B. The channel is seen in a cross section in the plane of the membrane. The four subunits are shown in different colors.
C. Another view in the same orientation as in part B shows only two of the four subunits. The channel contains five K+ binding sites (dashed). Four of the sites lie in the selectivity filter (yellow) and the fifth site lies in an inner chamber near the center of the channel. The four K+ binding sites of the selectivity filter are formed by successive rings of oxygen atoms (red) from five amino acid residues per subunit. Four of the rings are formed by carbonyl oxygen atoms from the main chain backbone of four consecutive amino acid residues—glycine (G), tyrosine (Y), glycine (G), and valine (V). A fifth ring of oxygen near the internal end of the selectivity filter is formed by the side-chain hydroxyl oxygen of threonine (T). Each ring contains four oxygen atoms, one from each subunit. Only the oxygen atoms from two of the four subunits are shown in this view. (Reproduced, with permission, from Zhou et al. 2001.)
D. A view of ion permeation through the channel illustrates the changes in occupancy of the various K+ binding sites. A pair of ions hops in concert between a pair of binding sites in the selectivity filter. In the initial state, the "outer configuration," a pair of ions is bound to sites 1 and 3. As an ion enters the inner mouth of the channel, the ion in the inner chamber jumps to occupy the innermost binding site of the selectivity filter (site 4). This causes the pair of ions in the outer configuration to hop outward, expelling an ion from the channel. The two ions now in the inner configuration (sites 2 and 4) can hop to binding sites 1 and 3, returning the channel to its initial state (the outer configuration), from which it can conduct a second K+ ion. (Adapted, with permission, from Miller 2001.)
The four inner α-helixes from each of the subunits line the cytoplasmic end of the pore. At the intracellular mouth of the channel these four helixes cross, forming a very narrow opening—the "smoke hole" of the teepee. The inner helixes are homologous to the S6 membrane-spanning segment of voltage-gated K+ channels. At the extracellular end of the channel the pair of transmembrane helixes from each subunit are connected by a region consisting of three elements: (1) a chain of amino acids that surrounds the mouth of the channel (the turret region); (2) an abbreviated α-helix (the pore helix) approximately 10 amino acids in length that projects toward the central axis of the pore; and (3) a stretch of 5 amino acids near the C-terminal end of the P-region that forms the selectivity filter (Figure 5–15B).
The shape and structure of the pore determine its ion-conducting properties. Both the inner and outer mouths of the pore are lined by acidic amino acids with negative charges that help attract cations from the bulk solution. Going from inside to outside, the pore consists of a medium wide tunnel 18 Å in length that leads into a wider (10 Å diameter) spherical inner chamber (Figure 5–15D). This chamber is lined predominantly by the side chains of hydrophobic amino acids. These relatively wide regions are followed by the very narrow selectivity filter, only 12 Å in length, which is rate-limiting for the passage of ions. A high ion throughput rate is ensured by the fact that the inner 28 Å of the pore, from the cytoplasmic entrance to the selectivity filter, lacks polar groups that could delay ion passage by binding and unbinding the ion.
An ion passing from the polar solution through the nonpolar lipid bilayer encounters the least energetically favorable region in the middle of the bilayer. The high energetic cost for a K+ ion to enter this region is minimized by two details of channel structure. The inner chamber is filled with water, which provides a highly polar environment, and the pore helixes provide a dipole whose electronegative carboxyl ends point toward this inner chamber (Figures 5–15C and 5–18A).
The high energetic cost incurred as a K+ ion sheds its waters of hydration is partially compensated by the presence of 20 oxygen atoms that line the walls of the selectivity filter and form favorable electrostatic interactions with the permeant ion. Each of the four subunits contributes four main-chain carbonyl oxygen atoms from the protein backbone and one side-chain hydroxyl oxygen atom to form a total of four binding sites for K+ ions. Each bound K+ ion is thus stabilized by interactions with a total of eight oxygen atoms, which lie in two planes above and below the bound cation. In this way the channel is able to compensate for the loss of the K+ ion's waters of hydration. The amino acid side chains of the selectivity filter, which are directed away from the central axis of the channel, help to stabilize the filter at a critical width, such that it provides optimal electrostatic interactions with K+ ions as they pass but is too wide for smaller Na+ ions to interact effectively with all eight carbonyl oxygens atoms at any point along the length of the filter (Figure 5–15C).
In light of the extensive interactions between a K+ ion and the channel, how does the KcsA channel manage its high rate of conduction? Although the channel contains a total of five potential binding sites for K+ ions—four in the selectivity filter and one in the inner chamber—X-ray analysis shows that the channel can be occupied by at most three K+ ions at any instant. One ion is normally present in the wide inner chamber, and two ions occupy two of the four binding sites within the selectivity filter (Figure 5–15D). Because of electrostatic repulsion, two K+ ions never simultaneously occupy adjacent binding sites within the selectivity filter; rather, a water molecule is always interspersed between K+ ions.
During conduction a pair of K+ ions within the selectivity filter hop in tandem between pairs of binding sites. If only one ion were in the selectivity filter it would be bound rather tightly, and the throughput rate for ion permeation would be compromised. But the mutual electrostatic repulsion between two K+ ions occupying nearby sites ensures that the ions will linger only briefly, thus resulting in a high overall rate of K+ conduction.
X-ray crystallographic analysis has also begun to provide insight into the conformational changes that underlie the opening and closing of K+ channels. Studies by Clay Armstrong in the 1960s suggested that a gate exists at the intracellular mouth of voltage-gated K+ channels of higher organisms. Small organic compounds such as tetraethylammonium can enter and block the channel only when this internal gate is opened by depolarization. We now know that this internal gate is the narrow opening formed by the crossing of the four α-helixes at the intracellular mouth of the channel. The small opening at the helix bundle crossing in KcsA revealed by X-ray crystallography turns out to be too narrow to allow ions to pass. Thus the X-ray crystal structure is that of a closed channel.
What do K+ channels look like when they are open? Although we do not know the answer for KcsA, MacKinnon and his colleagues determined the open structure for a related bacterial K+ channel, MthK. Each subunit of this channel has two transmembrane segments, similar to KcsA. Unlike KcsA, MthK has a cytoplasmic binding domain for Ca2+ and can be opened by high concentrations of internal Ca2+. MacKinnon and colleagues determined the structure of the open state by growing crystals of MthK in the presence of Ca2+. Remarkably, the inner helixes that form the tight bundle crossing in KcsA are bent in MthK at a flexible glycine residue that causes them to splay outward, forming an internal orifice that is dilated to 20 Å in diameter, wide enough to pass K+ as well as larger compounds such as tetraethylammonium (Figure 5–16). This mechanism is likely to be a general one because the inner helixes of many K+ channels of bacteria and higher organisms have a conserved glycine residue at this position. The presence of a bend at this glycine gating hinge was recently confirmed in the X-ray crystal structure of a mammalian voltage-gated K+ channel.
Gating of bacterial potassium channels.
Only two of four subunits are shown.
A. In the closed state the internal mouth of the pore formed by the inner helixes is too narrow to allow K+ to pass (based on the X-ray crystal structure of the KcsA channel).
B. In the open state the inner helixes are bent at a conserved glycine residue (red), widening the internal mouth of the pore (based on the X-ray crystal structure of the MthK channel). (Reproduced, with permission, from Yu et al. 2005; after Jiang et al. 2002.)
The Structural Basis of Chloride Selectivity Reveals a Close Relation Between Ion Channels and Ion Transporters
Ions move across cell membranes by active transport by ion transporters (or pumps) as well as diffusion through ion channels. Ion transporters are distinguished from ion channels because (1) they require a source of energy to actively transport ions against their electrochemical gradients and (2) they transport ions at rates much lower than those of ion channels, too low to support fast neuronal signaling. Nevertheless, some types of transporters and certain ion channels may have a similar structure according to studies of a bacterial membrane protein that transports protons and Cl−. These studies also yield insights into the structural basis for Cl− channel selectivity.
A large family of Cl− channels, the ClC channels, is widely expressed in neurons and other cells of vertebrates. In vertebrate skeletal muscle the ClC-1 channels are important in maintaining the resting potential. Mutations in the genes for these channels underlie certain inherited forms of myotonia; the loss of Cl− channel activity leads to a depolarizing after-potential following an action potential, resulting in repetitive firing of action potentials that produces abnormally prolonged muscle contraction. MacKinnon and his colleagues obtained a high- resolution X-ray crystal structure for a ClC protein from the bacterium Escherichia coli. Based on the close similarity of their amino acid sequences, it seems likely that the three-dimensional structures of the E. coli protein and vertebrate ClC channels would be very similar. Indeed, the E. coli ClC structure is consistent with the findings of a large number of previous studies of the effects of mutagenesis of vertebrate ClC channels.
It therefore came as a surprise when Chris Miller and colleagues found that the E. coli ClC is a transporter, not a channel. The E. coli ClC transporter uses the energy stored in a H+ gradient across the cell membrane to move Cl− against its electrochemical gradient from the inside of the cell to the outside in exchange for the transport of H+ from the outside of the cell to the inside, down its electrochemical gradient. This type of transporter is termed an ion exchanger. In the E. coli ClC two Cl− ions are transported in exchange for one proton. The rate of Cl− transport through the E. coli ClC by this exchange mechanism is 100- to 1,000-fold slower than that of vertebrate ClC channels.
The E. coli ClC protein, similar to vertebrate ClC channels, is a homodimer composed of two identical subunits. Each vertebrate ClC channel subunit forms a separate Cl− -conducting pore that gates independently from the other half (Figure 5–17A, B). The structure of the E. coli ClC transporter is much more complex than that of a K+ channel. Each subunit contains 18 α-helixes divided into related N-terminal and C-terminal halves, each containing nine helixes (Figure 5–17C, D). Surprisingly, the two halves are found in a head-to-head arrangement so that the helixes in each half have an opposite orientation in the membrane (helix 1 is related to helix 18, not helix 10). Unlike the pore of a K+ channel, which is widest in the central region, each pore of the E. coli ClC has an hourglass profile (Figure 5–18B). The neck of the hourglass, a tunnel 12 Å in length that forms the selectivity filter, is just wide enough to contain fully dehydrated Cl− ions.
The vertebrate ClC family of chloride channels and transporters are double-barrel channels with two identical pores.
A. Recordings of current through a single vertebrate Cl− channel show three levels of current: both pores closed (0), one pore open (1), and both pores open (2). (Reproduced, with permission, from Miller 1982.)
B. The ClC channels are dimers composed of two identical subunits, each forming a Cl−-selective pore. The channel is shown from the side (left) and looking down on the membrane from outside the cell (right). Each subunit contains its own ion transport pathway and gate. In addition, the dimer has a gate shared by both subunits (not shown). The drawing illustrates the functional properties of the channel.
C. The X-ray crystal structure of the Escherichia coli ClC Cl− /H+ exchanger in a side view (left) and top-down view (right). The ribbon diagram shows that each subunit is composed of a large number of α-helixes. Two Cl− ions (green spheres) are shown bound to each subunit: one ion is bound to the selectivity filter and a second is bound to an internal site closer to the cytoplasmic side of the membrane. (Reproduced, with permission, from Dutzler et al. 2002; Dutzler 2004.)
D. Left: The linear arrangement of the two cytoplasmic helixes (A and R) and 16-membrane helixes (B–Q) in a single subunit of the E. coli ClC transporter. The helixes from the N-terminal half of a subunit are shown in green, while the helixes from the C-terminal half are shown in cyan. (Reproduced, with permission, from Dutzler et al. 2002.) Right: The three-dimensional arrangement of the helixes in a single E. coli ClC subunit. The regions in red help form the Cl− permeation path. The Cl− ions at the selectivity filter (top) and an internal site (bottom) are illustrated as green spheres. A negative charge on the channel wall that may serve as the channel's gate is illustrated as a red sphere. (Reproduced, with permission, from Jentsch 2002.)
Comparison of general architectures of potassium and chloride channels.
(Reproduced, with permission, from Dutzler et al. 2002.)
A. In K+ channels the pore helixes (only two of four are shown here) are in a more or less parallel orientation, like the staves of a barrel. Their negatively charged dipoles point in the same direction to stabilize a K+ ion in the internal vestibule of the channel.
B. In Cl− channels the two pore helixes are in an antiparallel arrangement. Their positive dipole ends point toward the center of the channel to stabilize a Cl− ion in the middle of the channel. Only one subunit of the dimeric Cl− channel is shown.
Although the structures of the E. coli ClC transporter and K+ channel differ in significant respects, the two structures have evolved in four similar ways to achieve a high degree of ion selectivity. First, both can be occupied by multiple ions. The E. coli ClC contains three binding sites for Cl− ions within each selectivity filter. It appears that they can all be occupied simultaneously, thus creating a metastable state that ensures that the ions pass through the pore quickly. Second, in both cases the ion binding sites are formed by polar, partially charged atoms, not by fully ionized atoms. Thus the Cl− binding sites are formed by main chain amide nitrogen atoms, which bear a partial positive charge, and by side-chain hydroxyl groups. The binding energy provided by these polar groups is relatively weak, ensuring that the Cl− ions do not become too tightly bound. Third, in both structures permeant ions are stabilized in the center of the membrane by the partial charges of α helixes (Figure 5–18). In E. coli ClC the positively polarized (N-terminal) ends of two α-helixes dip partway into the membrane to lower the energetic barrier for Cl− ions within the nonpolar environment of the membrane. Fourth, in both cases wide water-filled vestibules at either end of the selectivity filter allow ions to approach the filter in a partially hydrated state (see Figure 5–18).
Thus we see that, although the K+ channel and E. coli ClC pores differ fundamentally in amino acid sequence and in secondary, tertiary, and quaternary structures, strikingly similar functional features have evolved in these two classes of membrane proteins to ensure both a high degree of ion selectivity and efficient throughput. These features have been conserved with surprising fidelity from prokaryotes through humans.
Why does the E. coli ClC function as a H+ /Cl− exchanger, whereas the vertebrate ClC proteins function as conventional channels? A likely explanation is that, unlike ion channels, the E. coli ClC does not have a continuous open pathway for ion movement from the outside of the membrane to the inside. Rather, like other pumps it is thought to have two gates, one external and one internal. Importantly, the two gates would never open simultaneously. Rather, ion movements and gate movements are presumed to be highly coupled in a tight cycle of reactions (Figure 5–19).
The functional difference between ion channels and pumps.
(Adapted, with permission, from Gadsby 2004.)
A. Channels have a continuous aqueous pathway for ion conduction across the membrane. This pathway can be occluded by the closing of a gate.
B. Pumps have two gates in series that control ion flux. The gates are never open simultaneously, but both can close to trap one or more ions in the pore. The type of pump illustrated here moves two different types of ions in opposite directions and is termed an exchanger or antiporter. Ion movement is tightly linked to a cycle of opening and closing of the two gates. When the external gate is open, one type of ion (red) leaves while the other type (blue) enters the channel (1). This triggers a conformational change, causing the external gate to shut, thereby trapping the incoming ion (2). A second conformational change then causes the internal gate to open, allowing the trapped ion to leave and a new ion to enter (3). A further conformational change opens the external gate, allowing the cycle to continue (4). With each cycle, one type of ion is transported from the outside to the inside of the cell, whereas the other type is transported from the inside to the outside of the cell. By coupling the movements of two or more ions, an exchanger can use the energy stored in the concentration gradient of one ion to actively transport another ion against its concentration gradient.
The relatively slow opening and closing of two gates in each cycle of ion transport, which takes place on a scale of milliseconds, explains why the rate of ion transfer through a transporter is several orders of magnitude slower than that of an ion channel. In the crystal structure of the E. coli ClC transporter the Cl− ion in the selectivity filter is trapped by a negatively charged side chain of a nearby glutamate residue on one side and an electronegative hydroxyl group from a serine residue on the other side. Protonation of this glutamate is likely to cause its side chain to rotate out of the way, permitting Cl− movement.
One or both of the E. coli ClC gates is thought to be missing in vertebrate ClC channels, providing an unobstructed pathway for the rapid diffusion of Cl − down its electrochemical gradient. Thus, despite the conservation of certain fundamental mechanisms in ClC proteins, relatively small structural changes give rise to two very distinct mechanisms of ion transport.