Berg J.M., Tymoczko J.L., Stryer L. Biochemistry

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proteins are shown in Table 13.1.

2. Ion channels exist in open and closed states. These channels undergo transitions from the closed state, incapable of

supporting ion transport, to the open state, through which ions can flow.

3. Transitions between the open and the closed states are regulated. Ion channels are divided into two classes: ligand-

gated channels and voltage-gated channels. Ligand-gated channels open and close in response to the binding of specific

chemicals, whereas voltage-gated channels open and close in response to the electrical potential across the membrane in

which they are found.

4. Open states of channels often spontaneously convert into inactivated states. Most ion channels do not remain in an

open state indefinitely but, instead, spontaneously transform into inactivated states that do not conduct ions. The

spontaneous transitions of ion channels from open to inactivated states act as built-in timers that determine the duration

of ion flow.

13.5.1. Patch-Clamp Conductance Measurements Reveal the Activities of Single

Channels

The study of ion channels has been revolutionized by the patch-clamp technique, which was introduced by Erwin Neher

and Bert Sakmann in 1976 (Figure 13.13). This powerful technique enables the measurement of the activity of a single

channel to be measured. A clean glass pipette with a tip diameter of about 1 µm is pressed against an intact cell to form a

seal. Slight suction leads to the formation of a very tight seal so that the resistance between the inside of the pipette and

the bathing solution is many gigaohms (1 gigaohm is equal to 10

ohms). Thus, a gigaohm seal (called a gigaseal)

ensures that an electric current flowing through the pipette is identical with the current flowing through the membrane

covered by the pipette. The gigaseal makes possible high-resolution current measurements while a known voltage is

applied across the membrane. In fact, patch clamping increased the precision of such measurements 100-fold. The flow

of ions through a single channel and transitions between the open and closed states of a channel can be monitored with

a time resolution of microseconds. Furthermore, the activity of a channel in its native membrane environment, even in an

intact cell, can be directly observed. Patch-clamp methods provided one of the first views of single biomolecules in

action. Subsequently, other methods for observing single molecules were invented, opening new vistas on biochemistry

at its most fundamental level.

13.5.2. Ion-Channel Proteins Are Built of Similar Units

How do ion channels, vital to a wide array of biological functions, operate at a molecular level? We will examine three

channels important in the propagation of nerve impulses: the ligand-gated channel; the acetylcholine receptor channel,

which communicates the nerve impulse between certain neurons; and the voltage-gated Na

and K

channels, which

conduct the nerve impulse down the axon of a neuron.

Nerve impulses are communicated across most synapses by small, diffusible molecules called neurotransmitters, of

which one is acetylcholine, referred to as a cholinergic neurotransmitter because it is derived from choline (Section

12.3.1). The presynaptic membrane of a synapse is separated from the postsynaptic membrane by a gap of about 50 nm,

called the synaptic cleft. The end of the presynaptic axon is filled with synaptic vesicles, each containing about 10

acetylcholine molecules (Figure 13.14). The arrival of a nerve impulse leads to the synchronous export of the contents of

some 300 vesicles, which raises the acetylcholine concentration in the cleft from 10 nM to 500 µM in less than a

millisecond. The binding of acetylcholine to the postsynaptic membrane markedly changes its ionic permeabilities

(Figure 13.15). The conductance of both Na

and K

increases greatly within 0.1 ms, leading to a large inward current

of Na

and a smaller outward current of K

. The inward Na

current depolarizes the postsynaptic membrane and

triggers an action potential (Section 13.5.3). Acetylcholine opens a single kind of cation channel, which is almost equally

permeable to Na

and K

. This change in ion permeability is mediated by the acetylcholine receptor.

The acetylcholine receptor is the best-understood ligand-gated channel. The activity of a single such channel is

graphically displayed in patch-clamp recordings of postsynaptic membranes of skeletal muscle (Figure 13.16). The

addition of acetylcholine is followed by transient openings of the channel. The current, i, flowing through an open

channel is 4 pA (picoamperes) when the membrane potential, V, is -100 mV. An ampere is the flow of 6.24 × 10

charges per second. Hence, 2.5 × 10

ions per second flow through an open channel.

The electric organ of Torpedo marmorata, an electric fish, is a choice source of acetylcholine receptors for study

because its electroplaxes (voltage-generating cells) are very rich in cholinergic postsynaptic membranes. The receptor is

very densely packed in these membranes (~20,000/µm

). An exotic biological material has been invaluable in the

isolation of acetylcholine receptors. Snake neurotoxins such as α -bungarotoxin (from the venom of a Formosan snake)

and cobratoxin block the transmission of impulses between nerve and muscle. These small (7-kd) basic proteins bind

specifically and very tightly to acetylcholine receptors and hence can be used as tags.

The acetylcholine receptor of the electric organ has been solubilized by adding a nonionic detergent to a

postsynaptic membrane preparation and purified by affinity chromatography on a column bearing covalently

attached cobratoxin. With the use of techniques presented in Chapter 4, the 268-kd receptor was identified as a pentamer

of four kinds of membrane-spanning subunits

, β, γ, and δ arranged in the form of a ring that creates a pore

through the membrane (Figure 13.17). The cloning and sequencing of the cDNAs for the four kinds of subunits (50 58

kd) showed that they have clearly similar sequences; the genes for the α, β, γ, and δ subunits arose by duplication and

divergence of a common ancestral gene. Each subunit has a large extracellular domain, followed at the carboxyl end by

four predominantly hydrophobic segments that span the bilayer membrane. Acetylcholine binds at the α

γ and α δ

interfaces. Electron microscopic studies of purified acetylcholine receptors demonstrated that the structure has

approximately fivefold symmetry, in harmony with the similarity of its five constituent subunits.

What is the basis of channel opening? A comparison of the structures of the closed and open forms of the channel would

be highly revealing, but such comparisons have been difficult to obtain. Cryoelectron micrographs indicate that the

binding of acetylcholine to the extracellular domain causes a structural alteration, which initiates rotations of the α-

helical rods lining the membrane-spanning pore. The amino acid sequences of these helices point to the presence of

alternating ridges of small polar or neutral residues (serine, threonine, glycine) and large nonpolar ones (isoleucine,

leucine, phenylalanine). In the closed state, the large residues may occlude the channel by forming a tight hydrophobic

ring (Figure 13.18). Indeed, each subunit has a bulky leucine residue at a critical position. The binding of acetylcholine

could allosterically rotate the membrane-spanning helices so that the pore would be lined by small polar residues rather

than by large hydrophobic ones. The wider, more polar pore would then be open to the passage of Na

and K

ions.

13.5.3. Action Potentials Are Mediated by Transient Changes in Na

and K

Permeability

We turn now from ligand-gated channels to voltage-gated channels, which are responsible for the propagation of nerve

impulses. A nerve impulse is an electrical signal produced by the flow of ions across the plasma membrane of a neuron

and is the fundamental means of communication in the nervous system. The interior of a neuron, like that of most other

cells, has a high concentration of K

and a low concentration of Na

. These ionic gradients are generated by an ATP-

driven pump (Section 13.2.1). In the resting state, the membrane potential is -60 mV. A nerve impulse, or action

potential, is generated when the membrane potential is depolarized beyond a critical threshold value (i.e., from -60 to -40

mV). The membrane potential becomes positive within about a millisecond and attains a value of about +30 mV before

turning negative again. This amplified depolarization is propagated along the nerve terminal (Figure 13.19)

Ingenious experiments carried out by Alan Hodgkin and Andrew Huxley revealed that action potentials arise from large,

transient changes in the permeability of the axon membrane to Na

and K

ions (see Figure 13.19A). Two kinds of

voltage-sensitive channels, one selectively permeable to Na

and the other to K

, were defined. The conductance of the

membrane to Na

changes first. Depolarization of the membrane beyond the threshold level leads to an opening of Na

channels. Sodium ions begin to flow into the cell because of the large electrochemical gradient across the plasma

membrane. The entry of Na

further depolarizes the membrane, and so more gates for Na

are opened. This positive

feedback between depolarization and Na

entry leads to a very rapid and large change in membrane potential, from about

-60 mV to +30 mV in a millisecond.

Sodium channels spontaneously close and potassium channels begin to open at about this time (see Figure 13.19B).

Consequently, potassium ions flow outward, and so the membrane potential returns to a negative value. The resting level

of -60 mV is restored in a few milliseconds as the K

conductance decreases to the value characteristic of the

unstimulated state. Only a very small proportion of the sodium and potassium ions in a nerve cell, of the order of one in a

million, flows across the plasma membrane during the action potential. Clearly, the action potential is a very efficient

means of signaling over large distances.

13.5.4. The Sodium Channel Is an Example of a Voltage-Gated Channel

Like the acetylcholine receptor channel, the sodium channel also was purified on the basis of its ability to bind a specific

neurotoxin. Tetrodotoxin, an organic compound isolated from the puffer fish, binds to sodium channels with great

avidity (K

1 nM). The lethal dose of this poison for an adult human being is about 10 ng. The sodium channel was

first purified from the electric organ of electric eel, which is a rich source of the protein forming this channel. The

isolated protein is a single chain of 260 kd.

The availability of purified protein enabled Shosaku Numa and coworkers to clone and sequence the cDNA for the

sodium channel from the electroplax cells of the eel electric organ and then from the rat. Subsequently, a large

number of sodium channel cDNAs have been cloned from other sources, and sequence comparisons have been made.

The eel and rat cDNA sequences are approximately 61% identical, which indicates that the amino acid sequence of the

sodium channel has been conserved over a long evolutionary period. Most interesting, the channel contains four internal

repeats, or homology units, having similar amino acid sequences, suggesting that gene duplication and divergence have

produced the gene for this channel. Hydrophobicity profiles indicate that each homology unit contains five hydrophobic

segments (S1, S2, S3, S5, and S6). Each repeat also contains a highly positively charged S4 segment; arginine or lysine

residues are present at nearly every third residue. Numa proposed that segments S1 through S6 are membrane-spanning

α helices (Figure 13.20). The positively charged residues in S4 segments act as the voltage sensors of the channel. The

purification of calcium channels and the subsequent cloning and sequencing of their cDNAs revealed that these proteins

are homologous to the sodium channels and have quite similar architectures; each protein comprises four imperfectly

repeated units, each of which has regions corresponding to segments S1 through S6.

We can thus note similarities between ligand-gated and voltage-gated channels. Like the acetylcholine receptor, the

sodium channel is constructed of similar units. The acetylcholine receptor has five units, whereas the sodium channel has

four units that have been fused into a single polypeptide chain. The acetylcholine receptor is composed of similar but

noncovalently attached subunits.

13.5.5. Potassium Channels Are Homologous to the Sodium Channel

The purification of potassium channels proved to be much more difficult because of their low abundance and the

lack of known high-affinity ligands comparable to tetrodotoxin. The breakthrough came in studies of mutant fruit

flies that shake violently when anesthetized with ether. The mapping and cloning of the gene, termed shaker, responsible

for this defect revealed the amino acid sequence encoded by a potassiumchannel gene. The availability of this gene

sequence has led to the cloning of potassium-channel cDNAs from many other organisms. Shaker cDNA encodes a 70-

kd protein that has regions that correspond to one of the homology units of the sodium channel containing the membrane-

spanning segments S1 through S6. Thus, a potassium-channel subunit is homologous to one of the repeated homology

units of the sodium and calcium channels. Consistent with this hypothesis, four potassium-channel subunits come

together to form a functional channel. Subsequently, other potassium channels were discovered, including some from

bacteria, which contain only the two membrane-spanning regions corresponding to segments S5 and S6. This and other

information pointed to the region between S5 and S6 as a key component of the ion-channel pore in the potassium

channel and in the sodium and calcium channels as well. The sequence relationships between these ion channels are

summarized in Figure 13.21.

13.5.6. The Structure of a Potassium Channel Reveals the Basis of Rapid Ion Flow with

Specificity

Structural Insights, The Potassium Channel, examines the structural basis of

the potassium channel's ion specificity and high conductivity in further detail.

Scientists were slowly discovering the likely structures of ion channels through a combination of patch-clamp methods,

site-directed mutagenesis, and other methods. However, progress was limited by the lack of a high- resolution three-

dimensional structure. The need was met by the determination of the structure of a bacterial potassium channel by x-ray

crystallography in 1998. The resulting structural framework is a source of insight into many aspects of ion-channel

function, including specificity and rapidity of ion flow.

As expected, the potassium channel is a tetramer of identical subunits, each of which includes two membrane-spanning

α helices. The four subunits come together to form a pore in the shape of a cone that runs through the center of the

structure (Figure 13.22). Beginning from the inside of the cell, the pore starts with a diameter of approximately 10 Å and

then constricts to a smaller cavity with a diameter of 8 Å. Both the opening to the outside and the central cavity of the

pore are filled with water, and a K

ion can fit in the pore without losing its shell of bound water molecules.

Approximately two-thirds of the way through the membrane, the pore becomes more constricted (3-Å diameter). At that

point, any K

ions must give up their water molecules and interact directly with groups from the protein. The channel

structure effectively reduces the thickness of the membrane from 34 Å to 12 Å by allowing the solvated ions to penetrate

into the membrane before the ions must directly interact with the channel (Figure 13.23).

For potassium ions to relinquish their water molecules, other polar interactions must replace those with water. The

restricted part of the pore is built from residues between the two transmembrane helices (which correspond to segments

S5 and S6 in the sodium channel). In particular, a five-amino-acid stretch within this region functions as the selectivity

filter that determines the preference for K

over other ions (Figure 13.24). The stretch has the sequence Thr-Val-Gly-Tyr-

Gly, which is nearly completely conserved in all K

channels and had already been identified as a signature sequence

useful for identifying potential K

channels. This region lies in a relatively extended conformation and is oriented such

that the peptide carbonyl groups are directed into the channel, facilitating interaction with the potassium ions.

Potassium channels are 100-fold as permeable to K

as to Na

. How is this high degree of selectivity achieved? The

narrow diameter (3 Å) of the selectivity filter of the potassium channel enables the filter to reject ions having a radius

larger than 1.5 Å. However, a bare Na

is small enough (Table 13.2) to pass through the pore. Indeed, the ionic radius of

is substantially smaller than that of K

. How then is Na

rejected?

We need to consider the free-energy cost of dehydrating the Na

and K

ions, given that they cannot pass through this

part of the channel bearing a retinue of water molecules. The key point is that the free-energy costs of dehydrating these

ions are considerable [Na

, 72 kcal mol

-1

(301 kJ mol

-1

), and K

, 55 kcal mol

-1

(203 kJ mol

-1

)]. The channel pays the

cost of dehydrating K

by providing compensating interactions with the carbonyl oxygen atoms lining the selectivity

filter. However, these oxygen atoms are positioned such that they do not interact very favorably with Na

, because it is

too small (Figure 13.25). The higher cost of dehydrating Na

would be unrecovered, and so Na

would be rejected. The

ionic radii of oxygen, potassium, and sodium are 1.4, 1.33, and 0.95 Å, respectively. Hence a ring of oxygen atoms

positioned so that the K

O distance is 2.73 Å (1.4 + 1.33 Å) would be optimal for interaction with K

compared with

the shorter Na

O bonds (0.95 + 1.4 = 2.35 Å) optimal for interaction with Na

. Thus, the potassium channel avoids

closely embracing Na

ions, which must stay hydrated and hence are impermeant.

13.5.7. The Structure of the Potassium Channel Explains Its Rapid Rates of Transport

In addition to selectivity, ion channels display rapid rates of ion transport. A structural analysis provides an appealing

explanation for this proficiency. The results of such studies revealed the presence of two potassium-binding sites in the

constricted regions of the potassium channel that are crucial for rapid ion flow. Consider the process of ion conductance.

One K

ion proceeds into the channel and through the relatively unrestricted part of the channel. It then gives up most or

all of its coordinated water molecules and binds to the first site in the selectivity filter region, a favorable binding site. It

can then jump to the second site, which appears to have comparable binding energy. However, the binding energy of the

second site presents a free-energy barrier, or trap, preventing the ion from completing its journey; there is no energetic

reason to leave the second ion-binding site. However, if a second ion moves through the channel into the first site, the

electrostatic repulsion between the two ions will destabilize the initially bound ion and help push it into solution (Figure

13.26). This mechanism provides a solution to the apparent paradox of high ion selectivity (requiring tight binding sites)

and rapid flow.

The structure determined for K

channels is a good start for considering the amino acid sequence similarities, as

well as the structural and functional relations, for Na

and Ca

channels because of their homology to K

channels. Sequence comparisons and the results of mutagenesis experiments have also implicated the region between

segments S5 and S6 in ion selectivity in the Ca

channels. In Ca

channels, one glutamate residue of this region in

each of the four units plays a major role in determining ion selectivity. The Na

channel's selection of Na

over K

depends on ionic radius; the diameter of the pore is sufficiently restricted that small ions such as Na

and Li

can pass

through the channel, but larger ions such as K

are significantly hindered (Figure 13.27).

Residues in the positions corresponding to the glutamate residues in Ca

channels are major components of the

selectivity filter of the Na

channel. These residues are aspartate, glutamate, lysine, and alanine in units 1, 2, 3, and 4,

respectively (the DEKA locus). Thus, the potential fourfold symmetry of the channel is clearly broken in this region,

providing one explanation of why Na

channels comprise single large polypeptide chains rather than a noncovalent

assembly of four identical subunits.

13.5.8. A Channel Can Be Inactivated by Occlusion of the Pore: The Ball-and-Chain

Model

The potassium channel and the sodium channel undergo inactivation within milliseconds of channel opening (Figure

13.28). A first clue to the mechanism of inactivation came from exposing the cytoplasmic side of either channel to

trypsin; cleavage by trypsin produced a trimmed channel that stayed persistently open after depolarization. A second clue

was the finding that alternatively spliced variants of the potassium channel have markedly different inactivation kinetics;

these variants differed from one another only near the amino terminus, which is on the cytoplasmic side of the channel.

A mutant Shaker channel lacking 42 amino acids near the amino terminus opened in response to depolarization but did

not inactivate (see Figure 13.28). Most revealing, inactivation was restored by adding a synthetic peptide corresponding

to the first 20 residues of the native channel.

These experiments strongly support the ball-and-chain model for channel inactivation that had been proposed years

earlier (Figure 13.29). According to this model, the first 20 residues of the potassium channel form a cytoplasmic unit

(the ball) that is attached to a flexible segment of the polypeptide (the chain). When the channel is closed, the ball rotates

freely in the aqueous solution. When the channel opens, the ball quickly finds a complementary site in the pore and

occludes it. Hence, the channel opens for only a brief interval before it undergoes inactivation by occlusion. Shortening

the chain speeds inactivation because the ball finds its target more quickly. Conversely, lengthening the chain slows

inactivation. Thus, the duration of the open state can be controlled by the length and flexibility of the tether.

I. The Molecular Design of Life 13. Membrane Channels and Pumps 13.5. Specific Channels Can Rapidly Transport Ions Across Membranes

Table 13.1. Relative permeabilities for selected ion channels

channel K

channel

Acetylcholine receptor Chloride channel

0.93 < 0.01 0.87 < 0.01

1.00 < 0.01 1.00 < 0.01

0.09 1.00 1.11 < 0.01

< 0.01 0.91

< 0.01 < 0.08 1.42

0.16 0.13 1.79

NOH

0.94 < 0.03 1.92

NNH

0.59 < 0.03

CNH

< 0.01 < 0.02

< 0.01 < 0.01 < 0.01 1.00

I. The Molecular Design of Life 13. Membrane Channels and Pumps 13.5. Specific Channels Can Rapidly Transport Ions Across Membranes

Figure 13.13. Patch-Clamp Modes. The patch-clamp technique for monitoring channel activity is highly versatile. A

high-resistance seal (gigaseal) is formed between the pipette and a small patch of plasma membrane. This configuration

is called cell attached. The breaking of the membrane patch by increased suction produces a low-resistance pathway

between the pipette and interior of the cell. The activity of the channels in the entire plasma membrane can be monitored

in this whole-cell mode. To prepare a membrane in the excised-patch mode, the pipette is pulled away from the cell. A

piece of plasma membrane with its cytosolic side now facing the medium is monitored by the patch pipette.

I. The Molecular Design of Life 13. Membrane Channels and Pumps 13.5. Specific Channels Can Rapidly Transport Ions Across Membranes

Figure 13.14. Schematic Representation of a Synapse.

I. The Molecular Design of Life 13. Membrane Channels and Pumps 13.5. Specific Channels Can Rapidly Transport Ions Across Membranes

Figure 13.15. Membrane Depolarization. Acetylcholine depolarizes the postsynaptic membrane by increasing the

conductance of Na

and K

I. The Molecular Design of Life 13. Membrane Channels and Pumps 13.5. Specific Channels Can Rapidly Transport Ions Across Membranes

Figure 13.16. Patch-Clamp Recordings of the Acetylcholine Receptor Channel. Patch-clamp recordings illustrate

changes in the conductance of an acetylcholine receptor channel in the presence of acetylcholine. The channel undergoes

frequent transitions between open and closed states. [Courtesy of Dr. D. Colquhoun and Dr. B. Sakmann.]

I. The Molecular Design of Life 13. Membrane Channels and Pumps 13.5. Specific Channels Can Rapidly Transport Ions Across Membranes

The torpedo (Torpedo marmorata, also known as the electric ray) has an electric organ, rich in acetylcholine receptors,

that can deliver a shock of as much as 200 V for approximately 1 s. [Yves Gladu/Jacana/ Photo Researchers.]

I. The Molecular Design of Life 13. Membrane Channels and Pumps 13.5. Specific Channels Can Rapidly Transport Ions Across Membranes

Figure 13.17. Schematic Representation of the Closed Form of the Acetylcholine Receptor Channel. In the closed

state, the narrowest part of the pore is occluded by side chains coming from five helices. [Courtesy of Dr. Nigel Unwin.]

I. The Molecular Design of Life 13. Membrane Channels and Pumps 13.5. Specific Channels Can Rapidly Transport Ions Across Membranes

Figure 13.18. Opening of the Acetylcholine Channel Pore. Large hydrophobic side chains (L) occlude the pore of the

closed form of the acetylcholine receptor channel. Channel opening is probably mediated by the tilting of helices that

line the pore. Large residues move away from the pore and small ones (S) take their place. [After N. Unwin. Neuron 3

(1989):665.]

I. The Molecular Design of Life 13. Membrane Channels and Pumps 13.5. Specific Channels Can Rapidly Transport Ions Across Membranes

Figure 13.19. Membrane Potential. Depolarization of an axon membrane results in an action potential. Time course of

(A) the change in membrane potential and (B) the change in Na

and K

conductances.

I. The Molecular Design of Life 13. Membrane Channels and Pumps 13.5. Specific Channels Can Rapidly Transport Ions Across Membranes

A puffer fish is regarded as a culinary delicacy in Japan. [Fred Bavendam/ Peter Arnold.]

I. The Molecular Design of Life 13. Membrane Channels and Pumps 13.5. Specific Channels Can Rapidly Transport Ions Across Membranes

Figure 13.20. The Sodium Channel. The Na

channel is a single polypeptide chain with four repeating units (I IV).

Each repeat probably folds into six transmembrane helices. The loops (shown in red) between helices 5 and 6 of each

domain form the pore of the channel.

I. The Molecular Design of Life 13. Membrane Channels and Pumps 13.5. Specific Channels Can Rapidly Transport Ions Across Membranes

Figure 13.21. Sequence Relationships of Ion Channels. Like colors indicate structurally similar regions of the sodium,

calcium, and potassium channels. These channels exhibit approximate fourfold symmetry.