Opening of Acetylcholine-Gated Cation Channels Leads to Muscle Contraction

The nicotinic acetylcholine receptor, a ligand-gated cation channel, admits both K⁺ and Na⁺. Although found in some neurons, this receptor is best known for its role in synapses between motor neurons and skeletal muscle cells. Patch-clamping studies on isolated outside-out patches of muscle plasma membranes have shown that acetylcholine causes opening of a cation channel in the receptor capable of transmitting 15,000 – 30,000 Na⁺ or K⁺ ions a millisecond.

Since the resting potential of the muscle plasma membrane is near E_K, the potassium equilibrium potential, opening of acetylcholine receptor channels causes little increase in the efflux of K⁺ ions; Na⁺ ions, on the other hand, flow into the muscle cell. The simultaneous increase in permeability to Na⁺ and K⁺ ions produces a net depolarization to about −15 mV from the muscle resting potential of −85 to −90 mV. This depolarization of the muscle membrane generates an action potential, which — like an action potential in a neuron — is conducted along the membrane surface via voltage-gated Na⁺ channels (see Figure 21-14). As detailed in Section 18.4, when the membrane depolarization reaches a specialized region, it triggers Ca²⁺ movement from its intracellular store, the sarcoplasmic reticulum, into the cytosol; the resultant rise in cytosolic Ca²⁺ induces muscle contraction (Figure 21-37).

Figure 21-37

Sequential activation of gated ion channels at a neuromuscular junction. Arrival of an action potential at the terminus of a presynaptic motor neuron induces opening of voltage-gated Ca²⁺ (more...)

Two factors greatly assisted in the characterization of the nicotinic acetylcholine receptor. First, this receptor can be rather easily purified from the electric organs of electric eels and electric rays; these organs are derived from stacks of muscle cells (minus the contractile proteins) and thus are richly endowed with this receptor. (In contrast, this receptor constitutes a minute fraction of the total membrane protein in most nerve and muscle tissues.) Second, α-bungarotoxin, a neurotoxin present in snake venom, binds specifically and irreversibly to nicotinic acetylcholine receptors. This toxin can be used in purifying the receptor by affinity chromatography and in localizing it. For instance, in autoradiographs of muscle-cell sections exposed to radioactive α-bungarotoxin, the toxin is localized in the plasma membrane of postsynaptic striated muscle cells immediately adjacent to the terminals of presynaptic neurons.

Careful monitoring of the membrane potential of the muscle membrane at a synapse with a cholinergic motor neuron has demonstrated spontaneous, intermittent, and random ≈2-ms depolarizations of about 0.5 – 1.0 mV in the absence of stimulation of the motor neuron. Each of these depolarizations is caused by the spontaneous release of acetylcholine from a single synaptic vesicle. Indeed, demonstration of such spontaneous small depolarizations led to the notion of the quantal release of acetylcholine (later applied to other neurotransmitters) and thereby led to the hypothesis of vesicle exocytosis at synapses. The release of one acetylcholine-containing synaptic vesicle results in the opening of about 3000 ion channels in the postsynaptic membrane, far short of the number needed to reach the threshold depolarization that induces an action potential. Clearly, stimulation of muscle contraction by a motor neuron requires the nearly simultaneous release of acetylcholine from numerous synaptic vesicles.

All Five Subunits in the Nicotinic Acetylcholine Receptor Contribute to the Ion Channel

The acetylcholine receptor from skeletal muscle is a pentameric protein with a subunit composition of α₂βγδ. Each molecule has a diameter of about 9 nm and protrudes about 6 nm into the extracellular space and about 2 nm into the cytosol (Figure 21-38a). The α, β, γ, and δ subunits have considerable sequence homology; on average, about 35 – 40 percent of the residues in any two subunits are similar. The complete receptor has a fivefold symmetry, and the actual cation channel is a tapered central pore, with a maximum diameter of 2.5 nm, formed by segments from each of the five subunits (Figure 21-38b).

Figure 21-38

Three-dimensional structure of the nicotinic acetylcholine receptor based on amino acid sequence data, computer-generated averaging of high-resolution electron micrographs, and information (more...)

The channel opens when the receptor cooperatively binds two acetylcholine molecules to sites located at the interfaces of the αδ and αγ subunits. Once acetylcholine is bound to a receptor, the channel is opened virtually instantaneously, probably within a few microseconds. Studies measuring the permeability of different small cations suggest that the open ion channel is, at its narrowest, about 0.65 – 0.80 nm in diameter, in agreement with estimates from electron micrographs. This would be sufficient to allow passage of both Na⁺ and K⁺ ions with their bound shell of water molecules (see Figure 2-14).

Although the structure of the central ion channel is not known in molecular detail, much evidence indicates that it is lined by five transmembrane M2 α helices, one from each of the five subunits. The M2 helices are composed largely of hydrophobic or uncharged polar amino acids, but negatively charged aspartate or glutamate residues are located at each end, near the membrane faces, and several serine or threonine residues are near the middle. If a single negatively charged glutamate or aspartate in one subunit is mutated to a positively charged lysine, and the mutant mRNA is injected together with mRNAs for the other three wild-type subunits into frog oocytes, a functional channel is expressed, but its ion conductivity — the number of ions that can cross it during the open state — is reduced. The greater the number of glutamate or aspartate residues mutated (in one or multiple subunits), the greater the reduction in conductivity. These findings suggest that aspartate and glutamate residues  — one residue from each of the five chains — form a ring of negative charges on the external surface of the pore that help to screen out anions and attract Na⁺ or K⁺ ions as they enter the channel (see Figure 21-38a). A similar ring of negative charges lining the cytosolic pore surface also helps select cations for passage.

The two acetylcholine-binding sites in the extracellular domain of the receptor lie ≈4 to 5 nm from the center of the pore. Binding of acetylcholine thus must trigger conformational changes in the receptor subunits that can cause channel opening at some distance from the binding sites. Receptors in isolated postsynaptic membranes can be trapped in the open or closed state by rapid freezing in liquid nitrogen. Images of such preparations suggest that the five M2 helices rotate relative to the vertical axis of the channel during opening and closing (Figure 21-39).

Figure 21-39

Schematic threedimensional models of the pore-lining M2 helices in the closed and opened states. In the closed state, the kink in the center of each M2 helix points inward, constricting the (more...)

Two Types of Glutamate-Gated Cation Channels May Function in a Type of “Cellular Memory”

The hippocampus is the region of the mammalian brain associated with many types of short-term memory. Certain types of hippocampal neurons, here simply called postsyn-aptic cells, receive inputs from hundreds of presynaptic cells. In long-term potentiation a burst of stimulation of a postsynaptic neuron makes it more responsive to subsequent stimulation by presynaptic neurons. For example, stimulation of a hippocampal presynaptic nerve with 100 depolarizations acting over only 200 milliseconds causes an increased sensitivity of the postsynaptic neuron that lasts hours to days. Changes in the responses of postsynaptic cells may underlie certain types of memory.

Two types of glutamate-gated cation channels in the postsynaptic neuron participate in long-term potentiation. Like other neurotransmitter-gated ion channels, both glutamate receptors have five subunits, each containing a pore-lining M2 helix; both are excitatory receptors, causing depolarization of the plasma membrane when activated. Because the two receptors were initially distinguished by their ability to be activated by the non-natural amino acid N-methyl-D-aspartate (NMDA), they are called NMDA glutamate receptors and non-NMDA glutamate receptors.

As illustrated in Figure 21-40, non-NMDA receptors are “conventional” in that binding of glutamate, released from the presynaptic cell, triggers their opening. NMDA glutamate receptors are different in two key respects. First, they allow influx of Ca²⁺ as well as Na⁺. Second, and more important, two conditions must be fulfilled for the ion channel to open: glutamate must be bound and the membrane must be partly depolarized. In this way, the NMDA receptor functions as a coincidence detector; that is, it integrates activity of the postsynaptic cell — reflected in its depolarized plasma membrane — with release of neurotransmitter from the presynaptic cell, generating a cellular response greater than that caused by glutamate release alone. Once a postsynaptic cell becomes “sensitized,” it takes fewer action potentials in the presynaptic neurons to induce a given depolarization in the postsynaptic neuron; in other words, the synapse “learns” to have an enhanced response to signals from the presynaptic cells.

Figure 21-40

Different properties of two types of glutamate receptors found in the hippocampus region in the brain. Because the ion channel in the NMDA receptor (green) normally is blocked by a Mg²⁺ ion, (more...)

Opening of NMDA receptors depends on membrane depolarization because of the voltage-sensitive blocking of the ion channel by a Mg²⁺ ion from the extracellular solution. A small depolarization of the membrane causes the Mg²⁺ ion to dissociate from the receptor, thereby making it possible for glutamate binding to open the channel. Mutagenesis of a single asparagine residue in the pore-lining M2 helix of the NMDA receptor abolishes the effect of Mg²⁺, indicating that Mg²⁺ binds in the channel.

Since activation of a single synapse, even at high frequency, generally causes only a small depolarization of the membrane of the postsynaptic cell, long-term potentiation is induced only when many synapses simultaneously stimulate a single postsynaptic neuron. Thus the requirement for membrane depolarization explains why long-term potentiation depends on the simultaneous activation of a large number of synapses on the postsynaptic cell.

GABA- and Glycine-Gated Cl⁻ Channels Are Found at Many Inhibitory Synapses

Synaptic inhibition in the vertebrate central nervous system is mediated primarily by two amino acids, glycine and γ-aminobutyric acid (GABA); the latter is formed from glutamate by loss of a carboxyl group. The concentration of GABA in the human brain is 200 — 1000 times higher than that of other neurotransmitters such as dopamine, norepinephrine, and acetylcholine. Glycine is the major inhibitory neurotransmitter in the spinal cord and brain stem; GABA predominates elsewhere in the brain. Both glycine and GABA activate ligand-gated Cl⁻ channels.

The opening of Cl⁻ channels tends to drive the membrane potential toward the Cl⁻ equilibrium potential E_Cl, which in general is slightly more negative than the resting membrane potential (see Figure 21-10). In other words, the membrane becomes slightly hyperpolarized. If many Cl⁻ channels are opened, the membrane potential will be held near E_Cl, and a much larger than normal increase in the Na⁺ permeability will then be required to depolarize the membrane. The effect of GABA or glycine on Cl⁻ permeability is induced rapidly (a fraction of a millisecond) but can last for a second or more, a long time compared with the millisecond required to generate an action potential. Thus GABA or glycine rapidly induces a fairly long-lasting inhibitory postsynaptic response.

GABA and glycine receptors have been purified, cloned, and sequenced. Although they are pentameric proteins, like the nicotinic acetylcholine receptor, GABA and glycine receptors are built of only one or two different types of subunits. Each subunit has a transmembrane M2 helix; these are thought to line the ion channel as in the nicotinic acetylcholine receptor. As mentioned previously, the negatively charged glutamate and aspartate side chains at the ends of the M2 helices in acetylcholine receptors may participate in selecting cations for passage (see Figure 21-38a). Strikingly, the M2 helices of the GABA and glycine receptor subunits have lysine or arginine residues at these positions; the positively charged side chains of these residues may attract Cl⁻ ions specifically and aid in repelling cations.

Cardiac Muscarinic Acetylcholine Receptors Activate a G Protein That Opens K⁺ Channels

We saw earlier that binding of acetylcholine to muscarinic acetylcholine receptors in cardiac muscle generates a slow inhibitory response (see Figure 21-32b). Stimulation of the cholinergic nerves in heart muscle, which causes a long-lived (several seconds) hyperpolarization of the membrane, is one of the principal ways by which the rate of heart muscle contraction is slowed.

The muscarinic acetylcholine receptor is a G protein – coupled receptor whose activation leads to opening of K⁺ channels and subsequent hyperpolarization of the plasma membrane. Like other G protein – coupled receptors, the muscarinic acetylcholine receptor has seven transmembrane α helices (see Figure 20-10). As depicted in Figure 21-41, binding of acetylcholine to the receptor activates a trimeric transducing G protein; the released G_βγ subunit then directly binds to and opens a particular K⁺ channel protein. That G_βγ directly activates the K⁺ channel has been shown by single-channel recording experiments in which purified G_βγ was added to the cytosolic face of a patch of heart muscle plasma membrane (see Figure 20-21b). Potassium channels opened immediately on addition of G_βγ in the absence of acetylcholine or other neurotransmitters. The K⁺ channels coupled to muscarinic acetylcholine receptors are tetrameric proteins similar in structure to those that maintain the resting membrane potential.

Figure 21-41

Acetylcholine-induced opening of K⁺ channels in the heart muscle plasma membrane. Binding of acetylcholine by muscarinic acetylcholine receptors triggers activation of a transducing G protein (more...)

The cardiac muscarinic receptor illustrates one way in which G protein – coupled receptors affect ion channels: the active G_βγ subunit binds to a channel protein. Activation of other G protein – coupled neurotransmitter receptors affects the activity of enzymes that synthesize or degrade intracellular second messengers; these, in turn, can affect the activity of channel proteins. To illustrate this type of receptor, we examine the catecholamine receptors.

Catecholamine Receptors Induce Changes in Second-Messenger Levels That Affect Ion-Channel Activity

Epinephrine and norepinephrine function as both systemic hormones and neurotransmitters. Norepinephrine is the transmitter at synapses with smooth muscles that are innervated by sympathetic autonomic motor neurons (see Figure 21-6). Stimulation of these peripheral neurons increases the activity of the heart and internal organs in “fight or flight” reactions. Norepinephrine is also found at synapses in the central nervous system. Epinephrine is synthesized and released into the blood by the adrenal medulla, an endocrine organ that has a common embryologic origin with neurons of the sympathetic system. Unlike neurons, the medulla cells do not develop axons or dendrites. Epinephrine, norepinephrine, and the related neurotransmitter dopamine are all synthesized from tyrosine and contain the catechol moiety; hence they are referred to as catecholamines (see Figure 21-28). Nerves that synthesize and use epinephrine or norepinephrine are termed adrenergic.

All known receptors for catecholamines are coupled to G proteins. Because different receptors are linked to different G proteins, their activation leads to changes in the levels of different intracellular second messengers. For instance, binding of norepinephrine to β-adrenergic receptors on nerve cells causes activation of G_s and an increase in cAMP synthesis, the same mechanism by which β-adrenergic receptors function in non-neuronal cells (see Figure 20-16). Other neuronal adrenergic receptors activate G_i, G_o, or other types of G proteins, resulting in a decrease in cAMP levels or increases in the levels of other intracellular second messengers, such as cGMP, inositol 1,4,5-trisphosphate (IP₃), diacylglycerol, and arachidonic acid (see Table 20-5). Some second messengers, such as cGMP and IP₃, act to directly open or close ion channels in neurons; IP₃, for example, opens Ca²⁺ channels in the membrane of the endoplasmic reticulum, causing an increase in cytosolic Ca²⁺. Other second messengers have a more indirect effect on ion channels, as exemplified by the serotonin receptor, another G protein coupled receptor.

A Serotonin Receptor Indirectly Modulates K⁺ Channel Function by Activating Adenylate Cyclase

Often an axon terminal of one neuron synapses with the axon terminal of another neuron. Such a modulatory synapse may either inhibit or stimulate the ability of the second axon terminal to release its neurotransmitter and signal a third cell. The operation of one such modulatory synapse in the sea slug Aplysia demonstrates the effect of an increase in cAMP on ion-channel function. In this example, a particular type of interneuron, called a facilitator neuron, forms a synapse with the axon terminal of a sensory neuron that stimulates a motor neuron by releasing glutamate. Stimulation of the facilitator neuron increases the ability of the sensory neuron to stimulate the motor neuron.

As illustrated in Figure 21-42, when the facilitator neuron is stimulated, it secretes serotonin, which binds to serotonin receptors on the sensory neuron (steps 1 and 2). This binding activates adenylate cyclase, triggering the synthesis of cAMP in the sensory neuron (step 3). Subsequent activation of cAMP-dependent protein kinase leads to phosphorylation of a voltage-gated K⁺ channel protein or an associated protein, thereby preventing opening of the K⁺ channels during an action potential (steps 4 and 5). This inhibition decreases the outward flow of K⁺ ions that normally repolarizes the membrane of the sensory neuron after an action potential reaches the axon terminal. The resulting prolonged membrane depolarization increases the influx of Ca²⁺ ions through voltage-gated Ca²⁺ channels (step 6). The increased Ca²⁺ level leads to greater exocytosis of glutamate-containing synaptic vesicles in the sensory neuron (step 7), and hence greater activation of the motor neuron (step 8) each time an action potential reaches the terminal.

Figure 21-42

Action of a serotonin modulatory synapse in the sea slug Aplysia punctata.. Serotonin secreted by an activated facilitator neuron binds to the G protein-coupled serotonin receptors, leading (more...)

As evidence for this model, direct administration of serotonin through a micropipette to the sensory neuron causes decreased efflux of K⁺ ions and prolongs depolarization of the membrane induced during an action potential. Also, the Aplysia sensory neuron is large enough that the active catalytic subunit of the cAMP-dependent protein kinase can be injected into it. Such treatment mimics the effect of applying serotonin to the outside of the cell. Additional supporting evidence that serotonin acts by means of cAMP and a protein kinase has come from patch-clamping studies on isolated inside-out pieces of sensory neuron plasma membrane (see Figure 21-20b). When both ATP and the purified active catalytic subunit of cAMP-dependent protein kinase are added to the cytosolic surface of the patches, the K⁺ channels close. Thus the protein kinase indeed acts on the cytosolic surface of the membrane to phosphorylate the channel protein itself or a membrane protein that regulates channel activity. We shall return to this particular synapse at the last section of this chapter, as these modifications in synapse efficiency are part of a simple learning response.

Some Neuropeptides Function as Both Transmitters and Hormones

The receptors for many of the small neuropeptides found in nervous tissue have been cloned; all are coupled to G proteins and have the characteristic seven membrane-spanning α helices. Thus, the intracellular signaling pathways induced by neuropeptides are the same as those induced by the classical neurotransmitters that activate G protein – coupled receptors.

Many neuropeptides function as synaptic neurotransmitters; others act in a paracrine fashion as “diffusible” hormones that affect many nearby neurons (see Figure 20-1). Yet other neuropeptides act as regulators of nerve cell growth and division. Many of the neuropeptides listed in Table 21-2 are found both in the brain and in non-neural tissues. However, in contrast to capillaries in other parts of the body, capillaries in the brain are essentially impermeable to peptides. Thus, any peptide hormones traveling through the body in the blood will be excluded from the brain: this constitutes the blood-brain barrier. Hormones in the blood do not “confuse” the functioning of the central nervous system.

Neurons that secrete peptide hormones, called neurosecretory cells, were first discovered in the hypothalamus. Secretion of peptide hormones by the anterior cells of the pituitary gland is controlled by the hypothalamus, which in turn is regulated by other regions of the brain. The hypothalamus is connected to the anterior pituitary by a special closed system of blood vessels. Hypothalamic neurons secrete hypothalamic peptide hormones into these vessels, and the hormones then bind to receptors on the anterior pituitary cells. One such hypothalamic hormone, thyrotropin- releasing hormone (TRH), stimulates secretion by the anterior pituitary of prolactin and thyrotropin. Another hypothalamic hormone, luteinizing hormone – releasing hormone (LHRH), causes other cells in the anterior pituitary to secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which are important in regulating the growth and maturation of oocytes in the ovary.

In contrast to serotonin and catecholamines, but like acetylcholine, neurohormones and neurotransmitters are used only once and then are degraded by extracellular proteases; they are not recycled.

SUMMARY

•  In general, neurotransmitter receptors that are ligand-gated ion channels mediate rapid postsynaptic responses, whereas G protein – coupled receptors mediate slow postsynaptic responses.
•  At the synapse of a motor neuron and striated muscle cell, binding of acetylcholine to nicotinic acetylcholine receptors triggers a rapid increase in permeability of the membrane to both Na⁺ and K⁺ ions, leading to depolarization, an action potential, and then contraction (see Figure 21-37).
•  The nicotinic acetylcholine receptor and other neurotransmitter receptors that are ligand-gated ion channels contain five subunits (see Figure 21-38). Each subunit contains a transmembrane α helix (M2) that lines the channel. Neurotransmitter binding to the receptor triggers a conformational change leading to channel opening.
•  Glutamate, the principal excitatory neurotransmitter in the mammalian brain, binds to two types of ligand-gated cation channels: NMDA and non-NMDA glutamate receptors. Activation of NMDA receptors requires both partial membrane depolarization and glutamate binding; these receptors may function in a type of “cellular memory.”
•  GABA and glycine are the principal inhibitory neurotransmitters; their receptors are ligand-gated Cl⁻ channels.
•  Binding of acetylcholine to muscarinic acetylcholine receptors in heart muscle causes dissociation of the coupled trimeric G protein; the released G_β,γ subunit binds to and opens a K⁺ channel protein (see Figure 21-41). The resulting influx of K⁺ ions hyperpolarizes the cell membrane, slowing heart contraction.
•  Stimulation of the G protein – coupled catecholamine receptors leads to an increase or decrease in cAMP or other intracellular second messengers. These and other G protein – coupled neurotransmitter receptors contain seven transmembrane α helices and induce signaling pathways similar to those in non-neuronal cells.
•  Stimulation of G protein – coupled serotonin receptors in sensory neurons of the sea slug Aplysia increases the ability of the sensory neurons to activate postsynaptic motor neurons. This modulatory effect results from closing of K⁺ channels induced by the rise in cytosolic cAMP following serotonin binding (see Figure 21-42).
•  Many small peptides released by neurons function as paracrine hormones as well as neurotransmitters, affecting both nearby secretory cells and adjacent neurons. The receptors for these neuropeptides are coupled to G proteins.