Thus far, ion channels have been discussed only in the most abstract sense. However, modern advances in molecular biology, such as the isolation, cloning, and mutagenesis of ion channels, and in cell physiology, such as patch clamping techniques that allow the observation of single ion channels, have provided scientists with remarkably detailed knowledge of the operation of ion channels. The crystallization of simple ion channels also has yielded important functional and structural information. Consequently, we know the exact amino acid composition of hundreds of ion channels, have detailed models about how these channels assemble in cellular membranes, and understand which amino acids are responsible for features of ion channels such as voltage gating, selective permeability, and the binding of pharmaceutical agents.
This section focuses on the voltage-gated–like (VGL) ion channel superfamily that includes but is not limited to voltage-gated Na+, Ca2+, and K+ channels (NaV, CaV, and KV). The extended family members also include Ca2+-activated K+ channels (KCa), cyclic nucleotide–modulated ion channels (cyclic nucleotide gated [CNG] and hyperpolarization-activated cyclic nucleotide gated [HCN]), transient receptor potential channels (TRP), inward rectifier K+ channels (Kir), and two-pore K+ channels (K2P). (Note that the term rectifier describes a channel that passes current much more efficiently in one direction; for example, an inward rectifier preferentially passes current into a cell.) Members of this gene family are principal players in regulating the electrical signals in neurons. We review these channels in considerable detail with the expectation that these proteins represent valuable targets for the development of future treatments for psychiatric and neurologic disorders. Another major family of ion channels, ligand-gated ion channels, which is responsible for translating chemical signals into electrical signals via generation of synaptic potentials, is discussed in subsequent chapters.
Structure of VGL Ion Channels
The fundamental features of an ion channel are its aqueous pore that controls ion permeation, the channel’s gating mechanism, and its modulation. The relationship among the >140 protein members of this ion channel family is built on the amino acid sequences of the minimum pore region 2–13. The founding members of this family are voltage-gated Na+ channels. Their principal α subunit consists of four internally homologous domains that surround a central pore. Each of the domains contains six membrane-spanning α helices (S1–S6), and a membrane reentrant loop between S5 and S6, also called SS1–SS2, that is believed to form a hairpin loop that dips down into the channel and forms the lining of the ion pore 2–14.
Representation of the amino acid sequence relations of the minimal pore regions of the voltage-gated ion channel superfamily. This global view of the >140 members of the structurally related ion channel genes highlights the seven major groups of ion channel families and their membrane topologies. Four-domain channels (CaV and NaV) are shown as blue branches, K+ selective channels are shown as red branches, CNG channels are shown as magenta branches, and TRP and related channels are shown as green branches. Background colors separate the ion channel proteins into related groups: light blue, CaV and NaV; light green, TRP channels; light red, potassium channels, except KV10–12, which have a cyclic nucleotide–binding domain and are more closely related to CNG and HCN channels; light orange, KV10–12 channels and cyclic nucleotide–modulated CNG and HCN channels. Minimal pore regions bounded by the transmembrane segments M1 or S5 and M2 or S6 were aligned and refined manually for 143 ion channel members; amino acid sequence positions with gaps in the alignment were omitted from the analyses. (Reproduced with permission from Yu FH, Catterall WA. The VGL-chanome: A protein superfamily for electrical signaling and ionic homeostasis. Science STKE. 2004;Oct 5;2004(253)re15.)
Structural similarities shared by voltage-dependent Na+, Ca2+, and K+ channels. Each channel has subunits whose domains contain six membrane-spanning segments. The α subunit in both Ca2+ and Na+ channels contains four domains. The subunit of the Shaker K+ channel contains only one domain. The region that connects segments 5 and 6 in each domain (SS1–SS2) is believed to form the pore of each channel. The segment 4 in each domain is believed to be the voltage sensor. The inactivation modules in Na+, K+, and Ca2+ channels are located at different regions of the channels. (Adapted with permission from Barchi RL. Sodium channel gene defects in the periodic paralyses. Curr Opin Neurobiol. 1992;2(5):631-637.)
Other voltage-gated channels display structures remarkably similar to those of the Na+ channel. Most similar are the principal α subunits of Ca2+ channels, which also contain four internally homologous domains, each of which possesses six putative membrane-spanning regions 2–14. Voltage-gated K+ channels appear to be composed of subunits that correspond to only one of the four internally homologous domains of the Na+ and Ca2+ channels. It is believed that four of these smaller subunits multimerize in the plasma membrane to form a channel that is similar in structure to the Na+ and Ca2+ channels. Several other families of ion channels (KCa, CNG, HCN, and TRP) also have this tetrameric structure. However, although the inwardly rectifying K+ channels are also tetramers, each of the subunits only has two transmembrane segments, named M1 and M2, which are analogous to the S5 and S6 segments of voltage-gated Na+, Ca2+, and K+ channels. On the other hand, two of these pore motifs are linked together to generate the two-pored K+ channels. In mammalian cells, each of these channels is composed of several subunits in addition to their primary α subunits; the role of these accessory proteins is discussed in 2–4.
2–4 Accessory Subunits of Channels
The major pore-forming α subunits of Na+, K+, and Ca2+ channels are capable of conducting their own voltage-dependent, ion-selective current, and commonly contain the regulatory motifs modified by second messenger signaling as well as the binding sites for pharmacologic agents. However, many of these principal α subunits are physically associated with accessory subunits that modify their expression, functional properties, and subcellular localization. Unlike the structurally similar principal pore-forming subunits, the accessory subunits exhibit considerable diversity in amino acid sequence, size, posttranslational modification, and postulated structure.
These accessory proteins are speculated to serve the following functions:
They can facilitate channel trafficking to the plasma membrane or stabilize channels in the membrane.
They can modulate activation and inactivation rates, normally toward faster gating kinetics.
They can shift voltage dependence, normally toward more hyperpolarized potentials.
Their phosphorylation can regulate channel properties 2–5.
They can influence ligand (toxins and pharmacologic agents) binding.
Importantly, mutations in some accessory subunits of ion channels are associated with familial diseases, such as epilepsy (eg, the neuronal Na+ channel β subunit and the neuronal Ca2+ channel β4 subunit) (Chapter 19) and long QT syndrome (the cardiac K+ channel β subunits) 2–1.
2–5 Phosphorylation of Ion Channels
Changes in the electrical properties of neurons, which are directly related to modifications of their ion channels, are crucial for the survival of an organism. In a threatening situation, for example, neurons that typically fire only a single action potential may respond by firing long trains of action potentials. The increased firing of such neurons can heighten awareness, provoke a stronger or more rapid behavioral response, or assist in forming a potent memory about a dangerous situation.
Among the most common means by which the excitability of neurons is altered is through the phosphorylation of ion channels, which involves the addition of free phosphate (–PO43–) groups to particular amino acid residues of a channel protein (Chapter 4). This process changes the stable conformational state of a channel and in turn alters the magnitude and time course of the ionic currents it conducts. Phosphorylation thereby provides an excellent basis for ion channel flexibility; in seconds or less, the activation of second messenger systems in a neuron can lead to the phosphorylation of channel proteins and to equally swift changes in the electrical properties of the neuron. Depending on the channel, the phosphorylation site, the location of the phosphorylated amino acid residue, and the internal milieu of the cell, this process can alter the following biophysical properties:
Inactivation kinetics. Protein kinase C phosphorylation of a single residue of an Na+ channel’s α subunit on the intracellular loop between transmembrane domains III and IV can slow the channel’s inactivation. Similarly, phosphorylation of the rapidly inactivating K+ channel known as KV3.4 by protein kinase C causes the channel’s inactivation to abate.
Current amplitude. Activation of protein kinase A increases the amplitude of K+ channel KV1.2 currents; activation of protein kinase C decreases the amplitude of currents through Na+ channels, and activation of protein kinase A increases the amplitude of currents in KCNQ2 channels (a type of KV channel). Mutations of the latter channel cause benign familial neonatal convulsions 2–1.
Voltage dependence of channel responses. Phosphorylation of L-type Ca2+ channels by protein kinase A not only slows channel inactivation but also shifts the channel’s voltage dependence to more negative potentials.
Accumulation of channel protein in the plasma membrane. Long-term protein kinase A treatment of oocytes expressing KV1.1 K+ channels increases the amplitude of currents expressed by these channels. This response is most likely caused by increased amounts of channel protein in the plasma membrane.
The effect of norepinephrine on hippocampal pyramidal cells illustrates how the phosphorylation of an ion channel protein can affect the behavior of a neuron, as shown in the figure. In the absence of norepinephrine, depolarization of a pyramidal cell results in a small number of action potentials. The activation of a Ca2+-activated K+ channel causes an after-hyperpolarization (AHP), which limits the number of action potentials that occur in response to a depolarizing stimulus. In the presence of norepinephrine, depolarization of the pyramidal cell leads to a much more prolonged train of action potentials. Norepinephrine binds to the β-adrenergic receptor, which activates adenylyl cyclase, and leads to an accumulation of cAMP and activation of protein kinase A (Chapter 4). Protein kinase A in turn phosphorylates Ca2+-activated K+ channels and blocks their activity. Accordingly, norepinephrine increases the firing of a neuron by removing the hyperpolarizing currents, or AHP, that inhibit its activity. (Adapted with permission from Madison DV, Nicoll RA. Actions of noradrenaline recorded intracellularly in rat hippocampal CA1 pyramidal neurons in vitro. J Physiol. 1986; Mar;372:221–244.)
Selectivity of Ion Channels
K+ channels are 100 to 1000 times more permeable to K+ than Na+. Na+ channels are 12 times more selective for Na+ than for any other ion, and some Ca2+ channels are 1000 times more selective for Ca2+ than for other cations. A channel’s ability to select one cation over another suggests remarkably sophisticated protein design, and how this is accomplished has been the focus of intense investigation.
Ions do not flow through ion channels like water through a pipe; such a model cannot explain how some channel pores are selective for Na+ and others are selective for K+. Instead, an ion binds transiently to one or more sites in a channel, with its permeability presumably determined by the amount of energy released during its binding to amino acid residues and by the energy required for its dissociation from some or all surrounding water molecules. The speed with which an ion can dissociate from a pore and escape from a channel is also likely to influence selectivity.
Crystallization of the bacterial KcsA K+ channel has provided great insight into how ionic selectivity may be accomplished in K+ channels. The selectivity filter is the narrowest part of the pore, formed by the SS1–SS2–like region of the KcsA channel, with a very conserved GYG sequence lying at the heart of the selectivity filter. Voltage-gated Na+ and Ca2+ channels probably use a different strategy to achieve selectivity. Both types of channels use negatively charged residues, and perhaps some backbone carbonyls as well, to coordinate the cation.
Na+ channels responsible for propagating action potentials, K+ channels responsible for terminating action potentials, and Ca2+ channels that admit Ca2+ to nerve terminals in response to action potentials are all voltage activated: they open when a membrane is depolarized. It is now known that voltage-activated ion channels display a gating charge, or a shift in the distribution of charge across the membrane, that occurs concomitantly with channel activation and is believed to result from the movement of a putative voltage sensor within the ion channel itself. The S4 transmembrane domain, marked by a unique pattern of positively charged amino acids, may behave as a gating particle 2–14. However, the exact conformational change that results from movement of the S4 domain and causes the opening of a channel remains controversial.
The addition of regulatory domains to the carboxyl terminus of Kir, KCa, CNG, and HCN channels yields gating by binding of small intracellular ligands such as Ca2+, ATP, and cyclic nucleotides or by interactions with protein ligands. Ligand binding to these domains is thought to rotate the S6 segment to open the pore. For KCa and HCN channels, ligand binding and membrane depolarization act in concert to open the pore.
In some cases, the closing of a voltage-gated ion channel is simply the opposite of its opening: the channel undergoes conformational changes in response to a particular membrane voltage and subsequently returns to its resting conformation when the membrane voltage change subsides. This process is called deactivation. For example, the closure of voltage-gated ion channels such as the delayed rectifier K+ channel, which restores an axon’s resting potential after an action potential, does not appear to require additional regulatory mechanisms.
In response to depolarization, however, a voltage-gated Na+ channel closes immediately after it is activated, even if the depolarization is maintained, such as during an action potential. This process of inactivation is distinctly different from that of deactivation, the return of the Na+ channel to its resting state. The “ball and chain” model of inactivation postulates the movement of a positively charged segment of a channel protein into an open ion channel, which prevents further conductance 2–14. Site-directed mutagenesis indicates that the ball and chain portion of the Shaker K+ channel (a prototype of KV channels discussed below) is most likely its N terminus, and that of the Na+ channel the cytoplasmic region between domains III and IV. In some Ca2+ channels, residues in the S6 segment of domain I, and the loop between domain I and II, appear to be functionally important in voltage-dependent inactivation. Some subtypes of Ca2+ channels also exhibit Ca2+-dependent inactivation, which involves the C-terminal region 2–14.
Several toxins and pharmaceutical agents act by modulating the process of inactivation. Some toxins selectively slow Na+ channel inactivation, causing these channels to remain open for a longer period of time; a class of scorpion toxins that act in this manner can lead to spastic paralysis, seizures, and death. Other substances, including some drugs used in the treatment of epilepsy (eg, phenytoin and carbamazepine), selectively stabilize the inactivated state of the Na+ channel, thereby decreasing neuronal excitability.
The voltage-dependent cation channels are similar enough in general architecture that it is possible to discuss their basic properties as a group. However, rapid advances in molecular biology, pharmacology, and electrophysiology have made it possible to distinguish hundreds of variations of ion channels. Distinct channels arise from different gene products, splice variants of the same gene transcript, posttranslational modifications, and combinations of individual channel subunits. The following sections of this chapter serve as an abbreviated catalogue of the various types and subtypes of voltage-gated cation channels and other types of cation channels from the VGL family 2–13. The properties, molecular composition, and medical relevance of each are summarized in the most general terms; more information about each channel type can be found in the references listed at the end of the chapter and from online resources.
Na+ channels are a relatively homogeneous group of ion channels. Variations do exist: in mammals, nine principal α subunit genes of Na+ channels have been cloned, and they show different tissue distribution patterns, and different sensitivities to the typical Na+ channel blocker tetrodotoxin. However, across species and tissue types, Na+ channel properties are remarkably similar in terms of voltage dependence and activation–inactivation kinetics.
Pharmacology and toxicology of sodium channels
Toxins that bind to Na+ channels are uncommon, but their effects can be dramatic. The isolation of the Na+ channel was initially possible because it binds tightly and specifically to toxins such as tetrodotoxin, a heterocyclic compound found in many marine species, including the Japanese puffer fish, the globe fish, and the blue-ringed octopus. Approximately 200 humans experience tetrodotoxin poisoning each year, in most cases after consuming improperly prepared puffer fish. Symptoms of tetrodotoxin poisoning include facial numbness, headache, and increasing paralysis and respiratory distress. Although victims are completely paralyzed, they may be conscious and lucid until shortly before death, which generally occurs within 4 to 6 hours. Among those poisoned, the mortality rate is approximately 50%; survival is usually attributed to rapid treatment (gastric lavage) or the victim’s ingestion of only a small amount of toxin. Because tetrodotoxin binds to the outside of the Na+ channel at the S5–S6 loop of the α subunit, site-directed mutagenesis of amino acids in this region greatly reduces its ability to bind to the channel (see 2–14). Changes in amino acid sequence in this region in certain Na+ channel subunits cause these channels to be resistant to tetrodotoxin to varying degrees.
Other Na+ channel toxins act not by blocking the channel but by activating it, usually by slowing or eliminating its voltage-dependent inactivation, which leads to the hyperexcitability of neurons and muscle tissue. Victims of these toxins, which have been isolated from scorpion and sea anemone species, may undergo convulsions and spastic paralysis.
Substances used to treat human disease generally have less dramatic effects on the Na+ channel. Phenytoin and carbamazepine, which are used to treat epilepsy, act on Na+ channels by slowing their recovery from an inactivated state. Because prolonged inactivation of these channels limits the firing rates of affected neurons, these drugs can prevent seizures. Local anesthetics, such as cocaine, and synthetic derivatives such as lidocaine and procaine, which are membrane permeable in their nonionized form, bind to the cytoplasmic side of the Na+ channel and also cause Na+ to bind to the inactivated state of the Na+ channel, creating a use-dependent blockade of the channel.
The sodium channel in disease
Some inherited neurologic disorders, including certain myotonias and periodic paralyses, are linked to NaV1.4 (also known as SkM1), the Na+ channel in adult skeletal muscle. Although myotonia, which is characterized by muscle excitability, and periodic paralysis, which is characterized by muscle hypoexcitability, seem to be diametrically opposed, the underlying Na+ channel defects are similar. Electrophysiologic studies indicate that both disorders involve slowed or impaired inactivation of the Na+ channel. In individuals who experience periodic paralysis, the defect in Na+ channel inactivation is severe enough that muscle remains at a depolarized potential and becomes refractory to further action potentials; in those who experience myotonia, the defect is less severe and results in repetitive firing. Mutations in neuronal and cardiac Na+ channel subunits that slow or impair the inactivation of Na+ channels are also linked to familial disorders such as familial neonatal seizures, generalized epilepsy, and long QT syndrome 2–1. Of great interest is the recent finding that certain sodium channel subunits, such as NaV1.7, NaV1.8, and NaV1.9, are preferentially expressed in peripheral sensory nerves and are critically important in the transmission of pain signals, raising the possibility that blockers of these channels might be useful in the treatment of pain syndromes (Chapter 11).
K+ channels generally act as a stabilizing force. Their diverse functions include setting a cell’s resting potential, repolarizing the cell after an action potential, and controlling an action potential’s rate of firing and shape. Proper K+ channel functioning is a critical determinant of neuronal activity, and altered expression levels of specific K+ channel subunits in response to prolonged periods of neuronal activation or inhibition play an important role in maintaining normal cell excitability, a process termed whole cell, nonsynaptic, or homeostatic plasticity. Mutations in K+ channels lead to a variety of disorders, ranging from ataxia to cardiac arrhythmias 2–1. A malfunctioning K+ channel generally causes some form of hyperexcitability in affected tissue; for example, the long QT syndrome, a delay in ventricular repolarization that can cause heart arrhythmias, results from a malfunction of the KV11.1 (HERG) K+ channel, which is responsible for repolarizing the ventricle after contraction. Indeed, binding to HERG channels is a common cause of cardiac side effects of many classes of medications and is now used as a routine screen during the drug discovery process. Mutations of the related KV7.2/7.3 (KCNQ2/3) K+ channel cause benign familial neonatal convulsions, which resolve as the affected child grows. Mutation of another K+ channel KV1.1, which is expressed at high levels in the cerebellum, is linked to a type of episodic ataxia believed to be caused by an abnormal increase in the firing of cerebellar cells. Tetraethylammonium (TEA), which selectively blocks most types of voltage-gated K+ channels, has been a useful research tool.
Potassium channel classifications
K+ channels can be placed into two broad classes, six TM (transmembrane domain) and two TM channels 2–3. Six TM channels include voltage-gated K+ channels (KV1–12 families) and Ca2+-activated K+ channels (KCa1–5 families). Two TM channels are inwardly rectifying K+ channels (Kir1–7 families). The differences in these channels reflect the variety of ways that they can affect the firing properties of a neuron.
2–3Blocking Agents for Potassium Channels ||Download (.pdf) 2–3 Blocking Agents for Potassium Channels
|Potassium Channel ||Acting From Outside ||Acting From Inside ||Membrane-Permeant || |
|6TM || |
| Delayed rectifier || |
Cs+, H+, Ba2+
TEA and QA
Cs+, Na+, Li+, Ba2+
| A || || || |
| K(Ca) || |
| || |
|2TM || |
| Inward rectifier || |
Cs+, Rb+, Na+
| KATP || || || |
Voltage-gated K+ channels subsequently have two different functional classes that are not categorized by subfamilies, delayed rectifiers and A-type channels. Delayed rectifiers undergo delayed activation after depolarization and inactivate slowly; they thereby facilitate repolarization by remaining open for a prolonged period of time. These channels also shape action potentials; blocking their activity increases the duration of action potentials that in turn depend solely on the inactivation of Na+ channels for repolarization. A-type channels, also referred to as Shaker-related channels (after the first K+ channel cloned from Drosophila), are transiently activated when a cell is depolarized after a period of hyperpolarization. They can function to decrease the frequency of action potential firing by prolonging the interspike interval.
Ca2+-gated K+ channels open in response to the binding of Ca2+, which usually occurs after a depolarization-induced influx of Ca2+. Generally, these channels can remain open for prolonged periods, such as a few seconds, and are responsible for several different types of long after-hyperpolarization (AHP), which is a hyperpolarization of the membrane that occurs after an action potential or series of action potentials. A long AHP can profoundly affect the firing pattern of a neuron; for instance, when a steady stimulus produces a train of action potentials, an AHP often is generated in response and can gradually slow the rate of firing. This phenomenon, known as spike-frequency adaptation, occurs throughout the nervous system and has many functional consequences.
SK channels (named for their small conductance) are a subtype of Ca2+-gated K+ channels that are important for AHPs; they have become attractive drug targets because of their roles in modulating neuronal firing. SK channels are blocked by apamin, a honeybee neurotoxin.
Inward rectifiers structurally resemble truncated Shaker-type channels that are missing S1–S4 segments; that is, they comprise only the putative pore-lining segments S5, S6, and SS1–SS2. As with the Shaker-type channels, four of these subunits are believed to multimerize to form a functional channel. Such rectifiers are described as anomalous because they open when the membrane is hyperpolarized and close as the membrane depolarizes. They most likely stabilize a membrane’s potential when the potential is near rest, without inhibiting depolarization. By conducting outward current in the voltage range just slightly positive to EK, they maintain a resting potential near EK; however, if the cell is sufficiently depolarized, they shut off, freeing the membrane to undergo further depolarization. This type of K+ channel is found in the heart, in striated muscle, and in neurons.
Seven subfamilies of inward rectifiers—Kir1–7—have been identified. Channels formed by members of the Kir3 subfamily are especially noteworthy because of the role that G proteins play in their regulation (Chapter 4). G protein–coupled Kir3 channels, termed GIRKs, are critical to many physiologic functions; for example, they mediate the slowing of heart rate by acetylcholine. The binding of acetylcholine to muscarinic acetylcholine receptors in the atrium activates a G protein and liberates its βγ complex, which in turn leads to the activation of a GIRK. The K+ current produced by this activation slows the depolarization of sinoatrial cells to their firing threshold. This process explains the positive ionotropic and chronotropic effects of muscarinic cholinergic antagonists such as atropine. GIRKs are also expressed in the brain where, among other functions, they mediate a neuron’s electrical responses to the activation of many types of G protein–coupled neurotransmitter receptors, especially those coupled with G proteins from the Gi family (Chapter 4).
K(ATP) channels (Kir6.2), another atypical member of the Kir family, play an important role in cellular and systemic metabolic regulation. Functionally, these channels are voltage insensitive; they close in the presence of high (100 μM to 1 mM) intracellular ATP concentrations, and open when these concentrations are low, resulting in a more hyperpolarized membrane potential. They may serve a protective function in neurons by reducing neural firing when neural energy reserves are depleted. Structurally, each of these ATP-sensitive K+ channels is a multimeric complex comprising four Kir6.2 subunits and one sulfonylurea receptor (SUR) protein. Sulfonylureas such as tolbutamide block K(ATP) channels and are used to treat adult-onset diabetes. The drugs act by increasing insulin secretion from pancreatic β cells. Mutations in Kir6.2 channels cause congenital hyperinsulinemia 2–1. Iptakalim is a structurally novel opener of K(ATP) channels, which shows promise as a neuroprotective agent in animal models. Minoxidil, used to restore hair growth, is also an activator of K(ATP) channels, although whether this action is related to its effects on hair growth remains unknown.
Ca2+ is an important signaling molecule that is present in low concentrations in extracellular fluid (1–5 mM) and in minute concentrations in most cell interiors (approximately 0.1–0.2 μM). The opening of Ca2+ channels is the critical link between cell depolarization and Ca2+ entry, which can result in local intracellular Ca2+ concentrations as great as 100 μM. The subsequent binding of Ca2+ to intracellular molecules leads to many biologic responses, including muscle contraction; the triggering of neurotransmitter release from nerve terminals (Chapter 3); the activation of second messenger systems that cause many changes, including alterations in gene expression (Chapter 4); and, in extreme cases, neuronal self-destruction (Chapters 18 and 20). Some Ca2+ channels also impart electrical properties to the cells in which they are expressed; for example, such cells may show Ca2+ spikes—action potentials in which the depolarizing current is carried predominantly by Ca2+.
Ca2+ channels are categorized in terms of their voltage dependence, kinetics (speed of activation and inactivation), and pharmacology. Ten Ca2+ channel genes have been isolated, many of which correspond to Ca2+ channel types that were originally classified according to their electrophysiologic properties 2–4.
2–4Calcium Channels ||Download (.pdf) 2–4 Calcium Channels
|Ca2+ Channel ||Ca2+ Current Type ||Primary Localizations ||Specific Blocker ||Functions|
|CaV1.1, 1.2, 1.3, 1.4 ||L || |
|DHPs || |
|CaV2.1 ||P/Q || |
|ω-Agatoxin || |
|CaV2.2 ||N || |
|ω-CTX-GVIA || |
|CaV2.3 ||R || ||SNX-482 || |
|CaV3.1, 3.2, 3.3 ||T || |
|Mibefradil || |
L-type (large current or “long open time”) Ca2+ channels are activated by large depolarizations (approximately −20 mV) and can remain open for a long time before inactivating (500 ms or more). Four major L-channel subunits have been cloned: CaV1.1–4. The CaV1.1 (α1S) subunit resides exclusively in skeletal muscle, where it is abundant in transverse tubules; CaV1.2 (α1C) subunits are found in cardiac muscle, smooth muscle, and brain; and CaV1.3 (α1D) subunits predominate in cochlear hair cells, and endocrine and kidney cells, but also reside in the brain. CaV1.4 (α1F) subunits are found only in the retina, with mutations in this gene linked to congenital stationary night blindness.
Clinically, these channels are targets for antianginal and antihypertensive drugs. L-type Ca2+ channel blockers, phenylalkylamines (eg, verapamil), dihydropyridines (eg, nifedipine), and benzothiazepines (eg, diltiazem), decrease myocardial contractile force and thereby reduce myocardial oxygen requirements, or reduce smooth muscle contractility and thereby decrease arterial and intraventricular pressure. L-type Ca2+ channels are located primarily on the cell bodies and proximal dendrites of neurons. They admit Ca2+ to the cell body during periods of strong depolarization, and this influx causes second messenger activation and changes in gene transcription. Yet substances that block these channels rarely have noticeable neurologic side effects, despite the fact that some—for example, nimodipine—can cross the blood–brain barrier. One possibility is that one of the neuronal L-type Ca2+ channels, CaV1.3 channels, are less sensitive to these typical L channel blockers.
T-type (tiny current or transient) Ca2+ channels are activated by depolarizations near resting potential; half-maximal activation of these channels occurs at approximately −40 mV. They exhibit voltage-dependent inactivation such that depolarization produced by their activation ultimately triggers their inactivation. Because of this self-limiting property, these channels are excellent oscillators; in fact, they are believed to provide a pacemaker current in thalamic neurons that generate the rhythmic cortical discharge associated with absence seizures (petit mal). Ethosuximide, which blocks T-type current, is an effective therapy for these seizures (Chapter 19). The cloning of T-type channel subunits CaV3.1–3 (α1G, H, I) should facilitate the development of drugs that interact more specifically with this important Ca2+ channel subtype.
The α subunits of these channels belong to one subfamily CaV2.1, 3 (α1A, B, and E). The CaV2 channels are high-voltage activated, not sensitive to L channel blockers, yet can be blocked with high affinity by peptide toxins derived from spiders and sea snails 2–4. This subfamily of Ca2+ channels is best known for their regulation of neurotransmitter and hormone release: Ca2+ flows into nerve terminals through these channels in a voltage-dependent manner that triggers this release (Chapter 3). P/Q (CaV2.1) and N (CaV2.2) channels contribute most of the Ca2+ influx that triggers neurotransmitter and hormone release. P/Q-type channels also control neurotransmitter release at neuromuscular junctions, whereas N-type channels control neurotransmitter release in sympathetic neurons. A synthetic peptide blocker of N-type Ca2+ channels (ziconotide), derived from a marine snail conotoxin, is under development for treating patients unresponsive to intrathecal opiates for relief of chronic pain (Chapter 11). CaV2.3 channels underlie a subpopulation of R (resistant)-type Ca2+ channels. The functions of CaV2.3 channels are less well defined, but may include neurotransmitter and hormone release and the generation of dendritic calcium transients.
Mutations of the human CaV2.1 (α1A) gene have been linked to familial hemiplegic migraine, spinocerebellar ataxia, and episodic ataxia. Although this gene is richly expressed in Purkinje cells, how the mutant P/Q channels cause the abnormalities seen in these disorders is still under study.
Cyclic Nucleotide–Regulated Channels
The family of cyclic nucleotide–regulated channels includes the CNG channels and the HCN channels. These channels are six TM segment channels, structurally similar to voltage-gated K+ channels. They are nonselective cation channels, in that monovalent ions (K+, Na+) can pass through with no discrimination, and divalent ions (Ca2+) can pass as well.
The activation of CNG channels is mediated by the direct binding of cGMP or cAMP to the C-terminal region of the channel protein. These channels are expressed in the cilia of olfactory neurons and in outer segments of rod and cone photoreceptors, where they transduce odorant signals and photons into electrical signals 2–6. Photoreceptor channels strongly discriminate between cGMP and cAMP, whereas the olfactory channels are almost equally sensitive to both ligands.
2–6 Signal Transduction in CNG Channels
The role that cGMP-gated channels play in converting light energy, in the form of photons, to an electrical signal that the nervous system can interpret is well understood. Photons activate the light-sensing pigment rhodopsin (which is highly homologous in structure to a G protein–coupled receptor) in the outer segments of rods in the retina. Photoactivated rhodopsin in turn activates a phosphodiesterase that causes the breakdown of cGMP (Chapter 4). This transient fall in the cytosolic concentration of cGMP results in the closure of tonically activated cGMP-gated channels, which causes hyperpolarization of the photoreceptor cell. Rods respond to this hyperpolarization by releasing less transmitter, thereby informing other cells in the retina that a light signal has been received.
A similar process transduces odorant signals. Activation of odorant receptors, which are G protein coupled, stimulates adenylyl cyclase and the production of cAMP (Chapter 4). The increase in cAMP activates cAMP-gated channels, which causes depolarization of the olfactory neurons. Resulting action potentials in these neurons signal efferent neurons that an olfactory stimulus has been received. In contrast to the channels involved in photoreception, which are activated preferentially by cGMP, olfactory channels can be opened by either cAMP or cGMP.
CNG channels are heterotetramers composed of homologous A subunits (CNGA1–4) and B subunits (CNGB1–3). Each subunit binds a single molecule of cAMP or cGMP. Although the binding of only one subunit can cause a channel to open, maximum activation of the channel requires the binding of all four subunits to cyclic nucleotide. Mutations in CNG genes are linked to retinitis pigmentosa and achromatopsia.
In mammals, the HCN family comprises four members (HCN1–4). They are expressed in neurons, cardiac pacemaking cells, and photoreceptors. In contrast to most other VGL channels, HCN channels open on hyperpolarization and close at positive potentials. The currents carried by these channels are also known as Ih, If, or Iq currents. cAMP and cGMP can bind to the C-terminal region of HCN channels and enhance channel activity by shifting the activation curve to more positive voltages. Because their activation causes the cell to depolarize, often beyond spike threshold, these channels help drive a repetitive cycle of rhythmic firing and, hence, are called “pacemaker channels.” Mutations in HCN4 are associated with sick sinus node disease 2–1.
Given the key role of HCN channels in cardiac pacemaking, these channels are promising pharmacologic targets for the development of drugs used in the treatment of cardiac arrhythmias and ischemic heart disease. For example, ivabradine, a heart rate–lowering agent, is a use-dependent blocker of HCN channels. Similar compounds include zetabradine and cilobradine. The effect of cAMP on these channels is exploited by several common drugs used to modify heart rate; for example, propranolol, a β-adrenergic antagonist, reduces cellular cAMP levels, which decreases heart rate in part by inhibiting Ih channels. Because the rhythmic activity of neurons is integral to functions such as arousal, maintenance of the sleep–wake cycle, and respiratory rate, these pacemaker channels are important targets for future drug development. Indeed, antagonists of these channels are currently undergoing evaluation for use in the treatment of a range of neuropsychiatric disorders.
TRP channels are named after their role in Drosophila phototransduction. Mutations in the channel caused the response to light to be transient, and resulted in a decrease in the level of light-induced Ca2+ influx. To date, approximately 28 genes have been identified in mammals encoding TRP channels. They are divided into six subfamilies (TRPC, TRPV, TRPM, TRPP, TRPA, TRPML), all of which are six TM domain channels that lack the complete set of positively charged residues in S4 that act as voltage sensors. TRP channels are permeable to Ca2+ and Na+, and, in some cases, Mg2+. No single activation mechanism is shared by all TRP channels. In general, TRP channels can be described as Ca2+-permeable cation channels with polymodal activation mechanisms, including receptor activation (ie, certain G protein–coupled receptors and receptor tyrosine kinases activate phospholipases that can subsequently modulate TRP channel activity), ligand activation (ie, exogenous small organic molecules, endogenous lipids or products of lipid metabolism, purine nucleotides, inorganic ions), and direct activation (ie, change of temperature, mechanical stimuli, conformational coupling to inositol triphosphate [IP3] receptors, channel phosphorylation). Because most TRP channels are responsive to multiple stimuli, they can integrate these stimuli, and couple them to downstream signal amplification cascades through elevation of intracellular Ca2+ and membrane depolarization.
TRP channels are of particular importance in sensory perception, including vision, taste, smell, hearing, mechanosensation, and thermosensation. As just one example, TRPV1 is activated by capsaicin, the active ingredient in hot pepper. TRP channels, particularly their involvement in pain, are discussed in greater detail in Chapter 11 (see 11–1). TRP channels also allow individual cells to sense changes in the local environment, such as alterations in fluid flow and mechanical stress. As cellular sensors, TRP channels are proposed to participate in various physiologic processes, such as regulating vessel tone, fertilization, Mg2+ absorption, and neurite outgrowth. Mutations in TRP channels are associated with diseases such as hypomagnesemia, polycystic kidney disease, and a neurodegenerative disease, mucolipidosis 2–1.
While this chapter has focused on cation channels, neurons and most other cells are also permeable to Cl−. Cl− channels are critical for many physiologic processes, and mutations of these channels have been implicated in a variety of diseases 2–1.
Cl− channels serve two primary functions. First, they dampen electrical excitability. In most cells, including neurons and myocytes, the intracellular concentration of Cl− is close to, but below, its electrochemical equilibrium. In a manner similar to that of K+ channels, Cl− channels provide a force—the concentration gradient of Cl−—that pulls the cell toward the equilibrium potential of Cl−, which is generally a hyperpolarized value. As a result, the inactivation of Cl− channels can lead to hyperexcitability; for example, mutations in the Cl− channel protein CLC-1 in skeletal muscle lead to muscle hyperexcitability, which results in myotonia. Similarly, the binding of strychnine to Cl−-conducting glycine receptors on, among other cells, motor neurons of the spinal cord can lead to violent convulsions and death (Chapter 5).
Cl− channels also control the osmotic flow of water across the cell membrane. Some Cl− channels that regulate osmotic balance are in fact activated by the swelling of a cell; activation of these channels allows Cl− to exit the swollen cell, accompanied by cations and water. These channels play an important role in secretory cells, such as those of the mucosal epithelium, and in the kidney. Indeed, the so-called loop diuretics, such as furosemide, block Cl− channels in the ascending loop of Henle and are widely used in the treatment of congestive heart failure.
As might be expected, the channels that permit Cl− to cross plasma membranes differ in molecular composition from the voltage-gated cation channels. Cl− channels comprise three molecular varieties 2–15. Ligand-gated Cl− channels are among the most important in the brain; they act as signaling proteins for the inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine and are discussed further in Chapter 5. CLC channels, which are believed to function as multimers of CLC proteins, are a family of homologous proteins characterized by 12 presumptive transmembrane domains (see 2–15). Nine CLCs have been cloned (CLC-1–7, CLC-Ka, and CLC-Kb); among them CLC-1, -2, -Ka, and -Kb reside on plasma membranes to control Cl− flux and membrane potential, and CLC-3–5, possibly 6 and 7 as well, are located on the membrane of intracellular vesicles and are thought to be important in maintaining the pH of these vesicles. Human mutations in CLC channels are linked to several diseases, including several renal disorders, neurodegeneration, and possibly epilepsy.
Structural models of three Cl− channel families. A. Topological model of γ-aminobutyric acid (GABA) and glycine receptor channels (see Chapter 5). B. CLC channel. C. CFTR channel. TM, transmembrane domains; NBF, nucleotide-binding fold; R, regulatory (phosphorylation) domain. (Adapted with permission from Jentsch TJ. Chloride channels: A molecular perspective. Curr Opin Neurobiol. 1996;6:304.)
Cystic fibrosis transmembrane conductance regulator (CFTR) channels are activated by the binding of ATP to two nucleotide-binding domains and by the phosphorylation of key serine residues on the regulatory domain (see 2–15; also see 2–5 for a discussion of ion channel phosphorylation). Like CLC channels, CFTRs are believed to possess 12 transmembrane domains; however, CFTR channels are distinguished from CLC channels by features such as their nucleotide-binding domains. The CFTR is among the most intensively studied ion channels because mutations of this channel cause cystic fibrosis, a relatively common hereditary disease that affects 1 in 3000 Caucasian newborns. In individuals with cystic fibrosis, the loss of CFTR Cl− channels in several types of epithelial cells limits the egress of Cl− ions into the lumen. Through mechanisms that are not completely understood, the disease also affects epithelial Na+ channels, whose malfunctioning causes the production of a thick, desiccated mucus. This abnormal secretion leads to obstruction of the biliary and pancreatic tracts and to a greatly increased incidence of pulmonary disease.