Ion Channels in Medicine

Frank Lehmann-Horn and Karin Jurkat-Rott,

Physiology, Ulm University, Ulm, Germany
doi: 10.1002/9780470048672.wecb260

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Article Contents
Ion Channels in Disease Biochemistry of Voltage-Gated Cation Channels Physiology of Voltage-Gated Cation Channels Hereditary Voltage-Gated Cation Channelopathies

Institute of Applied

Our current understanding of ion channels has been made possible by techniques such as electrophysiology and molecular biology, but genetics-based approaches inspired by associated diseases have pointed to regions of functional signicance on the ion channels. The ion channelopathies are caused by mutations of corresponding genes or autoantibodies. Although the channels form a highly diverse group (cation, anion, voltage-gated, ligand-gated) and are expressed in excitable and nonexcitable tissues, underlying mutations show recurrent patterns within functionally essential protein parts and lead to common clinical features and almost predictable mechanisms of pathogenesis. This knowledge will prove useful for development of treatment strategies for individuals with various genetic backgrounds and will contribute to both the effectiveness and safety of drugs in the future.

Hereditary Anion Channelopathies Tools and Techniques Used for the Study of Ion Channels Drug Treatment

Life s chemistry of aqueous solutions employs ions as carriers of cell signals. To overcome the impermeable lipid bilayer membrane, the cell invented transporters, channels, and pumps. Ion gradients are established by pumps and serve as batteries of potential energy. Ion channels are membrane-spanning proteins that possess a selectivity lter and a pore that usually is closed by gates in the resting state. The discharge occurs when ion channels open their gates and allow ions to ow down their electrochemical gradients. The cells depolarize when cations diffuse inward or when anions diffuse outward; the cells repolarize when the opposite occurs. Precise control of channel opening and closing is necessary for proper cell excitability and for the production of electrical signals and particularly the action potentials of nerve and muscle. Structures of importance like pore, selectivity lter, voltage sensors, ligand-binding sites, and opening and closing gates were highly conserved for more than 600 million years. Therefore, it is no surprise that an alteration of such functionally important structures can cause a disease. The episodic clinical features are provoked by environmental factors and caused by abnormal cell excitability, whereas the progressive manifestations that occur in some diseases (periodic paralysis, epilepsy, ataxia, and nephrocalcinosis) are caused by secondary cell degeneration.

disease (1, 2). Later, the term ion channelopathies was introduced to dene this class of diseases characterized by increased or decreased electrical cell excitability (3). Most channelopathies known to date do not lead to death, but rather they require an abnormal situation, a so-called trigger, to present with recognizable symptoms. Frequently, the attacks can be provoked by rest after physical activity or by exercise itself, hormones, stress, and certain types of food. Most channelopathies have a certain clinical pattern in common: Typically, the symptoms occur as episodic attacks lasting from minutes to days that show spontaneous and complete remission, onset in the rst or second decade of life, andfor unknown reasonshow amelioration at the age of 40 or 50 years. Surprisingly, many patients with channelopathies respond to acetazolamide, a carbonic anhydrase inhibitor. Most channelopathies show no chronic progression; however, a few exceptions exist. Channelopathies are caused by mutations or by autoantibodies. Although rare, they are important models for frequent disorders. As the hereditary channelopathies form a group of diseases too diverse to discuss in this short review, we refer to Table 1. In addition, we will describe in more detail some diseases

Ion Channels in Disease

The link to human disease came from applying electrophysiologic in vitro measurements on diseased muscle cells. These studies showed that an ion channel malfunction could cause a

of the largest group of hereditary voltage-gated cation channelopathies, the voltage-gated Na+ channelopathies of skeletal muscle, brain, and heart. Furthermore, we will give an example for an autoimmune ligand-gated cation channelopathy and an anion channelopathy. 1

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Ion Channels in Medicine

Table 1 Overview of hereditary human channelopathies Gene Locus Protein Disease Trait Change

Sodium channel SCN1A SCN1A SCN1B SCN2A SCN4A

2q24 2q24 19q13.1 2q23-24 17q23.1-25.3

Nav 1.1 Nav 1.1 1 subunit Nav 1.2 Nav 1.4

SMEI Familial hemiplegic migraine 3 GEFS+ Benign familial neonatal/infantile seizures Hyperkalemic periodic paralysis PC PAM Hypokalemic periodic paralysis 2 Long QT syndrome 3 Idiopathic Ventricular Fibrillations , Brugada Erythromyalgia, extreme paroxysmal pain Insensitivity to pain Liddles syndrome Pseudohypoaldosteronism I

D D D D

Loss (Gain) Loss Gain Gain

SCN5A

3p21

Nav 1.5

D D D D D D R

G/L Gain Loss Gain Loss Gain Loss

SCN9A

2q24

Nav 1.7

SCNN1B/G

16p12.2-p12.1

ENaC

Calcium channel CACNA1S CACNA1C CACNA1A CACNA1F CACNB4 RYR1 RYR2 PKD1

1q31-32 12p13.3 19p13.1 Xp11.23 2q22-23 19q13.1 1q42.1-43 16p13.3

Cav 1.1 Cav 1.2 Cav 2.1 Cav 1.4 4 subunit RYR1 RYR2 Polycystin-1

Hypokalemic periodic paralysis 1 Timothy syndrome Episodic ataxia 2, spinocerebellar ataxia 6 Familial hemiplegic migraine 1 Congenital stationary night blindness Generalized epilepsy, episodic ataxia 3 Malignant hyperthermia susceptibilty Central core disease, multiminicore disease Catecholinamergic polymorphic tachycardia Polycystic kidney disease, protein like Cav

D D D D R D D D/R D D

Unclear Gain Loss Gain Loss Gain Gain Gain Gain Unclear

Potassium channel KCNA1 12p13 KCNC3 19q13.3-4 KCNE1 21q22.1-22.2 KCNE2 KCNQ1 KCNQ2 KCNQ3 KCNQ4 KCNH2 KCNJ1 KCNJ2 KCNJ11 ABCC8 21q22.1 11p15.5 20q13.3 8q24.22-24.3 1p34 7q35-36 11q24 17q24.2 11p15.1 11p15.1

Kv1.1 Kv3.3 MinK MiRP1 Kv 7.1 Kv 7.2 Kv 7.3 Kv 7.4 Kv 11.1/HERG Kir 1.1 Kir2.1 Kir6.2 SUR1

Episodic ataxia 1 Spinocerebellar ataxia LQTS-5 Jervell and Lange-Nielsen LQT syndrome inducible, atrial brillation LQTS-1 Jervell and Lange-Nielsen Benign familial neonatal convulsions Dominant deafness LQTS-2 Antenatal variant of Bartter Andersen syndrome Short QT syndrome, atrial brillation Hyperinsulinemic hypoglycemia

D D D R D D R D D D D D D R

Loss G/L Loss Loss Loss Loss Loss Loss Loss Loss Loss Loss Gain Loss

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Ion Channels in Medicine

Table 1 (Continued ) Gene ABCC9 KCNMA1 Locus 12p12.1 10q22.3 Protein SUR2 KCa 1.1/BK Disease Dilated cardiomyopathy Epilepsy, paroxysmal dyskinesia Trait D D Change Loss Gain

Less-selective cation channels CNCG1 4p12-ce Chloride channel CLCN1 7q32-qter CLCN2 CLCN5 CLCN7 CLCNKB ABCC7 Glycine receptor GLRA1 3q27-28 Xp11.22 16p13 1p36 7q31.2

cGMP-gated

Retinitis pigmentosa

Loss

CLC1 CLC2 CLC5 CLC7 CLC-Kb CFTR

Thomsen myotonia Becker myotonia Idiopathic epileptic syndrome Dent disease Osteopetrosos Classic Bartter syndrome CF

D R D R D/R D R

Loss Loss G/L Loss Loss Loss Loss

5q31.2

GLRA1

Hyperekplexia

D/R

Loss

Nicotinic acetylcholine receptor CHRNA1 2q24-32 CHRNB1 17p12-11 CHRND 2q33-34 CHRNE 17 CHRNA2 8p21 CHRNA4 20q13.3 CHRNB2 1q21.3 GABA receptor GABRA1 5q34-35 GABRG2 5q31.1-33.1

nAChRA1 nAChRB1 nAChRD nAChRE nAChRA4 nAChRA4 nAChRB2

Congenital myasthenic syndrome

Nocturnal frontal lobe epilepsy

D/R D/R D/R D/R D D D

G/L G/L G/L G/L Gain Gain Gain

GABRA1 GABRG2

GEFS+ , Absence epilepsy with febrile seizures, juvenile myoclonic seizures

D D

Loss Loss

G , gain; L, loss; D , dominant; R , recessive. The names of the genes and their chromosomal location are given in column 1 and 2; the names of the corresponding proteins are listed in column 3 for voltage-gated (indicated by a subscribed v) and ligand-gated channels. Columns 4 and 5 list the diseases and their inheritances. The effects of the mutations on the function of the channel complex are categorized in column 6 as gain or loss of function. Gain stands for a gain that leads to a depolarization block. In general, gain-of-function mutations exert dominant effects if they dominate cell function, whereas loss-of-function mutations only cause dominant inheritance if the channel complex is multimeric (dominant negative effect) or if the second gene cannot compensate for it (haploinsufciency).

Biochemistry of Voltage-Gated Cation Channels

Basic motif of the main cation channel subunit, the so-called subunit, is a tetrameric association of a series of six transmembrane -helical segments, numbered S1S6, connected by both intracellular and extracellular loops, the interlinkers (Fig. 1). The subunit contains the ion-conducting pore and therefore determines main characteristics of the cation channel complex conveying ion selectivity, voltage sensitivity, pharmacology, and binding characteristics for endogenous and exogenous ligands. Although for Ca2+ and Na+ channels the subunit consists of a momomer, K+ channels form homotetramers or heterotetramers because each subunit consists only of one domain with six transmembrane helices Accessory subunits termed , , or do not share a common structure; some have one to several

transmembrane segments and others are entirely intracellular or extracellular. Functionally, they may inuence channel expression, trafcking, and gating. Voltage sensitive cation channels have at least one open state and at least two closed states, one from which the channel can be activated directly (the resting state) and one from which it cannot (the inactivated state). This implies that least two gates regulate the opening of the pore, an activation and an inactivation gate, both of which are usually mediated by the (subunit. Although activation is a voltage-dependent process, inactivation and the recovery from the inactivated state are time dependent. The voltage sensitivity of cation channels is conveyed by the S4 segments that are thought to move outward on depolarization and channel opening (4). During channel closing, not all voltage sensors move back at once, which generates a variety of closed states that explain the distribution of voltage sensor mutations to 3

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Ion Channels in Medicine

Voltage-gated Na+ channel I II III IV

Figure 1 Voltage-gated Na+ channel and associated disorders. The subunit consists of four highly homologous domains (repeats IIV) that contain six transmembrane segments each (S1S6). The S5 loops, S6 loops, and the transmembrane segments S6 form the ion selective pore, and the S4 segments contain positively charged residues that confer voltage dependence to the protein. Note the small, modulatory subunit. Mutations associated with channelopathies are indicated by conventional 1-letter abbreviations for the replaced amino acids.

phenotypes in Na+ channels. The ion-conducting pore is thought to be lined by the S5-S6 interlinkers that contain the selectivity lter. Although the localization of the activation gate may be within the pore, the inactivation gate has been shown to be located in different regions in the Na+ and K+ channels. The crystal structure of a channel claried the basis for selection of ions that can pass through the open channel pore and the mechanism by which the channel proteins sense changes in transmembrane voltage that control the open or closed conformational states of the channel (5). Thus, investigations of ion channel proteins employ fundamental physics to study the function of biologically critical proteins.

which the channels can be activated). In this simple, approximative model, transitions from one state into another are possible in both directions, which permits also the transition from the resting to the inactivated state at depolarization as well as the recovery from inactivation via the open state (for review see Reference (6)). Forward and backward rate constants for the transitions determine the probabilities of the various channel states.

Hereditary Voltage-Gated Cation Channelopathies

Hereditary diseases of voltage-gated ion channels cover the diverse elds of medicine myology, neurology, cardiology, and nephrology. As ion channels do not come alone, but rather in whole families of related proteins that conduct each ion type with slightly modied function and varying tissue expression patterns, the underlying mutations are restricted to single genes expressed in a specic tissue such as brain, skeletal muscle, cardiac muscle, sensory tissues, and secretory tissues (Table 1). Examples are myotonia, periodic paralyses, cardiac arrhythmia, long QT syndrome, migraine, episodic ataxia, epilepsy, and nephrocalcinosis. This trick is evolutionary: On one hand, it mediates many functions with the aid of one basic mechanism. On the other hand, it compensates for an eventually disturbed function by closely related channel siblings. The localization of the disease-causing mutations in the various channel proteins and their functional consequences can be similar in these disorders.

Physiology of Voltage-Gated Cation Channels

At the resting potential, the open probability of voltage-gated cation channels is extremely low, which indicates that very few channels open randomly. Depolarization causes channel activation by markedly increasing open probability. During maintained depolarization, open probability is reduced time-dependently and not voltage-dependently by channel inactivation, which leads to a closed state from which the channels cannot be reactivated immediately. Instead, inactivated channels require repolarization and a certain time for recovery from inactivation. On the other hand, repolarization of the membrane prior to the process of inactivation will deactivate the channel (i.e., reverse activation that leads to the closed resting state from 4

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Ion Channels in Medicine

Skeletal muscle voltage-gated Na+ channelopathies

Clinically, skeletal muscle Na+ channelopathies appear as recurring episodes of muscle stiffness or weakness triggered by typical circumstances such as cold, exercise, oral K+ load, or drugs. Muscle stiffness, termed myotonia, ameliorates by exercise and can be associated with transient weakness during quick movements that lasts only for seconds. It is the clinical phenotype brought about by uncontrolled repetitive ring of action potentials that lead to involuntary muscle contraction. On the other hand, the weakness is characterized by lack of action potentials or inexcitability. Several types of inherited myotonias have been observed because of mutations in the Na+ channel gene, SCN4A (Table 1, Fig. 1). Potassium-aggravated myotonia (PAM) may be distinguished clinically from other more frequent forms of Na+ channel myotonia by its sensitivity to K+ . On the other hand, paradoxical myotonia or paramyotonia (PC) worsens with exercise and cold, and it is followed by long spells of limb weakness that last from hours to days. Two other Na+ channel disorders are characterized by episodic types of weakness, with or without myotonia, and are distinguished by the serum K+ level during attacks of tetraplegia: hyperkalemic or hypokalemic periodic paralysis (HyperPP and HypoPP). All are autosomal and are transmitted dominantly. For PAM, PC, and HyperPP, the underlying Na+ channel pathogenesis mechanism is the same; mutations are present that cause a gating defect of the Na+ channel, which leads to slow or incomplete inactivationa so-called gain of function mutation (normally, Na+ channels are inactivating rapidly). This mutation results in an increased tendency of the muscle bers to depolarize. Although Na+ inux at slight depolarization

generates repetitive muscle action potentials and myotonia, stronger depolarizations lead to general inactivation of Na+ channels and to abolition of action potentials, which causes muscle weakness. Heterozygotes have both mutant and wild type channels, but the mutant channel determines the change in cell excitability, and hence, these disorders are dominant. The mutations (Fig. 1) are located mainly 1) in the voltage sensing S4 segment of domain IV, which is suggested to couple the inactivation to the activation process [PC; (7)]; 2) in the IIIIV interlinker, which is known to contain the inactivation gate [PC and PAM; (8)]; and 3) at several intracellularly-facing positions involved potentially as acceptor for the inactivation particle (Fig. 2) or involved in steric hindrance of the binding of acceptor and inactivation particle [PAM and HyperPP; (9, 10)]. Overlapping clinical phenotypes of the three disorders probably are caused by a similar increase in channel open probability (Fig. 3). The more severe membrane depolarization found in PC and HyperPP correlates with the higher transient intracellular Na+ accumulation, whereas depolarization and accumulation are small in PAM (11). Local anaesthetics and antiarrhythmic drugs of Class I, such as mexiletine and lidocaine derivatives, are useful antimyotonic agents for therapeutic treatment of PAM and PC. Their antimyotonic action occurs because they stabilize the inactivated state, which leads to a use-dependent block. The spontaneous weakness typical of HyperPP is not affected by mexiletine because no repetitive action potentials occur. However, for HyperPP, diuretics such as hydrochlorothiazide and acetazolamide can be useful; these drugs decrease the frequency and severity of paralytic episodes by lowering serum K+ and by other unknown mechanisms, perhaps, for example, by affecting pH or K+ channels.

Voltage-gated Cation Channel

Figure 2 Hinged-lid model of fast inactivation of Na+ channels. Birds eye view of the channel that consists of four similar repeats (IIV). The channel is shown cut and spread open between repeats I and IV to allow a view of the intracellular loop between repeats III and IV. The loop acts as the inactivation gate whose hinge GG (a pair of glycines) allows it to swing between two positions: the open channel state and the inactivated closed state where the inactivation particle IFM (the amino acids isoleucine, phenylalanine, and methionine) binds to its acceptor. WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.

Ion Channels in Medicine

WT

G1306V

V1589M

500 pA

500 pA

500 pA

5 ms

5 ms

3 pA

20 ms
Figure 3 Patch-clamp recordings from normal (WT), G1306V, and V1589 M channels expressed in human embryonic kidney cells. Upper panels: families of sodium currents recorded at various test potentials in the whole-cell mode show slowed decay and failure to return to baseline completely. Slowed inactivation is more pronounced with G1306V; the persistent inward sodium current is larger for V1589 M. Lower panels: traces of ve single-channel recordings each obtained by clamping the membrane potential to 20 mV. Mutant channels show re-openings, which are the reason for the macroscopic current alterations shown in the upper panels. Modied after Reference (8). Two examples of faulty inactivation of mutant Nav 1.4 Na+ channels of skeletal muscle associated with potassium-aggravated myotonia.

The disease in which mutations have only been found in voltage sensors, are hypokalemic periodic paralysis (HypoPP) types 1 and 2. In both types, episodes of generalized muscle weakness occur occasionally, often during the second half of the night after a day of intensive exercise. Another trigger is a carbohydrate-rich meal. Glucose and released insulin induce a rapid uptake of K+ into the muscle bers. The resulting hypokalemia correlates with the clinical expression of the paralytic attack and gave the disease its name. If no K+ is substituted, the weakness can last several hours or days until the serum level is normalized by a hypokalemia-induced rhabdomyolysis or K+ retention. As muscle strength is normal between attacks, at least in young patients, the underlying ion channel defect must be well compensated. The intermittent attacks of weakness in HypoPP lead to the requirement of trigger mechanisms. Insulin secretion, as after carbohydrate-rich meals, is one such trigger. Insulin activates the electrogenic Na+ /K+ -ATPase; insulin per se and the resulting decrease in [K+ ]o normally lead to a membrane hyperpolarization. In contrast to normal muscle, however, HypoPP-1 and HypoPP-2 muscle bers depolarize to 50 mV at a reduced [K+ ]o of 1 mmol/L and loose force (12). This explains the hypokalemic weakness of the patients. In HypoPP-1, mutations have been identied in the Cav 1.1 voltage sensors (S4 segments) of repeats II or IV (13, 14). HypoPP-2 mutations are located in S4 segments of Nav 1.4 repeats II and III [Fig. 1; (12, 15)]. The resulting changes in the pore currents were minor for HypoPP-1 and showed reduced function for HypoPP-1/2 rather than gain of function. Recent 6

results on K+ and Na+ channels indicate that voltage sensor mutations may create an accessory ion pathway that generates a hyperpolarization-activated cation leak independent of the main channel pore, and that this leak current is responsible for the pathogenesis of the disease (1618).

Neuronal voltage-gated Na+ channelopathies

Of the many neuronal voltage-gated Na+ channelopathies (Table 1), only generalized epilepsy with febrile seizures plus (GEFS+ ) and severe myoclonic epilepsy of infancy (SMEI) will be described in more detail. GEFS+ is an autosomal dominant childhood-onset syndrome that features febrile convulsions and a variety of afebrile epileptic seizure types within the same pedigree (19). SMEI is characterized by clonic and tonic-clonic seizures in the rst year of life that are often prolonged and associated with fever. Developmental stagnation with dementia occurs in early childhood. In contrast to GEFS+ , the syndrome usually is resistant to pharmacotherapy. Sometimes, patients with SMEI have a family history of febrile or afebrile seizures, and families exist in which GEFS+ and SMEI overlap, so that SMEI can be regarded as the most severe phenotype of the GEFS+ spectrum (20). The rst genetic defect in this group of diseases was discovered in a large GEFS+ pedigree (19). The authors identied a C121 W mutation that disrupts the disulde bridge of the 1 -subunit extracellular loop and leads to a loss of -subunit

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Ion Channels in Medicine

function. Subsequently, several groups found linkage to a cluster of genes that encode neuronal Na+ channel -subunits on chromosome 2q21-33 (Table 1). The rst two point mutations were detected in SCN1A to predict amino acid changes within the S4 segments of domains II and IV [T875 M, R1648H; (21)]. Many more SCN1A mutations have been described since then in addition to the mutations in the GABAA receptor (Table 1). A few of the Na+ channel mutations were expressed in human embryonic kidney cells or Xenopus oocytes that revealed both gainand loss-of-function mechanisms. Gain-of-function alterations were an acceleration of recovery from inactivation that shortened the refractory period after an action potential for R1648H (22). Increased persistent sodium currents predict membrane depolarization for T875 M, W1204 R, and R1648H (23); a hyperpolarizing shift in window current for W1204 R (24); and a reduced channel inactivation upon high frequency depolarizations for D188V (25).Whether the S4 mutations generate a hyperpolarization-activated cation leak through an accessory ion pathway has not yet been studied. In contrast, loss-of-function mechanisms were described in part for the same mutations such as enhanced fast and slow inactivation for T875 M and R1648H (22, 24) or a depolarizing shift of the steady-state activation curve for I1656 M and R1657 C (26), which all reduce the amount of available sodium channels. A complete loss-of-function was described for two other GEFS+ point mutations, V1353L and A1685V (26). Hence, loss-of-function mechanisms seem to predominate for GEFS+ , which is in agreement with the genetic ndings in SMEI as will be outlined below. In contrast to the point mutations found in GEFS+ families, most SMEI patients carry de novo nonsense mutations that predict truncated proteins without function (27). One SMEI point mutation was also shown to yield nonfunctional channels when expressed in human embryonic kidney cells (26). The sodium channel blocker lamotrigine, which is the only drug of this class that is in use in patients with idiopathic generalized epilepsies, deteriorates the clinical situation in SMEI patients; in particular, it can lead to an increased number of myoclonic seizures (28), similar to juvenile myoclonic epilepsy. This observation conrms that SMEI is a loss-of-function sodium channel disorder caused by haploinsufciency of SCN1A; from a genetic and clinical point of view, it is a severe allelic variant of GEFS+ . As a loss-of-function of a voltage-gated sodium channel decreases membrane excitability, it seems paradoxical that such mutations can cause epilepsy. However, when acting predominantly on inhibitory neurons, this effect could be responsible for the occurrence of hyperexcitability in neuronal circuits that induces epileptic seizures. The hypothesis that SCN1A is expressed selectively in inhibitory neurons has been recently conrmed (29, 30).

disturbed myocardial repolarization. Typical associated ventricular arrhythmias are Torsade de Pointes, in which the QRS complex twists around the isoelectric axis in the electrocardiogram. LQTS is a genetically heterogeneous disorder of usually dominant inheritance for which four causative genes have been identied: three K+ channel genes and one Na+ channel gene, SCN5A (Table 1). Triggering factors associated with arrhythmic events are different among genetic subsets of LQTS. LQT-3, the form caused by SCN5A mutations, often produce distinct clinical features that include bradycardia, and a tendency for cardiac events to occur during sleep or rest in often young and otherwise healthy individuals (31) whereas other LQTS manifest at high activity of the sympathetic nervous system with beta blockers being of benet. Expression of typical LQT-3 causing mutations such as the deletion of three amino acids in the loop that connects repeats III and IV, the inactivation gate of the channel (Fig. 1), revealed a defective fast inactivation characterized by a persistent sodium current during membrane depolarization (4). This persistent inward current is caused by channel bursting and reopenings as in the skeletal muscle sodium channel disorders described above. In addition to this gain of function, secondary indirect effects may occur in certain cases (32). The result is a prolongation of the cardiac action potential and refractory period, and the trigger-induced generation of arrhythmias that results in syncopes. Class I antiarrhythmics and local anesthetics such as mexiletine and ecainide proved to be effective because of their ability to prevent channel reopenings (33) whereas beta blockers are not effective (34). Mutations in SCN5A have also been associated with idiopathic ventricular brillation and with Brugada syndrome (35). A family history of sudden unexplained death is typical. Individuals with Brugada syndrome often exhibit a characteristic ECG pattern that consists of ST elevation in the right precordial leads, an apparent right bundle branch block, and normal QT intervals. They have an increased risk for potentially lethal polymorphic ventricular tachycardia. Administration of Class I antiarrhythmics and local anesthetics can expose this ECG pattern in latent cases. Accordingly, the proposed cellular basis of Brugada syndrome involves a primary reduction in myocardial sodium current. In fact, most Brugada mutations cause premature stop codons, frameshift errors, and splice site defects, and they are expected to cause nonfunctional channels. Heterologous expression of missense mutations revealed a reduced channel open probability also in agreement with a reduced channel function. Currently, an implantable cardioverter-debrillator is considered the only effective therapy to terminate ventricular arrhythmias in symptomatic patients with Brugada syndrome (36).

Cardiac voltage-gated Na+ channelopathies

Of all the episodic disorders known to be caused by ion channels, long QT syndrome (LQTS) is the most severe because of an increased risk of potentially fatal ventricular arrhythmias. The name is derived from the patients electrocardiogram, which shows an elongation of the QT interval as a result of

Acquired or autoimmune cation channelopathies

Many years before the term channelopathies had been established, muscle diseases were identied to be caused by autoantibodies against ligand- or voltage-gated ion channels. Often, these disorders occur in association with malignancy. For example, many patients with thymoma develop autoantibodies to the nicotinic acetylcholine receptor (nAChR) of the 7

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Ion Channels in Medicine

neuromuscular junction. Normally, ACh molecules can bind to nAChR, which is situated in the neuromuscular junction of the muscle cell membrane and is the rst cation channel cloned (37). ACh binding triggers opening of the ion channel and elicits an inward Na+ and Ca2+ current with fast rising and decaying phases. The inward current elicits with a high safety factor an action potential that propagates along the muscle ber membrane and activates excitation-contraction coupling. The myasthenia gravis antibodies cause the profound fatigue and weakness because of impairment in the safety factor of neuromuscular transmission from receptor block and internalization (38). The Lambert-Eaton syndrome is associated with small cell lung cancer and is caused by antibody binding to specic extracellular epitopes on the voltage-gated P/Q-type calcium channel glycoprotein of the peripheral motor neuron terminus. It is characterized by muscle fatigue (39). Peripheral nerve hyperexcitability (PNH) is a malfunction of the peripheral nerve and leads, in addition to other symptoms, to spontaneous skeletal muscle overactivity. In some patients, PNH is hereditary, whereas in most patients it is acquired and often an autoimmune channelopathy caused by serum antibodies to voltage-gated, dendrotoxin-sensitive K+ channels of peripheral nerves (40). As these autoimmune disorders full the criteria of channel disorders, this group of diseases has been included recently in the broader classication of channelopathies.

The patch clamp technique

Hodgkin and Huxley unraveled the ionic basis of nerve excitation in the squid giant axon by the rst detailed description of the processes of activation and inactivation of voltage-gated sodium and K+ currents that use the voltage-clamp technique (42). After this technique had become the principal tool for the study of channels for decades, a more recent development has revolutionized this eld. The patch clamp technique, developed by Hamill et al. (43), is a specic application of voltage clamping developed to record the current through a membrane patch conducted by a single channel molecule. The patch clamp technique allows for the direct electrical measurement of ion channel currents while simultaneously controlling the cells membrane potential. It uses a ne-tipped glass capillary to make contact with a patch of a cell membrane to form a G seal. Originally, this high resistance seal was yielded on skeletal muscle bers by enzymatic treatment that removed the basal membrane, the glycocalix, and the connective tissue. Thus, the treated preparation allowed Hamill et al. (43) to attach the glass pipette to the plasma membrane with a leak resistance of 10 to 50 M. Suction resulted in a G seal and enabled the measurements of currents in the 50-pA range with a small noise. Current variants of this technique make possible the application of solution on the exterior and interior of whole cells and on the membrane patches torn from the cell (outside-out or inside-out)every thinkable conguration of solution and ion channel orientation craved by the ion channel researcher. Usually, primary cultured cells or cell lines are preferred as they reveal a relatively clean surface membrane (44) and require no enzymatic treatment that damages the plasmamembrane. The patch clamp technique is now the gold standard measurement for characterizing and studying ion channels and is one of the most important methods applied to physiology. Single-channel recordings have shown that many channels (e.g., the voltage-gated Na+ channel) possess only two conductance levels: zero when the channel is closed and a constant conductance when the channel is open. After depolarization, a brief delay occurs before channels open. The intervals are not identical during each depolarization; in fact, the opening and closing of a given single channel is a random process even though the open probability depends on the voltage and is more sensitive to the voltage than an electronic device such as a transistor. After a subsequent short interval, the open time, the current jumps back to zero as the channels close. The stochastic nature can be understood by certain energy barriers that must be overcome before a channel can ip from one conformation (e.g., open) to another (e.g., closed). The energy needed for this purpose comes from the random thermal energy of the system. One can imagine that each time the channel molecule vibrates, bends or stretches, it has a chance to surmount the energy barrier. Each motion is like a binomial trial with a certain probability of success. Clearly, because the protein movements are on a picosecond time scale, but the channel stays open for milliseconds, the chance of success at each trial must be small, and many trials will be needed before the channel shuts. Usually, a normal Na+ channel does not reopen even though the depolarization may be pertained by the

Hereditary Anion Channelopathies

Cystic brosis (CF) is one of the most frequent genetic diseases with one case in 2000 to 4000 live births. It is a very serious disease with a mean life expectancy of 20 to 40 years. The patients suffer mostly from chronic pulmonal infections that lead to lung destruction, right heart insufciency, and heart failure. The defective gene product, the cystic brosis transmembrane conductance regulator (CFTR), was identied in 1991 (41). The complex molecule functions as Cl channel and as a regulator of other ion channels and transporters. A defect in CFTR leads to reduced NaCl and water secretion in the airways and in other epithelia. In addition, NaCl absorption is enhanced. As a result, the clearing of the airways is impeded, and chronic colonization by pathogenic bacteria such as Pseudomonas aeruginosa leads to airway destruction. CF is one of the rst genetic diseases in which genetic therapy is being attempted. However, the initial enthusiasm has been spoiled by the very limited success in animal models and the lack of convincing benet for the patient. Currently, new approaches for gene and even more so for classic therapy are under study. In addition, the CFTR molecule with all its complex functions is target of basic research. It is entirely feasible that the closer understanding of this molecule, its synthesis, maturation, and interaction with other transporters will lead to new and maybe unexpected therapeutic strategies.

Tools and Techniques Used for the Study of Ion Channels

8

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voltage clamp step for a certain time. Remarkably, the average behavior of a single channel is identical in time course to the macroscopic, whole-cell Na+ current. By combining the patch clamp with molecular cloning techniques, function and signicance of potentially important amino acid residues are elucidated rapidly. One common approach to this technique involves cloning the gene of interest and then inserting a designed or naturally occurring mutation into the clone by mutagenesis hoping that the mutation will produce measurable changes in channel function. Then, a heterologous expression system (e.g., a cell line of a different tissue), which does not express the gene of interest endogenously, is to be used to express the gene. This expression can be either transient, as in RNA-injected Xeno pus oocytes, or stable, as in viral-infected or transfected cells. Finally, the patch-clamp technique is used to characterize the function of a channel ensemble or of a single protein molecule.

Drug Treatment
The functional expression of the mutations in expression systems allows one to study the functional alterations of mutant channels and to develop new strategies for the therapy of ion channelopathies (e.g., by designing drugs that specically suppress the effects of malfunctioning channels). The limitations in throughput are overcome by the automation of the patch clamp technique by which it will become both cost-effective and fast and, at the same time, will enable the highest sensitivity and most accurate description of drug effect compared with any other ion channel drug screening method. Several strategies for the automation of the patch clamp technique have been pursued with set ups already available on the market. Currently, ion channel modulator drugs account for several billion U.S. dollars in worldwide sales, such as Cl channel activators (benzodiazepine, anxiolytics, and antiepileptics), K+ channel blockers (sulphonylurea antidiabetics, amiodarone-type anti-arrhythmics), Ca2+ channel blockers (verapamile-type antiarrhythmics), and Na+ channel blockers (lidocaine-type anesthetics, mexiletine-type antiarrhythmics, and antiepileptics such as lamotrigine and carbamazepine). As membrane-localized proteins, ion channels are accessible easily to drugs and, because of the variety of expression patterns and tissue-specic splicing, they likely have a restricted adverse-effect prole. This makes them ideal drug targets especially given the possibility of such precise observation of the drug effect on channel function by the patch clamp technique.

Acknowledgments
We thank for the continuous support of the German Research Foundation (DFG, JU470/1).

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Further Reading
Koch MC, Steinmeyer K, Lorenz C, Ricker K, Wolf F, Otto M, Zoll B, Lehmann-Horn F, Grzeschik KH, Jentsch TJ. The skeletal muscle chloride channel in dominant and recessive human myotonia. Science 1992;257:797800.

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