7 Molecular and Genetic Cardiovascular Medicine

Sonal Sharma, MD, Marcel E. Durieux, MD, PHD

1. The rapid development of molecular biologic and genetic techniques has greatly expanded the understanding of cardiac functioning, and they are beginning to be applied to the clinic.

2. Cardiac ion channels form the machinery behind the cardiac rhythm; cardiac membrane receptors regulate cardiac function.

3. Na⁺, K⁺, and Ca⁺⁺ channels are the main types involved in the cardiac action potential. Many subtypes exist, and their molecular structure is known in some detail, allowing a molecular explanation for phenomena such as voltage sensing, ion selectivity, and inactivation.

4. Muscarinic and adrenergic receptors, both of the G-protein–coupled receptor class, are the main regulators of cardiac function.

5. Adenosine plays important roles in myocardial preconditioning through an action on ATP-regulated K⁺ channels and is an effective antiarrhythmic drug by its action on G-protein–coupled adenosine receptors.

6. Volatile anesthetics significantly affect Ca⁺⁺ channels and muscarinic receptors.

7. Powerful genetic analysis techniques allow cardiovascular diagnosis through molecular approaches, but treatment through gene therapy has not yet become standard practice.

The past decades have witnessed what may be termed a revolution in the biomedical sciences, as molecular and genetic methodologies have suddenly jumped onto the clinical scene. Molecular biology originated in the 1950s, its birth most commonly identified with the description of the structure of deoxyribonucleic acid (DNA) by Watson and Crick.¹ For many years after, it was practiced almost exclusively in the research laboratory. Much of this research involved the laborious process of cloning: the identification of DNA molecules encoding specific proteins. Although at the time most people in the field realized that these advances would one day be of immense importance to clinical medicine, the exact place they would take was unclear.

Not generally appreciated was the rapidity with which molecular biology would advance. Now, more than a half century since the discovery of the structure of DNA, the human genome has been sequenced completely. Techniques for manipulating nucleic acids have been simplified enormously, and for many routine procedures, kits are available. The development of the polymerase chain reaction (PCR), a technique of remarkable simplicity and flexibility, has dramatically increased the speed with which many molecular biology procedures can be performed; in addition, it has allowed the invention of many new ones. Recent years have seen the development of techniques directed at screening large amounts of genetic material for changes associated with disease states. As a result of these and other developments, molecular biology has become a practical tool to study the expression and functioning of proteins in health and disease.

Cardiovascular medicine has been a major beneficiary of these advances. Not only have the electrophysiologic and pump function of the heart been placed on a firm molecular footing, but for a number of disease states, the pathophysiology has been determined, allowing progress in therapeutic development. Importantly, there is no indication that the pace of progress in molecular biology has slowed. If anything, the opposite is the case, and more dramatic advances may be expected in the years to come. Thus, techniques such as gene therapy may become available as therapeutic options in cardiac disease.

In this chapter, the most important aspects of molecular and genetic cardiovascular medicine are surveyed, with specific emphasis on medical issues relevant to the anesthesiologist. The myocyte membrane signaling proteins are of prime importance in this respect, and the two major classes—membrane channels and membrane receptors—are discussed. Simply stated, the channels form the machinery behind the cardiac rhythm, whereas the receptors are involved in regulation of cardiac function. This is, of course, an overgeneralization because close interactions among the various systems exist. In fact, these interactions have stimulated some of the more exciting areas of investigation in molecular cardiovascular medicine. In each of these sections, a brief overview of the general properties of the class of proteins is provided, and then several examples specific to the cardiovascular system are discussed. Each section ends with a discussion of some clinical correlates flowing from the material discussed.

The actions of anesthetics on these systems are also described. This is an area of active investigation. Much detail remains to be filled in, but it is clear now that anesthetics, at clinically relevant concentrations, interact with a number of cardiac signaling systems. Although it is too early to explain completely the cardiac effects of the various anesthetics through these mechanisms, there is no doubt that such interactions can be of significant clinical relevance. Considering the rapid pace of research in this area, rather than attempting to be all-inclusive, two examples in which a significant body of information is available, and in which clinical relevance appears likely, are emphasized. These examples are the cardiac Ca⁺⁺ channels and the muscarinic acetylcholine receptors.

The final section looks at the role of genetics in cardiovascular medicine, again with emphasis on developments of relevance to anesthesiology. The authors discuss techniques for genetic diagnostic screening, and their applications in the clinical setting. In addition, the potential of genetic therapy is also described briefly, an area that at this time has not yet made it into clinical practice.

In addition to providing an overview of the current state of knowledge, this chapter demonstrates a few of the many methodologies that have been used to obtain these results, to enable the reader to access the current literature with more ease. In quoting the literature, the authors have, therefore, chosen to provide references to many of the original articles describing techniques and findings, complemented by references to recent review articles to provide a current viewpoint.

The machinery behind the cardiac rhythm: ion channels

The cardiac action potential results from the flow of ions through ion channels, which are the membrane-bound proteins that form the structural machinery behind cardiac electrical excitability. In response to changes in electrical potential across the cell membrane, ion channels open and allow the passive flux of ions into or out of the cell along their electrochemical gradients. This flow of charged ions results in a current, which will alter the cell membrane potential toward the potential at which the electrochemical gradient for the ion is zero and is called the equilibrium potential (E) for the ion. Depolarization of the cell could, in principle, result from an inward cation current or an outward anion current; for repolarization, the reverse is true. In excitable cells, action potentials are mainly caused by the flow of cation currents. Membrane depolarization results principally from the flow of Na⁺ down its electrochemical gradient (E_Na is around +50mV), whereas repolarization results from the outward flux of K⁺ down its electrochemical gradient (E_K is around −90 mV). Opening and closing of ion channels selective for a single ion result in an individual ionic current. The integrated activity of many different ionic currents, each activated over precisely regulated potential ranges and at different times in the cardiac cycle, results in the cardiac action potential. Ion channels are usually highly (but not uniquely) selective for a single ion (e.g., K⁺ channels, Na⁺ channels). Channels may rectify, that is, pass current in one direction across the membrane more easily than the other. Electrical and chemical stimuli, which lead to opening and closing of the channel, cause a conformational change in the channel molecule (gating). The rate of change of channel conformation (gating kinetics) may be rapid, in which case the channel will open (activate) almost immediately (e.g., Na⁺ channels), or relatively slowly, which will result in a delay in channel activation (e.g., delayed rectifier K⁺ channels). After activation, ion channels may stay open until closed by another stimulus (e.g., repolarization of the membrane) or may close (inactivate) in the face of a continued stimulus. Inactivated channels will usually not reopen on repeat stimulation until they have recovered from inactivation (Box 7-1).

Patch Clamping

Much of the understanding of the molecular mechanisms behind the action potential has derived from the development and implementation of three techniques: patch clamping, a technique that allows recording of ion flow through individual channel molecules, voltage clamping of isolated cardiac cells, and cloning and heterologous expression of ion channel genes. Comparison of ionic currents recorded in isolated myocytes with currents recorded from cells expressing ion channel genes has resulted in the identification of many of the ion channel molecules that underlie the cardiac action potential.

The development of the voltage clamp technique in the early 1950s and its application to multicellular preparations of cardiac muscle allowed identification of the major ionic currents that underlie the cardiac action potential. The whole-cell currents recorded by the technique were smooth waveforms derived from summation of the activity of thousands of ion channels, and many different patterns of events at the single-channel level, arising from more than one molecular specie, can summate to produce identical whole-cell current waveforms. Patch clamping allows resolution of events at the single-channel level. In this technique, small patches of cell membrane (< 1 μm²) are isolated electrically and physically in the tip of a glass micropipette.² Single-channel events can then be resolved because there are only a few ion channel molecules present in the patch. Current flowing across the patch typically jumps between well-defined values corresponding to sudden opening or closing of the ion-conducting pore (Figure 7-1A). The whole-cell current will be the sum of the currents through all of the individual channels in the cell membrane; summation of the current flowing through a single channel during repeated stimuli will reproduce the macroscopic whole-cell current (see Figures 7-1A and B). As channel opening and closing in response to a stimulus are a stochastic phenomena, the regulation of ion flow, whether resulting from a change in membrane potential or from interaction with regulator molecules, is usually achieved by increasing the probability that the channel will be open. Thus, in Figure 7-1, which shows records from human cardiac muscle Na⁺ channels, the channels open (activate) a few milliseconds after depolarization because the probability that the channel will be open increases. Similarly, as the channels spontaneously close (inactivate), the open probability decreases.

Figure 7-1 Voltage clamp recordings from muscle Na⁺ channels.

A, Patch-clamp recordings. Individual Na⁺ channel openings are recorded as short-lived downward deflections. Note that openings are concentrated in the first part of the trace. The top five traces represent a single depolarizing clamp pulse. The sum of 100 such pulses (bottom trace) recreates the whole-cell current (B).

An ion current with distinct electrical and pharmacologic properties indicates the presence of a population of identical ion channel molecules. Application of molecular techniques has allowed the identification of many ion channel molecules, and thus a better understanding of the currents that underlie the cardiac action potential. An as-yet unrealized dream would be tailoring of pharmacologic agents that interact with specific channel types to shape the action potential.

Electrical Events Underlying the Cardiac Action Potential

Figure 7-2 shows a diagram of a cardiac action potential with a summary of the ionic currents flowing during each phase. As far as has been determined, the probable molecular identity of the ion channels that underlie these currents is also given. This section examines the biophysical properties of these currents; subsequent sections focus on possible molecular mechanisms underlying the biophysical phenomena.

Figure 7-2 Ionic currents underlying the cardiac action potential.

Currents are listed on the left, and the ion channel genes encoding the currents are listed on the right. 4-AP, 4-aminopyridine; CFTR, cystic fibrosis transmembrane regulator.

(From Roden DM, Lazzarra R, Rosen R, et al, for the SADS Foundation Task Force on LQTS: Multiple mechanisms in the long QT syndrome: Current knowledge, gaps and future directions. Circulation 94:1996, 1996.)

The Resting Membrane Potential and the Role of I_K1

The resting cardiac membrane potential is maintained close to the equilibrium potential for potassium (E_K) by a background, inwardly rectifying, highly selective K⁺ current (I_K1). Under physiologic conditions, the E_K is around −90 mV, and displacement of the membrane potential from this value should result in an inward or outward current through I_K1, thus returning the membrane potential toward E_K. However, although I_K1 passes significant inward current at potentials negative to E_K, at positive potentials, the channels display inward rectification (i.e., they pass inward current more easily than outward current), which limits outward current flow and reduces the tendency for I_K1 to hyperpolarize the membrane. This has obvious significance for an excitable cell; inward rectification ³ allows the initiation of action potentials and limits cellular K⁺ loss during the action potential. I_K1 is large in ventricular cells, smaller in atrial cells, and almost absent in nodal tissue.⁴ This explains why ventricular cells rest near E_K and have a high threshold for excitation, atrial cells have a more positive resting potential, and nodal cells have no defined resting potential.

Phase 0: Rapid Upstroke of the Cardiac Action Potential

The rapid upstroke of the cardiac action potential (phase 0) is caused by the flow of a large inward Na⁺ current (I_na) (Box 7-2).⁵ I_Na is activated by depolarization of the sarcolemma to a threshold potential of −65 to −70 mV. I_Na activation, and hence the action potential, is an all-or-nothing response. Subthreshold depolarizations have only local effects on the membrane. After the threshold for activation of fast Na⁺ channels is exceeded, Na⁺ channels open (i.e., I_Na activates) and Na⁺ ions enter the cell down their electrochemical gradient. This results in displacement of the membrane potential toward the equilibrium potential for Na⁺ ions, around +50 mV. I_Na activation is transient, lasting at most 1 to 2 msec because, simultaneous with activation, a second, slightly slower conformational change in the channel molecule occurs (inactivation), which closes the ion pore in the face of continued membrane depolarization (see Figures 7-1A and B). The channel cannot open again until it has recovered from inactivation (i.e., regained its resting conformation), a process that requires repolarization to the resting potential for a defined period. Thus, the channels cycle through three states: resting (and available for activation), open, and inactivated. Although the channel is inactivated, it is absolutely refractory to repeated stimulation. Stimuli that occur during recovery from inactivation will result in opening of fewer Na⁺ channels (because not all have recovered), and the resulting action potential will have a reduced maximal rate of depolarization and reduced conduction velocity. Na⁺ channels do not need to open to become inactivated. If the resting membrane potential depolarizes for a time, inactivation will occur in some channels and subsequent stimulation will result in an action potential of reduced amplitude and conduction velocity.

Phase 1: Early Rapid Repolarization

The early rapid repolarization phase of the action potential, which follows immediately after phase 0, results both from rapid inactivation of the majority of the Na⁺ current and from activation of a transient outward current (I_TO), carried mainly by K⁺ ions. On depolarization of the membrane, I_TO opens rapidly, over about 20 msec, before spontaneously inactivating. I_TO comprises two separate currents: the rapidly inactivating I_TO1, which is activated by depolarization and blocked by 4-aminopyridine, and the slowly inactivating I_TO2, which is activated by elevated intracellular Ca⁺⁺ (possibly explaining the observation that action potential duration tends to decrease with rapid heart rates and hypercalcemia)^6,⁷ (Figure 7-3).

Figure 7-3 Voltage clamp recordings from an atrial myocyte showing pharmacologic separation of I_TO1 (ito1) and I_TO2 (ito2).

In the presence of cobalt ions, only I_TO1 is seen. When cobalt is omitted and 4-aminopyridine (4-AP) is added, I_TO2 is revealed. Caffeine (Caf) eliminates I_TO2, leaving the underlying inward calcium current. In the presence of both 4-AP and cobalt ions, all outward currents are inhibited.

(From Wang Z, Fermini B, Nattel S: Delayed rectifier outward current and repolarization in human atrial myocytes. Circ Res 73:276, 1993.)

In addition to its effect on phase 1, I_TO, in combination with the delayed rectifier potassium currents (I_Kr and I_Ks) and I_K1, also contributes to membrane repolarization. Arrhythmogenic prolongation of the action potential in myocardial cells recovered from patients with myocardial hypertrophy,⁸ congestive cardiomyopathy,⁶ and from the border zone of myocardial infarction in animals⁹ appears to result from depression of I_TO.

Phases 2 and 3: Plateau Phase and Final Rapid Repolarization

The action potential plateau and final rapid repolarization are mediated by a balance between the slow inward current and outward, predominantly K⁺ current. During the plateau phase, membrane conductance to all ions falls and very little current flows. Potassium conductance is low because of inward rectification of I_K1 (i.e., inward current passes more easily than outward current), so little outward current flows despite the large outward electrochemical gradient for K⁺ ions and the delayed onset of the outwardly rectifying K⁺ currents (I_Ks, I_Kr, and I_Kur). The resulting small outward current is balanced by inward current, predominantly through L-type Ca⁺⁺ channels (I_Ca-L), but also via a slowly inactivating population of Na⁺ channels, and a small inward flux of chloride (Cl^–) ions, possibly carried by the cardiac variant of the ATP-dependent channel (abnormalities of which underlie cystic fibrosis).^10,¹¹ Phase 3, regenerative rapid repolarization, results from time-dependent inactivation of L-type Ca⁺⁺ current and increasing outward current through delayed rectifier K⁺ channels. The net membrane current becomes outward and the cell repolarizes.

Slow Inward Ca⁺⁺ Current

The slow inward current (I_Ca-L) is activated by depolarization of the cell to potentials less negative than −40 to −50 mV. In ventricular and atrial myocytes and in Purkinje fibers, I_Ca-L is activated by the regenerative depolarization caused by I_Na during phase 0 of the action potential. I_Ca-L does not contribute significantly to phase 0 because, in comparison with I_Na, it activates much more slowly (over about 10 msec) and is smaller in amplitude. I_Ca-L also inactivates slowly and, therefore, contributes the major inward current during the plateau of the action potential. I_Ca-L flows through L-type (long-lasting) Ca⁺⁺ channels, which are sensitive to block by dihydropyridines (e.g., nifedipine), and activation of contraction is related to the magnitude of the resulting calcium influx.¹² Gating of I_Ca-L is generally similar to I_Na in that channel opening and closing are dependent on membrane potential and time. Ca⁺⁺ channels are also, importantly, dynamically regulated by the autonomic nervous system.¹³ β agonists activate I_Ca-L (and hence increase myocardial contractility) indirectly by activating adenylyl cyclase via a guanosine triphosphate (GTP)–binding protein, G_s (Figure 7-4). The resulting increase in intracellular cyclic adenosine monophosphate (cAMP) activates protein kinase A (PKA), which phosphorylates the Ca⁺⁺ channel. Phosphorylated channels open in response to membrane depolarization; nonphosphorylated channels do not, so the effect of β-adrenergic stimulation is to increase the number of functional channels. The electrophysiologic effect of this is illustrated in Figure 7-5, which shows enhancement of the slow inward current by increase of adenosine monophosphate (AMP) level in single-channel Ca⁺⁺ channels and intact cells. β-Adrenergic effects on I_Ca-L are antagonized by acetylcholine, which, in myocardial cells, activates M₂ muscarinic receptors and inhibits adenylyl cyclase through activation of the GTP-binding protein G_i.

Figure 7-4 Autonomic regulation of ion currents.

ACh, acetylcholine; 5′AMP, adenosine 5′ monophosphate; β-AR, β-adrenergic receptor; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; Cyclase, adenylyl cyclase; Gi and Gs, guanosine triphosphate–binding proteins; ISO, isoproterenol; mAChR, M2 muscarinic receptor; Pi, pyrophosphate; R and C, regulatory and catalytic subunits, respectively, of protein kinase A.

Figure 7-5 Effects of elevated cyclic adenosine monophosphate (cAMP) on cardiac calcium channels.

A, Single-channel recordings; B, whole-cell currents. Left panels are control recordings; right panels show the effect of elevation of intracellular cAMP (in this case, induced by exposure to parathyroid hormone). Note the increased probability of channel opening and resulting increase in whole-cell current in the presence of increased cAMP.

(A, B, Modified from Rampe D, Lacerda AE, Dage RC, Brown AM: Parathyroid hormone: An endogenous modulator of cardiac calcium channels. Am J Physiol 261[6 Pt 2]:H1945, 1991.)

In the relatively depolarized pacemaker cells, which lack I_K1, I_Na is inactivated and the slow inward current is solely responsible for the upstroke of the action potential. I_Ca-L also can generate slowly propagated action potentials in diseased or damaged myocardial cells in which I_Na has been inactivated by depolarization. These slow responses, which may occur in the border zone of myocardial infarcts, are important because they may cause the slow conduction that can lead to reentrant arrhythmias.

Delayed Rectifier K⁺ Currents

Delayed rectifier K⁺ channels are present in all cardiac myocytes. They open slowly (over 200 to 300 msec) after depolarization of the membrane to the plateau level (−10 mV and greater), producing a K⁺-selective outward current, I_K. I_K does not inactivate on prolonged depolarization (unlike I_Na and I_Ca-L), and the channels close on repolarization of the membrane. Unlike I_K1, I_K displays outward rectification; that is, it passes outward current more easily than inward current. This is the expected behavior for a K⁺-selective channel because both the concentration and electrical gradients for potassium are outward. Thus, for any depolarizing displacement of membrane potential from E_K, the driving force will be larger in an outward direction. Similar to I_Ca-L, I_K is under autonomic control (see Figure 7-4). β-Adrenergic stimulation enhances I_K by a mechanism similar to that of the enhancement of I_Ca-L, thus ensuring repolarization of the cell in the face of increased inward Ca⁺⁺ current.¹⁴

Three components of I_K, carried by different channel molecules, can be distinguished. A rapidly activating component, I_Kr, is blocked by the compound E4031 (a Class III antiarrhythmic agent), which leaves a slower activating component, I_Ks, unaffected.¹⁵ This is illustrated in Figure 7-6, which also emphasizes the importance of I_K in the regulation of repolarization, and hence of action potential duration. A third component, the ultra-rapidly activated delayed rectifier, I_Kur, can be distinguished in atrial (but not ventricular) myocytes.¹⁶ This additional repolarizing current explains, in part, the enhancement of repolarization in atrial myocardium when compared with ventricle and Purkinje fibers.

Figure 7-6 A, Effect on action potential duration of blockade of I_Kr by E4031 in isolated guinea pig ventricular myocytes. After application of E4031, outward repolarizing currents are inhibited and action potential duration is increased. B, Voltage clamp records showing separation of I_Kr and I_Ks by E4031. The control delayed rectifier K⁺ current (C) is partially inhibited by E4031 (E), and the difference between the two currents (DIF) represents I_Ks.

(A, B, From Sanguinetti MC, Jurkiewicz NK: Two components of cardiac delayed rectifier K⁺ current. Differential sensitivity to block by class III antiarrhythmic agents. J Gen Physiol 96:195, 1990. Reproduced from The Journal of General Physiology by copyright permission of The Rockefeller University Press.)

Repolarization in Different Cardiac Tissue Types

Phase 3 repolarization in atrium and pacemaker tissues, but not in ventricular myocardium, is also enhanced by the presence of a large outward repolarizing K⁺ current (I_K[ACh]).^17,¹⁸ This potential independent current is activated indirectly by stimulation of muscarinic (M2-type) receptors by acetylcholine or purinergic (A-type) receptors by adenosine.¹⁹ This channel is potential independent and is activated via binding of an activated, membrane-bound GTP-binding protein (G_i), as discussed later.²⁰

There is variability in action potential duration between cells in normal ventricle.²¹ A gradient in action potential duration exists across the myocardium (from epicardium to endocardium), and specialized midmyocardial cells (M cells) have been identified, which exhibit prolongation of action potential duration at slow stimulation rates, possibly as a result of a decrease in I_Ks.

Phase 4: Diastolic Depolarization and I_f

Phase 4 diastolic depolarization, or normal automaticity, is a normal feature of cardiac cells in the sinus and atrioventricular (AV) nodes, but subsidiary pacemaker activity is also observed in the His-Purkinje system and in some specialized atrial and ventricular myocardial cells (see Chapter 4). Pacemaker discharge from the sinus node normally predominates because the rate of diastolic depolarization in the sinoatrial (SA) node is faster than in other pacemaker tissues. Pacemaker activity results from a slow net gain of positive charge, which depolarizes the cell from its maximal diastolic potential to threshold.

Pacemaker cells in the sinus node are relatively depolarized, with a maximal diastolic potential of −60 to −70 mV and a threshold potential of −40 mV. Rapid regenerative depolarization (phase 0) is dependent on opening of T-type and then L-type Ca⁺⁺ channels. Repolarization is dependent on activation of delayed rectifier K⁺ channels, and the maximum diastolic potential is around −80 mV. Pacemaker channels are activated by hyperpolarization to this potential and produce a slow inward Na⁺ current, I_f. This flows against slowly inactivating delayed rectifier K⁺ currents and results in diastolic depolarization.²² Because the current is nonselective among cations, its reversal potential lies between E_K and E_Na, at around −10 mV, and activation of I_f will tend to depolarize the cell toward this value. Similar to I_Ca-L, I_f is under autonomic control (see Figure 7-4) through GTP-dependent binding proteins G_s and G_i, which regulate cAMP production by adenylyl cyclase.^23,²⁴ β-Adrenergic stimulation shifts the voltage dependence of activation of I_f to more depolarized potentials, so for any hyperpolarizing stimulus, more I_f will be activated and diastolic depolarization will be enhanced. Acetylcholine has the opposite effect (Figure 7-7).

Figure 7-7 Effect of acetylcholine on spontaneous pacemaker activity in isolated sinoatrial (SA) node cells. Pacemaker activity in control solution is compared with activity in increasing doses of acetylcholine (ACh). Note the slowing of diastolic depolarization and resultant slowing of heart rate.

(From DiFrancesco D: Current I and the neuronal modulation of heart rate. In Zipes DP, Jaliffe J [eds]: Cardiac electrophysiology from cell to bedside. Philadelphia: WB Saunders Company, 1990, p 28.)

Molecular Biology of Ion Channels

The preceding sections have focused on the electrical events that underlie cardiac electrical excitability, and on the identification of cardiac ionic currents on the basis of their biophysical properties. Here the molecular structures behind these electrical phenomena are reviewed. The first step in understanding the molecular physiology of cardiac electrical excitability is to identify the ion channel proteins responsible for the ionic currents. Figure 7-2 gives the current classification of the ion channel responsible for each of the cardiac ionic currents. There are firm molecular candidates for voltage-gated Na⁺ and L-type Ca⁺⁺ channels. Similarly, channel molecules with properties similar to delayed rectifier K⁺ channels, the 4-aminopyridine–sensitive component of I_TO, the inward rectifier I_K1, the ligand-gated K⁺ channel I_K(ACh), and the pacemaker current I_f have been cloned. Figure 7-8 shows diagrams of the predicted membrane topology of some of these channels. Voltage-gated Na⁺, Ca⁺⁺, and K⁺ channels exist as conglomerates of molecules, consisting of a large α subunit and several accessory subunits (labeled β, δ, and γ in Figure 7-8). The α subunit alone is usually sufficient to induce channel activity in biologic membranes, but its activity is modulated by the presence of the accessory subunits. The diagrams in Figure 7-8 were deduced from hydrophobicity analysis of the primary structure of the major channel polypeptides. Regions of the polypeptides predicted to span the membrane are those that contain a high concentration of hydrophobic amino acids, whereas peptides linking these transmembrane sections are hydrophilic. Similarities among the various channels strongly suggest a common evolutionary ancestry. Na⁺ and Ca⁺⁺ channel α subunits (see Figures 7-8A and B) are strikingly similar, each consisting of four homologous transmembrane domains (labeled I to IV), linked by cytoplasmic peptides. Each homologous domain contains six linked membrane-spanning segments (labeled S1 to S6). These large polypeptides, containing more than 2000 amino acids, form a tetrameric structure and generate Na⁺ or Ca⁺⁺ channel activity in biologic membranes. Voltage-gated K⁺ channel α subunits, in contrast, are much smaller (see Figure 7-8C) and consist of a single transmembrane domain with six membrane-spanning segments, an arrangement similar to one of the individual domains of Na⁺ and Ca⁺⁺ channels. Four molecules are noncovalently linked in the membrane to produce a tetrameric structure, similar to a Na⁺ or Ca⁺⁺ channel, to produce K⁺ channel activity. The structure of the inwardly rectifying K⁺ channel molecules, I_K1 and I_K(ACh), is dissimilar to other K⁺ channels (see Figure 7-8C). The molecules are much less complex, having only two membrane-spanning segments, although these segments share considerable homology with the S5 and S6 segments of the classic voltage-gated K⁺ channel.

Figure 7-8 Diagrams of ion channel molecular structure.

A, Na⁺ channel. B, Ca⁺⁺ channel. C, K⁺ channels. ATP, adenosine triphosphate.

Voltage-gated ion channel activity requires that the channel molecule should sense and respond to changes in membrane potential, form an ion-selective membrane pore, and (in some cases) inactivate despite continuing depolarization. The molecular mechanisms for these phenomena are examined in separate sections later.

Molecular Mechanisms

The Voltage Sensor

Channel proteins respond to changes in electrical potential across the cell membrane by conformational changes (gating), which result from electrostatic interactions between charged portions of the molecule and the membrane electric field (Box 7-3). Gating of the channel is associated with a measurable flow of electrical charge through the membrane lipid bilayer (called gating current), as a zone of the molecule rich in electrical charge moves within the membrane.²⁵ This charge movement is linked to opening of the channel pore. The voltage sensor of voltage-dependent ion channels resides in the mobile S4 membrane-spanning segments, α-helical structures unusually rich in positively charged amino acids.²⁶ At rest, each of the positive charges in the S4 segment is balanced by fixed negative charges in other segments of the molecule. The resting membrane potential (negative inside) forces the (mobile) positive charges inward and the fixed negative charges outward. This dynamic equilibrium holds the channel pore closed. On depolarization, the force pulling the positive charge inward is relieved; positive charges (the S4 segments) are repelled outward and assume new partners with the fixed negative membrane charges. This charge movement comprises the gating current. If the depolarizing stimulus is short, repolarization of the membrane is followed by a gating current of equal and opposite magnitude as the S4 segment relaxes to its original position. If the depolarizing stimulus is prolonged, however, the movement of the S4 segments induces a conformational change in the channel molecule, which prohibits easy return to baseline. This conformational change in the channel molecule is manifested as activation (or channel opening), and this is closely coupled to channel closing (or inactivation; see later) in channels that inactivate. Thus, small changes in the membrane electric field cause conformational changes in the channel molecule, which result in opening (and closing) of the channel pore. An S4 segment rich in positive charge is a remarkably consistent feature of voltage-gated ion channels from a variety of different species and with a variety of ion selectivities. The dependence of channel activation on membrane potential is proportional to the density of positive charge in the S4 segment.

BOX 7-3 Molecular Mechanisms of Ion Channels

• Voltage sensor

• Gating mechanism (activation and inactivation)

• Ion pore

• Selectivity filter

Ion Channel Pore and Selectivity Filter

The presence of four homologous domains in voltage-gated Na⁺ and Ca⁺⁺ channels suggests that basic ion channel architecture consists of a transmembrane pore surrounded by the four homologous domains arranged symmetrically (see Figure 7-8). The membrane-spanning segments each form an α helix so that the walls of the pore will be derived from α-helical segments from each of the four domains. A pore formed from four such α helices would have limiting dimensions of 3 by 5 Angstrom units, similar to the size inferred for the Na⁺ channel pore by measurement of the permeability of cations of different sizes.^27,²⁸

The selectivity filter is formed by the S5 and S6 membrane-spanning segments of each domain together with their peptide linker.²⁹ As emphasized in Figure 7-8, unlike the hydrophilic extracellular linkers between other membrane-spanning segments, the S5/S6 linker is sufficiently hydrophobic to place it (at least partially) within the membrane lipid bilayer. The channel pore is lined both by the S5/S6 linker and the S5 and S6 membrane-spanning segments. Point mutations in the S5/S6 linker have dramatic effects on channel ion selectivity and reduce channel conductance to its primary ion. Extensive site-directed mutagenesis experiments of the S5/S6 linkers from a variety of channels suggest that they form a funnel that allows the passage of a specific ion into the pore. In Na⁺ channels, selectivity is imposed by two rings of negatively charged amino acids at the outer mouth of the funnel, which collect Na⁺ ions for transmission into the cell.²⁷

Channel Inactivation

Inactivation gating is the process by which ion channels close in the face of continuing depolarization. Inactivation is characteristic of voltage-gated Na⁺ and Ca⁺⁺ channels, as well as the K⁺ channels underlying I_TO. Inactivation begins after activation gating, as a second, slower conformational change in the molecule that halts the ion flux through the channel. Inactivation gating is thus closely coupled to activation gating, and ionic current flows only while both the activation and inactivation gates are open simultaneously. In Na⁺ channels, the inactivation gate is formed by the intracellular peptide linker between homologous domains III and IV (Figure 7-9A).³⁰ This peptide is postulated to act as a hinged lid, which moves upward to plug the ion pore (and thus halt current flow) shortly after membrane depolarization.²⁶ For the channel to recover from inactivation (i.e., to be ready to open in response to a new depolarizing stimulus), the III/IV linker peptide must resume its resting position, a process that requires hyperpolarization of the membrane to the resting potential for a finite period. Site-directed mutagenesis of the III/IV linker peptide has revealed a trio of hydrophobic amino acid residues (isoleucine, I; phenylalanine, F; and methionine, M), near to the domain III end of the peptide, which are crucial for normal channel inactivation. Replacement of just one of these residues (the phenylalanine) almost completely removes inactivation. These residues are postulated to latch on to a receptor in the channel pore to close the channel. The molecular basis of inactivation in K⁺ channels is rather different from Na⁺ channels. Because the four domains of K⁺ channels are formed by noncovalently linked molecules, there are no interdomain linkers to plug the channel pore. In K⁺ channels, a picture of an N-terminal ball-and-chain mechanism has emerged (see Figure 7-9B).³¹ The terminal 20 or so amino acids are very hydrophobic and are postulated to swing up and attach to the open pore. The next few amino acids contain a number of positively charged residues that draw the whole N-terminal end up to the membrane. These two domains act as a ball. The remaining amino acids, up to the beginning of the transmembrane S1 segment, act as a chain. If the chain is made longer, inactivation is slower, and vice versa.

Figure 7-9 Mechanism of inactivation of Na⁺ and K⁺ channels.

A, Hinged-lid mechanism of Na⁺-channel inactivation. B, Ball-and-chain mechanism of K⁺-channel inactivation. F, phenylalanine; I, isoleucine; M, methionine.

(A, B, From Catterall WA: Structure and function of voltage-gated ion channels. Annu Rev Biochem 64:493, 1995, by permission of the Annual Review of Biochemistry, Volume 64, ©1995, by Annual Reviews, Inc.)

Clinical Correlates

Ion Channels and Antiarrhythmic Drugs

Drug therapy of cardiac arrhythmias would ideally be targeted at an individual ionic current, tailoring the cardiac action potential in such a way that abnormal excitability was reduced but normal rhythmicity was unaffected. This remains an as-yet unrealized goal. The prototype antiarrhythmic agents (e.g., disopyramide and quinidine) have diverse effects on cardiac excitability and, similar to agents introduced more recently, frequently exhibit significant proarrhythmic activity with potentially fatal consequences. In the Cardiac Arrhythmia Suppression Trial (CAST), mortality among asymptomatic postmyocardial infarction patients was approximately doubled by treatment with the potent Na⁺-channel–blocking agents encainide and flecainide—an effect likely attributable to slowing of conduction velocity with a consequent increase in fatal reentrant arrhythmias.³² The results of the CAST prompted efforts to approach antiarrhythmic drug therapy by prolonging action potential duration (e.g., dofetilide)—a strategy with some support from animal studies, but one that also may cause proarrhythmia through induction of polymorphic ventricular tachycardia (the acquired long QT syndrome [LQTS]).³³ Drugs that prolong action potential duration all block I_Kr, and it is not clear that this therapeutic goal will result in arrhythmia control without induction of clinically significant proarrhythmia. The only drugs currently available that definitely prolong life by reducing fatal arrhythmias are β-blockers (e.g., ISIS-1, 1997), and these agents have no channel-blocking effects.

Ion Channels in Disease

Elucidation of the molecular mechanisms of the cardiac action potential is beginning to have direct impact on patient management. This is most obvious in patients with inherited genetic abnormalities of ion channels leading to cardiac sudden death, and two groups of diseases serve to illustrate this point: the LQTS and Brugada syndrome. An understanding of the molecular mechanism of cardiac electrical excitability is also starting to lead to the emergence of gene therapies and stem cell therapies that may in the future allow manipulation of cardiac rhythm and function.

Long QT Syndromes

This rare group of ion-channel abnormalities causes abnormal prolongation of the cardiac action potential, resulting in early afterdepolarizations (i.e., oscillations in the action potential during the plateau phase) and death from polymorphic ventricular tachycardia (torsades de pointes type). To date, six LQTS subtypes have been identified on the basis of the affected gene. They are numbered sequentially according to the date of discovery (Table 7-1), and all the loci so far identified encode ion channels except for LQT4. LQTS occurs because of disruption of cardiac repolarization, and in principle this can occur either from enhancement of inward depolarizing current or reduction of outward current. LQT3 is a gain-of-function mutation of the cardiac Na⁺ channel that results in failure of channel inactivation, most commonly from a deletion of three amino acids from the inactivation gate. LQT1 and LQT2 result from mutations of the delayed rectifier K channels that underlie I_Ks and I_Kr, and LQT5 and LQT6 result in reductions of I_Kr and I_Ks through mutations in channel accessory subunits. Loss of repolarizing current results in prolongation of the QT interval, and this leads to the syndrome. LQT4 is unique in that it results from a mutation in ankyrin B, an adapter protein that binds to the Na⁺ pump and Na⁺/Ca⁺⁺ exchanger. The resulting effects on cellular Ca⁺⁺ homeostasis cause ventricular arrhythmias.³⁴ Identification of the molecular substrate for LQTS will allow detection of the disease in asymptomatic carriers and may in the future allow targeted gene therapy; these issues are discussed in more detail later in the Genetic Cardiovascular Medicine section.

TABLE 7-1 Spectrum of Long QT Syndrome

Brugada Syndrome

The Brugada syndrome also is a group of ion-channel abnormalities that affect cardiac repolarization and result in cardiac sudden death. It is characterized by incomplete right bundle-branch block and persistent ST-segment elevation in the anterior precordial leads of the electrocardiogram (ECG).³⁵ Cases for which the genotype is available appear to result from channel mutations that reduce depolarizing Na⁺ current. This results in loss of the action potential dome, an effect that is most marked in the right ventricular epicardium where the transient outward current I_TO1 is strongly expressed (hence the ST-segment elevation in the anterior chest ECG leads). Early repolarization of the epicardial action potential results in a transmural repolarization gradient, and this can lead to reentry and sudden cardiac death.³⁶ In more than two thirds of patients with Brugada syndrome, the genetic locus is unknown, and much work remains to be done to elucidate the mechanism of this condition.