Jeanne M. Nerbonne

Chapter Outline

Summary

Electrophysiologic studies on isolated mammalian cardiac myocytes have identified the presence of multiple types of voltage-gated K⁺ (Kv) channel currents with distinct time- and voltage-dependent properties that contribute to determining action potential amplitudes, waveforms and durations. A number of non–voltage-gated, inwardly rectifying K⁺ (Kir) channels that contribute to action potential repolarization have also been functionally identified. The various types of Kv and Kir channels are differentially expressed in different cardiac cell types, contributing to cellular and regional differences in action potential waveforms and refractoriness. In the diseased myocardium, K⁺ current remodeling is evident and correlated with changes in action potential waveforms and durations, increased dispersion of repolarization and the development of substrates for reentrant arrhythmias. The cloning of Kv and Kir channel pore-forming (α) and accessory (β) subunits, as well as the subsequently identified two pore domain K⁺ (K2P) channel and small conductance Ca²⁺-dependent K⁺ (SK) channel subunits, has provided insights into the molecular basis of functional myocardial K⁺ channel diversity. In addition, a variety of experimental approaches have been, and continue to be, used to define the molecular determinants of native cardiac K⁺ channels and facilitated efforts focused on exploring the molecular mechanisms controlling the properties and the functional cell surface expression of these channels in the normal and in the diseased myocardium.

In atrial and ventricular myocytes, the action potential upstroke, attributed to inward currents through voltage-gated Na⁺ (Nav) channels, is rapid (Figure 3-1). In nodal tissues, which lack functional Nav channels, the upstroke of the action potential is substantially slower and is dominated by Ca²⁺ influx through voltage-gated Ca²⁺ (Cav) channels. There are also marked regional differences in action potential heights and durations, as well as in the time courses of action potential repolarization (see Figure 3-1). These differences, which affect the normal spread of excitation in the myocardium and influence the dispersion of repolarization in the ventricles, primarily reflect regional differences in the functional expression and the properties of the outward K⁺, as well as the inward (Nav and Cav) currents.¹

Cellular electrophysiologic studies have detailed the properties of the major inward (Nav and Cav) and outward (K⁺) currents that shape the waveforms of atrial and ventricular action potentials (Figure 3-2). In contrast to the cardiac Nav and Cav currents, there are multiple types of myocardial voltage-gated K⁺ (Kv) and non-voltage, inwardly rectifying K⁺ (Kir) channels (Table 3-1), many of which are differentially expressed, contributing to regional variations in myocardial action potential waveforms (see Figure 3-1) and refractoriness.^1–3 In addition, changes in the densities, distributions, and properties of Kv and Kir channels are evident in a variety of myocardial diseases, and these changes affect repolarization, influence propagation and decrease rhythmicity, effects that can produce substrates for the generation of life-threatening arrhythmias.¹ There is, therefore, considerable interest in defining the molecular mechanisms controlling the biophysical properties and the functional cell surface expression of these channels. A large number of Kv and Kir pore-forming α and accessory β (Table 3-2) subunits have been identified,^4,5 and considerable progress has been made in defining the relationships between these subunits and functional myocardial Kv and Kir channels.^1,6,7 Importantly, the studies completed to date have revealed that the molecular correlates of the various types of Kv and Kir channels distinguished electrophysiologically (see Table 3-1) are indeed distinct.¹ In the long term, defining the molecular compositions of myocardial Kv and Kir channels will also facilitate studies aimed at determining the molecular mechanisms controlling the marked regional differences in the expression of these channels in the normal myocardium, as well as the derangements in the expression or functioning of these channels that occur with myocardial disease. In this chapter, the electrophysiologic and molecular diversity of repolarizing myocardial Kv and Kir channels and the molecular determinants of native myocardial K⁺ channels will be reviewed.

Myocardial Voltage-Gated K⁺ Channels: Transient Outward Voltage-Gated K⁺ Channels

Kv currents, activated on membrane depolarization, influence myocardial action potential amplitudes and durations and, in most cells, two broad classes of Kv currents have been distinguished: transient outward K⁺ currents, I_to; and delayed, outwardly rectifying K⁺ currents, I_K (see Table 3-1). The transient currents (I_to) activate rapidly and underlie early (phase 1) repolarization, whereas the delayed rectifiers (I_K) determine the latter phase (phase 3) of action potential repolarization (see Figure 3-2) back to the resting membrane potential. These classifications are broad, however, and there are actually multiple types of transient (I_to) and delayed rectifier (I_K) Kv currents (see Table 3-1) expressed in cardiac cells.^1,3 Electrophysiologic and pharmacologic studies, for example, have clearly demonstrated that there are two types of transient outward K⁺ currents, now referred to as I_to,fast (I_to,f) and I_to,slow (I_to,s), and that these currents are differentially distributed.^6,7 The rapidly activating and inactivating transient outward K⁺ current, I_to,f, is also characterized by rapid recovery from steady-state inactivation, whereas I_to,s recovers slowly from inactivation.^6,7 In addition, I_to,f is readily distinguished from other Kv currents (including I_to,s) using Heteropoda toxin-2 or -3 (see Table 3-1).

Although originally identified in Purkinje fibers, I_to,f is a prominent repolarizing Kv current in atrial and ventricular myocytes, as well as in nodal cells, in most species.¹ There are, however, marked regional differences in I_to,f densities, with the highest densities typically in atrial myocytes. In addition, in mammalian ventricles, I_to,f and I_to,s are differentially distributed. In canine left ventricles (LVs), for example, I_to,f density is fivefold to sixfold higher in epicardial and midmyocardial, than in endocardial, cells.² There are also marked regional differences in I_to,f densities in adult mouse ventricles.^8–10 Specifically, I_to,f density is higher in right than in left ventricular myocytes, and within the LV, I_to,f densities are higher in apex than in base myocytes.^8–10 In the mouse, even greater Kv current heterogeneity is seen in cells isolated from the septum: all ventricular septum cells express I_to,s, and most (approximately 80%) also express I_to,f.⁸ When present, however, I_to,f density is significantly (p < 0.001) lower in septum than in RV or LV cells.⁸ I_to,f and I_to,s are also differentially expressed in ferret LV, and I_to,s is detected only in endocardial LV cells.¹¹ Despite heterogeneities in functional expression, the properties of I_to,f and I_to,s in different cardiac cell types (and species) are remarkably similar, leading to suggestions that the molecular compositions of the underlying (I_to,f and I_to,s) channels are also similar.³

Myocardial Voltage-Gated K⁺ Channels: Delayed Rectifier Voltage-Gated K⁺ Channels

Electrophysiologic and pharmacologic studies have also distinguished multiple types of cardiac delayed rectifier K⁺ currents, I_K (see Table 3-1). In atrial myocytes, for example, the dominant repolarizing K⁺ current is a rapidly activating, non-inactivating K⁺ current, I_Kur (I_K,ultrarapid), which is not detected in ventricular or nodal cells.¹ In most ventricular myocytes, there are two prominent components of delayed rectification, I_Kr (I_K,rapid) and I_Ks (I_K,slow), that are different from I_Kur in terms of time- and voltage-dependent properties.¹ The biophysical properties of I_Kr and I_Ks are distinct: I_Kr activates and inactivates rapidly, displays marked inward rectification, and is selectively blocked by class III anti-arrhythmics, including dofetilide and sotalol.¹² In contrast, I_Ks activates slowly and does not display inward rectification.¹²

Similar to the transient outward K⁺ currents, there are also marked regional differences in the functional expression of I_Ks and I_Kr in mammalian ventricular myocytes.1,2 The density of I_Ks in canine LV, for example, is higher in epicardial and endocardial than in M cells.² There are also regional differences in the functional expression of I_Kr and I_Ks channels in guinea pig LV.¹³ In cells isolated from the LV free wall, for example, the density of I_Kr is higher in subepicardial than in midmyocardial or subendocardial myocytes.¹³ At the base of the LV, in contrast, I_Kr and I_Ks densities are significantly lower in endocardial, than in either midmyocardial or epicardial, LV cells.¹³ Differences in Kv current densities contribute to the variations in action potential waveforms recorded in different regions (i.e., atria and ventricles, right and left ventricles, apex and base of the ventricles) of the heart, as well as in different layers (epicardial, midmyocardial, and endocardial) of the LV and RV walls.^1–3,8–13

In rodent ventricles, there are additional components of I_K with properties distinct from I_Ks and I_Kr (see Table 3-1). In mouse and rat ventricular myocytes, for example, there are novel delayed rectifier Kv currents that have been referred to as I_K, I_Kslow and I_ss (see Table 3-1).^1,3 Mouse ventricular I_K,slow was first identified as a rapidly activating and slowly inactivating K⁺ current with properties distinct from I_to,f, I_to,s and I_ss expressed in the same cells.¹⁴ In addition, I_K,slow was shown to be blocked selectively by micromolar concentrations of 4-aminopyridine (4-AP), which does not affect I_to,f or I_to,s.¹⁴ Subsequent work, however, revealed the presence of two components of mouse ventricular I_K,slow: I_K,slow1, which is blocked by µM 4-AP; and, I_K,slow2, which is blocked selectively by TEA.^15–18 In addition, it has been demonstrated that I_K,slow1 and I_K,slow2 reflect the expression of distinct molecular entities.^15–18 In contrast to the differential distribution of I_to,f and I_to,s, however, I_K,slow1, I_K,slow2, and I_ss appear to be uniformly expressed in mouse atrial and ventricular myocytes.^{8–10,15–19}

Inwardly Rectifying Myocardial K⁺ Channels Also Contribute to Repolarization

In addition to the Kv currents, Kir currents, specifically I_K1 and the ATP-dependent K⁺ current, I_KATP (Table 3-1), contribute to shaping myocardial action potential waveforms.^20–22 Similar to the Kv currents, the densities of the Kir currents vary in different regions of the heart (e.g., atria, ventricles, conducting tissue).^1,3 In contrast to the Kv currents, however, myocardial Kir current densities are similar in myocytes in different regions of the ventricles.¹ In mammalian atrial and ventricular myocytes, I_K1 plays a role in establishing resting membrane potentials and plateau potentials, and contributes to phase 3 repolarization (see Figure 3-2). The fact that the conductances of I_K1 channels are high at negative membrane potentials underlies the contribution of I_K1 to atrial and ventricular resting membrane potentials.²⁰ Although the voltage-dependent properties of I_K1 channels are such that conductance is low at potentials positive to approximately −40 mV, these channels nevertheless contribute outward K⁺ currents during the plateau phase of the action potential in ventricular cells,²⁰ as well as during phase 3 repolarization (see Figure 3-2), because the driving force on K⁺ is high at depolarized membrane potentials.

Myocardial ATP-dependent K⁺ channels are weak, inwardly rectifying channels that are inhibited by (elevated) intracellular ATP and activated by nucleotide diphosphates.²¹ In ventricular myocytes, activation of I_KATP channels during periods of hypoxia and ischemia results in action potential shortening, suggesting that these channels provide a link between cellular metabolism and membrane potential.^21,22 The opening of I_KATP channels appears to contribute to the cardioprotection resulting from ischemic preconditioning.²² In contrast with some ventricular Kv channels, I_KATP channels appear to be distributed uniformly in the right and left ventricles and through the thickness of the right or left ventricular walls. Interestingly, I_KATP channels are expressed at much higher densities than other sarcolemmal myocardial K⁺ channels,²¹ suggesting that action potentials will be shortened markedly when only a few I_KATP channels are activated.

Pore-Forming (α) Subunits of Myocardial Voltage-Gated K⁺ Channels

Kv channel pore-forming (α) subunits are six transmembrane-spanning domain proteins (Figure 3-3) with a region between the fifth and sixth transmembrane domains that contributes to the K⁺-selective pore.⁴ The positively charged fourth transmembrane domain in the Kv α-subunits (see Figure 3-3) is homologous to the corresponding regions in Nav and Cav channel α-subunits, placing them in the S4 superfamily of voltage-gated channels.^3,4 In contrast to Nav and Cav channels, in which only a single α-subunit is required to form a channel, functional Kv channels comprise four α-subunits (see Figure 3-3). Similar to the diversity of functional myocardial Kv channels (see Table 3-1), however, multiple Kv α-subunits have been identified. These subunits comprise several homologous Kv α-subunit subfamilies, Kv1.x, Kv2.x, Kv3.x, Kv4.x, Kv10.x, Kv11.x, and many members of these Kv α-subunit subfamilies are expressed in the mammalian heart.¹ In addition, further functional Kv channel diversity could arise through alternative splicing of transcripts and through the formation of heteromultimeric channels between two or more Kv α-subunit proteins in the same Kv α-subunit subfamily.^1,3,4

Additional subfamilies of Kv α-subunits were revealed with the cloning of the human eag-related (HERG) gene, KCNH2, subsequently identified as the locus of mutations underlying one form of familial long QT-syndrome, LQT2, and KCNQ1 (KvLQT1), the locus of mutations in another inherited long QT syndrome, LQT1.²³ Heterologous expression of KCNH2 (ERG1) reveals inwardly rectifying Kv currents²³ with properties similar to cardiac I_Kr (see Table 3-1). Although there are several ERG (KCNH) subfamily members, only KCNH2 (which encodes ERG1) appears to be expressed in the myocardium.¹ Heterologous expression of KCNQ1 (KvLQT1) alone reveals rapidly activating and non-inactivating Kv currents, whereas co-expression with the Kv accessory subunit, minK (see Table 3-2), produces slowly activating Kv currents that resemble the slow component of cardiac-delayed rectification, I_Ks.^1,3,23

Accessory/Auxiliary Subunits of Myocardial Voltage-Gated K⁺ Channels

In addition to the Kv α-subunits, a number of Kv channel accessory (Kv β) subunits have also been identified (see Table 3-2). The first of these subunits was KCNE1, which encodes a small (130-aa) protein (minK) with a single transmembrane spanning domain.²⁴ It appears that minK coassembles with KvLQT1 to form functional cardiac I_Ks channels.^23,24 Additional minK homologs, MiRP1 (KCNE2), MiRP2 (KCNE3), and MiRP3 (KCNE4) have also been identified (see Table 3-2), and it has been suggested that MiRP1 (KCNE2) functions as an accessory subunit coassembling with ERG1 to generate cardiac I_Kr.²⁵ It has also been reported that the MiRP subunits interact with multiple Kv α-subunit subfamilies and modify channel properties.²⁴ MiRP2, for example, coassembles with Kv3.4 in mammalian skeletal muscle,²⁶ and MiRP1 coassembles with Kv4.x α-subunits when coexpressed in heterologous cells.²⁷ These observations suggest that the MiRP (KCNE) accessory subunits can assemble with a variety of Kv α-subunits and contribute to the formation of multiple types of myocardial Kv channels. Direct experimental support for this hypothesis, however, has not been provided, and the roles of the various KCNE subunits in the generation of functional cardiac Kv^26,27 and other non-Kv²⁸ channels remain to be defined.

Another type of Kv channel accessory subunit was revealed with the isolation of low molecular weight (approximately 45 kD) cytosolic (Kvβ) subunits from brain.⁵ Three homologous Kv β-subunits, Kvβ1, Kvβ2, and Kvβ3 (see Table 3-2), as well as alternatively spliced transcripts, have been identified, and both Kvβ1 and Kvβ2 are expressed in heart.¹