Multiple voltage-gated K⁺ currents have been identified in the mammalian myocardium (Table 1). These currents are differentially expressed and contribute to the marked variations in the waveforms of action potentials in different regions of the heart (Figure 1). Two broad classes of voltage-gated K⁺ channel currents contributing to the repolarization phase of the action potential are:

Transient outward currents, Ito (Table 1) rapidly inactivating currents contribute to the early phase of repolarization two Ito subtypes have been identified: Ito,fast (Ito,f) and Ito,slow (Ito,s) Ito,f and Ito,s have distinct rates of inactivation and recovery from inactivation and different pharmacological properties underlie the initial, early phase (1) of action repolarization (Figure 1) Delayed rectifiers, IK (Table 1) typically activate later and inactivate slower (than Ito) on depolarization typically deactivate slowly on repolarization multiple types distinguished in myocardial cells, including IKr, IKs and IKur properties of the various IK (i.e., IKr, IKs, IKur) currents differ, suggesting that the molecular correlates of these currents are also distinct underlie the later phase (3) of repolarization (Figure 1 )underlie the later phase (3) of repolarization (Figure 1)

Importantly, the properties of the various currents (I_to,f, I_to,s, I_Kr, I_Ks and I_Kur, etc.; see Table 1) in different cardiac cell types and in different species are remarkably similar (Table 1), suggesting that the molecular correlates of these currents - which have been distinguished electrophysiologically and pharmacologically - in different cell types are also the same. However, this remains to be proven.

There is considerable interest in exploring the molecular mechanisms that regulate the functional expression of voltage-gated K⁺ channels in the myocardium under physiological and pathophysiological conditions. Pivotal to this effort has been recent studies aimed at identifying the molecular correlates of the different types of voltage-gated K⁺ channels in cardiac cells.

The first voltage-gated K⁺ channel pore-forming (a) subunit was cloned from the Shaker locus in Drosophila. The Shaker protein has six transmembrane domains, a highly charged S4 region that underlies the voltage-dependent gating properties of the channel, and a region between the fifth and sixth transmembrane domains that underlies the K⁺ selected pore. A number of C and N terminal splice variants of Shaker have been identified that give rise to K⁺ currents with distinct properties. Three additional, homologous genes, Shal, Shab and Shaw, which also encode voltage-gated (Kv) a subunits were subsequently cloned from Drosophila. Heterologous expression of these subunits also gives rise to voltage-gated K⁺ currents, albeit with distinct time- and voltage-dependent properties.

Molecular cloning techniques have been used to identify a number of mammalian homologues of Shaker, Shal, Shab and Shaw and importantly, in mammals, there are many members of each subfamily. Kva subunits of the Shaker subfamily are referred to as Kv1, and the genes are distinguished as Kv1.1, Kv1.2, and so forth (Table 2). The Shab subfamily is called Kv2, the Shaw subfamily Kv3, and the Shal subfamily Kv4 (Table 2). Functional voltage-gated K⁺ channels comprise four subunits and, in heterologous expression systems, members of the same subfamily can combine to form K⁺ channels with time and voltage-dependent properties different from the homomeric channels formed by the individual subunits alone. The role of heteromultimeric Kva subunit assembly in the generation of functional voltage-gated cardiac K⁺ channels, however, is unclear.

Additional, homologous subfamilies of Kva subunits, called ERG and KvLQT, have also been identified in the myocardium (Table 2). Importantly, ERG1 is the locus of mutations in long QT syndrome type 2 and mutations in KvLQT1 underlie long QT syndrome type 1. MiRP, and others are cytosolic, such as ß, KChIP and KChAP, proteins.

Molecular genetics is powerful tool for defining the roles of the various Kva subunits in the generation of functional voltage-gated K⁺ channels, as is evident in the case of ERG1, which underlies cardiac I_Kr, and KvLQT1, which underlies cardiac I_Ks. In the case of the other cardiac K⁺ currents, however, alternative experimental strategies are necessary to define these relationships. Work in Nerbonne's laboratory, for example, has focussed on exploiting in vivo approaches in mice, using transgenic and targeted deletion strategies, to identify the molecular correlates of the transient outward K⁺ currents, I_to,f and I_to,s.

Comparison of the properties of heterologously expressed Kva subunits and cardiac transient outward currents and analysis of the expression levels of the various Kva subunits in the myocardium, led to the hypotheses that K_V4 a subunits underlie I_to,f and that Kv1.4 underlies I_to,s. These hypotheses were tested in vivo using transgenic and targeted deletion strategies.

Electrical remodeling

In contrast to the dramatic effect on I_to,f, the densities and the properties of the other prominent voltage-gated outward K⁺ currents, I_K,slow and I_ss, in mouse ventricular cells are unaffected by Kv4.2W362F expression (Figure 2). Detailed analysis of the currents, however, suggested the presence of an additional, "novel" current component with decay kinetics slower than I_to,f and faster than I_K,slow in wild-type cells. Although this slow transient current could reflect effects of the mutant Kv4.2W362F on the properties of the wild type (I_to,f) channels, this seems unlikely given that no effects on kinetics are seen when the mutant subunit and wild-type Kv4 a subunits are co-expressed in heterologous systems. Rather, it appeared that a slow transient outward current was upregulated in Kv4.2W362F-expressing ventricular cells. Biochemical experiments revealed that Kv1.4 protein expression is increased in the ventricles of the Kv4.2W362F-expressing animals, whereas Kv1.2 and Kv2.1 protein expression levels in these animals are not significantly different from those determined in wild type animals. These results suggested a role for Kv1.4 in the generation of the slow transient K⁺ current in Kv4.2W362F-expressing cells.

Kv1.4 underlies I_to,s

Subsequent experiments revealed marked heterogeneity in the waveforms of the voltage-gated outward K⁺ currents in different regions of the mouse left ventricle (LV). In cells from the LV apex, for example, I_to,f is prominent and I_K,slow and I_ss are also expressed (Figure 2). The waveforms of the currents in cells isolated from the LV septum, however, are distinct (Figure 2). In most (~ 75 %) of the (septum) cells, I_Kslow, I_ss and I_to,f are expressed, although the density of I_to,f in these (LV septum cells) is significantly lower than in apex cells. In addition, in the septum cells lacking I_to,f, a slow transient current, referred to as I_to,s, was identified. Subsequent analyses revealed that I_to,s is expressed in all septum cells, i.e., in septum cells with I_to,f and in septum cells lacking I_to,f. Importantly, I_to,s is not detected in wild type apex cells. In addition, the properties of I_to,s in wild type septum cells are indistinguishable from the slow transient current described above that was identified in Kv4.2W362F-expresing transgenics cells. Interestingly, further experiments revealed that this current is only upregulated in Kv4.2W362F-expressing apex cells; I_to,s density is not increased in septum cells isolated from the Kv4.2W362F-expressing animals.

To determine the role of Kv1.4 in the generation of I_to,s, electrophysiological experiments were completed on ventricular myocytes isolated from animals with a targeted deletion in Kv1.4 (Kv1.4-/- animals). These experiments revealed that I_to,s is undetectable in (all) septum cells from the Kv1.4-/- mice (Figure 2). The properties and densities of the other currents, I_ss, I_K,slow and I_to,f in septum (and apex) cells, however, were unaffected by the loss of Kv1.4 (I_to,s). In addition and in contrast to the Kv4.2W362F-expressing transgenics, no electrical remodeling was seen in the Kv1.4-/- septum (or apex) cells.

To test the hypothesis that Kv1.4 upregulation underlies the appearance of slow transient current in Kv4.2W362F-expressing apex cells, they expressed Kv4.2W362F in the Kv1.4-/- background. Voltage-clamp recording from ventricular myocytes isolated from these animals revealed that I_to,f and I_to,s are eliminated (Figure 2). The waveforms of the currents in LV septum and apex cells from the crossed (Kv4.2W362F x Kv1.4-/-) animals are remarkably different from those recorded from wild-type LV cells (Figure 2). Interestingly, the currents in LV apex and septum cells from the crossed animals look remarkably similar when I_to,f and I_to,s are both eliminated (Figure 2), suggesting that the ventricles will have become remarkably homogeneous in terms of repolarization. In addition, analyses of the waveforms of the outward currents in the cells from the crossed animals revealed only the presence of I_K,slow and I_ss. Thus, in contrast to the Kv4.2W362F-expressing transgenics in which electrical remodeling is seen in (LV apex) cells when I_to,f is eliminated, there is no evidence of remodeling when I_to,f and I_to,s are both eliminated (Figure 2). Marked action potential prolongation, however, is seen in the cells isolated from the crossed animals and, in some cases, early after depolarizations were also noted.

A number of the other subunits have been shown by Nerbonne and other investigators to contribute to other types of voltage-gated channels. The functional roles of nearly all of the Kva subunits that encode the various types of cardiac voltage-gated K⁺ channels have been identified (Table 2). Notable exceptions are Kv1.7 and Kv 2.2, whose roles are presently unknown. A role for the Kv3 subfamily has recently been suggested in the generation of the ultrarapid component of outward rectification, I_Kur, in the canine myocardium, whereas Nerbonne's lab and the laboratory of Stanley Nattel in Montreal have shown that in both humans and rats, Kv1.5 underlies I_Kur. This is clearly an exception to the general rule that the various currents in different cell types and species are so similar it can be assumed the same subunits underlie them.