Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes

The amplitudes and the durations of action potentials in cardiac cells are largely determined by voltage-gated K+ channels and, in most cells, two types of voltage-gated K+ channel currents have been distinguished based on differences in time- and voltage-dependent properties and pharmacological sensitivities: (1) rapidly activating and inactivating, 4-aminopyridine-(4-AP)-sensitive K+ currents, referred to as Ito (transient outward); and (2) delayed, slowly inactivating and typically 4-AP-insensitive, K+ currents, referred to as IK (delayed rectifiers). These are broad classifications, however, and it is now clear that there are multiple components of IK, and that the densities, properties and functional roles of Ito and IK vary in cardiac cells isolated from different species, as well as in cells from different regions of the heart in the same species (Anumonwo et al. 1991; Antzelevitch et al. 1995; Barry & Nerbonne, 1996). There are also marked changes in the densities and the properties of voltage-gated K+ currents in the heart during normal development (Wetzel & Klitzner, 1996; Nerbonne, 1998) and in various myocardial disease states (Ten Eick et al. 1989; Boyden & Jeck, 1995; Nabauer & Kaab, 1998). For all of these reasons, there is considerable interest in defining the properties and the molecular correlates of cardiac Ito and IK channels, and in delineating the molecular mechanisms regulating the functional expression of these channels. Rapidly activating and inactivating Ca2+-independent, depolarization-activated transient outward, K+-selective currents, referred to as Ito1, Ito or It, have been identified in a number of cardiac cell types (Campbell et al. 1995; Barry & Nerbonne, 1996; Giles et al. 1996; Nerbonne, 1998). The time- and voltage-dependent properties of Ito in most cells are similar in that activation, inactivation and recovery from steady-state inactivation are all rapid (Campbell et al. 1995; Barry & Nerbonne, 1996; Giles et al. 1996). In rabbit atrial and ventricular myocytes, however, inactivation of Ito (It) is slow and recovery from steady-state inactivation is very slow, with complete recovery requiring seconds (Giles & Imaizumi, 1988; Fedida & Giles, 1991; Wang et al. 1999). Recently, it was also reported that there are regional differences in the properties of Ito in human (Nabauer et al. 1996), ferret (Brahmajothi et al. 1999), rat (Wickenden et al. 1999) and mouse (Xu et al. 1999) ventricles. The rates of Ito inactivation and recovery (from steady-state inactivation), for example, are significantly slower in human and ferret left ventricular endocardial (than epicardial) cells (Nabauer et al. 1996; Brahmajothi et al. 1999). In adult mouse ventricular myocytes, two transient outward K+ currents, similar to those characterized in the human and ferret, have also been distinguished; they have been referred to as Ito,fast (Ito,f) and Ito,slow (Ito,s) (Xu et al. 1999). In addition, Ito,f and Ito,s are differentially distributed in mouse ventricle: Ito,f is present in both left ventricular apex and septum cells, whereas Ito,s is only identified in left ventricular septum cells (Xu et al. 1999). Ferret epicardial Ito and mouse ventricular Ito,f have similar kinetic properties and both are blocked by nanomolar concentrations of Heteropoda toxins (HpTx) (Sanguinetti et al. 1997), whereas ferret endocardial Ito and mouse ventricular Ito,s are characterized by slow inactivation and recovery (from steady-state inactivation) kinetics and both currents are unaffected by HpTx (Brahmajothi et al. 1999; Xu et al. 1999). In ferret, there are also regional differences in the expression patterns of the voltage-gated (Kv), pore-forming (α) subunits Kv1.4, Kv4.2 and Kv4.3 (Brahmajothi et al. 1999). These observations have been interpreted as suggesting that distinct Kv α subunits underlie Ito in ferret left ventricular endocardial (Kv1.4) and epicardial (Kv4.2/Kv4.3) cells (Brahmajothi et al. 1999). Several recent studies have provided direct evidence to support a role for Kv α subunits of the Kv4 subfamily in the generation of the rapidly activating and inactivating current Ito,f in rat and mouse ventricular myocytes (Fiset et al. 1997b; Johns et al. 1997; Barry et al. 1998), although the molecular identity of mouse ventricular Ito,s has not been determined. To test directly the hypothesis that Kv1.4 underlies Ito,s in mouse left ventricular septum cells, electrophysiological experiments were completed on myocytes isolated from the left ventricles of mice with a targeted deletion of the Kv1.4 gene (Kv1.4−/−; London et al. 1998b). Analysis of whole-cell voltage-clamp recordings from Kv1.4−/− ventricular myocytes revealed that Ito,s in septum cells is selectively eliminated, whereas the currents in apex cells are unaffected by elimination of Kv1.4. In addition, in experiments conducted on left ventricular myocytes isolated from transgenic mice expressing a mutant Kv4.2 α subunit, Kv4.2W362F (Barry et al. 1998), Ito,f is eliminated in both septum and apex cells, whereas Ito,s in septum cells is unaffected. Analysis of the outward K+ currents in Kv4.2W362F-expressing left ventricular apex cells reveals the presence of a ‘novel’ current indistinguishable from Ito,s in septum cells. Importantly, however, Ito,s density in septum cells is unaffected by Kv4.2W362F expression. Western blot analysis also reveals that Kv1.4 protein expression is increased in the ventricles of Kv4.2W362F-expressing animals. Taken together, these results demonstrate that Kv1.4 underlies mouse ventricular Ito,s, and suggest that upregulation of Kv1.4 in apex cells underlies the electrophysiological remodelling observed in Kv4.2W362F-expressing transgenic mice.