Molecular determinants of cardiac transient outward potassium current (I(to)) expression and regulation

Abstract

Rapidly activating and inactivating cardiac transient outward K(+) currents, I(to), are expressed in most mammalian cardiomyocytes, and contribute importantly to the early phase of action potential repolarization and to plateau potentials. The rapidly recovering (I(t)(o,f)) and slowly recovering (I(t)(o,s)) components are differentially expressed in the myocardium, contributing to regional heterogeneities in action potential waveforms. Consistent with the marked differences in biophysical properties, distinct pore-forming (alpha) subunits underlie the two I(t)(o) components: Kv4.3/Kv4.2 subunits encode I(t)(o,f), whereas Kv1.4 encodes I(t)(o,s), channels. It has also become increasingly clear that cardiac I(t)(o) channels function as components of macromolecular protein complexes, comprising (four) Kvalpha subunits and a variety of accessory subunits and regulatory proteins that influence channel expression, biophysical properties and interactions with the actin cytoskeleton, and contribute to the generation of normal cardiac rhythms. Derangements in the expression or the regulation of I(t)(o) channels in inherited or acquired cardiac diseases would be expected to increase the risk of potentially life-threatening cardiac arrhythmias. Indeed, a recently identified Brugada syndrome mutation in KCNE3 (MiRP2) has been suggested to result in increased I(t)(o,f) densities. Continued focus in this area seems certain to provide new and fundamentally important insights into the molecular determinants of functional I(t)(o) channels and into the molecular mechanisms involved in the dynamic regulation of I(t)(o) channel functioning in the normal and diseased myocardium.

Figure 1. Functionally distinct components of I_to, I_to,f and I_to,s, are differentially expressed in ventricular myocytes

Representative whole-cell voltage-clamp recordings from adult mouse left ventricular apex (A) and interventricular septum (B) myocytes isolated from a wild type (WT) animal and a left ventricular apex cell (C) from a transgenic animal expressing a mutant Kv4.2 subunit, Kv4.2DN, that functions as a Kv4 subfamily specific dominant negative (and eliminates I_to,f). The recording conditions were optimized to allow the isolation of voltage-gated K⁺ (Kv) currents, and the voltage-clamp paradigm is illustrated to the right of the current records. The marked differences in the rates of recovery from inactivation of I_to,f and I_to,s and the predominance of I_to,f in WT apex cells (A) are clear in the current records shown; both the fast and the slow I_to components are expressed in WT septum cells (B). In contrast, I_to,f is not detected in Kv4.2DN left ventricular apex myocytes (C), and, in addition, I_to,s up-regulated in these cells. (D) The mean ± SEM normalized recovery data for I_to,f and I_to,s are plotted as function of the recovery time: I_to,f recovers very rapidly, whereas I_to,s recovery is slow. The amplitudes of the peak Kv currents and of I_to,f and I_to,s in individual cells were determined and normalized to the whole cell membrane capacitance (measured in the same cell) to obtain current densities and mean ± SEM peak (E), I_to,f (F) and I_to,s (G) densities are plotted. In Kv4.2DN myocytes, I_to,f is eliminated and only I_to,s is expressed, demonstrating the critical role for Kv4 α subunits in the generation of I_to,f channels. When the Kv4.2DN protein is expressed in the hearts of (Kv1.4−/−) mice in which the Kcna4 (Kv1.4) locus has been disrupted, I_to,s (in addition to I_to,f) is also eliminated, demonstrating the critical role for Kv1.4 in the generation of ventricular I_to,s channels. (Adapted with permission from [27,78,111])

Figure 2. Amino acid sequence and membrane topology of human Kv4.3

The linear amino acid sequence of human Kv4.3 is illustrated; the C-terminal insert in the long version of the protein (Kv4.3L) is highlighted. The tetramerization (T1) domain, the KChIP2 binding domain and Filamin C binding domain are also indicated. As discussed in the text, it has been reported that Kvβ subunit-mediated modulation of Kv4-encoded currents requires both the Kv4 N-terminus and C-terminus, although no specific information about putative Kvβ subunit binding domains is available. Several known and potential phosphorylation sites in the Kv4.3 sequence and the specific serine-threonine protein kinases, including protein kinase A (PKA), protein kinase C (PKC), protein kinase G (PKG), calcium-calmodulin dependent protein kinase II (CaMKII) and extracellular signal regulated kinase (ERK), meditating phosphorylation of these residues are also indicated; the color code is provided in the key the upper right corner of the Figure. Serine and threonine residues that are known or potential targets of multiple protein kinases are indicated accordingly, in multicolor.

Figure 3. Schematic illustration of a Kv4.3 channel with multiple accessory subunits

Cross section of a Kv4.3 channel in the membrane with the N-terminal tails and the S5-H loop-S6 (pore) assemblies (in blue) of (two) Kv4.3 α subunits drawn based on the published crystal structures of Kcsa [85,274], Kv1.2 [275,276] and the Kv4.3N terminus-KChIP1 complex [121]. Similarly, the KChIP2 structure and the KChIP2-Kv4.3 interaction illustrated are based on the published crystal structure of Kv4.3N terminus-KChIP1 complexes [122,123] and the DPP6 and minK structures are based on published DPP6 [277] and minK [278] structures, respectively. The Kvβ1 structure, derived from published crystallographic Kvβ2 data [279,280], is illustrated docked on the Kv4.3 N-terminus at a site distinct from (and non-overlapping with) the KChIP2 binding site. This configuration suggests that both the KChIP2 and the Kvβ1 subunits could be complexed to cardiac Kv4.3 α subunits in a 1:1:1: (4:4:4) stoichiometry in the generation of functional myocardial I_to,f channels.