The consequences of disrupting cardiac inwardly rectifying K+ current (IK1) as revealed by the targeted deletion of the murine Kir2.1 and Kir2.2 genes

Inwardly rectifying potassium channels have been identified in a wide range of tissues and are distinguished by their ability to allow the passage of inward current to a much greater extent than outward current. The attenuated outward flow of K+ through these channels at positive potentials is thought to occur because the internal pore of the channel becomes plugged by Mg2+ and positively charged polyamines (Matsuda et al. 1987; Vandenberg, 1987; Silver & DeCoursey, 1990; Matsuda, 1991; Ficker et al. 1994; Elam & Lansman, 1995; Fakler et al. 1995; Lopatin et al. 1995; Shyng et al. 1996). Cardiac tissue, especially atrial and ventricular myocytes, demonstrates an inward potassium current that is strongly and steeply rectifying and which has been termed IK1. This current is thought to be responsible for maintaining the negative resting potential seen in these cells (Nichols et al. 1996). In addition, because the channels responsible for IK1 close during depolarization, the conductance of cardiac myocytes decreases during the cardiac action potential. The rectification thereby conserves metabolic energy in a tissue that is constantly electrically active (Isomoto et al. 1997). Finally, the outward component of IK1 is thought to contribute to phase 3 repolarization of the cardiac action potential (Ibarra et al. 1991; Varro & Papp, 1992; Carmeliet, 1993; Deal et al. 1996). Although the physiological properties of IK1 have been extensively studied, its molecular composition is unknown. To date, seven families of genes encoding inward rectifiers have been identified. These Kir channels share a similar proposed subunit structure: a pore-forming loop with a single transmembrane segment on either side. Functional inward rectifier channels are thought to be made up of four subunits (Yang et al. 1995). In some cases, subunits encoded by separate genes can be assembled into a heteromultimeric channel. A notable example of such a channel is the acetylcholine-activated inward rectifier, which is composed of Kir3.1 and Kir3.4 subunits (Krapivinsky et al. 1995). However, not all gene products of the inward rectifier family appear to be able to co-assemble in vitro, and in many cases, including Kir2.1 and Kir2.2, functional channels appear to be formed by four identical subunits (Tinker et al. 1996). Attempts to pinpoint the molecular basis of IK1 have been hampered by the lack of suitable pharmacological agents. Most Kir channels are Ba2+ sensitive and there are no known toxins that distinguish them. Lately, transcript analysis in cardiac tissues has attempted to identify the inward rectifier responsible for the IK1 current (Dixon & McKinnon, 1994; Barry et al. 1995; Brahmajothi et al. 1996; Dixon et al. 1996). This analysis, in combination with comparisons of the biophysical characteristics of IK1 with those of cloned channels expressed in oocytes, has pointed to the Kir2 family. Within this family, Kir2.1 is the leading candidate because levels of its transcript correlate strongly with the amount of IK1 seen electrophysiologically (Brahmajothi et al. 1996). In the sinoatrial (SA) node where IK1 is absent, there is no Kir2.1 transcript. In the ventricle, where IK1 is present, Kir2.1, when compared to Kir2.2 or Kir2.3, has the highest level of transcript. However, these conclusions are tempered by the poor correlation seen between RNA and protein expression for some cardiac channels (Barry et al. 1995; Xu et al. 1996). Attempts to further pinpoint the molecular identity of IK1 by comparing its single channel conductance with that of Kir2.1, Kir2.2 or Kir2.3 are also problematic. The single channel conductances of Kir2.1, Kir2.2 and Kir2.3 are 22, 34 and 13 pS, respectively, with 150 mm external K+, while the reported predominant single channel conductances of IK1 range from 20 to 30 pS in 150 mm external K+ (Sakmann & Trube, 1984; Kurachi, 1985; Matsuda, 1988; Kubo et al. 1993; Takahashi et al. 1994; Morishige et al. 1994; Isomoto et al. 1997). Recently there has been more definitive evidence linking Kir2.1 to IK1 through genetic manipulation of cardiac cells in vitro. Nakamura (1998), using antisense oligonucleotides, partially suppressed IK1 in adult rat ventricular myocytes and thereby demonstrated that the Kir2.1 gene encodes at least one component of the native IK1. To further define the roles of the genes encoding Kir2.1 and Kir2.2 in the generation of IK1, we have engineered mice lacking the open reading frames encoding Kir2.1 and Kir2.2 (Zaritsky et al. 2000). This gene knockout strategy has allowed us to study the influence of the deletion of a single cardiac channel gene on the cellular and whole animal levels. In particular, we have examined the impact of the removal of these channels on individual currents as well as on action potential shape in isolated cardiac myocytes. In addition, we have attempted to correlate any changes seen at this level with changes seen in the behaviour of the heart in the intact animal.