K_IR2.x Proteins Constitute the Classical Inward Rectifier Current

The classical I_K1 channels are formed by K_IR2.x proteins, mainly as homotetramers but probably also as heterotetramers. This subfamily consists of five members of which K_IR2.1, 2.2, and 2.3, encoded by KCNJ2, KCNJ12, and KCNJ4, respectively, are most prominently expressed in the heart. The main subunits in the working myocardium are K_IR2.1 and to a lesser extent K_IR2.2. Expression levels of K_IR2.3 are much lower. K_IR2.x proteins are almost absent in nodal tissues.

Each monomer comprises a transmembrane (TM) and a cytoplasmic part. The transmembrane domain (TMD) consists of the two TM helices, the pore helix positioned between these, and a slide helix located in front of TM1. The cytoplasmic pore domain (CPD) is compiled by interaction of the amino- and carboxyl-termini. In the resulting channel, the four TM2 (inner) helices of each monomer face the central water-filled pore in the TMD, with the potassium selectivity filter (SF) positioned in close proximity to the cellular exterior (see Figure 13-2). It is believed that ion conduction may be regulated by two gates in series: one formed by the inner helices (TM2) of the TMD at the helix-bundle crossing and the other by the G loop at the apex of the CPD.²

Tetramerization of CPDs creates an extended pore region into the cytoplasm, separated from the TM pore region by the G-loops. Polyamine block of inwardly rectifying potassium (K_IR) channels underlies their key functional property of preferential conduction of inward K⁺ currents (inward rectification). Kinetic models describe polyamine block as a multistep process, incorporating sequentially linked “shallow” and “deep” binding steps of polyamines in the K_IR pore. Structurally, these shallow and deep binding steps are conceptualized as an initial weakly voltage-dependent binding in the cytoplasmic domain of the channel, followed by a steeply voltage-dependent step in which spermine migrates to a stable binding site in the TM pore (inner cavity). Five negatively charged residues within the cytoplasmic (E224, D259, E299, F254, and D255) and one in the TM pore regions (D172) are involved in interacting with the positively charged polyamines.⁵

K_IR2.x Channels Set the Resting Membrane Potential and Contribute to Repolarization

In the working myocardium, K_IR2.x-mediated I_K1 sets the resting membrane potential close to the potassium reversal potential and contributes outward potassium current during repolarization. With respect to the heart, neonatal K_IR2.1 knockout mice were bradycardic and displayed electrocardiogram (ECG) abnormalities such as lengthening of the PR (~50%) and QT (~50%) intervals. Isolated cardiomyocytes from K_IR2.1 knockout animals displayed (1) no I_K1; (2) action potential prolongation; and (3) spontaneous beating activity.⁹ In contrast, K_IR2.2 knockout mice were viable and lived through adulthood without ECG abnormalities. However, their cardiomyocytes displayed a 50% reduction in I_K1 densities. In humans, dominant negative KCNJ2 mutations are associated with Andersen-Tawil syndrome 1, characterized by periodic muscle paralysis, biventricular tachycardia and sometimes long QT times, and developmental bone abnormalities (see Table 13-1).¹⁰ Gain-of-function mutations in KCNJ2 have been associated with short QT syndrome, catecholaminergic polymorphic ventricular tachycardia, and congenital atrial fibrillation.^11–13 Currently, no disease-causing mutations in KCNJ12 and KCNJ4 have been described.

I_KACh Channels and Heart Rate Regulation

I_KACh slows heart rate and atrioventricular conduction in response to muscarinic M₂ receptor activation by ACh released from the vagus nerve. In this process, Gβγ directly interacts with and activates the channel that thereby antagonizes diastolic depolarization of the nodal cardiomyocytes. KCNJ3^–/– mice were viable but showed a lack of carbachol-induced I_KACh in atrial cardiomyocytes. Moreover, animals showed blunted responses to pharmacologic interventions directed at vagal-induced bradycardia.¹⁴ KCNJ5^–/– mice¹⁵ displayed loss of cardiac I_KAch. Mice were viable; in most studies their resting heart rate increased and animals showed a reduced negative chronotropic response after vagal stimulation. Furthermore, mice were unable to undergo rapid changes in heart frequency. In human heart function, KCNJ5 mutations have been associated with long QT syndrome, syncope, atrial fibrillation, and sudden cardiac death (see Table 13-1).¹⁶ No diseases related to mutations in KCNJ3 have been reported.

I_KATP Channels Confer Metabolic Status to Electrical Properties

The ATP-sensitive inward rectifier channels are metabolic sensors that couple the cardiac metabolic state to electrophysiologic properties. Under physiologic conditions, ATP favors closure, whereas ADP levels determine opening of the channels. Upon metabolic challenges, for example as a result of cardiac ischemia, decreased ATP/ADP ratios result in activation of the channel, thereby preserving normal resting potential and shortening the cardiac action potential and the period of cell contraction. These processes aid in ATP preservation during circumstances that challenge the vulnerable cardiac myocyte.

K_IR6.1 knockout mice die suddenly after the occurrence of ST elevation and subsequent third-degree atrioventricular block and cessation of heart rhythm, which may result from myocardial ischemia as a result of blunted coronary vasoregulation.¹⁷ The presence of K_IR6.1 in the cardiac conduction system may provide another or additional mechanism of cardiac pathology and conduction disturbances in these animals. Furthermore, K_IR6.1 knockout mice are more susceptible to endotoxin-mediated septic shock, resulting in ST elevation and cardiac death. In the hearts of K_IR6.2 knockout mice, a number of stressors induce calcium overload, followed by short-term abnormalities in cardiac rhythm (early afterdepolarization, premature ventricular contractions, and sinus node regulation) and hemodynamics (increased left ventricular end-diastolic pressure).^18,19 In the long term, cardiac maladaptive remodeling (exaggerated fibrosis and hypertrophy) finally results in impaired ejection fraction and congestive heart failure.

SUR1 knockout mice are fertile and phenotypically normal but lack atrial I_KATP.²⁰ Furthermore, ABCC8 null mutation increases resistance to cardiac, but not neuronal, ischemia-reperfusion injury.²¹ Knock out of SUR2 resulted in loss of I_KATP from all muscles, coronary vasospasms, increased blood pressure, and premature death from week 5 onward. Animals displayed slight cardiac hypertrophy and increased resistance to cardiac ischemia-reperfusion damage.²²

In humans, a gain-of-function mutation in KCNJ8 has been associated with early repolarization and Brugada syndrome, both characterized by specific (J-wave) ECG abnormalities and atrial fibrillation.23–26 KCNJ8 loss of function has been associated with sudden infant death syndrome.²⁷ ABCC9 mutations have been linked to atrial fibrillation, dilated cardiomyopathy, and Cantú syndrome, which causes cardiomegaly and pericardial effusion, among other effects (see Table 13-1).^28–30 Thus far, KCNJ11 and ABCC8 mutations have not been associated with cardiac phenotypes.

Effects of Drugs on K_IR Channels

The most commonly used blockers to examine the physiologic roles of K_IR channels in native cells and tissues are Ba²⁺ and Cs⁺. At micromolar concentration, Ba²⁺ blocks K_IR channels with relative specificity. Externally applied Ba²⁺ suppresses K_IR currents in a voltage-dependent manner and Ba²⁺ inhibits K_IR channels more strongly as the membrane is hyperpolarized.² However, experiments have shown that BaCl₂ infusion in vivo produces strong deleterious effects—mainly skeletal, cardiac, and smooth muscle dysfunction.⁵

In the intact heart, I_K1 blockers produce membrane depolarization (an effect that slows conduction velocity because of a voltage-dependent inactivation of Na⁺ channels), cause prolongation of the QT and QRS intervals on the surface ECG, and at higher concentrations cause ventricular ectopy and lethal ventricular arrhythmias. Conversely, it has been demonstrated that increasing inward rectifier currents stabilize reentrant arrhythmias. Recently, it has been shown that pharmacologic inhibition of inward rectifier currents by chloroquine induces antifibrillatory effects.³¹ Different antiarrhythmic (e.g., quinidine, amiodarone, propafenone, disopyramide) and noncardiac (e.g., terfenadine, astemizole) drugs at micromolar concentrations have been found to inhibit the classical cardiac I_K1. However, little is known about their mechanisms of inhibition.³²

Gambogic acid (GA) is a potent inducer of apoptosis that has been considered for anticancer therapy. At 10 µM, GA inhibited K_IR2.1 current differentially during acute applications that produced 30% of inhibition or during prolonged exposures (up to 3 hours), which produced 70% of inhibition. The IC₅₀ of K_IR2.1 inhibition by GA during prolonged exposures was 27 nM. GA–induced inhibition is not caused by changes in biogenesis or trafficking to the plasma membrane. It is a lipophilic molecule (LogP ~6.77) that can insert itself into the lipid bilayer and thereby might perturb association of K_IR2.1 with regulatory lipids and proteins that specifically facilitate the function of K_IR2.1 channels. It has been found that pretreatment of HEK293 cells with GA shifted K_IR2.1 and K_v2.1 channels from the Triton X-100–insoluble to the Triton X-100–soluble fraction. GA decreasing K_IR2.1 activity is consistent with K_IR2.1 being physiologically active in the context of Triton X-100–insoluble microdomains, but not of Triton X-100–soluble microdomains.³³ A different drug, the cell-permeable dienone phenol triterpene celastrol, a neuroprotective compound that is also a slow-acting compound, decreased the rate of ion channel transport and caused a reduction of channel density on the cell surface.

Muscarinic ACh receptors in the peripheral nervous system are found primarily on autonomic effector cells innervated by postganglionic parasympathetic nerves. In the heart, the muscarinic receptor M₂ is the predominant subtype, which is coupled to the G_i/o protein family. I_KACh channels can be activated by muscarinic receptor agonists like cholinomimetic choline esters (ACh, methacholine, carbachol, and bethanechol). The muscarinic receptor antagonist alkaloids such as atropine and synthetic and semisynthetic compounds prevented the effects of ACh by blocking its binding to muscarinic receptors.³⁴

Some antiarrhythmic drugs are known to antagonize at micromolar concentration actions of ACh on I_KACh. Evidence from different authors suggests that the mechanisms underlying the anti-ACh effect of antiarrhythmic drugs at micromolar levels are different among such drugs—that is, quinidine, pilsicainide, disopyramide, d,l-sotalol, propafenone, cibenzoline, and amiodarone mainly block the muscarinic ACh receptors—whereas flecainide, verapamil, and propafenone inhibit the K⁺

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