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⁺ channel itself as open channel blockers. Some relatively novel antiarrhythmic drugs have been found to inhibit I_KACh at nanomolar concentrations. Dronedarone inhibited I_KACh in single cells isolated from the sinoatrial node with an IC₅₀ of 63 nM.³⁵ The benzopyrene derivative NIP-151 potently inhibited the heterologously expressed (in HEK293 cells) channel K_IR3.1/3.4, with an IC₅₀ of 1.6 nM. In both cases, it was suggested that dronedarone and NIP-151 inhibited I_KACh channels by a direct inhibitory interaction with the channel protein or by disrupting the G-protein–mediated activation.³⁶

There are two major classes of therapeutic compounds that target the I_KATP channels: sulfonylureas and I_KATP channel openers (KCOs).² Sulfonylureas on pancreatic β cells bind to SUR1 channel subunits and block I_KATP channels, causing membrane depolarization and an influx of Ca²⁺ channels, stimulating insulin release and hypoglycemia.² KCOs activate I_KATP channels via their action on the SUR subunit of the channel. KCOs that activate cardiac I_KATP channels include cromakalim, pinacidil, nicorandil, and diazoxide. KCOs act by opening I_KATP channels by decreasing the threshold concentrations of intracellular nucleotides for opening of K_IR6.2/SUR2a channels, and they also increase the maximum amplitude.²

The rest of this chapter focuses on pharmacologic agents that inhibit or activate K_IR channels acting directly on the channel pore–forming K_IR subunits and their underlying blocking mechanisms.

Pore-Blocking Drugs

Several compounds that partially mimic polyamine binding to I_K1 channels have been identified over the last several years. Block of small molecules such as chloroquine, chloroethylclonidine, pentamidine, and quinacrine have been shown to inhibit I_K1 and heterologously expressed K_IR2.x channels in a voltage-dependent fashion.³⁷

In cardiac myocytes, in contrast to the voltage-independent mode of I_K1 inhibition induced by most of the tested drugs, the antimalarial drug chloroquine was found to inhibit I_K1 and I_KACh in a voltage-dependent manner, with less pronounced blockade at negative test potentials. In addition, unblock was achieved by hyperpolarizing pulses to potentials negative to the current reversal potential (Figure 13-3). This profile of voltage dependence is consistent with a positively charged molecule blocking the channel from the intracellular side and entering the pore to such an extent as to be subjected to the transmembrane electrical field. The blocking effects of chloroquine on I_K1 caused an increase in the duration of the ventricular action potential, depolarization of the resting membrane potential, and increase in Purkinje fibers’ automaticity.³⁸

Recent studies with small molecules such as chloroquine and pentamidine revealed the molecular basis by which these drugs block K_IR2.1 in a voltage-dependent manner. Through site-directed mutagenesis and molecular modeling studies, chloroquine and pentamidine were found to block the K_IR2.1 channel by plugging the center of the cytoplasmic conduction pathway via interference with negatively charged residues (E224, D259, and E299) and for chloroquine with an additional aromatic residue (F254), which have previously been shown to be involved in polyamine block (see Figure 13-2).^31,38–40 Comparative molecular modeling and ligand docking of the three-dimensional structures of quinidine and chloroquine in the intracellular domain of K_IR2.1 predicted that chloroquine effectively blocks potassium flow by binding at the center of the ion permeation vestibule of K_IR2.1. In contrast, quinidine binds the vestibular side, only partially blocking ion movement.⁴¹ An interesting finding was that neutralization of the D172 residue, which faces the transmembrane pore and is key in the polyamine block of K_IR2.1 (termed rectifying controller), does not affect chloroquine K_IR2.1-blocking potency. A gain-of-function mutation in KCNJ2 was recently reported to cause one form of short QT syndrome (SQT3). A missense mutation (D172N) located within the transmembrane ion conduction pathway of K_IR2.1 was found in a father and daughter with short QT interval.¹¹ The biophysical consequences of D172N mutation include a reduction in the degree of current rectification at depolarized potentials. Interestingly, chloroquine caused a dose- and voltage-dependent reduction in wild type (WT), D172N, and WT-D172N heteromeric K_IR2.1 current. As expected, the potency and kinetics of chloroquine block of D172N and WTD172N K_IR2.1 current were similar to WT. In silico modeling of the heterozygous WT-D172N K_IR2.1 condition predicted that 3 µM chloroquine would normalize inward rectifier K⁺ current magnitude, action potential duration, and effective refractory period. This suggested that therapeutic concentrations of chloroquine might lengthen cardiac repolarization in SQT3.^42,43

Chloroquine also blocked other cardiac inward rectifier channels. In patch-clamp experiments, chloroquine blocked I_K1 (IC₅₀ = 0.69 µM), I_KACh (IC₅₀ = 0.38 µM), and I_KATP (IC₅₀ = 0.51 µM) (see Figure 13-3). Comparative molecular modeling and ligand docking of chloroquine in the intracellular domains (cytoplasmic pore) of K_IR2.1, K_IR3.1, and K_IR6.2 suggested that chloroquine blocks or reduces potassium flow by interacting with negatively charged amino acids facing the ion permeation vestibule of the channel in question. However, these detailed analyses revealed important differences. The molecular modeling indicated that chloroquine binds more effectively to the cytoplasmic domains of K_IR2.1 and K_IR3.1 than to that of K_IR6.2.³¹ Chloroquine also seemed to interact with acidic residues in the cytoplasmic domain of K_IR3.1 and K_IR3.4; however, the negative electrostatic potential in the channel pore was lower than in K_IR2.1, resulting in partial channel block. In K_IR6.2, the molecular modeling and ligand docking of chloroquine in the intracellular domains (cytoplasmic pore) of 6.2 suggested that the drug binds to the side of a large cavity near the membrane side of the protein, leaving a pathway of 10 Å in diameter, which most likely will not impede the passage of potassium ions.

The honey bee venom–isolated toxin tertiapin and the oxidation-resistant tertiapin-Q derivative are potent inhibitors of K_IR3.1/3.4 and K_IR1.1 channels with nanomolar affinity. In isolated cardiac myocytes, tertiapin Q blocks I_KACh without affecting I_KATP, I_K1, and cardiac potassium voltage–dependent currents like I_to, I_Kr, or I_Ks and has shown to have antiarrhythmic efficacy in dogs. Tertiapin binds to the outer vestibule of the conduction pore formed by the linker between the first and second transmembrane (M1–M2) segments. An α-helical structure within the toxin interacts with a short sequence of aromatic residues located in the N-terminal part of the linker that confers high affinity for tertiapin.³⁵

The detailed information provided here provides the structural framework for the design of safer, alternative compounds that are devoid of K_IR channel–blocking properties. Conversely, these basic science insights may be useful in the development of novel and potentially useful antifibrillatory pharmacophores. The chloroquine-binding models for K_IR channels provide a starting point for feasible approaches in the generation of new therapies.

Drugs That Interfere in PIP₂–Channel Interaction

Several polyvalent cations, such as polyamines, trivalent metals, neomycin, and polylysine, inhibited K_IR channels by screening the negatively charged head groups of phosphatidylinositol 4,5-bisphosphate (PIP₂). PIP₂ comprises approximately 1% of plasma membrane phospholipids and is required in many plasma membrane processes, including the function of ion channels and transporters.44,45 Activation by PIP₂ is a common feature of all K_IR channels; after excision of an inside-out membrane patch from a cell expressing K_IR channels, current amplitude diminishes over time. Such a rundown of the current results from PIP₂ depletion from the cell membrane. The K⁺ currents can be restored partially by exposing the cytoplasmic face of the patch to PIP₂. Each PIP₂ molecule consists of an inositol head group and fatty-acid side chains inserted into the membrane. The negatively charged PIP₂ head groups are anchored just below the plane of the membrane by the lipid tails of the molecule. It has been proposed that the channel opens when the negative-head charges of PIP₂ interact with positively charged residues in the cytoplasmic domain, near the plasma membrane.^46,47

The x-ray crystal structure of a chicken K_IR2.2 channel in a complex with a short-chain (dioctanoyl) derivative of PIP₂ has been resolved recently.⁴⁸ A simple allosteric mechanism of gating control by PIP₂ appears to be responsible for opening the gate at the TM2 helix-bundle crossing. One PIP₂ molecule binds to each of the four channel subunits at an interface between the TMD and the CPD. On PIP₂ binding, the entire CTD translates 6 Å toward the TMD and becomes tethered to the TMD, and the inner helix gate starts to open. The PIP₂-binding site comprises amino acids from the TMD and the CPD, the acyl chains, glycerol backbone, and 1′ (phosphodiester) phosphate of PIP₂ interact with the TMD, whereas the inositol head group makes interactions with the CPD. The acyl chains insert into the membrane layer and interact with hydrophobic amino acids on both the inner and outer helices, whereas the 1′ phosphate makes interactions with amino acids forming the sequence arginine-tryptophan-arginine (amino acids 78-80 in K_IR2.2). The inositol ring of the head group is oriented toward the CPD, where the 4′ and 5′ phosphates are positioned to interact directly with K183, R186, K188, and K189 of the CPD. Other amino acids on the tether helix, including R190, participate in the formation of a hydrogen-bonding network that seems to strengthen the interaction between the tether helix and other regions of the CPD. Comparing the K_IR2.2 amino acids involved in binding to PIP₂ by sequence alignment with the other members of the K_IR channel family shows that the amino acids are highly conserved among the large family.

A variety of signaling partners influence K_IR channel activity by modulating the interaction of K_IR channels with PIP₂. Some of the results strongly suggest that the apparent affinity of channel-PIP₂ interactions is a key element in determining modulation of K_IR channels, whereby the strength of channel-PIP₂ interactions controls the sensitivity of K_IR channels to regulation by modulating factors. Strong interaction between PIP₂ and K_IR2.1 prevented interference from other modulatory factors like pH, protein kinase C, and membrane receptors such as M₁ (type-1 muscarinic) and epidermal growth factor receptors, whereas the weaker interaction between PIP₂ and K_IR2.3 was interfered further by those modulatory factors such that the function of K_IR2.3 or K_IR3.4* was reduced.46,47

The cationic amphiphilic drugs (CADs) represent one of the most abundant chemical groups used in pharmacotherapy.⁴⁹ In contrast to the diverse pharmacologic actions and therapeutic applications, these drugs share several common physicochemical properties. CADs typically contain a hydrophilic domain consisting of one or more primary or substituted nitrogen groups positively charged at physiologic pH and a hydrophobic domain consisting of an aromatic and/or aliphatic ring structure, which may be substituted with one or more halogen moieties. This results in polar hydrophilic cationic side-chain and apolar ring systems within one molecule. CADs bind to the negatively charged phosphoinositides. The cationic group of CADs is normally placed between the polar head groups of phospholipids, and the hydrophobic portion is directed toward the hydrophobic interior of the membrane; thus, the drug molecule intercalates between lipid molecules.

Several widely used CADs have been associated with inward rectifier current disturbances, and recent evidence points to interference of the channel–PIP₂ interaction as the underlying mechanism of action.50–55 The inhibitory potency of CADs on the different subfamilies of K_IR channels correlates with the sensitivity of the channel to rundown arising from PIP₂ depletion. Consistent with this, channels relatively resistant to rundown mediated by PIP₂ depletion, such as K_IR1.1, 2.1, and 2.2 (“high PIP₂ affinity”), are less sensitive to CADs, in contrast to K_IR2.3, 3.x, and 6.2, which are more sensitive.

Experimentally, interference of the drug with the interaction of K_IR2.x, K_IR3.1/3.4, and K_IR6.2/SUR2a channels and PIP₂ is suggested from four sources of evidence: (1) slow onset of current block when CADs are applied from either the inside or outside of the channel; (2) mutation of K_IR2.3 (I213L) and K_IR6.2 (C166S) increases their affinity for PIP₂ and lowers their sensitivity for CADs; (3) mutations of K_IR2.1 (L222I and K182Q), which decrease its affinity for PIP₂, increase its sensitivity for CADs (Figure 13-4); and (4) coapplication of CADs with PIP₂ lowers CAD-mediated current inhibition.^50–55

Bupivacaine was the first CAD in which the mechanism of inhibition of the inward rectifier channel K_IR3.2 by interference with the PIP₂–channel interaction was proposed. Bupivacaine is a local anesthetic drug with a long duration of action that produces excellent sensory anesthesia. Bupivacaine produced inhibitory effects on G protein–gated K_IR3.x channels expressed in Xenopus laevis oocytes but did not affect K_IR1.1 or K_IR2.1 channels. The effects of bupivacaine on K_IR3.x channels was independent of the method of channel activation, by activation of the muscarinic receptor or directly via coexpressed G protein G_βγ subunits or ethanol, which suggested that bupivacaine’s site of action was at the channel protein itself.⁵⁰ On the basis that K_IR1.1 and 2.1 are high-affinity PIP₂ binders and the K_IR3x subfamily channel shows a high sensitivity to PIP₂ depletion, it was proposed that bupivacaine antagonized the interaction of PIP₂ with K_IR3.x channels, thereby decreasing the current. Consistent with this hypothesis, it was found that K_IR3.2(I234L) and 3.1(M223L)/K_IR3.2(I234L) mutant channels, which have increased affinity for PIP₂, were less sensitive to bupivacaine.⁵⁰

The first-generation antihistamines, mepyramine and diphenhydramine, are H₁ competitive receptor antagonists. Both drugs reduced the K_IR2.3 current amplitude by 25% and 17% at 100-µM concentrations, whereas K_IR2.1 current was insensitive to both antihistamines.⁵⁶ Furthermore, K_IR3.4 current was inhibited by mepyramine similarly to K_IR2.3. The inhibitory effects of both of agents were voltage independent. Moreover, mepyramine-induced K_IR2.3 current inhibition was significantly reduced by a single-point mutation of K_IR2.3 (I213L), which enhanced its interaction with membrane PIP₂. These results suggested that membrane PIP₂ may likely be involved in the K_IR current inhibition by the first-generation antihistamines.

Quinacrine is an antimalarial agent that has been used for a number of additional indications, such as other parasitic infections, as an antifibrillatory agent and for treatment of autoimmune disorders. Like other CADs, quinacrine has a high lipophilicity (log P = 5.5) and interacts directly with membrane phospholipids to induce lipidosis. The side chain of the aminoacridine group of quinacrine (pKa = 10.3) is identical to the side chain of the aminoquinoline drug, chloroquine. Quinacrine differentially inhibited K_IR channels in the following order of potency: K_IR6.2 > K_IR2.3 > K_IR2.1. In addition, it was found in cardiac myocytes that quinacrine inhibited IK_ATP > IK₁ (see Figure 13-4). It was presented evidence that quinacrine displayed a double action toward K_IR2.1 and 2.3 channels, that is, interfering with PIP₂ interaction and direct pore block (see Figure 13-4). On K_IR6.2/S_UR2a channels, the inhibitory effects of quinacrine seemed to be only by interference in P_IP2-channel interaction.⁵⁵

Tamoxifen is a drug used in the treatment of hormone-responsive breast cancer and prevention of breast cancer in women at high risk. At clinically relevant concentrations, tamoxifen inhibited the inward rectifier potassium currents I_K1, I_KATP and I_KACh in feline atrial and ventricular myocytes in the following order of sensitivity: I_KACh > I_KATP > atrial I_K1 > ventricular I_K1. Tamoxifen also inhibited in that same order K_IR6.2/SUR2A and K_IR3.1/3.4 > K_IR2.3 > K_IR2.1 = K_IR2.2 in transfected HEK-293 cells.51,52 The order of sensitivity of the different K_IR channels, the slow inhibition time course, the independence of internal or external drug administration, and the voltage independence of the inhibition induced by tamoxifen suggested that tamoxifen probably inserts into the lipid membrane and might interfere with PIP₂–channel interactions. Evidence from three other sources—K_IR2.3(1213 L), K_IR6.2(C166S), and exogenous PIP₂—further suggested the interference of tamoxifen with the K_IR-PIP₂ interaction.

Mefloquine is an antimalarial drug that is used mainly as a prophylactic for malaria and in cases of chloroquine-resistant malaria. Mefloquine produced inhibition of I_KATP channels of pancreatic β cells. The effect is mediated by interaction of the drug with the K_IR6.2 subunit, rather than the regulatory SUR subunit. Mefloquine inhibited three different types of cardiac K_IR channels in the following order of potency, K_IR6.2/SUR2A > K_IR2.3 > K_IR2.1, as demonstrated by determination of the IC₅₀ for mefloquine inhibition of the respective K_IR channels.⁵³ Mefloquine also inhibited I_KATP and I_K1 in cardiac myocytes. Interestingly, the characteristics of the mefloquine inhibition of the K_IR channels were similar to those found with tamoxifen.^51,52 The effect on K_IR channels was concentration dependent but voltage independent and showed a slow time course inhibition. In addition, the effects were similar whether mefloquine was applied externally or internally, suggesting that the inhibitory effect was membrane delimited. In accordance, the K_IR2.3 (I213L) mutant was significantly less sensitive and in inside-out patches, continuous application of exogenous PIP₂ strikingly prevented the mefloquine inhibition.

Carvedilol, a β- and α-adrenoceptor blocker, is used to treat congestive heart failure, mild to moderate hypertension, and myocardial infarction. Carvedilol is a CAD with a high lipophilicity (log P = 3.8) and an α-hydroxyl secondary amine functional group (pKa 7.9). Ferrer et al. demonstrated that carvedilol inhibits K_IR2.3-carried current with at least 100-fold higher potency (IC₅₀ = 0.49 µM) than K_IR2.1 (IC₅₀ = 50 µM).⁵⁴ K_IR2.3 channels showed similar inhibition characteristics like tamoxifen and mefloquine, results that suggested that carvedilol, as other cationic amphiphilic drugs, inhibited K_IR2.3 channels by interfering with the PIP₂–channel interaction. Again, the effect was concentration dependent and voltage-independent, whereas the K_IR2.3 (I213L) mutation decreased the potency of block more than 20-fold (IC₅₀ = 11.1 µM), and in the presence of exogenous PIP₂, inhibition by carvedilol was strongly reduced (80 vs. 2% current inhibition).⁵⁴

The acidic compound thiopental is a short-acting barbiturate used intravenously for anesthesia induction. Recently, it has been shown that thiopental inhibited K_IR2.1, K_IR2.2, K_IR2.3, K_IR1.1, and K_IR6.2/SUR2A currents with similar potency; in whole-cell experiments, 30 µM thiopental decreased K_IR2.1, K_IR2.2, K_IR2.3, and K_IR1.1 currents to 55% ± 6%, 39% ± 8%, 42% ± 5%, and 49% ± 5%, respectively.⁵⁷ Evidence was presented showing that the thiopental effect on K_IR channels likely involves disruption of interactions between K_IR6.2/SUR2a and PIP₂. Thiopental inhibited all K_IR channels in a concentration-dependent and voltage-independent manner. In addition, the time course of thiopental inhibition was slow (T_1/2-4 minutes) and independent of external or internal drug application, suggesting that the inhibitory effect was membrane delimited. In addition, in the presence of exogenous PIP₂, inhibition by thiopental on K_IR channels was significantly decreased, suggesting an interference of PIP₂-K_IR channel interaction. It was proposed that the anionic drug thiopental may have a different mechanism of action on K_IR channels compared with polycations like neomycin or CADs. It was suggested that thiopental interacts with the various K_IR channels directly at a common site and it interfered with the K_IR channel–PIP₂ interaction. A candidate residue for thiopental binding may be R218 in K_IR2.1, which is conserved among all K_IR channels.⁵⁷ However, that hypothesis requires validation, and more work needs to be done to determine whether the inhibition of thiopental depends specifically on interference with K_IR channel–PIP₂ interactions.

Drugs That Induce I_K1 Activation

The multichannel-blocker antiarrhythmic drug flecainide exhibits effective control of ventricular arrhythmias associated with mutations in KCNJ2 associated with Andersen-Tawil syndrome. Recently, it was found that flecainide acutely increased human I_KIR2.1, mainly by reducing the polyamine blockade of the channel. In addition, incubation with flecainide increased functional K_IR2.1 channel density. It was proposed that flecainide reduces the inward rectification of the current at potentials positive to the potassium reversal potential. Flecainide interacts with C311 at the G loop of the CPD of the channel. It was also shown that incubation with flecainide increased expression of functional K_IR2.1 channels on the plasma membrane, an effect also determined by C311. Interestingly, flecainide pharmacologically rescues R67W, but not R218W, channel mutations found in patients with Andersen-Tawil syndrome.⁵⁸

The neurosteroid pregnenolone sulfate has been found to preferentially activate K_IR2.3 with a half-maximal concentration of 16 µM and a maximal current potentiation of approximately 80%. Pregnenolone appeared to be selective in that it had no effects on K_IR1.1, K_IR2.1, K_IR2.2, or K_IR3.1 currents. Preliminary data suggested that pregnenolone sulfate acts directly on K_IR2.3 at an extracellular site, although the precise binding site location and mechanism of action have not yet been defined.⁵⁹

1. Katz, B. Les constants electriques de la membrane du muscle. Arch Sci Physiol. 1949; 3:285–299.

2. Hibino, H, Inanobe, A, Furutani, K, et al. Inwardly rectifying potassium channels: Their structure, function, and physiological roles. Physiol Rev. 2010; 90:291–366.

3. Anumonwo, JM, Lopatin, AN. Cardiac strong inward rectifier potassium channels. J Mol Cell Cardiol. 2010; 48:45–54.

4. Flagg, TP, Enkvetchakul, D, Koster, JC, et al. Muscle K_ATP channels: recent insights to energy sensing and myoprotection. Physiol Rev. 2010; 90:799–829.

5. De Boer, TP, Houtman, MJC, Compier, M, et al. The mammalian K_IR2. x inward rectifier ion channel family: expression patterns and pathophysiology. Acta Physiol. 2010; 199:243–255.

6. Krapivinsky, G, Gordon, EA, Wickman, K, et al. The G-protein-gated atrial K⁺ channel I_KAch is a heteromultimer of two inwardly rectifying K⁺-channel proteins. Nature. 1995; 374:135–141.

7. Inagaki, N, Gonoi, T, Clement, JP, 4th., et al. Reconstitution of I_KATP: An inward rectifier subunit pulus the sulfonylurea receptor. Science. 1995; 270:1166–1170.

8. Dean, M, Annilo, T. Evolution of the ATP-Binding Cassette (ABC) transporter superfamily in vertebrates. Annu Rev Genom Hum Genet. 2005; 6:123–142.

9. Zaritsky, JJ, Redell, JB, Tempel, BL, et al. The consequences of disrupting cardiac inwardly rectifying K⁽⁺⁾ current (I_K1) as revealed by the targeted deletion of the murine K_IR2. 1 and K_IR2. 2 genes. J Physiol. 2001; 533:697–710.

10. Plaster, NM, Tawil, R, Tristani-Firouzi, M, et al. Mutations in K_IR2. 1 cause the developmental and episodic electrical phenotypes of Andersen’s syndrome. Cell. 2001; 105:511–519.

11. Priori, SG, Pandit, SV, Rivolta, I, et al. A novel form of short QT syndrome (SQT3) is caused by a mutation in the KCNJ2 gene. Circ Res. 2005; 96:800–807.

12. Vega, AL, Tester, DJ, Ackerman, MJ, et al. Protein kinase A-dependent biophysical phenotype for V227F-KCNJ2 mutation in catecholaminergic polymorphic ventricular tachycardia. Circ Arrhythm Electrophysiol. 2009; 2:540–547.

13. Hattori, T, Makiyama, T, Akao, M, et al. A novel gain-of-function KCNJ2 mutation associated with short-QT syndrome impairs inward rectification of K_IR2. 1 currents. Cardiovasc Res. 2012; 93:666–673.

14. Bettahi, I, Marker, CL, Roman, MI, et al. Contribution of the K_IR3. 1 subunit to the muscarinic-gated atrial potassium channel I_KACh. J Biol Chem. 2002; 277:48282–48288.

15. Wickman, K, Nemec, J, Gendler, SJ, et al. Abnormal heart rate regulation in GIRK4 knockout mice. Neuron. 1998; 20:103–114.

16. Yang, Y, Yang, Y, Liang, B, et al. Identification of a K_IR3. 4 mutation in congenital long QT syndrome. Am J Hum Genet. 2010; 86:872–880.

17. Miki, T, Suzuki, M, Shibasaki, T, et al. Mouse model of Prinzmetal angina by disruption of the inward rectifier K_IR6. 1. Nat Med. 2002; 8:466–472.

18. Saito, T, Sato, T, Miki, T, et al. Role of ATP-sensitive K⁺ channels in electrophysiological alterations during myocardial ischemia: A study using K_IR6. 2-null mice. Am J Physiol Heart Circ Physiol. 2005; 288:352–357.

19. Liu, XK, Yamada, S, Kane, GC, et al. Genetic disruption of K_IR6. 2, the pore-forming subunit of ATP-sensitive K⁺ channel, predisposes to catecholamine-induced ventricular dysrhythmia. Diabetes. 2004; 53:S165–S168.

20. Flagg, TP, Kurata, HT, Masia, R, et al. Differential structure of atrial and ventricular K_ATP: Atrial K_ATP channels require SUR1. Circ Res. 2008; 103:1458–1465.

21. Elrod, JW, Harrell, M, Flagg, TP, et al. Role of sulfonylurea receptor type 1 subunits of ATP-sensitive potassium channels in myocardial ischemia/reperfusion injury. Circulation. 2008; 117:1405–1413.

22. Stoller, D, Kakkar, R, Smelley, M, et al. Mice lacking sulfonylurea receptor 2 (SUR2) ATP-sensitive potassium channels are resistant to acute cardiovascular stress. J Mol Cell Cardiol. 2007; 43:445–454.

23. Haïssaguerre, M, Chatel, S, Sacher, F, et al. Ventricular fibrillation with prominent early repolarization associated with a rare variant of KCNJ8/K_ATP channel. J Cardiovasc Electrophysiol. 2009; 20:93–98.

24. Medeiros-Domingo, A, Tan, BH, Crotti, L, et al. Gain-of-function mutation S422L in the KCNJ8-encoded cardiac K_ATP channel K_IR6. 1 as a pathogenic substrate for J-wave syndromes. Heart Rhythm. 2010; 7:1466–1471.

25. Barajas-Martínez, H, Hu, D, Ferrer, T, et al. Molecular genetic and functional association of Brugada and early repolarization syndromes with S422L missense mutation in KCNJ8. Heart Rhythm. 2012; 9:548–555.

26. Delaney, JT, Muhammad, R, Blair, MA, et al. A KCNJ8 mutation associated with early repolarization and atrial fibrillation. Europace. 2012.

27. Tester, DJ, Tan, BH, Medeiros-Domingo, A, et al. Loss-of-function mutations in the KCNJ8-encoded K_IR6. 1 K_ATP channel and sudden infant death syndrome. Circ Cardiovasc Genet. 2011; 4:510–515.

28. Bienengraeber, M, Olson, TM, Selivanov, VA, et al. ABCC9 mutations identified in human dilated cardiomyopathy disrupt catalytic K_ATP channel gating. Nat Genet. 2004; 36:382–387.

29. Olson, TM, Alekseev, AE, Moreau, C, et al. K_ATP channel mutation confers risk for vein of Marshall adrenergic atrial fibrillation. Nat Clin Pract Cardiovasc Med. 2007; 4:110–116.

30. Harakalova, M, van Harssel, JJ, Terhal, PA, et al. Dominant missense mutations in ABCC9 cause Cantú syndrome. Nat Genet. 2012; 44:793–796.

31. Noujaim, SF, Stuckey, JA, Ponce-Balbuena, D, et al. Specific residues of the cytoplasmic domains of cardiac inward rectifier potassium channels are effective antifibrillatory targets. FASEB J. 2012; 4:4302–4312.

32. Tamargo, J, Caballero, R, Gómez, R, et al. Pharmacology of cardiac potassium channels. Cardiovasc Res. 2004; 62:9–33.

33. Zaks-Makhina, E, Li, H, Grishin, A, et al. Specific and slow inhibition of the K_IR2. 1 K⁺ channel by gambogic acid. J Biol Chem. 2009; 284:15432–15438.

34. Brown, JH, Laiken, N. Muscarinic receptors agonists and antagonists. In Brunton L, Chabner B, Knollman B, eds. : Goodman and Gilman’s The Pharmacological Basis of Therapeutics, ed 12, New York, McGraw-Hill, 2011.

35. Dobrev, D, Carlsson, L, Nattel, S. Novel molecular targets for atrial fibrillation therapy. Nat Rev Drug Discov. 2012; 11:275–291.

36. McBride, BF. The emerging role of antiarrhythmic compounds with atrial selectivity in the management of atrial fibrillation. J Clin Pharmacol. 2009; 49:258–267.

37. van der Heyden, MAF, Sánchez-Chapula, JA. Towards specific cardiac I_K1 modulators for in vivo application; old drugs point the way. Hearth Rhythm. 2011; 8:1076–1080.

38. Sanchez-Chapula, JA, Salinas-Stefanon, E, Torres-Jacome, J, et al. Blockade of currents by the antimalarial drug chloroquine in feline ventricular myocytes. J Pharm Exp Ther. 2001; 297:437–445.

39. Rodríguez-Menchaca, AA, Navarro-Polanco, RA, Ferrer-Villada, T, et al. The structural molecular basis of chloroquine block of the inward rectifier K_IR2. 1 channel. Proc Natl Acad Sci U S A. 2008; 105:1364–1368.

40. de Boer, TP, Nalos, L, Stary, A, et al. The anti-protozoal drug pentamidine blocks K_IR2. x-mediated inward rectifier current by entering the cytoplasmic pore region of the channel. Br J Pharmacol. 2010; 159:1532–1541.

41. Noujaim, SF, Stuckey, JA, Ponce-Balbuena, D, et al. Structural bases for the different antifibrillatory effects of chloroquine and quinidine. Cardiovasc Res. 2011; 89:1–8.

42. El Harchi, A, McPate, MJ, Zhang, YH, et al. Action potential clamp and chloroquine sensitivity of mutant K_IR2. 1 channels responsible for variant 3 short qt syndrome. J Mol Cel Cardiol. 2009; 47:743–747.

43. Lopez-Izquierdo, A, Ponce-Balbuena, D, Ferrer, T, et al. Chloroquine blocks a mutant K_IR2. 1 channel responsible for short QT syndrome and normalizes repolarization properties in silico. Cell Physiol Biochem. 2009; 24:153–160.

44. Fan, Z, Makielski, JC. Anionic phospholipids activate ATP-sensitive potassium channels. J Biol Chem. 1997; 272:5388–5395.

45. Hilgemann, DW, Feng, S, Nasuhoglu, C. The complex and intriguing lives of PIP₂ with ion channels and transporters. Sci STKE. 2001; RE19.

46. Du, X, Zhang, H, Lopes, C, et al. Characteristic interactions with phosphatidylinositol 4,5–bisphosphate determine regulation of K_IR channels by diverse modulators. J Biol Chem. 2004; 279:37271–37281.

47. Logothetis, DE, Jin, T, Lupyan, D, et al. Phosphoinositide-mediated gating of inwardly rectifying K⁺ channels. Pflugers Arch. 2007; 455:83–95.

48. Hansen, SB, Tao, X, MacKinnon, R. Structural basis of PIP2 activation of the classical inward rectifier K⁺ channel K_IR2. 2. Nature. 2011; 477:495–498.

49. Lundbæk, JA. Lipid bilayer–mediated regulation of ion channel function by amphiphilic drugs. J Gen Physiol. 2008; 131:421–429.

50. Zhou, W, Arrabit, C, Choe, S, et al. Mechanism underlying bupivacaine inhibition of G protein-gated inwardly rectifying K⁺ channels. Proc Natl Acad Sci U S A. 2001; 98:6482–6487.

51. Ponce-Balbuena, D, López-Izquierdo, A, Ferrer, T, et al. Tamoxifen inhibits K_IR2. x family of inward rectifier channels by interfering with PIP2-channel intaractions. J Pharm Exp Ther. 2009; 331:563–573.

52. Ponce-Balbuena, D, Moreno-Galindo, EG, López-Izquierdo, A, et al. Tamoxifen inhibits cardiac K_ATP and K_ACh currents in part by interfering with PIP₂–channel interaction. J Pharm Sci. 2010; 113:66–75.

53. López–Izquierdo, A, Ponce-Balbuena D. Moreno-Galindo, EG, et al. The antimalarial drug mefloquine inhibits cardiac inward rectifier K⁺ channels: Evidence for inference in PIP₂ –channel interaction. J Cardiovasc Pharmacol. 2011; 57:407–415.

54. Ferrer, T, Ponce-Balbuena, D, López-Izquierdo, A, et al. Carvedilol inhibits K_IR2. 3 channels by interference with PIP₂-channel interaction. Eur J Pharmacol. 2011; 668:72–77.

55. Lopez-Izquierdo, A, Aréchiga-Figueroa, IA, Moreno-Galindo, EG, et al. Mechanisms for K_IR channel inhibition by quinacrine: Acute pore block of K_IR2. x channels and interference in PIP₂ interaction with K_IR2. x and K_IR6. 2 channels. Pflügers Arch. 2011; 462:505–517.

56. Liu, B, Jia, Z, Geng, X, et al. Selective inhibition of K_IR currents by antihistamines. Eur J Pharmacol. 2007; 558:21–26.

57. López-Izquierdo, A, Ponce-Balbuena, D, Ferrer, T, et al. Thiopental inhibits function of different inward rectifying potassium channel isoforms by a similar mechanism. Eur J Pharmacol. 2010; 638:33–41.

58. Caballero, R, Dolz-Gaitón, P, Gómez, R, et al. Flecainide Increases K_IR2. 1 currents by interacting with Cysteine 311 decreasing the polyamine-induced rectification. Proc Nat Acad Sci U S A. 2010; 107:15631–15636.

59. Kobayashi, T, Washiyama, K, Ikeda, K. Pregnenolone sulfate potentiates the inwardly rectifying K channel K_IR2. 3. PLoS One. 2009; 4:e6311.