Structure and Function

Cellular and Membrane Localization

I_K1 currents, and thus the underlying Kir2.x subunits, display a distinct regional distribution in the heart. It has been shown that inward I_K1 is generally more prominent in ventricular myocytes and Purkinje fibers and is significantly smaller in atrial myocytes (with one known exception of the mouse heart). The density of inward I_K1 currents (i.e., currents normalized to membrane capacitance) is small in pacemaker cells of the sinoatrial node in mice and rats and essentially undetectable in the rabbit sinoatrial nodes. I_K1 cannot be detected in atrioventricular node of rabbit but relatively large I_K1 currents can be recorded in the guinea-pig atrioventricular node. Moreover, the density of inward I_K1 varies across the ventricular myocardium. For example, in the mouse heart inward, I_K1 is larger in apical myocytes compared with epicardial cells, and I_K1 is larger in right ventricular than in left ventricular myocytes.

Molecular biologic studies are also consistent with location-dependent expression of specific Kir2 isoforms. In particular, real-time reverse-transcriptase polymerase chain reaction analysis of Kir2 transcripts in the human heart showed the following relative expression levels: in Purkinje fibers, Kir2.1 > Kir2.3 > Kir2.2; and in the right ventricle, Kir2.1 > Kir2.2 > Kir2.3; and the sequence was reversed in right atrium, Kir2.3 > Kir2.2 > Kir2.1.¹⁴ Information on expression patterns of Kir2 subunits can also be gleaned from functional data using the unique properties of corresponding channels. For example, in cardiac atrial and ventricular myocytes, unitary conductance values display a wide spectrum ranging from 10 to 15 pS (as in Kir2.3 channels) to as high as 40 to 45 pS (as in Kir2.2 channels).

Evidence shows that I_K1 channels are located not only in the non–T-tubular component of sarcolemma of ventricular myocytes, but also in the intercalated discs and in the T-tubules. For example, accumulation and depletion of K⁺ only in T-tubules lead to changes in whole-cell I_K1.¹⁵ I_K1 accumulation/depletion phenomena are not observed in atrial cells, which essentially lack T-tubules and in ventricular myocytes, in which T-tubules are removed by osmotic shock.¹⁵

Alternatively, Kir2.1, Kir2.2, and Kir2.3 subunits were localized to the T-tubular membrane using immunolabeling with specific antibodies. Intercalated discs were not studied electrophysiologically in regard to the presence of I_K1 channels, although labeling with various Kir2 antibodies can clearly be observed at this location. Moreover, there is evidence that in canine ventricular and atrial myocytes, Kir2.3 subunits are expressed at higher levels in intercalated disc membranes relative to T-tubules.

Pharmacology and Regulation

Kir2.x and I_K1 channels can be regulated in a number of ways.² Most studies on adrenergic stimulation show that inward I_K1 currents are suppressed by activation of both α and β receptors, although opposite effects were also described. In addition, adrenergic regulation is clearly dependent on the type of receptors and subunit composition of the channel.

Both isoproterenol and forskolin inhibit I_K1 in human ventricular myocytes, suggesting involvement of protein kinase A (PKA)-mediated phosphorylation of underlying Kir2.x subunits. Molecular details of the phenomenon, however, are contradictory. For example, it has been shown that the application of a catalytic subunit of cyclic adenosine monophosphate (cAMP) dependent PKA leads to activation of Kir2.1 channels expressed in Xenopus oocytes, but to Kir2.1 inhibition when the channels they are expressed in a mammalian cell line (COS-7). The data on PKA regulation of native I_K1 channels is limited and somewhat controversial; however, most of the studies show that I_K1 channels are inhibited by exposure of the cytosolic side of the membrane to purified catalytic subunit of PKA. Studies in exogenous-expressing systems also showed the involvement of PKC in negative regulation of Kir2.1 channels. Consistent with the latter, experiments using human atrial myocytes show that α₁-adrenergic stimulation likely reduces I_K1 via a PKC-dependent mechanism. Kir channels are also targets for phosphorylation by tyrosine kinases. In Kir2.1 channels, the site of downregulation was targeted to a single Y242 residue in the C-terminus.¹⁶

PIP₂ is an important component in membrane-delimited second messenger signaling system and a powerful activator of Kir channels.¹⁷ There is significant evidence that PIP₂ regulates the channel gating primarily through specific electrostatic interactions with the cytosolic part of the channel (see Figure 4-2).⁶ It is also clear that various properties of Kir2 channels are modulated by PIP₂. For example, pH sensitivity of Kir2.3 channels is strongly dependent on the strength of channel-PIP₂ interaction.

Various common cations, such as Ca²⁺ and H⁺, contribute to regulation of Kir2 channels as well.² Intracellular Ca²⁺ blocks I_K1 channels in a voltage-dependent manner. There is evidence that a transient increase in intracellular Ca²⁺ during action potential can lead to significant blockage of I_K1.¹⁸ Regulation of I_K1 by intracellular pH (pH_i) is dependent on the species and the type of tissue. For example, rat and guinea pig ventricular I_K1 is not sensitive to physiologically relevant changes in pH_i. In contrast, I_K1 in sheep ventricular myocytes is inhibited by intracellular H⁺ with pKa of approximately 7.4. The difference in pH sensitivity is likely due to differences in subunit composition of I_K1. For example, channels composed of Kir2.3 subunits exhibit strong sensitivity to pH_i within a physiologically relevant range, whereas Kir2.1 and Kir2.2 channels are relatively insensitive to H⁺.

Pharmacologic tools for modulation of Kir2.x and I_K1 channels are limited. The most useful research tool for studying Kir2 channels is Ba²⁺ (inhibitory concentration of 50% [IC₅₀] for inward currents, 0.5 to 10 µM). Ba²⁺ action is subunit dependent. For example, Ba²⁺ blocks Kir2.2 channels fivefold to sevenfold more efficiently than Kir2.1 channels. In the early 1990s, a compound RP 58866 and its active enantiomer, terikalant, were shown to be selective blockers of I_K1 (IC₅₀, 5 to 20 µM). However, later studies revealed that terikalant also inhibits many other K⁺ channels, some with even higher potency (IC₅₀ in submicromolar range for I_Kr channels). Similarly, it was found that LY97119 compound (LY, a tertiary homolog of clofilium) blocks I_K1 in the low micromolar range, but it also blocked Ito (transient outward current) at submicromolar concentrations. Perhaps chloroquine (an antimalarial drug) is the most potent blocker of I_K1 with an IC₅₀ of approximately 0.5 µM. However, chloroquine does not discriminate among I_K1, I_KACh, and I_KATP, and it has been shown to affect other currents (e.g., I_Na) in the low micromolar range. Activators of Kir2 and I_K1 channels were also described. In particular, flecainide, a widely used antiarrhythmic drug, increases Kir2.1 currents by approximately 50% at a concentration of 1 µM, but has no effect on current carried through Kir2.2 and Kir2.3 channels.¹⁹ Arachidonic acid and the antiinflammatory agent tenidap were shown to specifically activate Kir2.3 channels, with a greater than twofold maximum increase in inward current with IC₅₀ of approximately 0.5 and 1.3 µM, respectively. Zacopride, a gastrointestinal prokinetic drug, was recently found to be a selective I_K1 channel agonist. The activating effect, however, is modest, with a maximum increase in I_K1 of approximately 34% at a concentration of 1 µM.

Channelopathies

There are at least four known channelopathies associated with I_K1 channels, all originating from mutations in KCNJ2: ATS, short QT (SQT) syndrome, familial atrial fibrillation (FAF) and catecholaminergic polymorphic ventricular tachycardia (CPVT) (Figure 4-3).²

ATS is characterized by a triad of pathologic clinical phenotypes including morphogenesis and functioning of skeletal and cardiac muscle. One of the prominent features of ATS is cardiac electrical abnormalities, including brief episodes of ventricular tachycardia, multi-focal ventricular ectopy induced by adrenergic stimulation and prolongation of QT interval. ATS1 is used to differentiate ATS patients carrying mutations in KCNJ2. Many ATS1 patients display clear QT prolongation and have been referred to, perhaps questionably in some cases, as LQT7.

LQT7/ATS1 mutations are numerous (see Figure 4-3) and result in nonfunctional channels when exogenously expressed in heterologous system.²⁰ Because affected patients are heterozygous for the mutant and wild type (WT) alleles, and the channel is composed of four subunits, a dominant-negative effect of mutated subunit can lead to reduced I_K1 current in cardiac myocytes (and other cells). The dominant-negative effect of LQT7/ATS1 mutations has been demonstrated using cloned channels. For example, coexpression of WT and D71V mutants results in barely measurable inward Kir2 currents compared with those produced by expression of WT subunits alone. The magnitude of dominant negative effect, however, is variable in different mutants.

SQT syndrome is characterized by an abnormally short QT interval (less than 300 ms) and increased risk of having fibrillation and sudden death. Currently, three forms of SQT syndrome have been described. SQT1 and SQT2 syndromes result from mutations in genes underlying two voltage-gated potassium channels, HERG (KCNH2) and I_Ks (KCNQ1). A third variant of SQT syndrome (SQT3) originating from mutation in KCNJ2 has been described recently.²¹ Genetic analysis revealed a charge-neutralizing substitution (D172N) in the critical place of the channel responsible for strong inward rectification (see Figure 4-2). Accordingly, coexpression of WT and D172N mutant subunits showed decreased rectification of heteromeric channel. Computer simulations showed that reduced rectification of I_K1 can explain some of the characteristic features of an electrocardiograph, such as tall and asymmetric T waves, observed in affected patients.

Recently, a second mutation in KCNJ2 associated with SQT syndrome has been identified (M301K).²² In contrast to D172N mutation, exogenous expression of M301K subunits alone did not produce measurable currents. However, coexpression of both WT and M301K subunits resulted in large K⁺ currents displaying significantly reduced inward rectification (relatively larger outward currents). Given that the M301K mutation resides on KCNJ2, a gene already associated with SQT3 syndrome, it is reasonable to classify M301K as another SQT3 mutation.

One study described association of single V93I mutation in KCNJ2 with familial atrial fibrillation.²³ Electrophysiologic experiments with cloned channels showed, in particular, that V93I mutation leads to a relative increase in the magnitude of the outward current, or a decrease in the strength of inward rectification. Regarding the inward rectification, the effect of V91I mutation on Kir2.1 current resembles that found in D172N mutant channels. However, patients carrying the V93I mutation display a normal QT interval in contrast to patients affected by D172N mutation.

Mutations in KCNJ2 were also linked to another type of excitability disorder—catecholaminergic polymorphic ventricular tachycardia (CPVT).²⁴ CPVT is a heritable disorder characterized by frequent ventricular arrhythmias and sudden cardiac death associated with physical activity or adrenergic stimulation. Four KCNJ2 mutations associated with CPVT have been identified (see Figure 4-3). Three of them are located in the N-terminus and one in the distal C-terminus of Kir2.1. The electrical phenotype of these mutations included prominent U waves, ventricular ectopy, and polymorphic ventricular tachycardia. In contrast to closely located mutations associated with LQT7/ATS1 syndrome, CPVT mutations were not associated with dysmorphic features or with skeletal muscle abnormalities. Electrophysiologic analysis of exogenously expressed mutants showed that they did not produce any measurable current. In addition, two of the mutations (T75M, R82W) displayed strong dominant negative effects when coexpressed with WT channels. T305A mutation did not exert a significant dominant-negative effect on inward currents, but strongly increased the strength of inward rectification, leading to a relative decrease in outward current. Finally, some mutations in KCNJ2 could not be easily classified into the four categories mentioned previously, and for this reason they are referred to as other in Figure 4-3. For example, C54F mutation was found in a patient who showed cardiac abnormalities (arrhythmia was not specified) upon corticosteroid intake.

Structure and Function

In the heart, Kir3.1/Kir3.4 channels are responsible for the effects of acetylcholine and adenosine, and they act through a coupling mechanism involving a receptor, a G protein (G_o/G_i family), and the potassium channel.2,25 Because channel gating requires a G protein, Kir3.1/Kir3.4 channels are considered a type of K_G channel.⁴ For channel activation, the G-protein–coupled receptor (Figure 4-4) can be a muscarinic (M2) or a purinergic (P1) receptor, which are activated by acetylcholine or adenosine, respectively. The ultimate result of channel activation is the opening of Kir3.1/Kir3.4 channels, which permits K⁺ efflux and consequently hyperpolarizes the cell membrane.

As in a typical potassium channel, ion selectivity in Kir3.x channels is conferred by the presence of the signature sequence (T-X-G-Y/F-G),4,26 and the mechanism of rectification involves an asparagine or an aspartate residue for interactions with the polyamines (details of rectification have been discussed previously). Thus, the mechanism of rectification is similar to that described for Kir2 channels, and Kir3.x channels belong to the class of strong inward rectifier channels.² Membrane topology of Kir3 channels is similar to that described for Kir2 channels (see Figure 4-2).

Exactly how do Kir3.1/Kir3.4 channels and G proteins interact to cause channel opening? First, a brief discussion of G proteins is necessary. G proteins are complexes consisting of an α (molecular weight [MW], ~40,000), a β (MW, ~35,000) and a γ (MW, ~8000) subunit, which transduce signals from membrane receptors (e.g., muscarinic [M2]) to an effector, such as a K_G channel.4,25 Four subfamily members of the guanosine diphosphate (GDP) G_α subunit dictate selectivity of signaling (activation of adenylate cyclase [G_αs], inhibition of adenylate cyclase [G_αi], and activation of phospholipase [G_q]).²⁵ Normally, G_α is bound to GDP in the absence of an agonist, and the G_α/GDP complex is coupled to the receptor and has low GTPase activity. With vagal stimulation and ligand-receptor interaction, GDP is exchanged for GTP and results in the uncoupling of G_βγ from G_α (see Figure 4-4, A). The released G_βγ subunits in turn activate the Kir3.1/Kir3.4 channels. There is a critical requirement for GTP for channel activation (see Figure 4-4, B). A variety of experiments have determined the precise molecular interactions involved in the G_βγ-induced Kir3.1/Kir3.4 channel gating, and have shown that the cytoplasmic region of the channel is intimately involved with gating. For example, crystal structure analyses of the cytoplasmic region of Kir3.1 suggest that the C-terminus of two neighboring Kir3 channels subunits bind to each other, and that an N-terminus is positioned between the two C-terminal domains.²⁷ The study also showed that for Kir3.1 channels, the cytoplasmic residues L262, L333, and E336 are important in gating, and the equivalent residues are H64 and L262 in Kir3.4 channels.

Cellular and Membrane Localization

There are tissue-dependent differences in expression of I_KACh in the myocardium, with a very high density of expression in nodal tissues.2,4 Following the cloning of genes underlying Kir3 channels in the early to middle 1990s, investigators have probed myocardial tissues to examine localization of the gene products. Overall, these studies reported an abundance of Kir3.1 and Kir3.4 mRNA in nodal and atrial, but not in ventricular tissues, which would be consistent with tissue distribution of I_KACh. In one such study,²⁸ a comprehensive analysis was performed using Western blot and immunofluorescence to examine channel distribution in sinus-nodal, atrial, and ventricular tissues, and showed similar results of expression pattern across species (rat, ferret, and guinea pig hearts). It was reported that, whereas there was minimal expression of Kir3.1 in the ventricles of all species tested, Kir3.1 and Kir 3.4 were highly expressed in atrial tissue of all the species. It was noted that although there were relatively high levels of expression in the atria, significant quantitative differences in Kir3.1 and Kir3.4 protein levels were found in the different species. Furthermore, it was demonstrated that Kir3.1 and M2 receptor colocalized in the sinoatrial node. In nodal and atrial tissue, immunofluorescence showed localization of Kir3.x/M2 receptors more on the outer (lateral) membranes than in T-tubular membranes.²⁸ Similar quantitative differences in expression have been reported for atrial versus ventricular tissues in the human myocardium.^28,29

Pharmacology and Regulation

It is well established that Kir3.1/Kir3.4 are generally insensitive to classical potassium channel blockers such as or 4-AP or TEA.⁴ However, experiments in cardiac myocytes isolated from rabbit hearts show that the toxin, tertiapin, is a selective blocker of I_KACh.³⁰ Channel block is highly potent, with an affinity in the nanomolar range. Cardiac Kir3.1/Kir3.4 channels are also inhibited by quinine, quinidine, and verapamil; however, affinities of the these drugs are in the micromolar range.⁴ Based on the results of experiments in heterologous (oocyte) expression systems, current through Kir3.x channels is enhanced by “intoxicating” concentrations of ethanol, and an approximately 43-aa stretch on the C-terminus has been identified as important for the ethanol effect.³¹ Similarly, in a heterologous expression system, Kir3.x channels displayed sensitivity to intracellular acidification, an inhibitory effect that was dependent on histidine residues in the N- and C-terminal regions of the channels.⁴ A variety of other agents have also been shown to modulate Kir3.x channels. For example, similar to all Kir channels, Kir3.1/Kir3.4 require PIP₂ for channel activity.⁴ Curiously, however, the PIP₂ effect is enhanced by other factors such as the G-protein G_βγ complex, as well as by intracellular cations. In general, Kir3.x channel activity is also sensitive to mechanical stretch, can be modified by phosphorylating agents, and is sensitive (negatively) to regulators of G-protein signaling.⁴

Channelopathies

Over four decades ago, a mouse with a striking locomotor deficiency (weaving) was described, and the defect has subsequently been traced to a naturally occurring gain-in-function mutation in the Kir3.2 channel.³² Additional neurologic defects attendant to this mutation earned the weaver mouse the title of the “most cantankerous rodent.”³³ There is relatively little information available on inherited cardiac channelopathies associated with Kir3.1/Kir3.4 channels. However, alterations in Kir3.1/Kir3.4 channel activity have been reported in certain cardiac rhythm abnormalities.³⁴ Electrical remodeling in atrial fibrillation patients has been shown to increase the constitutively active component of I_KACh, which could cause an abbreviation of action potential duration. More recently, a study was performed in a Long QT syndrome large Chinese family (49 individuals) with autosomal-dominant Long QT syndrome (LQTS).³⁵ The locus of the LQTS-associated gene was mapped to chromosome 11q23.3-24.3. A combination of biochemistry and cell electrophysiology in heterologous expression systems was used to demonstrate that a heterozygous G387R mutation on the Kir3.4 (KCNJ5) identified in all affected family members was responsible for reduced channel expression on the sarcolemma.

Structure and Function

Among all inward rectifiers, K_ATP channels are the most unique in their molecular architecture.³⁶ As in other Kir channels, the ion conduction pathway is provided by a tetrameric arrangement of pore-forming subunits, but additional auxiliary regulatory subunits are necessary for the channel to be fully functional (Figure 4-5, B). The two known isoforms of the pore-forming subunits are encoded by Kir6.1 (KCNJ8) and Kir6.2 (KCNJ11) genes. The two isoforms of auxiliary subunits are encoded by SUR1 (ABCC8) and SUR2 (ABCC9) genes (the SUR name originates from “sulfonylureas,” a class of drugs known to inhibit K_ATP channels by acting on the auxiliary subunit). SUR2 gene can be alternatively spliced at the very C-terminus (last 42 aa) leading to SUR2A and SUR2B isoforms. Genomic arrangements of SUR and Kir6 genes are unique as well. Specifically, SUR1 is followed by Kir6.2 in a close proximity on chromosome 11pp15.1, whereas while SUR2 is followed by Kir6.1 on chromosome 12p12.1. The consequences of this arrangement regarding the regulation at a genomic level are not clear at this time. Although heteromeric assembly of Kir6.x subunits produces functional channels in vitro, it remains unclear whether heteromeric Kir6.x complexes exist in native tissues. In contrast, both SUR1/Kir6.2 and SUR2/Kir6.2 channels likely exist in native tissues. Membrane topology and general organization of Kir6.x subunits is highly similar to that in well-characterized Kir2.x channels (see Figure 4-2).

K_ATP channels display weak rectification; however, it has been shown that just a single amino acid substitution in the so-called rectification controller region of inward rectifiers (see Figure 4-2, Kir2.1; N160D in Kir6.2) converts Kir6.2 into a strongly rectifying channel.³⁷ A defining property of K_ATP channels is their characteristic sensitivity to intracellular ATP (hence the name ATP-dependent K⁺ channels). Under normal conditions, both exogenously expressed Kir6.2/SUR2 channels and native channels in cardiac myocytes are inhibited by ATP in the micromolar range (10 to 100 µM) by direct binding to the channel rather than through phosphorylation mechanisms. In the absence of intracellular Mg²⁺ ions, adenosine diphosphate (ADP) and other nucleotides also inhibit the channel. Modeling studies suggests that the ATP binding pocket is located at the interface of the N and C terminus of each Kir6.x subunit; therefore, the K_ATP channel possesses four ATP binding sites.

The overall structure of auxiliary SUR subunit is presented in Figure 4-5, B. It is believed that SUR interacts with Kir6.x subunits to modulate channel gating through TMD0 domain and L0 linker region. Experimental data are consistent with the idea that intracellular ATP induces dimerization of nucleotide-binding domains NBD1 and NBD2, converting them into a catalytically active site for Mg²⁺-dependent hydrolysis of ATP (leading to MgATP). Hydrolysis of ATP is followed by a conformational change that is then transduced to the Kir6.x subunit; however, MgADP is an even more potent activator of the K_ATP channel. The latter puts the hydrolysis hypothesis into question. Accordingly, it has been suggested that NBDs can be locked in a post-hydrolytic state by MgADP and other nucleotides to sustain the active state of the channel.

Cellular and Membrane Localization

K_ATP channels are found in virtually every kind of cardiac tissues, but they are most prominent in cardiac myocytes and smooth muscle cells. Detailed experimental analysis reveals significant differences in various properties of native K_ATP channels, suggesting different subunit organization in every case.³⁸ A significant amount of evidence suggest that ventricular sarcolemmal K_ATP channels are likely composed of Kir6.2 and SUR2A subunits. In particular, functional sarcolemmal K_ATP channels are absent in Kir6.2 knockout mice, and various channel properties (including large single channel conductance, high sensitivity to pinacidil and cromakalim, and low sensitivity to diazoxide) are similar to those obtained from ventricular myocytes. The latter is also supported by a relatively low level of SUR1 (a pancreatic isoform) in the ventricles, and the finding that the activity of K_ATP channels is essentially unaffected in ventricular myocytes from SUR1 knockout mice. Recent experiments with mice showed, however, that SUR1 subunit of K_ATP channel may be a dominant isoform in atrial tissue. In particular, activity of K_ATP channels could not be detected in atrial myocytes isolated from SUR1 knockout mice, and the pharmacologic profile of atrial K_ATP channels is reminiscent of that conferred by SUR1 (higher sensitivity to diazoxide, lower to pinacidil) rather than SUR2 subunits. It remains unresolved whether this subunit composition exists in atrial tissue of other animals and humans.

K_ATP channels in smooth muscle display a number of properties distinct from that in cardiac myocytes, suggesting unique subunit composition.³⁶ Significantly smaller channel densities (up to 100-fold per cell), generally low single channel conductance (~30 pS), and a lack of channel activity upon excision of the membrane patch are some of the common features of smooth muscle K_ATP channels. Studies with exogenously expressing systems showed that cloned K_ATP channels originating from coexpression of Kir6.1 and SUR2B subunits resemble native K_ATP channels in smooth muscle, for the most part. A strongest support for Kir6.1/SUR2B composition of smooth muscle K_ATP channels comes from experiments with genetically modified mice. Specifically, K_ATP currents cannot be recorded in aortic smooth muscle cells isolated from either Kir6.1 or SUR2 knockout mice, whereas the activity of K_ATP channels is preserved in these cells in Kir6.2 knockout mice.

Mitochondrial K_ATP channels received a lot of attention since they were first described at this location in early 1990s. In contrast to sarcolemmal K_ATP channels, however, their molecular identity remains highly controversial.³⁶ Although exogenously coexpressed Kir6.1 and SUR1 subunits produce K_ATP channels with many properties resembling those found in mitochondria, the activity of mitochondrial K_ATP channels in Kir6.1 knockout mice was not affected. Recent promising developments in this area include the identification of mitochondria-specific short-form of SUR2 subunits generated by a nonconventional intraexonic splicing, which can underlie mitochondrial K_ATP channels.³⁹ There is also strong evidence that the pore-forming subunit of mitochondrial K_ATP channels is encoded by KCNJ1 (Kir1.1⁴⁰).

Pharmacology and Regulation

Pharmacology of K_ATP channels is extensive, and regulation is complex relative to other members of Kir family, which is in part due to the structural complexity of the channel.⁴ Cardiac K_ATP channels are regulated by a variety of mechanisms, and important quantitative details of their modulation surely depend on the specific subunit composition. The most prominent and characteristic mechanism involves regulation by intracellular nucleotides such as ATP and ADP. Intracellular ATP is a potent blocker of the channel while ADP (in the presence of Mg²⁺ ions) has an activating effect. An overwhelming level of ATP under normal conditions keeps the channel essentially shut (native channels in cardiac myocytes are half blocked by 50 to 100 µM ATP), whereas metabolic disturbances leading to both a drop in ATP levels and a consequent rise in ADP levels result in channel opening (in the presence of intracellular Mg²⁺ ions). Experiments with isolated membrane patches show that phospholipids, especially PIP₂, are potent activators of native (and cloned) K_ATP channels while their hydrolysis reduces channel activity. Regulation of K_ATP channels by PKA-dependent phosphorylation is well documented in smooth muscles, but the data are limited in cardiac myocytes. It has been shown that the activity of atrial K_ATP channels can be increased by stretch suggesting the involvement of channel-cytoskeleton interactions. Intracellular pH is another potent regulator of native K_ATP channels with acidification leading to increase in channel activity.

K_ATP channels can be inhibited or activated by a variety of drugs, all acting on the SUR subunit. Sulfonylureas such as acetohexamide, glipizide, glibenclamide, tolbutamide, and HMR 1098 are prominent inhibitors. Clinically, sulfonylureas are used exclusively for the treatment of type 2 diabetes, but they are also a useful research tool in work with cardiac preparations.

K_ATP channel openers (KCO) include pinacidil, cromakalim, rimakalim, nicorandil, diazoxide, and minoxidil sulfate. In contrast to sulfonylureas, KCOs are useful in treating cardiovascular disorders such as myocardial ischemia and congestive heart failure. KCOs display strong selectivity to subunit composition of K_ATP channels. In particular, K_ATP channels in pancreatic β-cells (SUR1 based) are strongly activated by diazoxide, but not affected by cromakalim or nicorandil while ventricular K_ATP channels (SUR2A based) are strongly activated by cromakalim or nicorandil but not by diazoxide. Smooth muscle K_ATP channels (SUR2B based) are activated by all these drugs. Mitochondrial K_ATP channels are known to be potently activated by diazoxide and inhibited by 5-hydroxydecanoate (5-HD).

Channelopathies

Mutations in genes underlying K_ATP channels have been associated with cardiovascular disorders. Specifically, F1524S and A1513T substitutions in the SUR2A gene (missense and frameshift, respectively) were linked to dilated cardiomyopathy. Both mutations were mapped to the locus of SUR2A, which is responsible for catalytic activity of NBD2 domains, and likely exert their actions through reduced activation of K_ATP channel. The other reported mutation in the SUR2A gene was associated with adrenergic atrial fibrillation originating in the vein of Marshall, a well-known location for this type of fibrillation. As in the previous case, the underlying missense mutation in the SUR2A gene (T1547I substitution) likely affects the functioning of NBD2 leading to a compromised channel regulation by adenine nucleotides. Recently, the novel gain-of-function mutation (S422L substitution) in the pore-forming Kir6.1 subunit (when coexpressed with SUR2A) was associated with ventricular fibrillation and linked to J-wave syndrome susceptibility.⁴¹

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