M₂-Receptor–Dependent I_K,ACh Facilitates Initiation and Maintenance of Atrial Fibrillation

In atrial myocytes, I_K,ACh channel activation leads to hyperpolarization of the resting membrane potential and shortening of the action-potential duration (APD).¹³ ACh-induced APD shortening and resting membrane potential hyperpolarization create an arrhythmogenic substrate facilitating the induction of atrial fibrillation (AF; see also chapter 45 of this book).¹⁴ It is well known that vagal nerve stimulation promotes AF in animal models and patients by facilitating the initiation and maintenance of reentry circuits.¹⁵ Reentry, the most established basic mechanism of AF, is described as continuous impulse propagation around a functional barrier or an anatomical obstacle. An important requirement for reentry is that the initially activated tissue zone regains excitability while the electrical impulse propagates around the reentry circuit, explaining why reduced effective refractory period or decreased conduction velocity can provide a substrate for AF maintenance. APD shortening induced by I_K,ACh activation reduces effective refractory period, thereby favoring AF initiation by vagal stimulation. In knockout mice lacking the Kir3.4 channel subunit, M-receptor stimulation does not induce AF, clearly suggesting that the AF facilitating effects of vagal nerve activation are exclusively mediated by I_K,ACh.¹⁶ In addition, mathematical modeling studies suggest that increases in an inward-rectifier K⁺ current such as I_K,ACh have a significant role in AF promotion.¹⁷ Because of their ability to hyperpolarize atrial cardiomyocytes and remove voltage-dependent Na⁺-current (I_Na) inactivation, enhanced inward-rectifier K⁺ currents are more effective in stabilizing and accelerating AF-sustaining reentry circuits (rotors) than are changes in other ionic currents (e.g., reduced L-type Ca²⁺ currents) that produce a similar degree of APD shortening. In vivo evidence has been obtained to support the validity of these modeling data.¹⁸

Interestingly, AF-related atrial remodeling is associated with reduced maximum activation of I_K,ACh upon M-receptor stimulation,13,19–22 which might partly result from reduced expression levels of Kir3.1 and Kir3.4 subunits in AF patients.^13,21 The reduced agonist-dependent I_K,ACh in AF patients could be a protective mechanism against the profibrillatory effects of vagal nerve stimulation.

Left-to-right atrial gradients of inward-rectifier K⁺ currents contribute to AF pathophysiology. There is clinical and experimental evidence that certain cases of paroxysmal AF (pAF) and chronic AF (cAF) are maintained by high-frequency reentrant sources (rotors) with a consistent left-to-right dominant frequency gradient, particularly in pAF.23–25 Because increased inward-rectifier currents enhance rotor frequency, the left-to-right atrial gradient in the basal inward-rectifier current present in pAF, but not in cAF or sinus rhythm (SR), can contribute to these dominant frequency gradients.²² In a sheep model of ACh-mediated, pacing-induced AF, the left-to-right (LA-RA) dominant frequency gradient parallels a left-to-right I_K,ACh gradient, supporting the hypothesis that an unequal LA-RA distribution of inward-rectifier K⁺ currents can contribute to AF maintenance.²⁶ However, in atria from patients with SR, there is a right-to-left atrial gradient of I_K,ACh current that is absent in pAF and cAF.²² The lack of RA-dominant agonist-activated I_K,ACh could have a permissive role for LA-dominant drivers in pAF and cAF, particularly in vagal contexts.

Agonist-Independent, Constitutively Active I_K,ACh Can Contribute to Atrial Fibrillation Maintenance

It is well recognized that I_K,ACh can possess agonist-independent “resting” activity, with a much lower opening frequency than agonist-induced I_K,ACh.²⁷ Constitutive I_K,ACh current (I_K,ACh,c) may underlie a major part of basal K⁺ conductance in SA node cells, which lack I_K1, creating an important role in regulating heart rate.²⁸ In the normal heart, constitutive I_K,ACh activity is low in atrial myocytes, but can increase substantially with cardiac pathology. Agonist-independent I_K,ACh,c activity increases in atrial myocytes from patients and animal models of AF, whereas maximum M-receptor activation of I_K,ACh is reduced (see Figure 38-1, C).^19,20,22,29 I_K,ACh,c might contribute to APD shortening, which is a hallmark of the AF-related electrical remodeling¹⁴ (see also Chapter 45). Therefore, agonist-independent I_K,ACh,c is expected to increase atrial vulnerability to tachyarrhythmias and to promote persistence of AF. Accordingly, inhibition of I_K,ACh with the highly-selective I_K,ACh blocker tertiapin reverses the APD abbreviation and prevents the AF promotion in dogs with atrial tachycardia remodeling (ATR) that also develop increased I_K,ACh,c.³⁰ I_K,ACh is almost absent in ventricles; therefore, it is a promising atrial-selective anti-AF target that lacks proarrhythmic side effects in the ventricles.³¹

In whole-cell patch-clamp experiments, electrophysiological properties of I_K,ACh (i.e., current-voltage relationship) are comparable with other inward-rectifier currents such as I_K1 or I_K,ATP; therefore, single-channel recordings are often used as a direct index of increased constitutive I_K,ACh,c activity in atrial myocytes from patients with cAF and dogs with ATR.19,32–34 Due to their short opening-times and a characteristic single-channel conductance of approximately 40 pS I_K,ACh single-channel openings are clearly different from openings of I_K1 or I_K,ATP (Table 38-1). Figure 38-1 shows representative recordings of I_K,ACh in the presence and absence of a muscarinic-receptor agonist in atrial myocytes from patients with SR and cAF. Inclusion of the nonselective M-receptor agonist carbachol (10 µM) in the pipette solution strongly activated I_K,ACh in both groups, causing frequent channel openings. In the absence of M-receptor agonists, constitutive I_K,ACh,c openings are apparent in cAF, whereas they occur only sporadically in myocytes from SR patients.¹⁹

In dogs subjected to AF mimicking, atrial tachycardia remodeling I_K,ACh also develops agonist-independent constitutive I_K,ACh,c activity, suggesting that the development of I_K,ACh,c in patients with cAF can result from the high atrial rate rather than from the underlying heart disease.29,30,34 In addition, the alterations in single-channel properties of I_K,ACh,c are comparable between dogs with ATR and cAF patients, suggesting a common molecular basis.³⁴ The complex regulation of I_K,ACh points to several possible mechanisms that could contribute to the development of I_K,ACh,c. The following discussion summarizes the current knowledge about the molecular regulation of agonist-dependent I_K,ACh in atrial myocytes, particularly focusing on the putative mechanisms underlying constitutive I_K,ACh activity in AF.

M₂-Receptor–Dependent I_K,ACh

Inward-rectifier I_K,ACh channels are activated through stimulation of appropriate G_i-protein–coupled receptors (M₂-receptors), resulting in the dissociation of heterotrimeric G_i-proteins and consecutive binding of Gβγ-subunits to the channel (see Figure 38-2, A, B).³⁸ Heterotrimeric G-proteins are composed of two functional units, the guanosine-5′-diphosphate and guanosine-5′-triphosphate (GDP/GTP) binding Gα-subunit and the Gβγ-dimer. Upon stimulation of G-protein–coupled receptors, GDP (bound to the Gα-subunit under resting conditions) is released and replaced by GTP. The resulting conformational changes lead to a dissociation of Gα and Gβγ subunits, which then initiate and regulate multiple intracellular pathways. Intrinsic GTPase activity of the Gα-subunit results in GTP hydrolysis into GDP followed by reassembly of the G-protein subunits and reestablishment of the initial inactive state.³⁹ Gα-subunits are subdivided into Gα_s-subunits, which stimulate adenylate cyclases, Gα_i-subunits, which inhibit adenylate cyclases, and Gα_q-subunits, which activate phospholipase-C (see later). I_K,ACh channels are activated by direct binding of βγ-subunits originating from G_i-protein.^4,38 It is unclear whether Gβγ-subunits originating from G_s– or G_q-proteins also activate I_K,ACh channels under physiological conditions.

Although Gα_i-subunits do not directly activate I_K,ACh channels, they certainly have an important role in I_K,ACh regulation. Under resting conditions, the GDP-bound Gα_i-subunit is associated with the Kir3.1 channel subunit, thereby creating a “preformed complex” between the heterotrimeric G-protein and the I_K,ACh channel.⁴⁰ In this condition, Gβγ-subunits are always in close proximity to the channel and can thus effectively and rapidly activate I_K,ACh upon Gα_i-dissociation. The interaction between Gα_I and Kir3.1 is therefore an important contributor to the specificity between receptor stimulation and I_K,ACh activation. In addition, GDP-bound Gα_i can act as a Gβγ-scavenger, continuously chelating Gβγ-subunits from I_K,ACh channels, thereby preventing agonist-independent I_K,ACh activity.⁴¹ It is unknown whether reduced Gα_i-expression or a higher Gβγ-subunit availability contribute to the enhanced constitutive I_K,ACh activity in patients with AF.

The cycling of G_i-proteins is fine-tuned by multiple mechanisms. Regulator of G-protein signaling (RGS) proteins, which accelerate the GTPase activity of the Gα-protein subunit, accelerate the inactivation kinetics of receptor-activated I_K,ACh (see Figure 38-3, B).⁴² [Na⁺]_i also accelerates the G-protein cycle,^43,44 although direct binding of Na⁺ to Kir3.4 could activate I_K,ACh channels (see Figure 38-3, A).^45–47,47a In addition, nucleoside diphosphate kinases (NDPKs), which classically transfer phosphates from ATP to GDP by a ping-pong mechanism involving the formation of a high-energy phosphate intermediate on histidine-118, might also contribute to the replenishment of the GTP necessary for the G-protein cycle (see Figure 38-2, B, C).³⁹

Constitutively Active I_K,ACh

Previous studies showed that the M-receptor antagonist atropine does not abolish constitutive I_K,ACh,c activity in atrial myocytes from cAF patients or ATR-dogs, suggesting that a receptor-independent mechanism might be implicated in the formation of constitutive I_K,ACh,c.¹⁹ Whether increased receptor-independent dissociation of Gα_i– and Gβγ-subunits contributes to generation of constitutive I_K,ACh,c in cAF patients is not known. Neither pertussis toxin, which uncouples G_i-proteins from the receptor, nor the absence of GTP, which is necessary for the action of G-proteins, affected the I_K,ACh-like current component of the basal inward-rectifier K⁺ current in atrial myocytes from ATR dogs and atrial myocytes from cAF patients,^19,29 suggesting that receptor-independent dissociation of Gα_i– and Gβγ-subunits might not be a major contributor to constitutive I_K,ACh,c in AF. However, a fraction of G-protein βγ-subunits forms complexes with the B-isoform of NDPKs and these complexes may cause receptor-independent G-protein activation (see Figure 38-2, B).³⁹ This process involves NDPK-B–mediated phosphorylation of the Gβ-subunit at histidine-266. Subsequently the phosphate is transferred onto GDP bound to the Gα-subunit, and the formed GTP leads to receptor-independent G-protein activation. Because NDPK-B accumulates in the atrial membrane fraction of cAF patients⁴⁸ and is involved in I_K,ACh regulation,⁴⁹ this receptor-independent mechanism of G-protein activation could be a major contributor to the development of constitutive I_K,ACh,c in patients with cAF, although the NDPK hypothesis has also been challenged as a possible explanation for agonist-independent channel activation.⁵⁰

M₂-Receptor Dependent I_K,ACh

I_K,ACh channels require PIP₂ to maintain physiological properties and removal of PIP₂ completely runs down the I_K,ACh current.⁵¹ Under resting conditions, the interaction between I_K,ACh-channels and PIP₂ is weak, and I_K,ACh activity is low. Binding of Gβγ-subunits to I_K,ACh channels strengthen the PIP₂-channel subunit interaction, strongly increasing I_K,ACh activity (see Figure 38-3, A). In atrial myocytes, the membrane PIP₂ levels close to the I_K,ACh channel are dynamically regulated by Gα_q-protein–coupled receptors, the stimulation of which results in PIP₂ breakdown by activating the PIP₂ hydrolyzing enzyme phospholipase-C (see Figure 38-3, A). I_K,ACh is also regulated by G_q-protein–coupled α₁-adrenergic-, angiotensin-II type-1 (AT1)-, and endothelin-A (ET_A) receptors, suggesting that GIRK-channels integrate hormone and neurotransmitter signals from different pathways.^52,53 In addition, there is also evidence for the existence of Gα_q-coupled M₁– and M₃-receptors in atria.^54,55 Activation of Gα_q-coupled M₁– and M₃-receptors and the associated PIP₂ depletion are suggested to contribute to the fast desensitization of M₂-receptor–activated I_K,ACh current (see Figure 38-3, B).^56,57 However, the existence of functional non–M₂-receptors in atria is still controversial.^58,59

M₂-Receptor–Dependent I_K,ACh

The GIRK channel forms a macromolecular complex that is composed of the catalytic subunits of protein kinase A (PKA) and C (PKC), the Ca²⁺/calmodulin-regulated protein kinase II (CaMKII) and the type-1 and type-2A protein phosphatases (PP1 and PP2A).⁶¹ In addition, the Kir3.1 and Kir3.4 channel subunits possess phosphorylation sites for PKA, PKC, CaMKII, and possibly for PKG,^61,62 suggesting that channel phosphorylation status might play a crucial role in the regulation of I_K,ACh activity (see Figure 38-3, C).

Inhibition of CaMKII, PKC, and PKG reduced agonist-activated I_K,ACh current in human atrial myocytes, suggesting that CaMKII-, PKC- and PKG-mediated channel phosphorylation may stabilize the binding of Gβγ-subunits to the channel, thereby increasing I_K,ACh.²⁰ The regulation of I_K,ACh by PKC is isoform specific. The PKC family contains many isoforms, including conventional isoforms (cPKCs), which are activated by Ca²⁺ and DAG, and novel isoforms (nPKCs), which require DAG but not Ca²⁺ for activation.⁶³ There are at least five major PKC isoforms in the heart: cPKCα, cPKCβI, cPKCβII, nPKCδ, and nPKCε. Purified cPKC isoforms strongly reduce GTPγS-activated I_K,ACh channel, whereas nPKC isoforms have the opposite effect.³³ The relative expression-levels of stimulatory and inhibitory PKC isoforms can vary depending on species, diseases, or other conditions, potentially accounting for discrepant results between neonatal rat and canine atrial myocytes, in which PKC activation inhibits I_K,ACh and human atrial myocytes, for which PKC appears to stimulate I_K,ACh.^61,64 PKA activity is expected to be reduced via Gα_i-coupled M₂-receptor activation consequent to ACh-application.

Upon application of ACh, direct binding of liberated Gβγ-subunits to the I_K,ACh channel causes rapid activation of I_K,ACh, followed by desensitization with a typical biphasic pattern (see Figure 38-3, middle).⁶² Although other mechanisms such as RGS proteins and PIP₂ depletion owing to activation of Gq-protein–coupled M₁/M₃-receptors could also contribute, it has been suggested that the fast phase of desensitization is due to channel dephosphorylation,^65,66 whereas the slower (intermediate) phase involves progressive M-receptor phosphorylation by a G-protein–coupled receptor type-2 kinase, which uncouples the receptor from the G-protein (see Figure 38-3, C).⁶⁷

Because there is evidence for increased PKA, CaMKII, and PKC activity in cAF patients,20,68 I_K,ACh phosphorylation might also be involved in disease-associated remodeling of I_K,ACh. However, inhibition of CaMKII, PKC, PKG, and PKA does not affect M-receptor–activated I_K,ACh in patients with cAF.²⁰ In addition, activities of PP1 and PP2A are higher in cAF than in control SR patients and do not translate into altered protein phosphorylation.^69–71 Thus, the lack of contribution of the these kinases to M-receptor–activated I_K,ACh in cAF might result from opposing increases in channel dephosphorylation. Alternatively, the channel could be hyperphosphorylated because of abnormal control of kinase–phosphatase signaling in the macromolecular complex,⁶¹ and inhibition of a single kinase might not reduce the channel’s phosphorylation state below the threshold required to impair agonist-activated I_K,ACh. Further work is needed to verify these hypotheses.

Constitutively Active I_K,ACh,c

Because activation of I_K,ACh requires ATP, modified phosphorylation-dependent channel regulation could contribute to constitutive I_K,ACh,c activity.62,66 In single-channel patch-clamp experiments, excision of the patch and removal from the intact cell produces a cell-free piece of sarcolemma attached to the pipette and containing the I_K,ACh channel (inside-out configuration), with the cytosolic side facing the bath solution. Under these conditions, I_K,ACh,c open probability is strongly reduced, suggesting that an intracellular component necessary for constitutive I_K,ACh,c activity is washed out.³³ In the presence of phosphatase inhibitors, excision of the patch only slightly reduces the open probability of constitutive I_K,ACh,c. These experiments point to a crucial role of phosphorylation in I_K,ACh,c. Inhibition of PKC reduces basal current, which contains constitutive I_K,ACh,c activity, in patients with cAF but not SR, further suggesting that PKC-mediated phosphorylation of I_K,ACh channels is involved in maintaining constitutive I_K,ACh,c.²⁰ The expression level of PKCε, which stimulates I_K,ACh,³³ is increased in patients with cAF, whereas PKCα, PKCβI, and PKCδ remains unchanged.²⁰ These data indicate the involvement of PKC isoform imbalance in the development of agonist-independent I_K,ACh,c. Consistent with this hypothesis, Makary et al.³³ showed that changes in the balance between stimulatory nPKC and inhibitory cPKC isoforms contribute to increased constitutive I_K,ACh,c activity in AF.³³ The cPKC (inhibitory) phenotype predominates in control dogs, whereas the nPKC (stimulatory) phenotype prevails in ATR dogs (Figure 38-4).³³ This idea is supported by a reduced total expression of PKCα (cPKC isoform) and increased membrane translocation of PKCε (nPKC isoform), in atrial myocytes from ATR dogs. In control myocytes, blocking cPKC isoforms increases constitutive I_K,ACh,c, suggesting tonic cPKC-mediated inhibition of I_K,ACh because of predominance of inhibitory PKCα isoforms. cPKC inhibition has no effect in ATR dogs. In contrast, blocking PKCε does not affect constitutive I_K,ACh,c in control myocytes, but it reduces I_K,ACh,c in ATR myocytes to levels seen in control dogs. These data indicate that a reduced tonic inhibitory effect of PKCα and an increased stimulatory effect of PKCε are the major determinants of enhanced constitutive I_K,ACh,c activity in AF.

Rapid in vitro pacing of isolated atrial myocytes from control dogs reproduces the phenotype seen in ATR dogs, with a PKC isoform switch and increased constitutive I_K,ACh,c activity, demonstrating that the PKC isoform switch in ATR could be attributable to the rapid atrial activity per se.³³ In addition, the cellular in vitro pacing experiments revealed that PKCα downregulation likely results from atrial tachycardia-induced Ca²⁺ loading and subsequent activation of the Ca²⁺-dependent protease calpain, which might increase the degradation of PKCα isoforms. In contrast, the increased membrane translocation of PKCε in ATR myocytes seems to be Ca²⁺ independent (see Figure 38-4), suggesting that two independent mechanisms combine to cause constitutive I_K,ACh,c activity in AF. Although there is only indirect evidence that phosphorylation-dependent mechanisms contribute to the increase of constitutive I_K,ACh,c in human AF,²⁰ these posttranslational mechanisms could provide interesting new therapeutic targets for atrial-selective and pathology-specific treatment of AF.

1. Loewi, O. Über humorale Übertragbarkeit der Herznervenwirkung. Pflügers Arch. 1921; 189(1):239–242.

2. Noma, A, Trautwein, W. Relaxation of the ACh-induced potassium current in the rabbit sinoatrial node cell. Pflügers Arch. 1978; 377(3):193–200.

3. Krapivinsky, G, Gordon, EA, Wickman, K, et al. The G-protein-gated atrial K⁺ channel I_K,ACh is a heteromultimer of two inwardly rectifying K⁺-channel proteins. Nature. 1995; 374(6518):135–141.

4. Hibino, H, Inanobe, A, Furutani, K, et al. Inwardly rectifying potassium channels: their structure, function, and physiological roles. Physiol Rev. 2010; 90(1):291–366.

5. Yamada, M, Inanobe, A, Kurachi, Y. G protein regulation of potassium ion channels. Pharmacol Rev. 1998; 50(4):723–760.

6. Bender, K, Wellner-Kienitz, MC, Inanobe, A, et al. Overexpression of monomeric and multimeric GIRK4 subunits in rat atrial myocytes removes fast desensitization and reduces inward rectification of muscarinic K⁺ current (I_K,ACh). Evidence for functional homomeric GIRK4 channels. J Biol Chem. 2001; 276(31):28873–28880.

7. Dobrev, D, Wettwer, E, Himmel, HM, et al. G-Protein beta(3)-subunit 825T allele is associated with enhanced human atrial inward rectifier potassium currents. Circulation. 2000; 102(6):692–697.

8. Kurachi, Y, Nakajima, T, Sugimoto, T. On the mechanism of activation of muscarinic K⁺ channels by adenosine in isolated atrial cells: involvement of GTP-binding proteins. Pflugers Arch. 1986; 407(3):264–274.

9. Himmel, HM, Meyer Zu Heringdorf, D, Graf, E, et al. Evidence for Edg-3 receptor-mediated activation of I_K,ACh by sphingosine-1-phosphate in human atrial cardiomyocytes. Mol Pharmacol. 2000; 58(2):449–454.

10. Verkerk, AO, Geuzebroek, GS, Veldkamp, MW, et al. Effects of acetylcholine and noradrenalin on action potentials of isolated rabbit sinoatrial and atrial myocytes. Front Physiol. 2012; 3:174.

11. Gaborit, N, Le Bouter, S, Szuts, V, et al. Regional and tissue specific transcript signatures of ion channel genes in the non-diseased human heart. J Physiol. 2007; 582(Pt 2):675–693.

12. Koumi, S, Wasserstrom, JA. Acetylcholine-sensitive muscarinic K⁺ channels in mammalian ventricular myocytes. Am J Physiol. 1994; 266(5 Pt 2):H1812–H1821.

13. Dobrev, D, Graf, E, Wettwer, E, et al. Molecular basis of downregulation of G-protein-coupled inward rectifying K⁺ current (I_K,ACh) in chronic human atrial fibrillation: decrease in GIRK4 mRNA correlates with reduced I_K,ACh and muscarinic receptor-mediated shortening of action potentials. Circulation. 2001; 104(21):2551–2557.

14. Wakili, R, Voigt, N, Kaab, S, et al. Recent advances in the molecular pathophysiology of atrial fibrillation. J Clin Invest. 2011; 121(8):2955–2968.

15. Chen, PS, Tan, AY. Autonomic nerve activity and atrial fibrillation. Heart Rhythm. 2007; 4(3 Suppl):S61–S64.

16. Kovoor, P, Wickman, K, Maguire, CT, et al. Evaluation of the role of I_K,ACh in atrial fibrillation using a mouse knockout model. J Am Coll Cardiol. 2001; 37(8):2136–2143.

17. Pandit, SV, Berenfeld, O, Anumonwo, JM, et al. Ionic determinants of functional reentry in a 2-D model of human atrial cells during simulated chronic atrial fibrillation. Biophys J. 2005; 88(6):3806–3821.

18. Katsouras, G, Sakabe, M, Comtois, P, et al. Differences in atrial fibrillation properties under vagal nerve stimulation versus atrial tachycardia remodeling. Heart Rhythm. 2009; 6(10):1465–1472.

19. Dobrev, D, Friedrich, A, Voigt, N, et al. The G protein-gated potassium current I_K,ACh is constitutively active in patients with chronic atrial fibrillation. Circulation. 2005; 112(24):3697–3706.

20. Voigt, N, Friedrich, A, Bock, M, et al. Differential phosphorylation-dependent regulation of constitutively active and muscarinic receptor-activated I_K,ACh channels in patients with chronic atrial fibrillation. Cardiovasc Res. 2007; 74(3):426–437.

21. Voigt, N, Rozmaritsa, N, Trausch, A, et al. Inhibition of I_K,ACh current may contribute to clinical efficacy of class I and class III antiarrhythmic drugs in patients with atrial fibrillation. Naunyn Schmiedebergs Arch Pharmacol. 2010; 381(3):251–259.

22. Voigt, N, Trausch, A, Knaut, M, et al. Left-to-right atrial inward rectifier potassium current gradients in patients with paroxysmal versus chronic atrial fibrillation. Circ Arrhythm Electrophysiol. 2010; 3(5):472–480.

23. Lazar, S, Dixit, S, Marchlinski, FE, et al. Presence of left-to-right atrial frequency gradient in paroxysmal but not persistent atrial fibrillation in humans. Circulation. 2004; 110(20):3181–3186.

24. Sanders, P, Berenfeld, O, Hocini, M, et al. Spectral analysis identifies sites of high-frequency activity maintaining atrial fibrillation in humans. Circulation. 2005; 112(6):789–797.

25. Swartz, MF, Fink, GW, Lutz, CJ, et al. Left versus right atrial difference in dominant frequency, K⁺ channel transcripts, and fibrosis in patients developing atrial fibrillation after cardiac surgery. Heart Rhythm. 2009; 6(10):1415–1422.

26. Sarmast, F, Kolli, A, Zaitsev, A, et al. Cholinergic atrial fibrillation: I_K,ACh gradients determine unequal left/right atrial frequencies and rotor dynamics. Cardiovasc Res. 2003; 59(4):863–873.

27. Sakmann, B, Noma, A, Trautwein, W. Acetylcholine activation of single muscarinic K⁺ channels in isolated pacemaker cells of the mammalian heart. Nature. 1983; 303(5914):250–253.

28. Ito, H, Ono, K, Noma, A. Background conductance attributable to spontaneous opening of muscarinic K⁺ channels in rabbit sino-atrial node cells. J Physiol. 1994; 476(1):55–68.

29. Ehrlich, JR, Cha, TJ, Zhang, L, et al. Characterization of a hyperpolarization-activated time-dependent potassium current in canine cardiomyocytes from pulmonary vein myocardial sleeves and left atrium. J Physiol. 2004; 557(Pt 2):583–597.

30. Cha, TJ, Ehrlich, JR, Chartier, D, et al. Kir3-based inward rectifier potassium current: potential role in atrial tachycardia remodeling effects on atrial repolarization and arrhythmias. Circulation. 2006; 113(14):1730–1737.

31. Dobrev, D, Nattel, S. New antiarrhythmic drugs for treatment of atrial fibrillation. Lancet. 2010; 375(9721):1212–1223.

32. Voigt, N, Makary, S, Nattel, S, et al. Voltage-clamp-based methods for the detection of constitutively active acetylcholine-gated I_K,ACh channels in the diseased heart. Methods Enzymol. 2010; 484:653–675.

33. Makary, S, Voigt, N, Maguy, A, et al. Differential protein kinase C isoform regulation and increased constitutive activity of acetylcholine-regulated potassium channels in atrial remodeling. Circ Res. 2011; 109(9):1031–1043.

34. Voigt, N, Maguy, A, Yeh, YH, et al. Changes in I_K,ACh single-channel activity with atrial tachycardia remodelling in canine atrial cardiomyocytes. Cardiovasc Res. 2008; 77(1):35–43.

35. Heidbüchel, H, Vereecke, J, Carmeliet, E. Three different potassium channels in human atrium. Contribution to the basal potassium conductance. Circ Res. 1990; 66(5):1277–1286.

36. Sato, R, Hisatome, I, Wasserstrom, JA, et al. Acetylcholine-sensitive potassium channels in human atrial myocytes. Am J Physiol. 1990; 259(6 Pt 2):H1730–H1735.

37. Koumi, S-i, Backer, CL, Arentzen, CE. Characterization of inwardly rectifying K⁺ channel in human cardiac myocytes : alterations in channel behavior in myocytes isolated from patients with idiopathic dilated cardiomyopathy. Circulation. 1995; 92(2):164–174.

38. Logothetis, DE, Kurachi, Y, Galper, J, et al. The beta gamma subunits of GTP-binding proteins activate the muscarinic K⁺ channel in heart. Nature. 1987; 325(6102):321–326.

39. Wieland, T. Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein betagamma dimers: consequences on G protein activation and stability. Naunyn Schmiedebergs Arch Pharmacol. 2007; 374(5-6):373–383.

40. Riven, I, Iwanir, S, Reuveny, E. GIRK channel activation involves a local rearrangement of a preformed G protein channel complex. Neuron. 2006; 51(5):561–573.

41. Yamada, M, Ho, YK, Lee, RH, et al. Muscarinic K⁺ channels are activated by beta gamma subunits and inhibited by the GDP-bound form of alpha subunit of transducin. Biochem Biophys Res Commun. 1994; 200(3):1484–1490.

42. Doupnik, CA, Davidson, N, Lester, HA, et al. RGS proteins reconstitute the rapid gating kinetics of gbetagamma-activated inwardly rectifying K⁺ channels. Proc Natl Acad Sci U S A. 1997; 94(19):10461–10466.

43. Rishal, I, Keren-Raifman, T, Yakubovich, D, et al. Na⁺ promotes the dissociation between Galpha GDP and Gbeta gamma, activating G protein-gated K⁺ channels. J Biol Chem. 2003; 278(6):3840–3845.

44. Yakubovich, D, Rishal, I, Dascal, N. Kinetic modeling of Na⁺-induced, Gbetagamma-dependent activation of G protein-gated K⁺ channels. J Mol Neurosci. 2005; 25(1):7–19.

45. Ho, IH, Murrell-Lagnado, RD. Molecular mechanism for sodium-dependent activation of G protein-gated K⁺ channels. J Physiol. 1999; 520(Pt 3):645–651.

46. Mintert, E, Bosche, LI, Rinne, A, et al. Generation of a constitutive Na⁺-dependent inward-rectifier current in rat adult atrial myocytes by overexpression of Kir3. 4. J Physiol. 2007; 585(Pt 1):3–13.

47. Rosenhouse-Dantsker, A, Sui, JL, Zhao, Q, et al. A sodium-mediated structural switch that controls the sensitivity of Kir channels to PtdIns(4,5)P(2). Nat Chem Biol. 2008; 4(10):624–631.

47a. Voigt, N, Heijman, J, Trausch, A, et al. Impaired Na⁺-dependent regulation of acetylcholine-activated inward-rectifier K⁺ current modulates action potential rate dependence in patients with chronic atrial fibrillation. J Mol Cell Cardiol. 2013.

48. Abu-Taha, I, Voigt, N, Nattel, S, et al. Nucleoside diphosphate kinase B is a novel receptor-independent activator of G-protein signaling in clinical and experimental atrial fibrillation. Heart Rhythm. 2012; 9(5 (Supplement):S397.

49. Kaibara, M, Nakajima, T, Irisawa, H, et al. Regulation of spontaneous opening of muscarinic K⁺ channels in rabbit atrium. J Physiol. 1991; 433:589–613.

50. Sorota, S, Chlenov, M, Du, XY, et al. ATP-dependent activation of the atrial acetylcholine-induced K⁺ channel does not require nucleoside diphosphate kinase activity. Circ Res. 1998; 82(9):971–979.

51. Huang, CL, Feng, S, Hilgemann, DW. Direct activation of inward rectifier potassium channels by PIP₂ and its stabilization by Gbetagamma. Nature. 1998; 391(6669):803–806.

52. Cui, S, Ho, WK, Kim, ST, et al. Agonist-induced localization of Gq-coupled receptors and G protein-gated inwardly rectifying K⁺ (GIRK) channels to caveolae determines receptor specificity of phosphatidylinositol 4,5-bisphosphate signaling. J Biol Chem. 2010; 285(53):41732–41739.

53. Choisy, SC, James, AF, Hancox, JC. Acute desensitization of acetylcholine and endothelin-1 activated inward rectifier K⁺ current in myocytes from the cardiac atrioventricular node. Biochem Biophys Res Commun. 2012.

54. Wang, H, Han, H, Zhang, L, et al. Expression of multiple subtypes of muscarinic receptors and cellular distribution in the human heart. Mol Pharmacol. 2001; 59(5):1029–1036.

55. Perez, CC, Tobar, ID, Jimenez, E, et al. Kinetic and molecular evidences that human cardiac muscle express non-M2 muscarinic receptor subtypes that are able to interact themselves. Pharmacol Res. 2006; 54(5):345–355.

56. Jan, LY, Jan, YN. Heartfelt crosstalk: desensitization of the GIRK current. Nat Cell Biol. 2000; 2(9):E165–E167.

57. Kobrinsky, E, Mirshahi, T, Zhang, H, et al. Receptor-mediated hydrolysis of plasma membrane messenger PIP₂ leads to K⁺-current desensitization. Nat Cell Biol. 2000; 2(8):507–514.

58. Hellgren, I, Mustafa, A, Riazi, M, et al. Muscarinic M3 receptor subtype gene expression in the human heart. Cell Mol Life Sci. 2000; 57(1):175–180.

59. Oberhauser, V, Schwertfeger, E, Rutz, T, et al. Acetylcholine release in human heart atrium: influence of muscarinic autoreceptors, diabetes, and age. Circulation. 2001; 103(12):1638–1643.

60. Grandi, E, Pandit, SV, Voigt, N, et al. Human atrial action potential and Ca²⁺ model: sinus rhythm and chronic atrial fibrillation. Circ Res. 2011; 109(9):1055–1066.

61. Nikolov, EN, Ivanova-Nikolova, TT. Coordination of membrane excitability through a GIRK1 signaling complex in the atria. J Biol Chem. 2004; 279(22):23630–23636.

62. Medina, I, Krapivinsky, G, Arnold, S, et al. A switch mechanism for G beta gamma activation of I_K,ACh. J Biol Chem. 2000; 275(38):29709–29716.

63. Steinberg, SF. Structural basis of protein kinase C isoform function. Physiol Rev. 2008; 88(4):1341–1378.

64. Yeh, YH, Ehrlich, JR, Qi, X, et al. Adrenergic control of a constitutively active acetylcholine-regulated potassium current in canine atrial cardiomyocytes. Cardiovasc Res. 2007; 74(3):406–415.

65. Kim, D. Mechanism of rapid desensitization of muscarinic K⁺ current in adult rat and guinea pig atrial cells. Circ Res. 1993; 73(1):89–97.

66. Shui, Z, Boyett, MR, Zang, WJ. ATP-dependent desensitization of the muscarinic K⁺ channel in rat atrial cells. J Physiol. 1997; 505(Pt 1):77–93.

67. Shui, Z, Boyett, MR, Zang, WJ, et al. Receptor kinase-dependent desensitization of the muscarinic K⁺ current in rat atrial cells. J Physiol. 1995; 487(Pt 2):359–366.

68. Voigt, N, Li, N, Wang, Q, et al. Enhanced sarcoplasmic reticulum Ca²⁺ leak and increased Na⁺-Ca²⁺ exchanger function underlie delayed afterdepolarizations in patients with chronic atrial fibrillation. Circulation. 2012; 125(17):2059–2070.

69. Christ, T, Boknik, P, Wohrl, S, et al. L-type Ca²⁺ current downregulation in chronic human atrial fibrillation is associated with increased activity of protein phosphatases. Circulation. 2004; 110(17):2651–2657.

70. El-Armouche, A, Boknik, P, Eschenhagen, T, et al. Molecular determinants of altered Ca²⁺ handling in human chronic atrial fibrillation. Circulation. 2006; 114(7):670–680.

71. Greiser, M, Halaszovich, CR, Frechen, D, et al. Pharmacological evidence for altered src kinase regulation of I_Ca,L in patients with chronic atrial fibrillation. Naunyn Schmiedebergs Arch Pharmacol. 2007; 375(6):383–392.

72. Dobrev, D, Carlsson, L, Nattel, S. Novel molecular targets for atrial fibrillation therapy. Nat Rev Drug Discov. 2012; 11(4):275–291.

73. Kurachi, Y, Nakajima, T, Ito, H, et al. AN-132, a new class I anti-arrhythmic agent, depresses the acetylcholine-induced K⁺ current in atrial myocytes. Eur J Pharmacol. 1989; 165(2-3):319–322.

74. Kurachi, Y, Nakajima, T, Sugimoto, T. Quinidine inhibition of the muscarine receptor-activated K⁺ channel current in atrial cells of guinea pig. Naunyn Schmiedebergs Arch Pharmacol. 1987; 335(2):216–218.

75. Furutani, K, Ohno, Y, Inanobe, A, et al. Mutational and in silico analyses for antidepressant block of astroglial inward-rectifier Kir4. 1 channel. Mol Pharmacol. 2009; 75(6):1287–1295.

76. Whorton, MR, MacKinnon, R. Crystal structure of the mammalian GIRK₂ K⁺ channel and gating regulation by G proteins, PIP₂, and sodium. Cell. 2011; 147(1):199–208.

77. Jin, W, Klem, AM, Lewis, JH, et al. Mechanisms of inward-rectifier K⁺ channel inhibition by tertiapin-Q. Biochemistry. 1999; 38(43):14294–14301.

78. Jin, W, Lu, Z. A novel high-affinity inhibitor for inward-rectifier K⁺ channels. Biochemistry. 1998; 37(38):13291–13299.

79. Jin, W, Lu, Z. Synthesis of a stable form of tertiapin: a high-affinity inhibitor for inward-rectifier K⁺ channels. Biochemistry. 1999; 38(43):14286–14293.

80. Ramu, Y, Klem, AM, Lu, Z. Short variable sequence acquired in evolution enables selective inhibition of various inward-rectifier K⁺ channels. Biochemistry. 2004; 43(33):10701–10709.

81. Ramu, Y, Xu, Y, Lu, Z. Engineered specific and high-affinity inhibitor for a subtype of inward-rectifier K⁺ channels. Proc Natl Acad Sci U S A. 2008; 105(31):10774–10778.

82. 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. 2010; 24(11):4302–4312.