Cholinergic and Constitutive Regulation of Atrial Potassium Channel

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Cholinergic and Constitutive Regulation of Atrial Potassium Channel

Dobromir Dobrev, Niels Voigt and Stanley Nattel

Chapter Outline

G-Protein Cycle May Contribute to the Generation of Constitutive I_K,ACh

Regulation of I_K,ACh Gating by PIP₂

Constitutively Active I_K,ACh

Phosphorylation-Dependent I_K,ACh Regulation

I_K,ACh Channels as Potential Therapeutic Targets in Atrial Fibrillation

Stimulation of the vagal nerve, the principal cardiac arm of the parasympathetic nervous system, reduces heart rate and slows conduction in the atrioventricular node, thereby tuning the heart rate to “rest-and-digest” activities. In 1921, Otto Loewi found that the vagal effects on the heart are mediated by release of acetylcholine (ACh) from the parasympathetic synapses, and ACh became the first neurotransmitter ever discovered.¹ However, it took more than 50 years until it was suggested that ACh activates a specific population of K⁺ channels (ACh-gated I_K,ACh; Figure 38-1, A) leading to hyperpolarization of the cell membrane, thereby decreasing pacemaker activity in sinoatrial (SA) node cells.² It was also found that I_K,ACh conductance is voltage dependent, with high K⁺ conductance at hyperpolarized membrane potentials (at which the current is typically inward) and small conductance at depolarized membrane potentials associated with outward current. This typical current-voltage (IV) relationship designates I_K,ACh as an inward-rectifier K⁺ current (see Figure 38-1, B and C) similar to I_K1, which is active in the absence of any receptor agonists, and I_K,ATP, which is activated by reduced intracellular ATP levels. Cardiac I_K,ACh channels are heterotetramers, usually consisting of two Kir3.1 and two Kir3.4 channel subunits.³

Figure 38-1 Basal and muscarinic (M₂)-receptor activated inward-rectifier potassium (K⁺) currents in sinus rhythm (SR) and chronic atrial fibrillation (cAF). A, Left panel, Physiologically G-protein–activated inward-rectifier K⁺ (GIRK) channels are supposed to be closed in the absence of M-receptor agonists and only the basal inward-rectifier potassium current I_K1 is active. B and C, Representative single-channel (upper tracings) and whole-cell recordings (lower tracings) in patients with SR (B) and cAF (C). The single-channel recordings were performed at –100 mV in a cell-attached configuration. Filled and empty arrow heads indicate closed and open levels of I_K,ACh, respectively. In the absence of M-receptor agonists (basal conditions), myocytes from the cAF patients (C) exhibit both I_K1 (#) and constitutive I_K,ACh openings (*), whereas the latter is a rare event in myocytes from patients with SR (B). Besides increased expression of I_K1 channels, constitutive I_K,ACh,c activity can contribute to the increased whole-cell basal inward-rectifier current in cAF (compare left lower tracings in B and C) (Replotted with permission from 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 112:3697–3706, 2005. Figure was produced using Servier Medical Art.)

I_K,ACh channels are activated by ACh binding to type-2 muscarinic receptors (M₂-receptors), which causes dissociation of inhibitory G_i-proteins, thereby increasing I_K,ACh open probability via direct interaction of G-protein βγ-subunits with the channel (Figure 38-2, A, B).^4,5 After current activation by M₂-receptor stimulation, atrial I_K,ACh shows a characteristic biphasic desensitization that starts within a few seconds (Figure 38-3, middle). Although both Kir3.1 and Kir3.4 channel homomers produce similar peak-current densities upon M₂-receptor stimulation when expressed in HEK-cells, I_K,ACh desensitization was observed in Kir3.1 homomers only. Moreover, the desensitization process depends on the presence of the Kir3.1 subunit within the channel complex.⁶ Besides channel subunit composition, kinetics of G-protein cycle, membrane content of the anionic phospholipid phosphatidylinositol-4,5-bisphosphate (PIP₂), and phosphorylation processes also contribute I_K,ACh regulation and desensitization (see Figure 38-3).

Figure 38-2 Muscarinic (M₂)-receptor and nucleoside diphosphate kinase (NDPK)-dependent activation of G_i-proteins. A, Agonist binding to M2-receptors triggers GDP/GTP (guanosine-5′-triphosphate and guanosine-5′-diphosphate) exchange at the Gα_i-subunit of the heterotrimeric G_i-proteins. Consecutive dissociation of the Gα_i– and Gβγ-subunits leads to activation of I_K,ACh by binding of the Gβγ-subunit to the I_K,ACh channel (B). Intrinsic GTPase activity of the Gα_i-subunit hydrolyses GTP into GDP resulting in reassociation of the heterotrimeric G-protein and restorage of the initial state. NDPKs provide the GTP, which is necessary for G-protein dissociation, by a phosphotransfer from ATP to GDP. C, In the subpopulation of heterotrimeric G_i-proteins complexed with NDPK B, a phosphotransfer from ATP to His118 in NDPK-B, and subsequently onto His266 of the Gβ, results in a high energetic phosphate, which promotes the formation of GTP and leads to receptor independent G-protein activation. This can contribute to the development of receptor-independent constitutive I_K,ACh activity. (Figure was produced using Servier Medical Art.)

Figure 38-3 Mechanisms contributing to I_K,ACh desensitization. Upon muscarinic (M₂)-receptor activation, I_K,ACh is activated by direct binding of liberated Gβγ-subunits to the I_K,ACh channel, followed by a biphasic desensitization (fast and intermediate desensitization). A, The binding of Gβγ-subunits to the I_K,ACh channel subunits Kir3.1 and Kir3.4 strengthens their interaction with cell membrane–located phosphatidyl inositol 4,5-bisphosphate (PIP₂), thereby increasing I_K,ACh open probability. Similarly, increased intracellular Na⁺ also strengthens the interaction of PIP₂ with the channel leading to receptor-independent current activation. In contrast, stimulation of Gq-coupled M1/3-receptors activates phospholipase-C (PLC), thereby lowering the PIP₂ membrane content resulting in I_K,ACh inhibition and contributing to fast I_K,ACh desensitization. B, Regulator of G-protein signaling proteins (RGS) accelerate the GTP hydrolysis rate of the Gα_i-subunit, thereby contributing to faster desensitization and reducing agonist-independent constitutive I_K,ACh,c activity (see also Figure 38-2, A). C, The phosphorylation of muscarinic (M) receptors and I_K,ACh channels is controlled by various kinases and phosphatases, whereby channel phosphorylation increases I_K,ACh, while concomitant phosphorylation of M-receptors reduces I_K,ACh. Whereas channel dephosphorylation is supposed to contribute to the fast phase (green), the intermediate phase (blue) involves progressive receptor phosphorylation by a G-protein–coupled receptor kinase (GRK), which uncouples the receptor from the G-protein. (Figure was produced using Servier Medical Art.)

Apart from canonical M₂-receptor–mediated activation of I_K,ACh, purinergic A₁,7,8 and sphingolipid Edg-3⁹ receptors (also coupled to G_i-proteins) can activate cardiac I_K,ACh channels. Based on their regulation and biophysics, I_K,ACh channels are also designated G-protein–activated inwardly rectifying K⁺ (GIRK) channels. In contrast to ventricular tissue, I_K,ACh channels are highly expressed in the atria, including SA and atrioventricular nodes, where they contribute to vagal regulation of heart rate (negative chronotropic effect) and conduction from the atria to the ventricles (negative dromotropic effects).^10–12

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

Table 38-1

Single-Channel Characteristics of Inward-Rectifier K⁺ Currents in Human Atrial Myocytes: Acetylcholine/Carbachol (10 µM) Activated I_K,ACh in Patients with Sinus Rhythm

G_S, single-channel conductance; P_open, channel open probability; τ_open/τ_close, time constant for open and closed time distributions.

In pico siemens (pS)/S = 1 V/A.

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.

G-Protein Cycle Can Contribute to the Generation of Constitutive I_K,ACh

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