Selective activation of heteromeric SK channels contributes to action potential repolarization in mouse atrial myocytes

doi:10.1016/j.hrthm.2015.01.027

Heart Rhythm

Volume 12, Issue 5, May 2015, Pages 1003-1015

https://doi.org/10.1016/j.hrthm.2015.01.027 Get rights and content

Background

Activation of small conductance calcium-activated potassium (SK) channels is proposed to contribute to repolarization of the action potential in atrial myocytes. This role is controversial, as these cardiac SK channels appear to exhibit an uncharacteristic pharmacology.

Objectives

The objectives of this study were to resolve whether activation of SK channels contributes to atrial action potential repolarization and to determine the likely subunit composition of the channel.

Methods

The effect of 2 SK channel inhibitors was assessed on outward current evoked in voltage clamp and on action potential duration in perforated patch and whole-cell current clamp recording from acutely isolated mouse atrial myocytes. The presence of SK channel subunits was assessed using immunocytochemistry.

Results

A significant component of outward current was reduced by the SK channel blockers apamin and UCL1684. Block by apamin displayed a sensitivity indicating that this current was carried by homomeric SK2 channels. Action potential duration was significantly prolonged by UCL1684, but not by apamin. This effect was accompanied by an increase in beat-to-beat variability and action potential triangulation. This pharmacology was matched by that of expressed heteromeric SK2-SK3 channels in HEK293 cells. Immunocytochemistry showed that atrial myocytes express both SK2 and SK3 channels with an overlapping expression pattern.

Conclusion

Only proposed heteromeric SK2-SK3 channels are physiologically activated to contribute to action potential repolarization, which is indicated by the difference in pharmacology of evoked outward current and prolongation of atrial action potential duration. The effect of blocking this channel on the action potential suggests that SK channel inhibition during cardiac function has the potential to be proarrhythmic.

Introduction

Approximately 70 million people are believed to suffer from atrial fibrillation (AF), with patients having an increased risk of stroke.1, 2 AF can be treated with potassium channel blockers that are capable of prolonging repolarization of the cardiac action potential and refractory period.³ However, currently used potassium channel blockers exhibit unwanted ventricular side effects that are potentially proarrhythmic. Therefore, there is a strong interest in developing atrial selective drugs by targeting an ion channel that is preferentially expressed in atrial myocytes.⁴

Evidence exists that AF can be affected by inhibitors of small conductance calcium–activated potassium (SK) channels.5, 6, 7 SK channels are voltage-independent channels that are inhibited by apamin, an octapeptide toxin from the honey bee Apis mellifera.8, 9 Three subtypes of SK channels have been cloned (SK1-3), all of which are sensitive to apamin¹⁰ and are present in the mouse and human heart. SK channel messenger RNA (mRNA) is abundant in both atria and ventricles, with a slightly higher level of SK1 and 2 mRNA transcripts being observed in mouse atria compared with ventricles.11, 12 This suggests that the inhibition of SK channel activity could produce atrial-selective effects. SK channel protein levels change after AF-related electrical remodeling.¹³ However, the loss of SK channel function by knockout was proarrhythmic, while inhibition has been shown to be antiarrhythmic in models of AF.5, 6, 7, 14 The functional role of SK channel activation is complicated further by inconsistent data of the effect of apamin. Apamin substantially prolonged the action potential duration (APD) in mouse and human atrial myocytes,¹² while having no effect on APD in dog or rat atrial myocytes.¹⁵ In a rat model of AF, administration of the organic SK channel blocker UCL1684 shortened the duration of AF substantially more than did apamin.⁷ In addition, a guinea pig ex vivo model of AF showed that SK channel inhibition by UCL1684 was antiarrhythmic, a response not replicated with apamin.⁵

Unlike existing organic blockers, apamin is the only SK channel inhibitor to discriminate between SK channel subtypes.¹⁰ We report that UCL1684 blocked a larger component of outward current than did apamin in mouse atrial myocytes. Apamin hardly affected APD, while UCL1684 significantly slowed the repolarization phase. These data suggest that more than 1 population of SK channels exist in mouse atrial myocytes. We show that caution would have to be applied to any therapeutic potential of atrial SK channel block as this promoted beat-to-beat variability in APD, which is an accurate marker for drug-induced repolarization-related arrhythmias.16, 17

Section snippets

Atrial and ventricular myocyte isolation

Animal studies reported in this study were performed according to the Guide for the Care and Use of Laboratory Animals. Mouse myocytes were dissociated from 25 to 30 g C57BL6 male mice using a modified version of a previously published protocol.¹⁸ Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (200 mg/kg), plus 30 µL of heparin (25,000 IU/mL). The heart was excised and mounted on the cannula of a whole heart perfusion apparatus and retrogradely perfused via the

Functional SK channels in atrial myocytes

Resolving the possible role of SK channel activation in atrial myocytes has been complicated by an inconsistent effect of blockers. A number of studies have used NS8593 to resolve the effects of SK channel activation.5, 6, 7, 23, 24 Step depolarizations positive to −20 mV evoked outward current that was partly sensitive to the application of a supramaximal concentration of either apamin (100 nM) or UCL1684 (100 nM) (Figures 1A and 1B). In contrast, NS8593 (10 µM) blocked significantly more

Discussion

Apamin is commonly used to discriminate between SK channel subtypes because it exhibits reasonable selectivity when blocking homomeric SK channels. SK2 is the most sensitive (IC₅₀ ~ 70 pM) followed by SK3 (IC₅₀ ~ 0.63–6 nM), with SK1 being the least sensitive (IC₅₀ ~ 1–8 nM).¹⁰ The rat isoform of SK1 is apamin insensitive.¹⁰ Significant progress has been made in understanding how apamin discriminates between homomeric SK channel subtypes. Apamin binds with high affinity to the outer pore⁹ and

Conclusion

The data in this study are consistent with the presence of 2 types of SK channels in mouse atrial myocytes, with one type proposed to be homomeric SK2 channels and the second proposed to be heteromeric. It is suggested that the heteromeric channel is composed of SK2-SK3 channel subunits and is physiologically activated to contribute to AP repolarization. The effect of block of this channel on the AP suggests that SK channel inhibition during cardiac function has the potential to be

Acknowledgments

We are indebted to Ms Lisa Yang and Mr Sohaib Sarwar for contributing data on the selectivity of NS8953 and UCL1684.

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Inhibition of small conductance Ca²⁺-activated K⁺ channels terminates and protects against atrial fibrillation

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Cited by (27)

ROS in diabetic atria regulate SK2 degradation by Atrogin-1 through the NF-κB signaling pathway
2024, Journal of Biological Chemistry
One of the independent risk factors for atrial fibrillation is diabetes mellitus (DM); however, the underlying mechanisms causing atrial fibrillation in DM are unknown. The underlying mechanism of Atrogin-1–mediated SK2 degradation and associated signaling pathways are unclear. The aim of this study was to elucidate the relationship among reactive oxygen species (ROS), the NF-κB signaling pathway, and Atrogin-1 protein expression in the atrial myocardia of DM mice. We found that SK2 expression was downregulated comitant with increased ROS generation and enhanced NF-κB signaling activation in the atrial cardiomyocytes of DM mice. These observations were mimicked by exogenously applicating H₂O₂ and by high glucose culture conditions in HL-1 cells. Inhibition of ROS production by diphenyleneiodonium chloride or silencing of NF-κB by siRNA decreased the protein expression of NF-κB and Atrogin-1 and increased that of SK2 in HL-1 cells with high glucose culture. Moreover, chromatin immunoprecipitation assay demonstrated that NF-κB/p65 directly binds to the promoter of the FBXO32 gene (encoding Atrogin-1), regulating the FBXO32 transcription. Finally, we evaluated the therapeutic effects of curcumin, known as a NF-κB inhibitor, on Atrogin-1 and SK2 expression in DM mice and confirmed that oral administration of curcumin for 4 weeks significantly suppressed Atrogin-1 expression and protected SK2 expression against hyperglycemia. In summary, the results from this study indicated that the ROS/NF-κB signaling pathway participates in Atrogin-1–mediated SK2 regulation in the atria of streptozotocin-induced DM mice.
Does the small conductance Ca2+-activated K+ current I<inf>SK</inf> flow under physiological conditions in rabbit and human atrial isolated cardiomyocytes?
2023, Journal of Molecular and Cellular Cardiology
The small conductance Ca²⁺-activated K⁺ current (I_SK) is a potential therapeutic target for treating atrial fibrillation.
To clarify, in rabbit and human atrial cardiomyocytes, the intracellular [Ca²⁺]-sensitivity of I_SK, and its contribution to action potential (AP) repolarisation, under physiological conditions.
Whole-cell-patch clamp, fluorescence microscopy: to record ion currents, APs and [Ca²⁺]_i; 35–37°C.
In rabbit atrial myocytes, 0.5 mM Ba²⁺ (positive control) significantly decreased whole-cell current, from −12.8 to −4.9 pA/pF (P < 0.05, n = 17 cells, 8 rabbits). By contrast, the I_SK blocker apamin (100 nM) had no effect on whole-cell current, at any set [Ca²⁺]_i (∼100–450 nM). The I_SK blocker ICAGEN (1 μM: ≥2 x IC₅₀) also had no effect on current over this [Ca²⁺]_i range. In human atrial myocytes, neither 1 μM ICAGEN (at [Ca²⁺]_i ∼ 100–450 nM), nor 100 nM apamin ([Ca²⁺]_i ∼ 250 nM) affected whole-cell current (5–10 cells, 3–5 patients/group). APs were significantly prolonged (at APD₃₀ and APD₇₀) by 2 mM 4-aminopyridine (positive control) in rabbit atrial myocytes, but 1 μM ICAGEN had no effect on APDs, versus either pre-ICAGEN or time-matched controls. High concentration (10 μM) ICAGEN (potentially I_SK-non-selective) moderately increased APD₇₀ and APD₉₀, by 5 and 26 ms, respectively. In human atrial myocytes, 1 μM ICAGEN had no effect on APD_30–90, whether stimulated at 1, 2 or 3 Hz (6–9 cells, 2–4 patients/rate).
I_SK does not flow in human or rabbit atrial cardiomyocytes with [Ca²⁺]_i set within the global average diastolic-systolic range, nor during APs stimulated at physiological or supra-physiological (≤3 Hz) rates.
Preferential formation of human heteromeric SK2:SK3 channels limits homomeric SK channel assembly and function
2023, Journal of Biological Chemistry
Citation Excerpt :
Representative cells were selected for Figures 1 & 2 following imaging of at least 6 areas of coverslips during each transfection condition and transfections were repeated three times to ensure consistency. Glass coverslips were placed into a recording chamber and superfused at room temperature with a solution of composition (in mM): NaCl (138), KCl (4), HEPES (10), D-glucose (10), MgCl2 (1.2), CaCl2 (2.5); and titrated to pH 7.4 with NaOH. Glass patch pipettes (WPI, 1B150F-3) were pulled to a resistance of 2-3.5MΩ and filled with intracellular solution containing (in mM): K-aspartate (97), KCl (20), Na2-ATP (1.5), HEPES-Na (10), EGTA (10), CaCl2 and MgCl2; and titrated to pH 7.2 with KOH.
Three isoforms of small conductance, calcium-activated potassium (SK) channel subunits have been identified (SK1-3) that exhibit a broad and overlapping tissue distribution. SK channels have been implicated in several disease states including hypertension and atrial fibrillation, but therapeutic targeting of SK channels is hampered by a lack of subtype-selective inhibitors. This is further complicated by studies showing that SK1 and SK2 preferentially form heteromeric channels during co-expression, likely limiting the function of homomeric channels in vivo. Here, we utilized a simplified expression system to investigate functional current produced when human (h) SK2 and hSK3 subunits are co-expressed. When expressed alone, hSK3 subunits were more clearly expressed on the cell surface than hSK2 subunits. hSK3 surface expression was reduced by co-transfection with hSK2. Whole-cell recording showed homomeric hSK3 currents were larger than homomeric hSK2 currents or heteromeric hSK2:hSK3 currents. The smaller amplitude of hSK2:hSK3-mediated current when compared with homomeric hSK3-mediated current suggests hSK2 subunits regulate surface expression of heteromers. Co-expression of hSK2 and hSK3 subunits produced a current that arose from a single population of heteromeric channels as exhibited by an intermediate sensitivity to the inhibitors apamin and UCL1684. Co-expression of the apamin-sensitive hSK2 subunit and a mutant, apamin-insensitive hSK3 subunit [hSK3(H485N)], produced an apamin-sensitive current. Concentration-inhibition relationships were best fit by a monophasic Hill equation, confirming preferential formation of heteromers. These data show that co-expressed hSK2 and hSK3 preferentially form heteromeric channels and suggest that the hSK2 subunit acts as a chaperone, limiting membrane expression of hSK2:hSK3 heteromeric channels.
Impact of I<inf>SK</inf> Voltage and Ca2+/Mg2+-Dependent Rectification on Cardiac Repolarization
2020, Biophysical Journal
Citation Excerpt :
The amino acids associated with both forms of rectification are conserved in all three mammalian SK channel isoforms (SK1, SK2, and SK3) by PubMed BLAST analysis (33,34). Although most of the studies of SK channel rectification in heterologous expression systems used the rSK2 isoform (30–32), homomeric human SK1 (35) and SK3 (36,37) also show inward rectification. Both intrinsic and Ca2+/Mg2+-dependent rectification may play significant roles in modulating cardiac repolarizations.
Cardiac small conductance Ca²⁺-activated K⁺ (SK) channels are activated solely by Ca²⁺, but the SK current (I_SK) is inwardly rectified. However, the impact of inward rectification in shaping action potentials (APs) in ventricular cardiomyocytes under β-adrenergic stimulation or in disease states remains undefined. Two processes underlie this inward rectification: an intrinsic rectification caused by an electrostatic energy barrier from positively charged amino acids at the inner pore and a voltage-dependent Ca²⁺/Mg²⁺ block. Thus, Ca²⁺ has a biphasic effect on I_SK, activating at low [Ca²⁺] yet inhibiting I_SK at high [Ca²⁺]. We examined the effect of I_SK rectification on APs in rat cardiomyocytes by simultaneously recording whole-cell apamin-sensitive currents and Ca²⁺ transients during an AP waveform and developed a computer model of SK channels with rectification features. The typical profile of I_SK during AP clamp included an initial peak (mean 1.6 pA/pF) followed by decay to the point that submembrane [Ca²⁺] reached ∼10 μM. During the rest of the AP stimulus, I_SK either plateaued or gradually increased as the cell repolarized and submembrane [Ca²⁺] decreased further. We used a six-state gating model combined with intrinsic and Ca²⁺/Mg²⁺-dependent rectification to simulate I_SK and investigated the relative contributions of each type of rectification to AP shape. This SK channel model replicates key features of I_SK recording during AP clamp showing that intrinsic rectification limits I_SK at high V_m during the early and plateau phase of APs. Furthermore, the initial rise of Ca²⁺ transients activates, but higher [Ca²⁺] blocks SK channels, yielding a transient outward-like I_SK trajectory. During the decay phase of Ca²⁺, the Ca²⁺-dependent block is released, causing I_SK to rise again and contribute to repolarization. Therefore, I_SK is an important repolarizing current, and the rectification characteristics of an SK channel determine its impact on early, plateau, and repolarization phases of APs.
Role of SK channel activation in determining the action potential configuration in freshly isolated human atrial myocytes from the SKArF study
2019, Biochemical and Biophysical Research Communications
Citation Excerpt :
In contrast, small organic molecule inhibitors such as UCL1684 only bind the outer pore turret, with it being proposed that the lack of binding to the extracellular loop prevents these molecules from selecting between SK channel subtypes [19]. UCL1684 has been found to be very selective for SK channels, not affecting a number of ion channel currents that underlie the atrial action potential [20]. SK channel subunits of different identities can co-assemble to form functional heteromeric channels that display atypical pharmacology [11,20].
Inhibition of SK channel function is being pursued in animal models as a possible therapeutic approach to treat atrial fibrillation (AF). However, the pharmacology of SK channels in human atria is unclear. SK channel function is inhibited by both apamin and UCL1684, with the former discriminating between SK channel subtypes. In this proof-of-principle study, the effects of apamin and UCL1684 on right atrial myocytes freshly isolated from patients in sinus rhythm undergoing elective cardiac surgery were investigated. Outward current evoked from voltage clamped human atrial myocytes was reduced by these two inhibitors of SK channel function. In contrast, membrane current underlying the atrial action potential was affected significantly only by UCL1684 and not by apamin. This pharmacology mirrors that observed in mouse atria, suggesting that mammalian atria possess two populations of SK channels, with only one population contributing to the action potential waveform. Immuno-visualization of the subcellular localization of SK2 and SK3 subunits showed a high degree of colocalization, consistent with the formation of heteromeric SK2/SK3 channels. These data reveal that human atrial myocytes express two SK channel subtypes, one exhibiting an unusual pharmacology. These channels contribute to the atrial action potential waveform and might be a target for novel therapeutic approaches to treat supraventricular arrhythmic conditions such as atrial fibrillation.
Are SK channels a logical target for treating ventricular arrhythmias? First, do no harm
2015, Trends in Cardiovascular Medicine

View all citing articles on Scopus

: This work was supported by a grant from the British Heart Foundation (grant nos. FS/10/68 and PG/11/97, to Dr James and Dr Hancox; grant no. PG/11/24, to Dr Hancox).

: Present address of Dr Weatherall: MediTech Media Ltd, London EC1V 9RU, United Kingdom.

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Selective activation of heteromeric SK channels contributes to action potential repolarization in mouse atrial myocytes

Background

Objectives

Methods

Results

Conclusion

Introduction

Section snippets

Atrial and ventricular myocyte isolation

Functional SK channels in atrial myocytes

Discussion

Conclusion

Acknowledgments

Heart Rhythm

Pharmacol Ther

Drug Discov Today

J Biol Chem

Prog Neurobiol

J Biol Chem

J Mol Cell Cardiol

J Pharmacol Toxicol Methods

J Mol Cell Cardiol

J Biol Chem

J Biol Chem

Heart Rhythm

Heart Rhythm

Pathophysiological mechanisms of atrial fibrillation: a translational appraisal

Physiol Rev

Inhibition of small conductance Ca2+-activated K+ channels terminates and protects against atrial fibrillation

Circ Arrhythm Electrophysiol

Effects on atrial fibrillation in aged hypertensive rats by Ca2+-activated K+ channel inhibition

Hypertension

The duration of pacing-induced atrial fibrillation is reduced in vivo by inhibition of small conductance Ca2+-activated K+ channels

J Cardiovasc Pharmacol

Structure-function relationships and site of action of apamin, a neurotoxic polypeptide of bee venom with an action on central nervous system

Biochemistry

Differential expression of small-conductance Ca2+-activated K+ channels SK1, SK2, and SK3 in mouse atrial and ventricular myocytes

Am J Physiol Heart Circ Physiol

Early electrical remodeling in rabbit pulmonary vein results from trafficking of intracellular SK2 channels to membrane sites

Cardiovasc Res

Ablation of a Ca2+-activated K+ channel (SK2 channel) results in action potential prolongation in atrial myocytes and atrial fibrillation

J Physiol

Inhibition of small conductance Ca²⁺-activated K⁺ channels terminates and protects against atrial fibrillation

Effects on atrial fibrillation in aged hypertensive rats by Ca²⁺-activated K⁺ channel inhibition

The duration of pacing-induced atrial fibrillation is reduced in vivo by inhibition of small conductance Ca²⁺-activated K⁺ channels

Differential expression of small-conductance Ca²⁺-activated K⁺ channels SK1, SK2, and SK3 in mouse atrial and ventricular myocytes

Ablation of a Ca²⁺-activated K⁺ channel (SK2 channel) results in action potential prolongation in atrial myocytes and atrial fibrillation