Selective activation of heteromeric SK channels contributes to action potential repolarization in mouse atrial myocytes
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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2024, Journal of Biological ChemistryDoes the small conductance Ca<sup>2+</sup>-activated K<sup>+</sup> current I<inf>SK</inf> flow under physiological conditions in rabbit and human atrial isolated cardiomyocytes?
2023, Journal of Molecular and Cellular CardiologyPreferential formation of human heteromeric SK2:SK3 channels limits homomeric SK channel assembly and function
2023, Journal of Biological ChemistryCitation 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.
Impact of I<inf>SK</inf> Voltage and Ca<sup>2+</sup>/Mg<sup>2+</sup>-Dependent Rectification on Cardiac Repolarization
2020, Biophysical JournalCitation 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.
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 CommunicationsCitation 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].
Are SK channels a logical target for treating ventricular arrhythmias? First, do no harm
2015, Trends in Cardiovascular Medicine
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.