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ScienceDirect Pharmacology of voltage-gated potassium channel Kv1.5 — impact on cardiac excitability Erich Wettwer1 and Heinrich Terlau2 Voltage activated potassium (Kv) channels are intensely investigated targets within the pharmacological strategies to treat cardiac arrhythmia. For atrial fibrillation (AF) substances inhibiting the ultra rapid outward rectifying Kv current (IKur) and its underlying Kv1.5 channel have been developed. Here we describe potential limitations of this approach with respect to critical parameters of Kv channel pharmacology. In healthy tissue IKur/Kv1.5 inhibition can unexpectedly lead to action potential shortening with corresponding arrhythmogenic effects. In tissue with chronic AF, electrical remodeling occurs which is accompanied with changes in ion channel expression and composition. As a consequence atrial tissue exhibits a different pharmacological fingerprint. New strategies to obtain more mechanistic insight into drug target interaction are needed for better understanding the pharmacological potential of IKur/Kv1.5 inhibition for AF treatment. Addresses 1 Department of Pharmacology and Toxicology, Technische Universita¨t Dresden, Medizinische Fakulta¨t Carl Gustav Carus, Fetscherstraße 74, 01307 Dresden, Germany 2 Institute of Physiology, University of Kiel, Hermann-Rodewald-Straße 5, 24118 Kiel, Germany Corresponding author: Terlau, Heinrich ([email protected])
Current Opinion in Pharmacology 2014, 15:115–121 This review comes from a themed issue on Cardiovascular and renal Edited by Gregory J Kaczorowski and Olaf Pongs For a complete overview see the Issue and the Editorial Available online 13th March 2014 1471-4892/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.coph.2014.02.001
Introduction In the heart the electrical excitability of the cardiomyocytes is directly coupled and converted into the mechanical action of the cardiac muscle. The electrical activity as reflected in the action potential (AP) waveform of the cardiomyocytes is a result of an extremely well tuned concert of the activity of different ion channel proteins (for review see: [1]). Among those voltage-gated potassium (Kv) channels play a fundamental role within the AP repolarization phase(s). In the human atrium Kv currents present a fast inactivating transient outward Kv current (Ito) and an ‘ultra rapid delayed rectifier’ IKur with relatively slow inactivation properties. Ito leads to a very early phase of repolarization and in combination with IKur to a www.sciencedirect.com
prominent notch in the action potential shape. In addition, ‘delayed’ rectifying Kv currents which are at least composed of a rapid (IKr) and a slowly activating ‘delayed rectifier’ current (IKS) Kv current components are present in human atrial cardiomyocytes [2]. The different Kv channels underlying the different repolarization phases of cardiac action potentials are more or less well known and well described [2]. Kv1.5 is known to be very abundant in atrial tissue and it is widely accepted that the molecular bases of IKur is predominantly KCNA5 (Kv1.5) [3–5]. Hence, Kv1.5 channels play an important role for AP repolarization of atrial human cardiomyocytes. In accordance with the large variability in the action potential waveforms in different mammalian species the ion channel composition can, however, vary substantially between different species [6,7]. Atrial fibrillation is the most common cardiac arrhythmia afflicting humans with a prevalence increasing with age [8]. Potential treatments of atrial fibrillation are attempts to reduce arrhythmic foci or suppress conduction of arrhythmic activity. On the basis of our current knowledge of the pathophysiology of atrial fibrillation several diverse targets have been proposed and are discussed as potentially important in the pharmacotherapy of this disease (for review see: [9,10,11,12]). Since IKur plays exclusively in atrial cardiomyocytes a significant role for the very fast and late AP repolarization phases, the potential of Kv1.5 as a target for drugs with antiarrhythmic action has been intensely investigated (for review see: [13]). In this brief review we focus on the potential benefit of drugs interfering with IKur and its underlying K+-channel Kv1.5. Especially, we will discuss limitations of current approaches to tackle atrial fibrillation using blockers of Kv1.5 and will give an outlook for potential improvements in Kv1.5 pharmacology.
Physiological characteristics of Kv1.5 and IKur Functional expression of Kv1.5 results in rapid activating and slowly inactivating Kv currents that are sensitive to 4Aminopyridine (4-AP) but not affected by other ‘classical’ Kv current blockers like tetraethylammonium (TEA), dendrotoxin (DTX) or charybtotoxin (CTX) (overview: [14,15]). Current characteristics like inactivating kinetics exhibit strong temperature dependency and are modulated by accessory subunits ([16,17]; see also Figure 1). Northern blot analysis revealed that in rat Kv1.5 mRNA is Current Opinion in Pharmacology 2014, 15:115–121
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Figure 1
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Kv1.5 currents at 248C and 378C from mouse fibroblasts (ltk-) permanently transfected with human HK2 channel. Voltage-clamp experiments. (a) Original current recordings at 238C. Voltage clamp protocol see inset, protocol repeated at 0.2 Hz. Cell c96, Cm 19 pF. (b) Current recordings at 378C from same cell as in (a), same protocol as in (a). Bath solution (in mM): KCl 5.4, NaCl 150.0, HEPES 10.0, MgCl2 2.0, CaCl2 2.0, glucose 11.1, pH 7.4 adjusted with NaOH. Electrode solution (in mM): KCl 140.0, EGTA 10.0, MgCl2 4.0, HEPES 10.0, CaCl2 4.98, Na2ATP 4.0, pH 7.3 adjusted with KOH. Note the difference in the inactivation characteristics of the currents. Unpublished data: EW. For details of HK2 (Kv1.5) cloning see Snyders et al. [15].
expressed in many different tissues, for example, heart, kidney, skeletal muscle, lung, brain, pituitary and aorta (see: [14,18]). In cardiac tissue Kv1.5 is much more abundantly expressed in atrium than in ventricle and a detailed localization of Kv1.5 in cardiac tissue is described in [17]. The use of antisense oligonucleotides revealed that Kv1.5 is responsible for most of IKur in human atrium [5] and today it is widely accepted that Kv1.5 is the major underlying channel of IKur. Point mutations in KCNA5 correlated with increased or decreased IKur can enhance AF susceptibility [19].
reviewed [13]. Although since over 20 years a large body of experimental data on newly developed and classical compounds has been published [13], unequivocal proof, that antagonists of Kv1.5 currents have antiarrhythmic activity in patients, is still missing.
Pharmacology of Kv1.5
The effects of Kv1.5 blockers on atrial APs were, however, inconsistent. Intracellular AP recordings from multicellular human atrial specimen produced an unexpected result: instead of the expected AP prolongation, AVE 0118 shortened APs in preparations from patients in sinus rhythm (SR), and only prolonged the dramatically altered AP in preparations from patients in chronic atrial fibrillation (AF) [23]. Similar results were reported for 4-AP and recently developed specific Kv1.5 blockers like the Xention compounds XEN-D0101 and XEN-D0103 ([24,25]; see also Figure 2).
With the first identification of IKur as ‘ultra rapid outward current’ by Wang et al. [3] and the cloning and characterisation of Kv1.5 of human atria and its preferential expression in atrial tissue [20] this current and K+-channel was proposed as a potentially new pharmacological target for treating atrial fibrillation [3,21]. The underlying therapeutic principle seemed evident. Kv1.5 current inhibition was expected to produce an increase in atrial AP duration and consequently increase the refractory period of fibrillating atrium. Thus, specific block of the Kv1.5 channel, despite its widespread expression in non-cardiac tissue, seemed an extremely promising idea in the development of new antiarrhythmics in AF therapy with the important benefit of missing arrhythmogenic action potential prolonging ‘Class III’-effects in the ventricle. This hypothesis was supported by the effects of the first ‘selective’ Kv1.5 blocker, 4-AP (for chemical structure of substances see Figure 3). 4-AP inhibits IKur currents in the micromolar concentration range [3,20] and exerts a prolongation of AP duration recorded from isolated human atrial myocytes [3]. On the basis of these experimental results numerous compounds were developed by pharmaceutical companies as recently comprehensively Current Opinion in Pharmacology 2014, 15:115–121
One of the first intensely studied Kv1.5 blockers, AVE0118 (although with substantial additional effects on the transient outward current Ito, Kv4.3), demonstrated a clear antiarrhythmic potency in a pig model of atrial fibrillation [22].
The more sophisticated insight from this finding is that the effect of Kv1.5 channel block is dependent on the ion channel composition of a cell and therefore species and disease dependent [26]. Retrospectively, the very first and thorough investigations on human atrial specimen already demonstrated an — at that time unexplained — AP-shortening by 4-AP [27,28]. Recently Parvi et al. [29] published a clinical study with the selective Kv1.5 blocker MK-0448 on — this is a potential drawback — healthy volunteers. In this study the effects were insignificant questioning the antiarrhythmic principle of Kv1.5 channel block. Vernakalant is the www.sciencedirect.com
Pharmacology of IKur/Kv1.5 and cardiac excitability Wettwer and Terlau 117
Figure 2
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Effects of the selective Kv1.5 blocker XEN-D0101 on action potentials from human right atrial specimen and from isolated atrial myocytes. (a) Representative action potential recordings with sharp microelectrodes from human atrial appendages from patients in sinus rhythm (left), chronic atrial fibrillation (middle) and from left ventricle (right). Control registrations (black) and 20 min after the addition of XEN-D0101 at concentrations of 0.3 mM, 1,0 mM and 3 mM. Note the shortening of APD90 in SR and the prolongation in AF, no effect in ventricle. Stimulation at 1 Hz. (b) Outward current measurements in single isolated atrial myocytes. Representative voltage-clamp current registrations from a myocytes of SR patient (left) and AF patient (right). Holding potential 60 mV, clamp step to +50 mV. Control trace (black) and 2 min after the addition of 10 mM XEN-D0101 (red). Dashed line (left) denotes zero current level. Note the reduced control currents in AF (electrical remodeling) and incomplete current block with drug. Experiments performed at 378C. Modified from Ford et al. [25] with permission. For experimental details see Ford et al. [25]. See also: [46].
most recent drug development introduced into AF therapy 2010 [30]. However, its effectiveness is most probably due to the strong frequency dependent block of Na-channels and much less to blocking effects on Kv1.5 [10,31]. Therefore, it is presently difficult to judge the usefulness of Kv1.5 blockade in AF pharmacotherapy. The observed AP shortening in SR would be arrhythmogenic. Conversely, AP prolongation in AF would probably be antiarrhythmic. However, the dramatic changes in ion channel expression and composition that occur in AF, need to be taken into account to assess the final effect of Kv1.5 blockade on rhythm (Figure 2a). Kv1.5 expression was found to be reduced as well as outward IKur currents from single myocytes (Figure 2b) [25,26]. Block of a current that is already downregulated is likely an ineffective treatment. Instead of following the approach to cure the already remodeled atrium it is also conceivable to prevent AF to become chronic. Interestingly, it was very recently reported that the selective Kv1.5 blockers XEND0103 and MK-0448 show a frequency dependent effect on refractory period in SR with shortening of AP duration www.sciencedirect.com
at low frequencies but prolongation of AP duration and effective refractory period at high stimulation frequencies (Loose et al.: AHA Meeting 2013, abstract). In this approach, periods of fibrillation could be prevented and postpone the manifestation of AF in the elderly patient. Clinical studies are mandatory to prove the validity of this concept.
Outlook/perspectives Many different groups of substances interacting with Kv1.5/IKur have and are being developed for a pharmacological treatment of AF. Yet a detailed mechanistic insight on how a substance interacting with Kv1.5 may lead to potentially beneficial effects during AF is still quite poor. There are several aspects which ought to be considered. Ion channel composition
Although it is assumed that the molecular substrate of IKur is Kv1.5 there are discrepancies between the pharmacological profile of Kv1.5 in expression systems compared to IKur in atrial cardiomyocytes. The IC50 for Kv1.5 inhibition by 4-AP is about one order of magnitude lower (ca. Current Opinion in Pharmacology 2014, 15:115–121
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Figure 3
N
N
O N
O
N N
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N O
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Chemical structures of selected agents affecting IKur.
200 mM, [15]) than the IC50 value for IKur inhibition in human atrial cardiomyocytes (IC50 < 10 mM, [24,32]; see also Table 1). It is therefore questionable if Kv1.5 is the only molecule forming the channel protein responsible for IKur. Most likely, Kv1.5 like other ion channels is localized at the plasma membrane in multiprotein complexes. For example, accessory proteins like the Kvb subunits are also expressed in human atria and might not only affect the biophysical properties of Kv1.5 like inactivation time constants or current-voltage
relationships but also the pharmacological properties of Kv1.5 [17]. An additional and/or alternative explanation for discrepancies in pharmacological properties of IKur/ Kv1.5 would be the potential existence of at least a proportion of Kv1.5 within heteromeric Kv-channels. For CNS neurons it is well described that heteromeric Kv-channel complexes exist [33]. The physiological role of these heteromeric channel complexes is far less understood which mainly reflects the experimental difficulties in investigating native heteromeric Kv channels.
Table 1 Pharmacological parameters of selected agents affecting IKur 4-Aminopyridine
AVE0118
IC50 value Kv1.5 IKur
181 6b
IKr hERG IKS KCNQ1/KCNE1 Ito Kv4.3 INa
f
c
MK-0448
Xention D0101
d
e
Xention D0103
Vernakalant a
g
7 0.2 c
0.009 0.011
0.24 0.4
0.025 na
13 0.8–9
mM range
10 a
110
13
na
21
mM range
10% at 10 a
0.79
na
na
na
1000 b
1.8 c
2.3
4.2
na
30
na
na
10 ne
> 100
na
19
IC50 values expressed in mM producing half maximum inhibition; na: no data available, ne: no effect. From [11]; bfrom [32]; cfrom [23]; dfrom [29]; efrom [25]; ffrom [15]; gfrom [46].
a
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Pharmacology of IKur/Kv1.5 and cardiac excitability Wettwer and Terlau 119
Substance selectivity
A complication for the mechanistic analysis of the substances used to block IKur is the poor selectivity of these substances like 4-AP that besides with Kv1.5 also interacts with other Kv channels like Kv2, Kv3 or Kv4 channels (see: [14], Table 1). In principle any potential beneficial effect in AF pharmacotherapy observed from non-selective substances might not be related to the binding to Kv1.5 but could also result from the interaction with a different target involved in cardiac excitability. Overall, affecting several targets might be more effective in changing the electrophysiological properties of atrial cardiomyocytes than a specific block of only one target. The poor specificity of certain Kv binding substances in part might be correlated with the low molecular weight and the corresponding small size of these molecules. High molecular weight substances like peptides known to bind to the pore region of Kv channels can achieve a higher specificity due to multiple interaction sites within the ion channel pore (for pharmacological strategies see: [34]). To this end a more intense molecular modeling is desirable for an optimization of Kv1.5 channel pharmacology. Most recently new approaches like co-crystallizations of and/or solid state NMR shed some light on the structural bases for interaction of drugs on ion channels [35,36]. State dependent binding
The electrical remodeling which occurs during AF is correlated with upregulation and/or downregulation of the activity of a variety of ion channel proteins (for review see: [26]). This in turn might alter the interaction of a substance affecting IKur/binding to Kv1.5. For a whole variety of substances interacting with voltage activated channels it is known that at least a certain degree of state dependent binding occurs (best known for local anaesthetics; [37]). This implies that the gating of the ion channel protein (i.e. open, close, inactivated state of the channel) affects binding of a substance to its target site. It has even been demonstrated that the functional effect of a biological substance in theory can be the opposite depending on the state of the targeted channel protein (closed state: current blocker; C-type inactivated state: current enhancer; [38]). For 4-AP well-known as a Kv channel ‘blocker’ it has been shown that in neurons 4AP can result in a current increase [39] and a current enhancing effect can also be observed for the classical Kv channel ‘blocker’ TEA (HT: unpublished results). Accordingly, state dependent binding also has to be taken into account for substances affecting IKur. This raises the possibility for different drug–target interactions of a substance to the targeted open, closed or inactivated state of the Kv1.5 channel. Therefore the view that a ‘blocker’ always leads to a certain physiological effect might be at least partially wrong due to the state dependence in the drug ion channel interaction. Note that the electrical remodeling during chronic AF is likely associated with changes in the ion channel gating. This raises the www.sciencedirect.com
possibility for different biological actions of the binding of a substance to the targeted Kv1.5. Long term effects
Besides the direct physiological effects upon ion channel binding also long term effects of substances interacting with Kv channels might be relevant for AF treatment. In addition to its immediate effects on ion channel conductance, ion channel ‘antagonists’ may also effect ion channel expression, for example, half lives at the plasma membrane, ion channel trafficking and gene expression. This type of long term effect has been reported for the effects of pentamidine on functional hERG channel density [40,41]. It is desirable to have studies investigating also these aspects for IKur/Kv1.5 targeting substances in order to improve the treatment of AF with substances interacting with Kv1.5 channels. To this end mathematical model calculations of the human atrial AP may be of considerable help in understanding effects of dynamic drug interactions with Kv1.5 (see: [42], see also: [43]). Combination of different techniques like scanning mutagenesis, molecular dynamic simulation (MDS) and electrophysiology [44] or homology modeling of Kv1.5 [45] will give us more insight into the molecular but also pathophysiological processes underlying AF. Overall, a better knowledge of the interplay between the ion channel composition, target specificity, state dependent binding and long term (patho-)physiology will be essential for new pharmacological strategies in AF treatment.
Acknowledgements We wish to thank Ursula Ravens, Technische Universita¨t Dresden, Department of Pharmacology and Toxicology, Dresden, Germany for intense and constructive discussions.
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the basis for a new mechanism of Kv channel-specific inhibition. Nat Chem Biol 2013, 9:507-513. By using alanine-scanning mutagenesis, electrophysiological recordings and molecular modeling the author identify a side pocket in the vicinity of the central pore cavity of Kv1 channels to which a small-molecule inhibitor (Psora-4) can bind. Occupancy of this side pocket together with occupancy to the central pore cavity is important for the subtype specificity for different Kv1 channels of this small-molecule inhibitor but also important for high-affinity binding and use-sependence. 45. Tikhonov DB, Zhorov BS: Homology modeling of Kv1.5 channel block by cationic and electroneutral ligands. Biochim Biophys Acta 2013 http://dx.doi.org/10.1016/j.bbamem.2013.11.019. 46. Loose S, Muller J, Wettwer E, El-Haou S, Jackson C, Tang R, Milnes J, Ravens U, Ford J: Positive frequency-dependent effects of highly selective Kv1.5 blockers (XEN-D0103 & MK0448) in right atrial trabeculae from patients in sinus rhythm. Circulation 2013, 128:A18323 (abstract).
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ScienceDirect Pharmacology of voltage-gated potassium channel Kv1.5 — impact on cardiac excitability Erich Wettwer1 and Heinrich Terlau2 Voltage activated potassium (Kv) channels are intensely investigated targets within the pharmacological strategies to treat cardiac arrhythmia. For atrial fibrillation (AF) substances inhibiting the ultra rapid outward rectifying Kv current (IKur) and its underlying Kv1.5 channel have been developed. Here we describe potential limitations of this approach with respect to critical parameters of Kv channel pharmacology. In healthy tissue IKur/Kv1.5 inhibition can unexpectedly lead to action potential shortening with corresponding arrhythmogenic effects. In tissue with chronic AF, electrical remodeling occurs which is accompanied with changes in ion channel expression and composition. As a consequence atrial tissue exhibits a different pharmacological fingerprint. New strategies to obtain more mechanistic insight into drug target interaction are needed for better understanding the pharmacological potential of IKur/Kv1.5 inhibition for AF treatment. Addresses 1 Department of Pharmacology and Toxicology, Technische Universita¨t Dresden, Medizinische Fakulta¨t Carl Gustav Carus, Fetscherstraße 74, 01307 Dresden, Germany 2 Institute of Physiology, University of Kiel, Hermann-Rodewald-Straße 5, 24118 Kiel, Germany Corresponding author: Terlau, Heinrich ([email protected])
Current Opinion in Pharmacology 2014, 15:115–121 This review comes from a themed issue on Cardiovascular and renal Edited by Gregory J Kaczorowski and Olaf Pongs For a complete overview see the Issue and the Editorial Available online 13th March 2014 1471-4892/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.coph.2014.02.001
Introduction In the heart the electrical excitability of the cardiomyocytes is directly coupled and converted into the mechanical action of the cardiac muscle. The electrical activity as reflected in the action potential (AP) waveform of the cardiomyocytes is a result of an extremely well tuned concert of the activity of different ion channel proteins (for review see: [1]). Among those voltage-gated potassium (Kv) channels play a fundamental role within the AP repolarization phase(s). In the human atrium Kv currents present a fast inactivating transient outward Kv current (Ito) and an ‘ultra rapid delayed rectifier’ IKur with relatively slow inactivation properties. Ito leads to a very early phase of repolarization and in combination with IKur to a www.sciencedirect.com
prominent notch in the action potential shape. In addition, ‘delayed’ rectifying Kv currents which are at least composed of a rapid (IKr) and a slowly activating ‘delayed rectifier’ current (IKS) Kv current components are present in human atrial cardiomyocytes [2]. The different Kv channels underlying the different repolarization phases of cardiac action potentials are more or less well known and well described [2]. Kv1.5 is known to be very abundant in atrial tissue and it is widely accepted that the molecular bases of IKur is predominantly KCNA5 (Kv1.5) [3–5]. Hence, Kv1.5 channels play an important role for AP repolarization of atrial human cardiomyocytes. In accordance with the large variability in the action potential waveforms in different mammalian species the ion channel composition can, however, vary substantially between different species [6,7]. Atrial fibrillation is the most common cardiac arrhythmia afflicting humans with a prevalence increasing with age [8]. Potential treatments of atrial fibrillation are attempts to reduce arrhythmic foci or suppress conduction of arrhythmic activity. On the basis of our current knowledge of the pathophysiology of atrial fibrillation several diverse targets have been proposed and are discussed as potentially important in the pharmacotherapy of this disease (for review see: [9,10,11,12]). Since IKur plays exclusively in atrial cardiomyocytes a significant role for the very fast and late AP repolarization phases, the potential of Kv1.5 as a target for drugs with antiarrhythmic action has been intensely investigated (for review see: [13]). In this brief review we focus on the potential benefit of drugs interfering with IKur and its underlying K+-channel Kv1.5. Especially, we will discuss limitations of current approaches to tackle atrial fibrillation using blockers of Kv1.5 and will give an outlook for potential improvements in Kv1.5 pharmacology.
Physiological characteristics of Kv1.5 and IKur Functional expression of Kv1.5 results in rapid activating and slowly inactivating Kv currents that are sensitive to 4Aminopyridine (4-AP) but not affected by other ‘classical’ Kv current blockers like tetraethylammonium (TEA), dendrotoxin (DTX) or charybtotoxin (CTX) (overview: [14,15]). Current characteristics like inactivating kinetics exhibit strong temperature dependency and are modulated by accessory subunits ([16,17]; see also Figure 1). Northern blot analysis revealed that in rat Kv1.5 mRNA is Current Opinion in Pharmacology 2014, 15:115–121
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Figure 1
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Kv1.5 currents at 248C and 378C from mouse fibroblasts (ltk-) permanently transfected with human HK2 channel. Voltage-clamp experiments. (a) Original current recordings at 238C. Voltage clamp protocol see inset, protocol repeated at 0.2 Hz. Cell c96, Cm 19 pF. (b) Current recordings at 378C from same cell as in (a), same protocol as in (a). Bath solution (in mM): KCl 5.4, NaCl 150.0, HEPES 10.0, MgCl2 2.0, CaCl2 2.0, glucose 11.1, pH 7.4 adjusted with NaOH. Electrode solution (in mM): KCl 140.0, EGTA 10.0, MgCl2 4.0, HEPES 10.0, CaCl2 4.98, Na2ATP 4.0, pH 7.3 adjusted with KOH. Note the difference in the inactivation characteristics of the currents. Unpublished data: EW. For details of HK2 (Kv1.5) cloning see Snyders et al. [15].
expressed in many different tissues, for example, heart, kidney, skeletal muscle, lung, brain, pituitary and aorta (see: [14,18]). In cardiac tissue Kv1.5 is much more abundantly expressed in atrium than in ventricle and a detailed localization of Kv1.5 in cardiac tissue is described in [17]. The use of antisense oligonucleotides revealed that Kv1.5 is responsible for most of IKur in human atrium [5] and today it is widely accepted that Kv1.5 is the major underlying channel of IKur. Point mutations in KCNA5 correlated with increased or decreased IKur can enhance AF susceptibility [19].
reviewed [13]. Although since over 20 years a large body of experimental data on newly developed and classical compounds has been published [13], unequivocal proof, that antagonists of Kv1.5 currents have antiarrhythmic activity in patients, is still missing.
Pharmacology of Kv1.5
The effects of Kv1.5 blockers on atrial APs were, however, inconsistent. Intracellular AP recordings from multicellular human atrial specimen produced an unexpected result: instead of the expected AP prolongation, AVE 0118 shortened APs in preparations from patients in sinus rhythm (SR), and only prolonged the dramatically altered AP in preparations from patients in chronic atrial fibrillation (AF) [23]. Similar results were reported for 4-AP and recently developed specific Kv1.5 blockers like the Xention compounds XEN-D0101 and XEN-D0103 ([24,25]; see also Figure 2).
With the first identification of IKur as ‘ultra rapid outward current’ by Wang et al. [3] and the cloning and characterisation of Kv1.5 of human atria and its preferential expression in atrial tissue [20] this current and K+-channel was proposed as a potentially new pharmacological target for treating atrial fibrillation [3,21]. The underlying therapeutic principle seemed evident. Kv1.5 current inhibition was expected to produce an increase in atrial AP duration and consequently increase the refractory period of fibrillating atrium. Thus, specific block of the Kv1.5 channel, despite its widespread expression in non-cardiac tissue, seemed an extremely promising idea in the development of new antiarrhythmics in AF therapy with the important benefit of missing arrhythmogenic action potential prolonging ‘Class III’-effects in the ventricle. This hypothesis was supported by the effects of the first ‘selective’ Kv1.5 blocker, 4-AP (for chemical structure of substances see Figure 3). 4-AP inhibits IKur currents in the micromolar concentration range [3,20] and exerts a prolongation of AP duration recorded from isolated human atrial myocytes [3]. On the basis of these experimental results numerous compounds were developed by pharmaceutical companies as recently comprehensively Current Opinion in Pharmacology 2014, 15:115–121
One of the first intensely studied Kv1.5 blockers, AVE0118 (although with substantial additional effects on the transient outward current Ito, Kv4.3), demonstrated a clear antiarrhythmic potency in a pig model of atrial fibrillation [22].
The more sophisticated insight from this finding is that the effect of Kv1.5 channel block is dependent on the ion channel composition of a cell and therefore species and disease dependent [26]. Retrospectively, the very first and thorough investigations on human atrial specimen already demonstrated an — at that time unexplained — AP-shortening by 4-AP [27,28]. Recently Parvi et al. [29] published a clinical study with the selective Kv1.5 blocker MK-0448 on — this is a potential drawback — healthy volunteers. In this study the effects were insignificant questioning the antiarrhythmic principle of Kv1.5 channel block. Vernakalant is the www.sciencedirect.com
Pharmacology of IKur/Kv1.5 and cardiac excitability Wettwer and Terlau 117
Figure 2
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Effects of the selective Kv1.5 blocker XEN-D0101 on action potentials from human right atrial specimen and from isolated atrial myocytes. (a) Representative action potential recordings with sharp microelectrodes from human atrial appendages from patients in sinus rhythm (left), chronic atrial fibrillation (middle) and from left ventricle (right). Control registrations (black) and 20 min after the addition of XEN-D0101 at concentrations of 0.3 mM, 1,0 mM and 3 mM. Note the shortening of APD90 in SR and the prolongation in AF, no effect in ventricle. Stimulation at 1 Hz. (b) Outward current measurements in single isolated atrial myocytes. Representative voltage-clamp current registrations from a myocytes of SR patient (left) and AF patient (right). Holding potential 60 mV, clamp step to +50 mV. Control trace (black) and 2 min after the addition of 10 mM XEN-D0101 (red). Dashed line (left) denotes zero current level. Note the reduced control currents in AF (electrical remodeling) and incomplete current block with drug. Experiments performed at 378C. Modified from Ford et al. [25] with permission. For experimental details see Ford et al. [25]. See also: [46].
most recent drug development introduced into AF therapy 2010 [30]. However, its effectiveness is most probably due to the strong frequency dependent block of Na-channels and much less to blocking effects on Kv1.5 [10,31]. Therefore, it is presently difficult to judge the usefulness of Kv1.5 blockade in AF pharmacotherapy. The observed AP shortening in SR would be arrhythmogenic. Conversely, AP prolongation in AF would probably be antiarrhythmic. However, the dramatic changes in ion channel expression and composition that occur in AF, need to be taken into account to assess the final effect of Kv1.5 blockade on rhythm (Figure 2a). Kv1.5 expression was found to be reduced as well as outward IKur currents from single myocytes (Figure 2b) [25,26]. Block of a current that is already downregulated is likely an ineffective treatment. Instead of following the approach to cure the already remodeled atrium it is also conceivable to prevent AF to become chronic. Interestingly, it was very recently reported that the selective Kv1.5 blockers XEND0103 and MK-0448 show a frequency dependent effect on refractory period in SR with shortening of AP duration www.sciencedirect.com
at low frequencies but prolongation of AP duration and effective refractory period at high stimulation frequencies (Loose et al.: AHA Meeting 2013, abstract). In this approach, periods of fibrillation could be prevented and postpone the manifestation of AF in the elderly patient. Clinical studies are mandatory to prove the validity of this concept.
Outlook/perspectives Many different groups of substances interacting with Kv1.5/IKur have and are being developed for a pharmacological treatment of AF. Yet a detailed mechanistic insight on how a substance interacting with Kv1.5 may lead to potentially beneficial effects during AF is still quite poor. There are several aspects which ought to be considered. Ion channel composition
Although it is assumed that the molecular substrate of IKur is Kv1.5 there are discrepancies between the pharmacological profile of Kv1.5 in expression systems compared to IKur in atrial cardiomyocytes. The IC50 for Kv1.5 inhibition by 4-AP is about one order of magnitude lower (ca. Current Opinion in Pharmacology 2014, 15:115–121
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Figure 3
N
N
O N
O
N N
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AVE 0118 O
N O
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OH F
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RSD1235 Vernakalant Brinavess®
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Chemical structures of selected agents affecting IKur.
200 mM, [15]) than the IC50 value for IKur inhibition in human atrial cardiomyocytes (IC50 < 10 mM, [24,32]; see also Table 1). It is therefore questionable if Kv1.5 is the only molecule forming the channel protein responsible for IKur. Most likely, Kv1.5 like other ion channels is localized at the plasma membrane in multiprotein complexes. For example, accessory proteins like the Kvb subunits are also expressed in human atria and might not only affect the biophysical properties of Kv1.5 like inactivation time constants or current-voltage
relationships but also the pharmacological properties of Kv1.5 [17]. An additional and/or alternative explanation for discrepancies in pharmacological properties of IKur/ Kv1.5 would be the potential existence of at least a proportion of Kv1.5 within heteromeric Kv-channels. For CNS neurons it is well described that heteromeric Kv-channel complexes exist [33]. The physiological role of these heteromeric channel complexes is far less understood which mainly reflects the experimental difficulties in investigating native heteromeric Kv channels.
Table 1 Pharmacological parameters of selected agents affecting IKur 4-Aminopyridine
AVE0118
IC50 value Kv1.5 IKur
181 6b
IKr hERG IKS KCNQ1/KCNE1 Ito Kv4.3 INa
f
c
MK-0448
Xention D0101
d
e
Xention D0103
Vernakalant a
g
7 0.2 c
0.009 0.011
0.24 0.4
0.025 na
13 0.8–9
mM range
10 a
110
13
na
21
mM range
10% at 10 a
0.79
na
na
na
1000 b
1.8 c
2.3
4.2
na
30
na
na
10 ne
> 100
na
19
IC50 values expressed in mM producing half maximum inhibition; na: no data available, ne: no effect. From [11]; bfrom [32]; cfrom [23]; dfrom [29]; efrom [25]; ffrom [15]; gfrom [46].
a
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Pharmacology of IKur/Kv1.5 and cardiac excitability Wettwer and Terlau 119
Substance selectivity
A complication for the mechanistic analysis of the substances used to block IKur is the poor selectivity of these substances like 4-AP that besides with Kv1.5 also interacts with other Kv channels like Kv2, Kv3 or Kv4 channels (see: [14], Table 1). In principle any potential beneficial effect in AF pharmacotherapy observed from non-selective substances might not be related to the binding to Kv1.5 but could also result from the interaction with a different target involved in cardiac excitability. Overall, affecting several targets might be more effective in changing the electrophysiological properties of atrial cardiomyocytes than a specific block of only one target. The poor specificity of certain Kv binding substances in part might be correlated with the low molecular weight and the corresponding small size of these molecules. High molecular weight substances like peptides known to bind to the pore region of Kv channels can achieve a higher specificity due to multiple interaction sites within the ion channel pore (for pharmacological strategies see: [34]). To this end a more intense molecular modeling is desirable for an optimization of Kv1.5 channel pharmacology. Most recently new approaches like co-crystallizations of and/or solid state NMR shed some light on the structural bases for interaction of drugs on ion channels [35,36]. State dependent binding
The electrical remodeling which occurs during AF is correlated with upregulation and/or downregulation of the activity of a variety of ion channel proteins (for review see: [26]). This in turn might alter the interaction of a substance affecting IKur/binding to Kv1.5. For a whole variety of substances interacting with voltage activated channels it is known that at least a certain degree of state dependent binding occurs (best known for local anaesthetics; [37]). This implies that the gating of the ion channel protein (i.e. open, close, inactivated state of the channel) affects binding of a substance to its target site. It has even been demonstrated that the functional effect of a biological substance in theory can be the opposite depending on the state of the targeted channel protein (closed state: current blocker; C-type inactivated state: current enhancer; [38]). For 4-AP well-known as a Kv channel ‘blocker’ it has been shown that in neurons 4AP can result in a current increase [39] and a current enhancing effect can also be observed for the classical Kv channel ‘blocker’ TEA (HT: unpublished results). Accordingly, state dependent binding also has to be taken into account for substances affecting IKur. This raises the possibility for different drug–target interactions of a substance to the targeted open, closed or inactivated state of the Kv1.5 channel. Therefore the view that a ‘blocker’ always leads to a certain physiological effect might be at least partially wrong due to the state dependence in the drug ion channel interaction. Note that the electrical remodeling during chronic AF is likely associated with changes in the ion channel gating. This raises the www.sciencedirect.com
possibility for different biological actions of the binding of a substance to the targeted Kv1.5. Long term effects
Besides the direct physiological effects upon ion channel binding also long term effects of substances interacting with Kv channels might be relevant for AF treatment. In addition to its immediate effects on ion channel conductance, ion channel ‘antagonists’ may also effect ion channel expression, for example, half lives at the plasma membrane, ion channel trafficking and gene expression. This type of long term effect has been reported for the effects of pentamidine on functional hERG channel density [40,41]. It is desirable to have studies investigating also these aspects for IKur/Kv1.5 targeting substances in order to improve the treatment of AF with substances interacting with Kv1.5 channels. To this end mathematical model calculations of the human atrial AP may be of considerable help in understanding effects of dynamic drug interactions with Kv1.5 (see: [42], see also: [43]). Combination of different techniques like scanning mutagenesis, molecular dynamic simulation (MDS) and electrophysiology [44] or homology modeling of Kv1.5 [45] will give us more insight into the molecular but also pathophysiological processes underlying AF. Overall, a better knowledge of the interplay between the ion channel composition, target specificity, state dependent binding and long term (patho-)physiology will be essential for new pharmacological strategies in AF treatment.
Acknowledgements We wish to thank Ursula Ravens, Technische Universita¨t Dresden, Department of Pharmacology and Toxicology, Dresden, Germany for intense and constructive discussions.
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Current Opinion in Pharmacology 2014, 15:115–121
