Class III antiarrhythmic drugs amiodarone and dronedarone impair KIR2.1 backward trafficking

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. KIR2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (IK1), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late-endosome/lysosome system. Here we defined the potential interference in KIR2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited IK1 in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK-KWGF cells, both drugs dose- and time-dependently increased KIR2.1 expression (2.0 ± 0.2-fold with amiodarone: 10 μM, 24 hrs; 2.3 ± 0.3-fold with dronedarone: 5 μM, 24 hrs) and late-endosomal/lysosomal KIR2.1 accumulation. Increased KIR2.1 expression level was also observed in the presence of Nav1.5 co-expression. Augmented KIR2.1 protein levels and intracellular accumulation were also observed in COS-7, END-2, MES-1 and EPI-7 cells. Both drugs had no effect on Kv11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at −120 mV, 5 μM) enhanced IKIR2.1 upon 24-hrs treatment, whereas dronedarone tended to increase IKIR2.1 and it did not reach significance (43.8 ± 5.5%, P = 0.26 at −120 mV; 2 μM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced IK1 by inhibiting KIR2.1 degradation

Class III antiarrhythmic drugs amiodarone and dronedarone impair KIR2.1 backward trafficking

Drug-induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. KIR2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (IK 1), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late-endosome/lysosome system. Here we defined the potential interference in KIR2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited IK 1 in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK-KWGF cells, both drugs dose- and time-dependently increased KIR2.1 expression (2.0 ± 0.2-fold with amiodarone: 10 μM, 24 hrs; 2.3 ± 0.3-fold with dronedarone: 5 μM, 24 hrs) and late-endosomal/lysosomal KIR2.1 accumulation. Increased KIR2.1 expression level was also observed in the presence of Nav1.5 co-expression. Augmented KIR2.1 protein levels and intracellular accumulation were also observed in COS-7, END-2, MES-1 and EPI-7 cells. Both drugs had no effect on Kv11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at −120 mV, 5 μM) enhanced IKIR 2.1 upon 24-hrs treatment, whereas dronedarone tended to increase IKIR 2.1 and it did not reach significance (43.8 ± 5.5%, P = 0.26 at −120 mV; 2 μM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced IK 1 by inhibiting KIR2.1 degradation

Targeting Potassium Channel Trafficking in Cardiac Arrhythmia

Ion channel trafficking, this is the transport of ion channel proteins within the heart cell, is an underestimated cause of severe cardiac arrhythmias. Importantly, it is often missed in initial cardiac safety assessment during the drug development process. The main focus of this thesis is exploring the role of cardiac potassium channel trafficking in arrhythmia and new means to counteract the latter. The inward rectifier KIR2.x and delayed rectifier Kv11.1 (hERG) channels were emphasized. In Chapter 2, we listed several inherited diseases that relate to altered ion channel function and linked it to channel mistrafficking and recognized mutation clusters. In Chapter 3, we also reviewed current animal models applied in cardiac arrhythmia studies to find appropriate models to enable the transition of ion-channel trafficking studies from in vitro to in vivo systems. The role of ion channel trafficking from several aspects, for example, pharmacological activation and structure identification, was evaluated in Chapter 4 - 6. In Chapter 7, we further identified the functional properties of several Pentamidine analogues, a typical Kv11.1 trafficking inhibitor. Finally, in Chapter 8, we summarized and discussed the results and conclusions from previous chapters. To conclude, potassium channel trafficking is a valid and important target for treating cardiac arrhythmia, using a pharmacological approach

Disease Associated Mutations in KIR Proteins Linked to Aberrant Inward Rectifier Channel Trafficking

The ubiquitously expressed family of inward rectifier potassium (KIR) channels, encoded by KCNJ genes, is primarily involved in cell excitability and potassium homeostasis. Channel mutations associate with a variety of severe human diseases and syndromes, affecting many organ systems including the central and peripheral neural system, heart, kidney, pancreas, and skeletal muscle. A number of mutations associate with altered ion channel expression at the plasma membrane, which might result from defective channel trafficking. Trafficking involves cellular processes that transport ion channels to and from their place of function. By alignment of all KIR channels, and depicting the trafficking associated mutations, three mutational hotspots were identified. One localized in the transmembrane-domain 1 and immediately adjacent sequences, one was found in the G-loop and Golgi-export domain, and the third one was detected at the immunoglobulin-like domain. Surprisingly, only few mutations were observed in experimentally determined Endoplasmic Reticulum (ER)exit-, export-, or ER-retention motifs. Structural mapping of the trafficking defect causing mutations provided a 3D framework, which indicates that trafficking deficient mutations form clusters. These &ldquo;mutation clusters&rdquo; affect trafficking by different mechanisms, including protein stability

Identification of a PEST Sequence in Vertebrate KIR2.1 That Modifies Rectification

K IR2.1 potassium channels, producing inward rectifier potassium current (I K1), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal K IR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. K IR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within K IR2.1 sequences using the "epestfind" webtool. WT and ΔPEST K IR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. K IR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine K IR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the K IR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in K IR2.2, K IR2.3, and K IR2.6 proteins. Deletion of the PEST domain in California kingsnake and human K IR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T1/2 from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical I K1, however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of K IR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of I K1 channels

Identification of a PEST Sequence in Vertebrate KIR2.1 That Modifies Rectification

K IR2.1 potassium channels, producing inward rectifier potassium current (I K1), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal K IR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. K IR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within K IR2.1 sequences using the "epestfind" webtool. WT and ΔPEST K IR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. K IR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine K IR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the K IR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in K IR2.2, K IR2.3, and K IR2.6 proteins. Deletion of the PEST domain in California kingsnake and human K IR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T1/2 from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical I K1, however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of K IR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of I K1 channels

Glibenclamide and HMR1098 normalize Cantú syndrome-associated gain-of-function currents

Cantú syndrome (CS) is caused by dominant gain-of-function mutation in ATP-dependent potassium channels. Cellular ATP concentrations regulate potassium current thereby coupling energy status with membrane excitability. No specific pharmacotherapeutic options are available to treat CS but IKATP channels are pharmaceutical targets in type II diabetes or cardiac arrhythmia treatment. We have been suggested that IKATP inhibitors, glibenclamide and HMR1098, normalize CS channels. IKATP in response to Mg-ATP, glibenclamide and HMR1098 were measured by inside-out patch-clamp electrophysiology. Results were interpreted in view of cryo-EM IKATP channel structures. Mg-ATP IC50 values of outward current were increased for D207E (0.71 ± 0.14 mmol/L), S1020P (1.83 ± 0.10), S1054Y (0.95 ± 0.06) and R1154Q (0.75 ± 0.13) channels compared to H60Y (0.14 ± 0.01) and wild-type (0.15 ± 0.01). HMR1098 dose-dependently inhibited S1020P and S1054Y channels in the presence of 0.15 mmol/L Mg-ATP, reaching, at 30 μmol/L, current levels displayed by wild-type and H60Y channels in the presence of 0.15 mmol/L Mg-ATP. Glibenclamide (10 μmol/L) induced similar normalization. S1054Y sensitivity to glibenclamide increases strongly at 0.5 mmol/L Mg-ATP compared to 0.15 mmol/L, in contrast to D207E and S1020P channels. Experimental findings agree with structural considerations. We conclude that CS channel activity can be normalized by existing drugs; however, complete normalization can be achieved at supraclinical concentrations only

Glibenclamide and HMR1098 normalize Cantú syndrome-associated gain-of-function currents

Cantú syndrome (CS) is caused by dominant gain-of-function mutation in ATP-dependent potassium channels. Cellular ATP concentrations regulate potassium current thereby coupling energy status with membrane excitability. No specific pharmacotherapeutic options are available to treat CS but IKATP channels are pharmaceutical targets in type II diabetes or cardiac arrhythmia treatment. We have been suggested that IKATP inhibitors, glibenclamide and HMR1098, normalize CS channels. IKATP in response to Mg-ATP, glibenclamide and HMR1098 were measured by inside-out patch-clamp electrophysiology. Results were interpreted in view of cryo-EM IKATP channel structures. Mg-ATP IC50 values of outward current were increased for D207E (0.71 ± 0.14 mmol/L), S1020P (1.83 ± 0.10), S1054Y (0.95 ± 0.06) and R1154Q (0.75 ± 0.13) channels compared to H60Y (0.14 ± 0.01) and wild-type (0.15 ± 0.01). HMR1098 dose-dependently inhibited S1020P and S1054Y channels in the presence of 0.15 mmol/L Mg-ATP, reaching, at 30 μmol/L, current levels displayed by wild-type and H60Y channels in the presence of 0.15 mmol/L Mg-ATP. Glibenclamide (10 μmol/L) induced similar normalization. S1054Y sensitivity to glibenclamide increases strongly at 0.5 mmol/L Mg-ATP compared to 0.15 mmol/L, in contrast to D207E and S1020P channels. Experimental findings agree with structural considerations. We conclude that CS channel activity can be normalized by existing drugs; however, complete normalization can be achieved at supraclinical concentrations only

LUF7244, an allosteric modulator/activator of K v11.1 channels, counteracts dofetilide-induced torsades de pointes arrhythmia in the chronic atrioventricular block dog model

Background and Purpose: K v11.1 (hERG) channel blockade is an adverse effect of many drugs and lead compounds, associated with lethal cardiac arrhythmias. LUF7244 is a negative allosteric modulator/activator of K v11.1 channels that inhibits early afterdepolarizations in vitro. We tested LUF7244 for antiarrhythmic efficacy and potential proarrhythmia in a dog model. Experimental Approach: LUF7244 was tested in vitro for (a) increasing human I Kv11.1 and canine I Kr and (b) decreasing dofetilide-induced action potential lengthening and early afterdepolarizations in cardiomyocytes derived from human induced pluripotent stem cells and canine isolated ventricular cardiomyocytes. In vivo, LUF7244 was given intravenously to anaesthetized dogs in sinus rhythm or with chronic atrioventricular block. Key Results: LUF7244 (0.5–10 μM) concentration dependently increased I Kv11.1 by inhibiting inactivation. In vitro, LUF7244 (10 μM) had no effects on I KIR2.1, I Nav1.5, I Ca-L, and I Ks, doubled I Kr, shortened human and canine action potential duration by approximately 50%, and inhibited dofetilide-induced early afterdepolarizations. LUF7244 (2.5 mg·kg −1·15 min −1) in dogs with sinus rhythm was not proarrhythmic and shortened, non-significantly, repolarization parameters (QTc: −6.8%). In dogs with chronic atrioventricular block, LUF7244 prevented dofetilide-induced torsades de pointes arrhythmias in 5/7 animals without normalization of the QTc. Peak LUF7244 plasma levels were 1.75 ± 0.80 during sinus rhythm and 2.34 ± 1.57 μM after chronic atrioventricular block. Conclusions and Implications: LUF7244 counteracted dofetilide-induced early afterdepolarizations in vitro and torsades de pointes in vivo. Allosteric modulators/activators of K v11.1 channels might neutralize adverse cardiac effects of existing drugs and newly developed compounds that display QTc lengthening