CO2/HCO3- modulates K+ and Ca2+ currents in glomus cells of the carotid body.
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Animals↗
Biomedical subjects
E Ficker
Publications and source records attributed to E Ficker.
Molecular determinants of inactivation and dofetilide block in ether a-go-go (EAG) channels and EAG-related K(+) channels.
The major subunit of the cardiac delayed rectifier current I(Kr) is encoded by the human ether a-go-go related gene (HERG). HERG/I(Kr) channels are blocked selectively by class III antiarrhythmic methanesulfonanilide drugs such as dofetilide. The binding site for methanesulfonanilides is believed to be similar for nonantiarrhythmic drugs such as antihistamines, antibiotics, and antipsychotics. To gain further insight into the binding site, we examined the minimal structural changes necessary to transform low-affinity binding of dofetilide by the related bovine ether a-go-go channel bEAG to high-affinity binding of HERG. Previously, it was shown that high-affinity binding in HERG required intact C-type inactivation; the bovine ether a-go-go K(+) channel (bEAG), unlike HERG, is noninactivating. Therefore, we introduced C-type inactivation into noninactivating bEAG using site-directed mutagenesis. Two point mutations in the pore region, T432S and A443S, were sufficient to produce C-type inactivation. Low concentrations of dofetilide produced block of bEAG T432S/A443S; unlike HERG, block was almost irreversible. Substitution of an additional amino acid in transmembrane domain S6 made the block reversible. Dofetilide blocked the triply mutated bEAG T432S/A443S/A453S with an IC(50) value of 1.1 microM. The blocking potency was 30-fold greater than bEAG WT and about one third that of HERG WT. We conclude that high affinity methanesulfonanilide binding to HERG channels is strongly dependent on C-type inactivation.
Amino Acid Substitution↗
High-affinity blockade of human ether-a-go-go-related gene human cardiac potassium channels by the novel antiarrhythmic drug BRL-32872.
Human ether-a-go-go-related gene (HERG) potassium channels are one primary target for the pharmacological treatment of cardiac arrhythmias by class III antiarrhythmic drugs. These drugs are characterized by high antiarrhythmic efficacy, but they can also initiate life-threatening "torsade de pointes" tachyarrhythmias. Recently, it has been suggested that combining potassium and calcium channel blocking mechanisms reduces the proarrhythmic potential of selective class III antiarrhythmic agents. BRL-32872 is a novel antiarrhythmic drug that inhibits potassium and calcium currents in isolated cardiomyocytes. In our study, we investigated the effects of BRL-32872 on cloned HERG channels heterologously expressed in Xenopus oocytes. Using the two-microelectrode voltage clamp technique, we found that BRL-32872 caused a high-affinity, state-dependent block of open HERG channels (IC(50) = 241 nM) in a frequency-dependent manner with slow unbinding kinetics. Inactivated channels mainly had to open to be blocked by BRL-32872. The HERG S620T mutant channel, which has a strongly reduced degree of inactivation, was 51-fold less sensitive to BRL-32872 block, indicating that BRL-32872 binding was enhanced by the inactivation process. In an additional approach, we studied HERG channels expressed in a human cell line (HEK 293) using the whole-cell patch-clamp technique. BRL-32872 inhibited HERG currents in HEK 293 cells in a dose-dependent manner, with an IC(50) value of 19.8 nM. We conclude that BRL-32872 is a potent blocker of HERG potassium channels, which accounts for the class III antiarrhythmic action of BRL-32872.
Animals↗
Retention in the endoplasmic reticulum as a mechanism of dominant-negative current suppression in human long QT syndrome.
Mutations in the cardiac potassium channel HERG (KCNH2) cause chromosome 7-linked long QT syndrome (LQT2) characterized by a prolonged QT interval, recurrent syncope and sudden cardiac death. Most mutations in HERG exhibit "loss of function" phenotypes with defective channels either inserted into the plasma membrane or retained in the endoplasmic reticulum. "Loss of function" mutations reduce I(Kr), the cardiac delayed rectifier current encoded by HERG, due to haploinsufficiency or suppression of wild-type function by a dominant-negative mechanism. One explanation for dominant-negative current suppression is that mutant subunits render tetrameric channel complexes non-conducting on co-assembly. In the present paper we describe an alternative mechanism for this phenomenon. We show (1) that the dominant-negative HERG mutation A561V is retained in the endoplasmic reticulum and (2) that wild-type channels are tagged for retention in the ER by co-assembly with trafficking deficient A561V subunits. Thus, in HERG A561V dominant-negative suppression of wild-type function is the result of an acquired trafficking defect.
Animals↗
Chemosensing at the carotid body. Involvement of a HERG-like potassium current in glomus cells.
Currently, it is not clear what type of K+ channel(s) is active at the resting membrane potential (RMP) in glomus cells of the carotid body (CB). HERG channels produce currents that are known to contribute to the RMP in other neuronal cells. The goal of the present study was to determine whether CB glomus cells express HERG-like (HL) K+ current, and if so, to determine whether HL currents regulate the RMP. With high [K+]o, depolarizing voltage steps from -85 mV revealed a slowly deactivating inward tail current indicative of HL K+ current in whole-cell, voltage clamped glomus cells. The HL currents were blocked by dofetilide (DOF) in a concentration-dependent manner (IC50 = 13 nM) and high concentrations (1 and 10 mM) of Ba2+. The steady-state activation properties of the HL current (Vh = -45 mV) suggest that it is active at the RMP in glomus cells. Whole-cell, current clamped glomus cells exhibited a RMP of -48 mV. 150 nM DOF caused a significant (14 mV) depolarizing shift in the RMP. In isolated glomus cells, [Ca2+]i increased in response to DOF (1 microM). In an in-vitro CB preparation, DOF increased basal sensory discharge in a concentration-dependent manner and significantly attenuated the sensory response to hypoxia. These results suggest that the HERG-like current is responsible for controlling the RMP in glomus cells of the rabbit CB, and that it is involved in the chemosensory response to hypoxia of the CB.
Animals↗
Novel characteristics of a misprocessed mutant HERG channel linked to hereditary long QT syndrome.
Hereditary long QT syndrome (hLQTS) is a heterogeneous genetic disease characterized by prolonged QT interval in the electrocardiogram, recurrent syncope, and sudden cardiac death. Mutations in the cardiac potassium channel HERG (KCNH2) are the second most common form of hLQTS and reduce the delayed rectifier K(+) currents, thereby prolonging repolarization. We studied a novel COOH-terminal missense mutation, HERG R752W, which segregated with the disease in a family of 101 genotyped individuals. When the mutant cRNA was expressed in Xenopus oocytes it produced enhanced rather than reduced currents. Simulations using the Luo-Rudy model predicted minimal shortening rather than prolongation of the cardiac action potential. Consequently, a normal or shortened QT interval would be expected in contrast to the long QT observed clinically. This anomaly was resolved by our observation that the mutant protein was not delivered to the plasma membrane of mammalian cells but was retained intracellularly. We found that this trafficking defect was corrected at lower incubation temperatures and that functional channels were now delivered to the plasma membrane. However, trafficking could not be restored by chemical chaperones or E-4031, a specific blocker of HERG channels. Therefore, HERG R752W represents a new class of trafficking mutants in hLQTS. The occurrence of different classes of misprocessed channels suggests that a unified therapeutic approach for altering HERG trafficking will not be possible and that different treatment modalities will have to be matched to the different classes of trafficking mutants.
Action Potentials↗
HERG-Like potassium current regulates the resting membrane potential in glomus cells of the rabbit carotid body.
Direct evidence for a specific K(+) channel underlying the resting membrane potential in glomus cells of the carotid body has been absent. The product of the human ether-a-go-go-related gene (HERG) produces inward rectifier currents that are known to contribute to the resting membrane potential in other neuronal cells. The goal of the present study was to determine whether carotid body glomus cells express HERG-like K(+) current, and if so, to determine whether a HERG-like current regulates the resting membrane potential. Freshly dissociated rabbit glomus cells under whole cell voltage clamp exhibited slowly decaying outward currents that activated 20-30 mV positive to the resting membrane potential. Raising extracellular K(+) revealed a slowly deactivating inward tail current indicative of HERG-like K(+) current. HERG-like currents were not found in cells resembling type II cells. The HERG-like current was blocked by dofetilide (DOF) in a concentration-dependent manner (IC(50) = 13 +/- 4 nM, mean +/- SE) and high concentrations of Ba(2+) (1 and 10 mM). The biophysical and pharmacological characteristics of this inward tail current suggest that it is conducted by a HERG-like channel. The steady-state activation properties of the HERG-like current (V(h) = -44 +/- 2 mV) suggest that it is active at the resting membrane potential in glomus cells. In whole cell, current-clamped glomus cells (average resting membrane potential, - 48 +/- 4 mV), DOF, but not tetraethylammonium, caused a significant (13 mV) depolarizing shift in the resting membrane potential. Using fluorescence imaging, DOF increased [Ca(2+)](i) in isolated glomus cells. In an in-vitro carotid body preparation, DOF increased basal sensory discharge in the carotid sinus nerve in a concentration-dependent manner. These results demonstrate that glomus cells express a HERG-like current that is active at, and responsible for controlling the resting membrane potential.
Animals↗
Molecular determinants of dofetilide block of HERG K+ channels.
The human ether-a-go-go-related gene (HERG) encodes a K+ channel with biophysical properties nearly identical to the rapid component of the cardiac delayed rectifier K+ current (IKr). HERG/IKr channels are a prime target for the pharmacological management of arrhythmias and are selectively blocked by class III antiarrhythmic methanesulfonanilide drugs, such as dofetilide, E4031, and MK-499, at submicromolar concentrations. By contrast, the closely related bovine ether-a-go-go channel (BEAG) is 100-fold less sensitive to dofetilide. To identify the molecular determinants for dofetilide block, we first engineered chimeras between HERG and BEAG and then used site-directed mutagenesis to localize single amino acid residues responsible for block. Using constructs heterologously expressed in Xenopus oocytes, we found that transplantation of the S5-S6 linker from BEAG into HERG removed high-affinity block by dofetilide. A point mutation in the S5-S6 linker region, HERG S620T, abolished high-affinity block and interfered with C-type inactivation. Thus, our results indicate that important determinants of dofetilide binding are localized to the pore region of HERG. Since the loss of high-affinity drug binding was always correlated with a loss of C-type inactivation, it is possible that the changes observed in drug binding are due to indirect allosteric modifications in the structure of the channel protein and not to the direct interaction of dofetilide with the respective mutated site chains. However, the chimeric approach was not able to identify domains outside the S5-S6 linker region of the HERG channel as putative candidates involved in drug binding. Moreover, the reverse mutation BEAG T432S increased the affinity of BEAG K+ channels for dofetilide, whereas C-type inactivation could not be recovered. Thus, the serine in position HERG 620 may participate directly in dofetilide binding; however, an intact C-type inactivation process seems to be crucial for high-affinity drug binding.
Amino Acid Sequence↗
Regulation of the human ether-a-gogo related gene (HERG) K+ channels by reactive oxygen species.
Human ether-a-gogo related gene (HERG) K+ channels are key elements in the control of cell excitability in both the cardiovascular and the central nervous systems. For this reason, the possible modulation by reactive oxygen species (ROS) of HERG and other cloned K+ channels expressed in Xenopus oocytes has been explored in the present study. Exposure of Xenopus oocytes to an extracellular solution containing FeSO4 (25-100 microM) and ascorbic acid (50-200 microM) (Fe/Asc) increased both malondialdehyde content and 2',7'-dichlorofluorescin fluorescence, two indexes of ROS production. Oocyte perfusion with Fe/Asc caused a 50% increase of the outward K+ currents carried by HERG channels, whereas inward currents were not modified. This ROS-induced increase in HERG outward K+ currents was due to a depolarizing shift of the voltage-dependence of channel inactivation, with no change in channel activation. No effect of Fe/Asc was observed on the expressed K+ currents carried by other K+ channels such as bEAG, rDRK1, and mIRK1. Fe/Asc-induced stimulation of HERG outward currents was completely prevented by perfusion of the oocytes with a ROS scavenger mixture (containing 1,000 units/ml catalase, 200 ng/ml superoxide dismutase, and 2 mM mannitol). Furthermore, the scavenger mixture also was able to reduce HERG outward currents in resting conditions by 30%, an effect mimicked by catalase alone. In conclusion, the present results seem to suggest that changes in ROS production can specifically influence K+ currents carried by the HERG channels.
Animals↗
[Multi-point contact (MPC) osteosynthesis plate. 1: Animal experiment histomorphologic studies in the Göttingen minipig].
Based on the results of clinical and animal studies as reported in the literature, the subimplant cortex becomes porous underneath conventional osteosynthesis plates with a flat surface. To solve this problem, we developed an implantable plate which creates multiple contact points between plate and bone, called the multi-point contact or MPC plate. In an experimental animal study conducted on 16 Göttingen minipigs we investigated the bone reaction beneath 2 different types of osteosynthesis plates: the conventional type with a flat interface versus the multi-point contact type. Both epiperiostal and subperiostal plating was performed on pig's intact tibiae. After an implantation period of 16 weeks, the results were documented and compared. It was shown that the osteal remodeling activity of the cortical bone adjacent to the plate increased under both plates up to the twelfth week, but declined towards the end of the study period. Compared to the MPC plate, a conspicuous remodeling front accompanied by porosis of the cortical bone adjacent to the plate was found underneath the conventional osteosynthesis plates with a flat surface-to-bone interface. The different subimplant reactions between the 2 plates can be best explained by the fact that intracortical implant-induced viscoelastic osteocyte diffusion is better under the MPC plate, whereas it is impaired under the conventional flat plate.
Animals↗
Age-dependent variations in potassium sensitivity of A-currents in rat hippocampal neurons.
Hippocampal pyramidal neurons were either cultured from prenatal rats or acutely isolated from the brain of newborn and juvenile rats. The influence of lowering the concentration of the extracellular potassium concentration ([K+]o) on isolated fast transient outward K+ currents (I(A)) was studied in these neurons using the patch clamp technique in the whole cell configuration. With respect to the response of I(A) to lowering [K+]o, three types of cells were observed. The first subpopulation of neurons was characterized by a complete suppression of I(A) over the whole voltage range under potassium-free solutions (type A neurons). A second proportion of cells showed an increase of I(A) at test pulses below -0 mV and a decrease of I(A) at voltages above -0 mV (type B neurons). In a third group of neurons, amplitudes of I(A) increased at all potentials tested during omission of potassium ions from the extracellular superfusate (type C neurons). Whereas type A and type B neurons were preferentially found in freshly plated cultures and newborn rats, the majority of type C cells was detected in long-term cultures and in animals of older ages. Thus, hippocampal A-currents lose their sensitivity to extracellular potassium ions during early ontogenesis.
Aging↗
Regulation by spermine of native inward rectifier K+ channels in RBL-1 cells.
Polyamines have been shown to participate in the rectification of cloned inwardly rectifying potassium channels, a class of potassium channel proteins that conducts inward current more readily than outward current. Here, basophil leukemia cells were used to determine the effects of polyamines on a native, inwardly rectifying potassium current. Rat basophil leukemia cells were cultured in the presence of two different polyamine biosynthesis inhibitors, and both the electrophysiological properties and the polyamine levels were monitored. Treatment with alpha-difluoromethylornithine, a specific ornithine decarboxylase inhibitor, resulted in no significant change of electrophysiological properties. In contrast, treatment with 5'-[(Z)-4-amino-2-butenyl]- methyl-amino-5'-deoxyadenosine (MDL73811), an inhibitor of S-adenosylmethionine decarboxylase, resulted in increased outward currents through inwardly rectifying potassium channels while intracellular putrescine was markedly increased and spermidine and spermine levels were decreased. Fluctuations of intracellular polyamine concentrations as imposed by MDL73811 were directly translated in an altered cell excitability. Based on these results we conclude that the rectification properties of native inwardly rectifying potassium channels are largely controlled by intracellular spermine.
Animals↗
Electrophysiological properties of neurones in cultures from postnatal rat dentate gyrus.
Electrophysiological properties of neurofilament-positive neurones in dissociated cell cultures were prepared at postnatal days 4-5 from rat dentate gyrus and studied using the whole-cell patch-clamp technique. These cells expressed a fast-inactivating, 0.5 microM tetrodotoxin-sensitive Na+ current; a high-voltage-activated (HVA) Ca2+ current, which was 30 microM Cd(2+)- and partially 2 microM nicardipine-sensitive; and an inward rectifier current, which was sensitive to extracellularly applied 1 mM Cs+. The outward current pattern was composed of a delayed rectifier-like outward current sensitive to 20 mM tetraethylammonium (TEA) and a fast-inactivating, Ca(2+)-dependent outward current. This transient Ca(2+)-dependent K+ outward current was identified by a subtraction procedure. K+ currents recorded under conditions of blocked Ca2+ currents (after rundown of the HVA Ca2+ current or blocked by extracellularly applied Cd2+) were subtracted from control currents. By comparison with the current pattern of identified dentate granule cells, it is concluded that the investigated cell type originated from interneurones or projection neurones of the dentate hilus.
Animals↗
C-terminus determinants for Mg2+ and polyamine block of the inward rectifier K+ channel IRK1.
Critical loci for ion conduction in inward rectifier K+ channels are only now being discovered. The C-terminal region of IRK1 plays a crucial role in Mg2+i blockade and single-channel K+ conductance. A negatively charged aspartate in the putative second transmembrane domain (position 172) is essential for time-dependent block by the cytoplasmic polyamines spermine and spermidine. We have now localized the C-terminus effect in IRK1 to a single, negatively charged residue (E224). Mutation of E224 to G, Q and S drastically reduced rectification. Furthermore, the IRK1 E224G mutation decreased block by Mg2+i and spermidine and, like the E224Q mutation, caused a dramatic reduction in the apparent single-channel K+ conductance. The double mutation IRK1 D172N+ E224G was markedly insensitive to spermidine block, displaying an affinity similar to ROMK1. The results are compatible with a model in which the negatively charged residue at position 224, E224, is a major determinant of pore properties in IRK1. By means of a specific interaction with the negatively charged residue at position 172, D172, E224 contributes to the formation of the binding pocket for Mg2+ and polyamines, a characteristic of strong inward rectifiers.
Amino Acid Sequence↗
Pharmacological implications of inward rectifier K+ channels regulation by cytoplasmic polyamines.
The powerful combination of molecular biology and electrophysiology has allowed extraordinary progress in the field of ion channel structure-function. In fact, only 10 years have passed since the first amino acid sequence of a voltage-dependent ion channel, the Na+ channel, was deduced [1], and already the structural domains involved in ion channel permeation, block and gating have been identified in many channel types. Despite this progress, in most cases the correlation between specific domains and ion channel function is still speculative at present, due to the absence of direct structural information [2]. In this review we will describe recent progress in the field of structure-function of one class of K+ channels, the inward rectifiers (IRKs). In particular, we will review the sequences of structure-function experiments which have led to the discovery of a novel regulation of IRKs by cytoplasmic organic polycationic substances like polyamines (PAs). This discovery represents a paradigm for how structure-function information has preceded and made possible the identification of physiological mechanisms of ion channel regulation. Owing to the important role played by IRKs in the regulation of resting membrane potential, a major determinant of cellular transport and volume [3], and to the established link between PAs and cell growth and division, the direct regulation of IRKs by PAs assumes a critical importance for the pharmacological control of cell growth and neoplastic transformation.
Animals↗
Comparison of voltage-dependent potassium currents in rat pyramidal neurons acutely isolated from hippocampal regions CA1 and CA3.
1. The properties of voltage-gated potassium currents were studied in acutely isolated rat hippocampal pyramidal cells from area CA1 and CA3 at postnatal ages of day 6-8, 9-14, and 26-29 (P6-8, P9-14, and P26-29) with the use of the whole cell version of the patch-clamp technique. 2. The outward current pattern of all cells under investigation could be separated in a fast transient A current (IA) and a delayed rectifier-like current (IK). 3. In both preparations, IA activated and inactivated rapidly. Vh describing steady-state inactivation was -84.5 mV in CA3 cells and -85.5 mV in CA1 cells. The activation behavior was characterized by Vh = -23.8 mV in CA3 cells and -27.2 mV in CA1 cells. The removal of inactivation was monoexponential both in CA1 and CA3 neurons with time constants of 32.1 and 28.5 ms, respectively. IA was insensitive to tetraethylammonium (TEA), dendrotoxin (300 nM), and mast cell degranulating peptide (200 nM), but could be blocked with 5 mM 4-aminopyridine (4-AP) by approximately 80%. In both preparations, A currents did not depend on Ca2+ influx. 4. Delayed rectifier currents (IK) in CA1 and CA3 pyramidal neurons decayed along a double exponential time course. Steady-state inactivation was described by Vh = -79.5 mV in CA3 cells and -76.0 mV in CA1 cells. The activation curves were characterized by midpoints of -3.8 mV in CA3 cells and of -1.4 mV in CA1 cells. The removal of inactivation was monoexponential in CA1 and CA3 neurons with time constants of 210.3 and 202.4 ms, respectively. All kinetic properties were identical for the differentially decaying components of IK. In CA1 cells IK was blocked by TEA at +30 mV with an IC50 of 0.98 mM. In CA3 cells the corresponding IC50 value was 1.05 mM. About 20% of IK were insensitive to TEA. IK was partially blocked by approximately 30% with 100 microM 4-AP. Mast cell degranulating peptide (100-200 nM) and dendrotoxin (50-300 nM) had no effect on IK. 6. Perfusion of charybdotoxin (30 nM), Cd2+ (300 microM), La3+ (10 microM), or Ca(2+)-free solutions resulted in the isolation of a small noninactivating outward current component. Around 10% of IK appeared to be Ca2+ dependent in CA1 neurons. In CA3 pyramidal cells Ca(2+)-dependent outward currents seemed to be somewhat larger with approximately 20%. 7. In CA1 as well as in CA3 cells, the kinetic and pharmacological properties of IA and IK remained stable during postnatal development. However, the contribution of IA and IK to the whole cell current varied with age. IA was more prominent in CA1 cells of age group P6-8 than in age-matched CA3 cells. CA3 cells had smaller A currents and larger delayed rectifier currents than CA1 pyramidal cells. Current densities of IA and IK were analyzed during development to assess changes in the expression of these currents. With increasing postnatal age, the expression of IA was downregulated in both preparations. This effect was more pronounced in CA3 than in CA1 cells. In contrast, IK was upregulated during the same developmental period. This increase in the expression of IK was with approximately 300% much larger in CA1 cells than in CA3 cells with only approximately 50%.
4-Aminopyridine↗
Cloned human inward rectifier K+ channel as a target for class III methanesulfonanilides.
Methanesulfonanilide derivatives such as dofetilide are members of the widely used Class III group of cardiac antiarrhythmic drugs. A methanesulfonanilide-sensitive cardiac current has been identified as IKr, the rapidly activating component of the repolarizing outward cardiac K+ current, IK. IKr may be encoded by the human ether-related gene (hERG), which belongs to the family of voltage-dependent K+ (Kv) channels having six putative transmembrane segments. The hERG also expresses an inwardly rectifying, methanesulfonanilide-sensitive K+ current. Here we show that hIRK, a member of the two-transmembrane-segment family of inward K+ rectifiers that we have cloned from human heart, is a target for dofetilide. hIRK currents, expressed heterologously in Xenopus oocytes, are blocked by dofetilide at submicromolar concentrations (IC50 = 533 nmol/L at 40 mV and 20 degrees C). The drug has no significant blocking effect on the human cardiac Kv channels hKv1.2, hKv1.4, hKv1.5, or hKv2.1. The block is voltage dependent, use dependent, and shortens open times in a manner consistent with open-channel block. While steady state block is strongest at depolarized potentials, recovery from block is very slow even at hyperpolarized potentials (tau = 1.17 seconds at -80 mV). Thus, block of hIRK may persist during diastole and might thereby affect cardiac excitability.
Animals↗
Spermine and spermidine as gating molecules for inward rectifier K+ channels.
Inward rectifier K+ channels pass prominent inward currents, while outward currents are largely blocked. The inward rectification is due to block by intracellular Mg2+ and a Mg(2+)-independent process described as intrinsic gating. The rapid loss of gating upon patch excision suggests that cytoplasmic factors participate in gating. "Intrinsic" gating can be restored in excised patches by nanomolar concentrations of two naturally occurring polyamines, spermine and spermidine. Spermine and spermidine may function as physiological blockers of inward rectifier K+ channels and "intrinsic" gating may largely reflect voltage-dependent block by these cations.
Animals↗