Biomedical subjects
Publications and source records attributed to Olaf Pongs.
In this work, we used a panel prokaryote/eukaryote K+ channel chimeras to generate K+ channel arrays. Their behaviour in solution was compared with that when spotted on a nitrocellulose-supported film and their responses to selective high affinity ligands, including polypeptide toxins and TEA, were studied.
Beta-adrenergic receptor-mediated cAMP or protein kinase A (PKA)-dependent modulation of cardiac potassium currents controls ventricular action potential duration (APD) at faster heart rates. HERG (KCNH2) gene mutations are associated with congenital long-QT syndrome (LQT2) and affect IKr activity, a key determinant in ventricular repolarization. Physical activity or emotional stress often triggers lethal arrhythmias in LQT2 patients. Beta-adrenergic stimulation of HERG channel activity is amplified and prolonged in vitro by the adaptor protein 14-3-3epsilon. In LQT2 families, we identified three novel heterozygous HERG mutations (G965X, R1014PfsX39, V1038AfsX21) in the C-terminus that led to protein truncation and loss of a PKA phosphorylation site required for binding of 14-3-3epsilon. When expressed in CHO cells, the mutants produced functional HERG channels with normal kinetic properties. We now provide evidence that HERG channel regulation by 14-3-3epsilon is of physiological significance in humans. Upon co-expression with 14-3-3epsilon, mutant channels still bound 14-3-3epsilon but did not respond with a hyperpolarizing shift in voltage dependence as seen in wild-type channels. Co-expression experiments of wild-type and mutant channels revealed dominant-negative behavior of all three HERG mutations. Simulations of the effects of sympathetic stimulation of HERG channel activity on the whole-cell action potential suggested a role in rate-dependent control of APD and an impaired ability of mutant cardiac myocytes to respond to a triggered event or an ectopic beat. In summary, the attenuated functional effects of 14-3-3epsilon on C-terminally truncated HERG channels demonstrate the physiological importance of coupling beta-adrenergic stimulation and HERG channel activity.
OBJECTIVE: Nitric oxide (NO)-induced vasorelaxation involves activation of large conductance Ca2+-activated K+ channels (BK). A regulatory BKbeta1 subunit confers Ca2+, voltage, and NO/cGMP sensitivity to the BK channel. We investigated whether endothelial function and NO/cGMP signaling is affected by a deletion of the beta1-subunit. METHODS AND RESULTS: Vascular superoxide in BKbeta1-/- was measured using the fluorescent dye hydroethidine and lucigenin-enhanced chemiluminescence. Vascular NO formation was analyzed using electron paramagnetic resonance (EPR), expression of NADPH oxidase subunits, the endothelial NO synthase (eNOS), the soluble guanylyl cyclase (sGC), as well as the activity and expression of the cyclic GMP-dependent kinase I (cGK-I) were assessed by Western blotting technique. eNOS, sGC, cGK-I expression and acetylcholine-induced NO production were unaltered in Bkbeta1-/- animals, whereas endothelial function was impaired and the activity of the cGK-I was reduced. Vascular O2- and expression of the NADPH oxidase subunits p67phox and Nox1 were increased. Endothelial dysfunction was normalized by the NADPH oxidase inhibitor apocynin. Potassium chloride- and iberiotoxin-induced depolarization mimicked the effect of BKbeta1-deletion by increasing vascular O2- in an NADPH-dependent fashion. CONCLUSIONS: The deletion of BKbeta1 causes endothelial dysfunction by increasing O2- formation via increasing activity and expression of the vascular NADPH oxidase.
The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane--similar to the catalytic function of the active site of an enzyme--and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel-scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton-proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein-inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin-K+ channel interactions.
The slow IKS K+ channel plays a major role in repolarizing the cardiac action potential and consists of the assembly of KCNQ1 and KCNE1 subunits. Mutations in either KCNQ1 or KCNE1 genes produce the long-QT syndrome, a life-threatening ventricular arrhythmia. Here, we show that long-QT mutations located in the KCNQ1 C terminus impair calmodulin (CaM) binding, which affects both channel gating and assembly. The mutations produce a voltage-dependent macroscopic inactivation and dramatically alter channel assembly. KCNE1 forms a ternary complex with wild-type KCNQ1 and Ca(2+)-CaM that prevents inactivation, facilitates channel assembly, and mediates a Ca(2+)-sensitive increase of IKS-current, with a considerable Ca(2+)-dependent left-shift of the voltage-dependence of activation. Coexpression of KCNQ1 or IKS channels with a Ca(2+)-insensitive CaM mutant markedly suppresses the currents and produces a right shift in the voltage-dependence of channel activation. KCNE1 association to KCNQ1 long-QT mutants significantly improves mutant channel expression and prevents macroscopic inactivation. However, the marked right shift in channel activation and the subsequent decrease in current amplitude cannot restore normal levels of IKS channel activity. Our data indicate that in healthy individuals, CaM binding to KCNQ1 is essential for correct channel folding and assembly and for conferring Ca(2+)-sensitive IKS-current stimulation, which increases the cardiac repolarization reserve and hence prevents the risk of ventricular arrhythmias.
Solid-state NMR (ssNMR) represents a spectroscopic method to study membrane protein structure and dynamics in lipid bilayers. We present two-dimensional correlation experiments conducted on a fully [13C,15N] labeled version of a chimeric potassium (KcsA-Kv1.3) channel. Data obtained by using two different ion concentrations suggest a structural conservation of the selectivity filter region. SsNMR experiments conducted at two different temperatures point to differential molecular dynamics of the channel.
Association of Shal gene-related voltage-gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C-terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine-scanning mutagenesis within the proximal 40 amino acid portion and a structure-based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C-terminus was truncated at various positions. Wild-type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2-induced modulation of Kv4.2 currents was studied with whole-cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N-terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C-terminal deletions also had marked effects on KChIP2-dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C-terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose 'anchoring' at T1- and C-terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.
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In humans, mutations in the KCNQ2 or KCNQ3 potassium-channel genes are associated with an inherited epilepsy syndrome. We have studied the contribution of KCNQ/M-channels to the control of neuronal excitability by using transgenic mice that conditionally express dominant-negative KCNQ2 subunits in brain. We show that suppression of the neuronal M current in mice is associated with spontaneous seizures, behavioral hyperactivity and morphological changes in the hippocampus. Restriction of transgene expression to defined developmental periods revealed that M-channel activity is critical to the development of normal hippocampal morphology during the first postnatal weeks. Suppression of the M current after this critical period resulted in mice with signs of increased neuronal excitability and deficits in hippocampus-dependent spatial memory. M-current-deficient hippocampal CA1 pyramidal neurons showed increased excitability, reduced spike-frequency adaptation, attenuated medium afterhyperpolarization and reduced intrinsic subthreshold theta resonance. M channels are thus critical determinants of cellular and neuronal network excitability, postnatal brain development and cognitive performance.
In order to find new peptide inhibitors for voltage-dependent potassium (Kv) channels, we examined the effects of venom from Theraphosa leblondi on Kv channel-mediated currents with the whole-cell patch-clamp technique. Both A-type currents in cultured hippocampal neurons and A-type currents recorded from HEK 293 cells transiently expressing recombinant Kv4.2 channels were selectively inhibited by T. leblondi venom. No venom activity was observed on recombinant Kv1.3, Kv1.4, Kv2.1 or Kv3.4 channels. We purified and sequenced three novel homologous peptides from this venom, which are related to previously identified Kv4 channel-specific peptide inhibitors and were named T. leblondi toxin (TLTx) 1, 2 and 3. The mode of action of TLTx1 on recombinant Kv4.2 channels was studied in more detail. TLTx1 inhibited Kv4.2-mediated currents with an IC50 of approximately 200 nM, and macroscopic current inactivation was slowed in the presence of TLTx1. Notably, TLTx1 also caused a shallower voltage dependence of Kv4.2 peak conductance and a shift of the activation midpoint to more positive potentials (DeltaV1/2 = +35 mV). TLTx1 caused a noticable slowing of Kv4.2 activation kinetics, and Kv4.2 deactivation kinetics were accelerated by TLTx1 as infered from Rb+ tail current measurements. Chimeric Kv2.1(4.2L3-4) channels, in which the linker region between S3 and S4 of the TLTx1-insensitive Kv2.1 channel was replaced by the corresponding Kv4.2 domain, were sensitive to TLTx1. Apparently, TLTx1 can act as a gating modifier of Kv4.2 channels.
We have found a novel nonsense mutation in the C-terminus of HERG in a four-generation Chinese family with long QT syndrome and investigated the molecular mechanism of this mutation in vitro. Six family members, including the proband, were clinically affected. Syncope and ventricular tachycardia of torsades de pointes were triggered by startling or emotional stress, and beta-adrenergic blockade treatment was ineffective. Haplotype analysis showed that only LQT2 markers cosegregated with the disease, and sequence analysis revealed a substitution of T with C at nucleotide position 2770 of the HERG gene (U04270), which creates a stop codon at amino acid position 863 (R863X) of the HERG protein, leading to a deletion of 296 amino acids. Whole cell patch clamp studies showed that the R863X HERG could not induce time-dependent current. Coexpression of R863X with wild-type HERG showed reduced current densities and accelerated voltage-dependent inactivation of HERG channels. Subcellular localization of R863X-EGFP revealed that the mutant did not traffic to the cell surface. These data suggest that R863X failed to form functional HERG channels, contributing to a prolongation of the QT interval and long QT syndrome with a dominant phenotype. These findings provide new insights into the structure-function relationships of the HERG C-terminus.
We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Delta40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K+. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Delta40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Delta40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation.
Ca(v)1.2 and Ca(v)1.3 L-type Ca(2+) channels (LTCCs) are believed to underlie Ca(2+) currents in brain, pancreatic beta cells, and the cardiovascular system. In the CNS, neuronal LTCCs control excitation-transcription coupling and neuronal plasticity. However, the pharmacotherapeutic implications of CNS LTCC modulation are difficult to study because LTCC modulators cause cardiovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Ca(v)1.2 alpha 1 subunits (Ca(v)1.2DHP-/-) without affecting function and expression. This allowed separation of the DHP effects of Ca(v)1.2 from those of Ca(v)1.3 and other LTCCs. DHP effects on pancreatic beta cell LTCC currents, insulin secretion, cardiac inotropy, and arterial smooth muscle contractility were lost in Ca(v)1.2DHP-/- mice, which rules out a direct role of Ca(v)1.3 for these physiological processes. Using Ca(v)1.2DHP-/- mice, we established DHPs as mood-modifying agents: LTCC activator-induced neurotoxicity was abolished and disclosed a depression-like behavioral effect without affecting spontaneous locomotor activity. LTCC activator BayK 8644 (BayK) activated only a specific set of brain areas. In the ventral striatum, BayK-induced release of glutamate and 5-HT, but not dopamine and noradrenaline, was abolished. This animal model provides a useful tool to elucidate whether Ca(v)1.3-selective channel modulation represents a novel pharmacological approach to modify CNS function without major peripheral effects.
Depolarizing voltage steps activate voltage-dependent K(+) (Kv) channels by moving the voltage sensor, which triggers a coupling reaction leading to the opening of the pore. We constructed chimeric channels in which intracellular regions of slowly activating Kv2.1 channels were replaced by respective regions of rapidly activating Kv1.2 channels. Substitution of either the N-terminus, S4-S5 linker, or C-terminus generated chimeric Kv2.1/1.2 channels with a paradoxically slow and approximately exponential activation time course consisting of a fast and a slow component. Using combined chimeras, each of these Kv1.2 regions further slowed activation at the voltage of 0 mV, irrespective of the nature of the other two regions, whereas at the voltage of 40 mV both slowing and accelerating effects were observed. These results suggest voltage-dependent interactions of the three intracellular regions. This observation was quantified by double-mutant cycle analysis. It is concluded that interactions between N-terminus, S4-S5 linker, and/or C-terminus modulate the activation time course of Kv2.1 channels and that part of these interactions is voltage dependent.
Frequenin, also known as neuronal calcium sensor-1 (NCS-1), is an N-myristoylated Ca2+-binding protein that has been conserved in both sequence and three-dimensional fold during evolution. We demonstrate using both genetic and biochemical approaches that the observed structural conservation between Saccharomyces cerevisiae frequenin (Frq1) and human NCS-1 is also reflected at the functional level. In yeast, the sole essential target of Frq1 is the phosphatidylinositol 4-kinase isoform, Pik1; both FRQ1 and PIK1 are indispensable for cell viability. Expression of human NCS-1 in yeast, but not a close relative (human KChIP2), rescues the inviability of frq1 cells. Furthermore, in vitro, Frq1 and NCS-1 (either N-myristoylated or unmyristoylated) compete for binding to a small 28-residue motif near the N terminus of Pik1. Site-directed mutagenesis indicates that the binding determinant in Pik1 is a hydrophobic alpha-helix and that frequenins bind to one side of this alpha-helix. We propose, therefore, that the function of NCS-1 in mammals may closely resemble that of Frq1 in S. cerevisiae and, hence, that frequenins in general may serve as regulators of certain isoforms of phosphatidylinositol 4-kinase.
OBJECTIVES: To identify the underlying genetic basis of a Chinese pedigree with Long QT syndrome, the causally related genes were screened in a family and the functional consequence of the identified gene mutation was evaluated in vitro. METHODS: Mutations in the five defined Long QT syndrome related genes were screened with polymerase chain reaction and single-strand conformation polymorphism methods and direct sequencing. The electrophysiological properties of the identified mutation were characterized in the Xenopus oocyte heterologous expression system. RESULTS: A novel missense mutation, G to A at position 154 in the KCNE1 gene was identified in a Chinese Long QT syndrome family, which leads to an amino acid substitution of arginine (R) for glycine (G) at position 52 (G52R-KCNE1). Of 26 family members (one DNA was not available), seven were mutation carriers and two of them with normal electrocardiogram. Compared with wild-type KCNE1/KCNQ1 channels, coexpression of G52R-KCNE1 with KCNQ1 in Xenopus oocytes did not amplify the KCNQ1 current amplitudes and slightly changed the activation kinetics of the KCNQ1 channels. Coexpression of KCNQ1 together with wild type KCNE1 and G52R-KCNE1 reduced the wild-type I(ks) current amplitude by 50%, whereas other biophysical properties of the I(ks) were not altered. CONCLUSIONS: Our findings indicate that glycine52 in the transmembrane domain is critical for KCNE1 function. The mutant G52R-KCNE1 has a dominant negative effect on I(ks) current, which reduces the I(ks) current amplitude and leads to a prolongation of the cardiac action potential. This could underlie the molecular mechanism of ventricular arrhythmias and sudden death in those patients.
Oxygen-sensitive K(+) channels are important elements in the cellular response to hypoxia. Although much progress has been made in identifying their molecular composition, the structural components associated to their O(2)-sensitivity are not yet understood. Recombinant Kv1.2 currents expressed in Xenopus oocytes are inhibited by a decrease in O(2) availability. On the contrary, heterologous Kv2.1 channels are O(2)-insensitive. To elucidate the protein segment responsible for the O(2)-sensitivity of Kv1.2 channels, we analyzed the response to anoxia of Kv1.2/Kv2.1 chimeric channels. Expression of chimeric Kv2.1 channels each containing the S4, the S1-S3 or the S6-COOH segments of Kv1.2 polypeptide resulted in a K(+) current insensitive to anoxia. In contrast, transferring the S5-S6 segment of Kv1.2 into Kv2.1 produced an O(2)-sensitive K(+) current. Finally, mutating a redox-sensitive methionine residue (M380) of Kv1.2 polypeptide did not affect O(2)-sensitivity. Thus, the pore and its surrounding regions of Kv1.2 polypeptide confer its hypoxic inhibition. This response is independent on the redox modulation of methionine residues in this protein segment.
Many important physiological processes involve changes in cell volume, e.g. the transport of salt and water in epithelial cells and the contraction of cardiomyocytes. In this study, we show that voltage-gated KCNQ1 channels, which are strongly expressed in epithelial cells or cardiomyocytes, and KCNQ4 channels, expressed in hair cells and the auditory tract, are tightly regulated by small cell volume changes when co-expressed with aquaporin 1 water-channels (AQP1) in Xenopus oocytes. The KCNQ1 and KCNQ4 current amplitudes precisely reflect the volume of the oocytes. By contrast, the related KCNQ2 and KCNQ3 channels, which are prominently expressed in neurons, are insensitive to cell volume changes. The sensitivity of the KCNQ1 and KCNQ4 channels to cell volume changes is independent of the presence of the auxiliary KCNE1-3 subunits, although modulated by KCNE1 in the case of KCNQ1. Incubation of the oocytes in cytochalasin D and experiments with truncated KCNQ1 channels suggest that KCNQ1 channels sense cell volume changes through interactions between the cytoskeleton and the N-terminus of the channel protein. From our results we propose that KCNQ1 and KCNQ4 channels play an important role in cell volume control, e.g. during transepithelial transport of salt and water.