Biomedical subjects

Olaf Pongs

Publications and source records attributed to Olaf Pongs.

24 records · Page 2Linked to original sources

PubMed2003

Novel gene hKCNE4 slows the activation of the KCNQ1 channel.

The KCNE genes encode small, single transmembrane domain peptides that associate with pore-forming potassium channel subunits to form mixed complexes with unique characteristics. We have identified a novel member of the human KCNE gene family, hKCNE4. The hKCNE4 gene encodes 170 amino acid protein and is localized to chromosome 2q35-36. The protein sequence shows 90% homology to mouse KCNE4 and 38% identity to human KCNE1. Northern blot analysis revealed that hKCNE4 is expressed strongly in heart, skeletal muscle, and kidney, less in placenta, lung, and liver, and weakly in brain and blood cells. Electrophysiological study showed that hKCNE4 modulates the activation of the KCNQ1 channel.

Amino Acid Sequence↗

PubMed2003

Pacemaker channel dysfunction in a patient with sinus node disease.

The cardiac pacemaker current I(f) is a major determinant of diastolic depolarization in sinus nodal cells and has a key role in heartbeat generation. Therefore, we hypothesized that some forms of "idiopathic" sinus node dysfunction (SND) are related to inherited dysfunctions of cardiac pacemaker ion channels. In a candidate gene approach, a heterozygous 1-bp deletion (1631delC) in exon 5 of the human HCN4 gene was detected in a patient with idiopathic SND. The mutant HCN4 protein (HCN4-573X) had a truncated C-terminus and lacked the cyclic nucleotide-binding domain. COS-7 cells transiently transfected with HCN4-573X cDNA indicated normal intracellular trafficking and membrane integration of HCN4-573X subunits. Patch-clamp experiments showed that HCN4-573X channels mediated I(f)-like currents that were insensitive to increased cellular cAMP levels. Coexpression experiments showed a dominant-negative effect of HCN4-573X subunits on wild-type subunits. These data indicate that the cardiac I(f) channels are functionally expressed but with altered biophysical properties. Taken together, the clinical, genetic, and in vitro data provide a likely explanation for the patient's sinus bradycardia and the chronotropic incompetence.

Aged↗

PubMed2003

Inhibition of human TREK-1 channels by bupivacaine.

UNLABELLED: Human TWIK-related K(+) channels (TREK-1) stabilize the membrane potential (mp) of neurons and have a major role in the regulation of membrane excitability. In view of their physiological significance, interaction of bupivacaine with TREK-1 channels may be clinically important. Our aim was to characterize with the patch-clamp technique the properties of human TREK-1 channels and the effects of bupivacaine on these channels expressed in Chinese hamster ovary (CHO) cells. Transfection of CHO cells with TREK-1 channels (CHO(TREK-1) cells) hyperpolarized the mp from -33 +/- 13 to -78 +/- 4 mV. The channels were stimulated by intracellular acidosis. Inhibition of TREK-1 channels by bupivacaine was reversible, concentration-dependent, voltage-independent, and increased with intracellular acidosis. Bupivacaine depolarized the mp of CHO(TREK-1) cells in a reversible and concentration-dependent manner. Concentrations for channel inhibition and membrane depolarization were not linearly related (50% inhibitory concentration value for channel inhibition 370 +/- 20 micro M, Hill coefficient 1.8 +/- 0.1, n = 51; 50% inhibitory concentration value for membrane depolarization 856 +/- 14 micro M, Hill coefficient 2.4 +/- 0.1, mean +/- SEM, n = 27). The results suggest that protonated bupivacaine elicits the observed effects via a site of interaction accessible from the intracellular space. Inhibition of TREK-1 channels and consecutive depolarization of the cell membrane by bupivacaine may contribute to blockade of neuronal signal conduction during regional anesthesia. IMPLICATIONS: The interaction of bupivacaine with human TREK-1 channels was studied with the patch-clamp technique. Bupivacaine inhibited TREK-1 channels and depolarized the membrane potential of cells expressing TREK-1 channels in a concentration-dependent and reversible manner. Both effects may contribute to conductance block caused by bupivacaine.

Acidosis↗

PubMed2002

Engineering-specific pharmacological binding sites for peptidyl inhibitors of potassium channels into KcsA.

The bacterial potassium channel, KcsA, can be modified to express a high-affinity receptor site for the scorpion toxin kaliotoxin (KTX) by substituting subregion I in the P region of KcsA with the one present in the human voltage-gated potassium channel Kv1.3 [Legros, C., Pollmann, V., Knaus, H. G., Farrell, A. M., Darbon, H., Bougis, P. E., Martin-Eauclaire, M. F., and Pongs, O. (2000) J. Biol. Chem. 275, 16918-16924]. This approach opened the way to investigate whether sequence differences in subregion I of Kv1 channels correlate with the distinct pharmacological profiles of peptide inhibitors. A panel of six chimeras between KcsA and human Kv1.1-6 were constructed, expressed in Escherichia coli, purified to homogeneity, and assessed in filter binding assays using either monoiodo-tyrosine-KTX ([(125)I]KTX) or monoiodo-tyrosine-hongotoxin(1)(A19Y/Y37F) ([(125)I]HgTX(1)(A19Y/Y37F)). The KcsA-Kv1.X chimeras were found to have lower affinities for these ligands than the corresponding mammalian Kv1.X channels, indicating that other parts of the channels may contribute to binding or that subtle structural differences exist between these channels. The properties of the KcsA-Kv1.X chimeras were also characterized in surface plasmon resonance experiments. KcsA-Kv1.3 chimeras were immobilized on the surface of a sensor chip for determining, in real time, binding of the peptides. KTX binding properties to immobilized KcsA-Kv1.3 chimera were similar to those determined by filtration techniques. Taken together, our results demonstrate that the pharmacological profile of peptide toxins can be incorporated into KcsA-Kv1.X chimeras containing the subregion I of the corresponding mammalian Kv1.X channels. This innovative approach may facilitate the high-throughput screening of ligand libraries aimed at the discovery of novel potassium channel modulators.

Alanine↗

PubMed2002

Identification and functional characterization of a novel KCNE2 (MiRP1) mutation that alters HERG channel kinetics.

Long-QT syndrome (LQTS) may cause syncope and sudden death due to cardiac tachyarrhythmia. Chromosome 7-linked LQTS (LQT2) has been correlated with mutations in the human ether-a-go-go-related gene (HERG). HERG forms voltage-gated K channels that may be associated with Mink-related peptide 1 (MiRP1), an auxiliary beta-subunit. The channels mediate currents that resemble native I(Kr). Mutations in the KCNE2 gene encoding MiRP1 may also cause LQTS. In this study, the frequency of mutations in KCNE2 of 150 unrelated LQTS patients without known genotype and of 100 controls was analyzed using single-strand conformation polymorphism analysis and direct sequencing. We identified a novel missense mutation, V65 M, in the KCNE2 gene of a 17-year-old female with syncope and LQTS. Expression studies in Chinese hamster ovary cells revealed that mutant and wild-type MiRP1 co-localized with HERG subunits and formed functional channels. However, mutant HERG/MiRP1(V65M) channels mediated currents with an accelerated inactivation time course compared with wild-type channels. The accelerated inactivation time course of HERG/MiRP1(V65M) channels may decrease I(Kr) current density of myocardial cells, thereby impairing the ability of myocytes to repolarize in response to sudden membrane depolarizations such as extrasystoles.

Animals↗

PubMedDate not supplied

Differential modulation of Kv1 channel-mediated currents by co-expression of Kvbeta3 subunit in a mammalian cell-line.

The effect of Kvbeta3 subunit co-expression on currents mediated by the Shaker-related channels Kv1.1 to Kv1.6 in Chinese hamster ovary (CHO) cells was studied with patch-clamp techniques. In the presence of Kvbeta3, differences in the voltage dependence of activation for Kv1.1, Kv1.3 and Kv1.6 were detected, but not for Kv1.2- and Kv1.4-mediated currents. Co-expression of Kvbeta3 did not cause a significant increase in current density for any of the tested channels. In contrast to previous studies in Xenopus oocyte expression system, Kvbeta3 confered a rapid inactivation to all except Kv1.3 channels. Also, Kv1.6 channels that possess an N-type inactivation prevention (NIP) domain for Kvbeta1.1, inactivated rapidly when co-expressed with Kvbeta3. Onset and recovery kinetics of channel inactivation distinctly differed for the various Kv1alpha/Kvbeta3 subunit combinations investigated in this study. The results indicate that the choice of expression system may critically determine Kvbeta3 inactivating activity. This suggests that the presence of an inactivating domain and a receptor in a channel pore, although necessary, may not be sufficient for an effective rapid N-type inactivation of Kv1 channels in heterologous expression systems.

Animals↗