Biomedical subjects

Michael Pasternack

Publications and source records attributed to Michael Pasternack.

12 recordsLinked to original sources

PubMed2006

The action of the novel gastrointestinal prokinetic prucalopride on the HERG K+ channel and the common T897 polymorph.

The human ether-à-go-go related gene (HERG) encodes the alpha-subunit of a delayed rectifier potassium channel important in the repolarisation of the cardiac action potential. Excessive action potential prolongation through HERG channel inhibition is associated with a risk of torsade de pointes arrhythmias and is a major challenge for drug development. The acute effects of the novel prokinetic prucalopride were examined on heterologously expressed HERG channels in human embryonic kidney (HEK) 293 cells using the whole-cell patch-clamp technique. Prucalopride inhibited HERG channels in a concentration-dependent manner with an IC(50) of 4.1 microM. Prucalopride significantly slowed channel deactivation and recovery from inactivation, accelerated and altered the extent of inactivation. Similar concentration-dependency and kinetic changes were observed with the minor T897 polymorphic HERG variant. Prucalopride block was frequency-independent due to rapid state-dependent block, with binding occurring in the open and inactivated states. Though prucalopride blocks HERG channels this is unlikely to be significant at clinically relevant concentrations.

Benzofurans↗

PubMed2006

Evidence for a functional role of GABA receptors in the rat mature hippocampus.

Both gamma-aminobutyric acid (GABA)(C) receptor subunit mRNA and protein are expressed in the stratum pyramidale in the CA1 area of the adult rat hippocampus, but so far no conclusive evidence about functional hippocampal GABA(C) receptors has been presented. Here, the contribution of GABA(C) receptors to stimulus-evoked postsynaptic potentials was studied in the hippocampal CA1 area with extracellular and intracellular recordings at the age range of 21-47 postnatal days. Activation of GABA(C) receptors with the specific agonist cis-4-aminocrotonic acid (CACA) suppressed postsynaptic excitability and increased the membrane conductance. The GABA(C) receptor antagonist 1,2,5,6-tetrahydropyridine-4-ylmethylphosphinic acid (TPMPA), but not the GABA(A) receptor antagonist bicuculline, inhibited the effects of CACA. GABA-mediated long-lasting depolarizing responses evoked by high-frequency stimulation of local inhibitory interneurons in the CA1 area in the presence of ionotropic glutamate receptor and GABA(B) receptor blockers were prolonged by TPMPA, indicating that GABA(C) receptors are activated under these conditions. For weaker stimulation, the effect of TPMPA was enhanced after GABA uptake was inhibited. Our data demonstrate that GABA(C) receptors can be activated by endogenous synaptic transmitter release following strong stimulation or under conditions of reduced GABA uptake. The lack of GABA(C) receptor activation by less intensive stimulation under control conditions suggests that these receptors are extrasynaptic and activated via spillover of synaptically released GABA.

Action Potentials↗

PubMed2005

Downregulation of the HERG (KCNH2) K(+) channel by ceramide: evidence for ubiquitin-mediated lysosomal degradation.

The HERG (KCNH2) potassium channel underlies the rapid component of the delayed rectifier current (I(kr)), a current contributing to the repolarisation of the cardiac action potential. Mutations in HERG can cause the hereditary forms of the short-QT and long-QT syndromes, predisposing to ventricular arrhythmias and sudden cardiac death. HERG is expressed mainly in the cell membrane of cardiac myocytes, but has also been identified in cell membranes of a range of other cells, including smooth muscle and neurones. The mechanisms regulating the surface expression have however not yet been elucidated. Here we show, using stable HERG-expressing HEK 293 cells, that ceramide evokes a time-dependent decrease in HERG current which was not attributable to a change in gating properties of the channel. Surface expression of the HERG channel protein was reduced by ceramide as shown by biotinylation of surface proteins, western blotting and immunocytochemistry. The rapid decline in HERG protein after ceramide stimulation was due to protein ubiquitylation and its association with lysosomes. The results demonstrate that the surface expression of HERG is strictly regulated, and that ceramide modifies HERG currents and targets the protein for lysosomal degradation.

Cells, Cultured↗

PubMed2005

GABA receptor rho subunit expression in the developing rat brain.

Ionotropic GABA(C) receptors are composed of rho1, rho2 and rho3 subunits. Although the distribution of rho subunit mRNAs in the adult brain has been studied, information on the developmental regulation of different rho subunits in the brain is scattered and incomplete. Here, GABA(C) receptor rho subunit expression was studied in the developing rat brain. In situ hybridization on postnatal brain slices showed rho2 mRNA expression from newborn in superficial gray layer (SGL) of superior colliculus (SuC), and from the first postnatal week in the hippocampal CA1 region and pretectal nucleus of the optic tract. rho2 mRNA was also expressed in the adult dorsal lateral geniculate nucleus. Quantitative RT-PCR revealed expression of all three rho subunits in the hippocampus and superior colliculus from the first postnatal day. In the hippocampus, rho2 mRNA expression clearly dominated over rho1 and rho3, whereas in the superior colliculus, rho1 mRNA expression levels were similar to rho2. In both areas, a clear up-modulation of rho2 and rho3 mRNA during the first postnatal week was detected. GABA(C) receptor protein expression was confirmed in adult hippocampus, superior colliculus and dorsal lateral geniculate nucleus by immunohistochemistry. Our results demonstrate for the first time the expression of all three rho subunit mRNAs in several regions of the developing and adult rat brain. Our quantitative data allows assessment of putative subunit combinations in the superior colliculus and hippocampus. From the selective distribution of rho subunits, it may be hypothesized that GABA(C) receptors are specifically involved in aspects of visual image motion processing in the rat brain.

Animals↗

PubMed2005

Functional characterization of rat rho2 subunits expressed in HEK 293 cells.

GABA(C) receptors are thought to be homo- or heteropentamers composed of rho1, rho2 and rho3 subunits. Previous work on rat rho2 subunits expressed in Xenopus oocytes has suggested that they do not form functional homo-oligomeric GABA(C) receptors, but do combine with rho1 or rho3 subunits to form hetero-oligomers. These findings are difficult to interpret because both human and mouse rho2 subunits do form functional homo-oligomeric receptors. Also, many regions of the rat brain express solely rho2 subunit transcripts which, according to presently available evidence, would not result in expression of functional GABA(C) receptors. We show here that homomeric rat rho2 receptors can be expressed in HEK 293 cells. Homo-oligomeric rat rho2 receptors expressed in mammalian cells matured slowly and displayed small but detectable GABA-induced currents with slow kinetics. Rat rho2 receptors also had a decreased sensitivity to picrotoxin and a marked sensitivity to the GABA(C) receptor agonist cis-aminocrotonic acid. Our results demonstrate for the first time the expression of functional homomeric rat rho2 receptors, and suggest that rho(2) subunits may contribute to brain function, including in areas not expressing other rho subunits.

Animals↗

PubMed2004

Ceramide 1-phosphate enhances calcium entry through voltage-operated calcium channels by a protein kinase C-dependent mechanism in GH4C1 rat pituitary cells.

Sphingomyelin derivatives modulate a multitude of cellular processes, including the regulation of [Ca2+]i (the intracellular free calcium concentration). Previous studies have shown that these metabolites often inhibit calcium entry through VOCCs (voltage-operated calcium channels). In the present study, we show that, in pituitary GH4C1 cells, C1P (C2-ceramide 1-phosphate) enhances calcium entry in a dose-dependent manner. The phospholipase C inhibitor U73122 attenuated the response. C1P invoked a small, but significant, increase in the formation of inositol phosphates. Pre-treatment of the cells with pertussis toxin was without an effect on the C1P-evoked increase in [Ca2+]i. The effect of C1P was critically dependent on extracellular calcium, since no increase in [Ca2+]i was observed when cells in a calcium-free buffer were stimulated with C1P. Furthermore, if the cells were retreated with 300 nM of the VOCC inhibitor nimodipine, the effect of C1P was almost totally abolished. In addition, ceramide C8-1-phosphate evoked an increase in [Ca2+]i, but the onset of the response was slow compared with that of C1P. In cells treated with 1 mM thapsigargin for 15 min, C1P still evoked an increase in [Ca2+]i. In patch-clamp experiments in the whole-cell mode, C1P enhanced calcium entry through the VOCCs compared with vehicle-treated cells. Dialysis of the cells with C1P did not enhance the calcium current. On-cell patch-clamp experiments showed an enhanced probability of the VOCCs being open (P(open)) in the presence of C1P. Inhibition of PKC (protein kinase C) with GF109203X and down-regulation of PKC with PMA attenuated the C1P-evoked increase in [Ca2+]i. Furthermore, down-regulation of PKC abolished the effect of C1P on P(open). This is the first report showing that a sphingomyelin derivative enhances calcium entry through VOCCs.

Animals↗

PubMed2004

Tumor necrosis factor alpha and ceramide depolarise the resting membrane potential of thyroid FRTL-5 cells via a protein kinase Czeta-dependent regulation of K+ channels.

Tumor necrosis factor alpha (TNFalpha) alters the electrophysiological properties of many cell types. In thyroid cells however, the effects have not yet been elucidated. Here, we report the effect of TNFalpha and its second messenger ceramide on the resting membrane potential (RMP) of thyroid FRTL-5 cells. In patch-clamp experiments, we showed that TNFalpha and ceramide depolarise the RMP by inhibiting an acid-sensitive inwardly rectifying potassium current. This depolarisation depended on the activation of protein kinase Czeta (PKCzeta), because it can be blocked by calphostin C, a PKC-inhibitory peptide and a specific inhibitor peptide for PKCzeta. The activation of PKCzeta was confirmed by Western blotting, in which a stimulation with TNFalpha led to the translocation of PKCzeta to the particulate fraction. We conclude that TNFalpha and ceramide depolarise the RMP of thyroid FRTL-5 cells by attenuating a Ba(2+)- and acid-sensitive potassium conductance via activation of PKCzeta.

Animals↗

PubMed2003

Functional characterization of the common amino acid 897 polymorphism of the cardiac potassium channel KCNH2 (HERG).

OBJECTIVE: To determine whether the amino acid 897 threonine (T) to lysine (K) polymorphism of the KCNH2 (HERG) potassium channel influences channel performance or patient phenotype. METHODS: The phenotypic effects of this polymorphism were investigated in vitro by electrophysiological experiments in HEK-293 cells and in vivo by exercise electrocardiography in a group of LQTS patients carrying the same genetically proven KCNQ1 mutation. RESULTS: When expressed in HEK-293 cells, the 897T isoform of the KCNH2 channel exhibited changes in inactivation and deactivation properties, and a smaller current density than the more common 897K isoform. Western blot experiments indicated that the decreased current density associated with 897T was caused by reduced channel expression. During a maximal exercise test in 39 LQT1 patients carrying an identical KCNQ1 mutation (G589D) and showing a prolonged QT interval (>440 ms), QT intervals were longer in patients carrying the 897T allele than in those homozygous for the 897K allele. CONCLUSIONS: The K897T variation has an effect on channel function and clinical phenotype. Our data warrant further investigations into the significance of this polymorphism in drug-induced and inherited LQTS.

Adolescent↗

PubMed2003

Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain.

The ligand-binding domains of AMPA receptor subunits carry two conserved N-glycosylation sites. In order to gain insight into the functional role of the corresponding N-glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand-binding characteristics, structural stability, cell-surface expression, and channel properties of homomeric GluR-D (GluR4) receptor and its soluble ligand-binding domain (S1S2). GluR-D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand-binding properties were observed between the 'wild-type' S1S2 and non-glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N-glycosylation. Purified glycosylated and non-glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full-length homomeric GluR-D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild-type GluR-D. In outside-out patches, GluR-D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand-binding domain glycans do not play any major role in receptor-ligand interactions, do not impart a stabilizing effect on the ligand-binding domain, and are not critical for the formation and surface localization of homomeric GluR-D AMPA receptors in HEK293 cells.

Amino Acid Substitution↗

PubMed2002

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor channels lacking the N-terminal domain.

Ionotropic glutamate receptor (iGluR) subunits contain a approximately 400-residue extracellular N-terminal domain ("X domain"), which is sequence-related to bacterial amino acid-binding proteins and to class C G-protein-coupled receptors. The X domain has been implicated in the assembly, transport to the cell surface, allosteric ligand binding, and desensitization in various members of the iGluR family, but its actual role in these events is poorly characterized. We have studied the properties of homomeric alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)-selective GluR-D glutamate receptors carrying N-terminal deletions. Our analysis indicates that, surprisingly, transport to the cell surface, ligand binding properties, agonist-triggered channel activation, rapid desensitization, and allosteric potentiation by cyclothiazide can occur normally in the complete absence of the X domain (residues 22-402). The relatively intact ligand-gated channel function of a homomeric AMPA receptor in the absence of the X domain indirectly suggests more subtle roles for this domain in AMPA receptors, e.g. in the assembly of heteromeric receptors and in synaptic protein interactions.

Allosteric Site↗

PubMed2002

Ceramide 1-(2-cyanoethyl) phosphate enhances store-operated Ca2+ entry in thyroid FRTL-5 cells.

Sphingolipid derivatives cause diverse effects towards the regulation of intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in a multitude of nonexcitable cells. In the present investigation, the effect of C-8 ceramide-1-(2-cyanoethyl) phosphate (C1CP) on store-operated Ca(2+) (SOC) entry was investigated. C1CP evoked a modest increase in [Ca(2+)](i). The increase was inhibited by the SOC channel antagonist 1-(beta-[3-(4methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SKF96365) but not by overnight pretreatment of the cells with pertussis toxin. C1CP did not invoke the production of inositol phosphates. When cells were stimulated with both C1CP and thapsigargin, the thapsigargin-invoked increase in [Ca(2+)](i) was enhanced in comparison to control cells. When Ca(2+) was added to cells treated with both C1CP and thapsigargin in a Ca(2+)-free buffer, the increase in [Ca(2+)](i) was enhanced in comparison to control cells. In patch-clamp experiments, C1CP hyperpolarized the membrane potential (E(m)) of the cells and attenuated the thapsigargin-invoked depolarization of the E(m). The effects of C1CP came, in part, as a result of a decreased conductance of the cell membrane towards Cl(-) ions, as C1CP in a Cl(-)-free solution also enhanced Ca(2+) entry. Barium 2-cyanoethylphosphate (Ba2Cy), which also contains the 2-cyanoethyl group, did not modulate thapsigargin-invoked changes in [Ca(2+)](i) nor did it modulate the E(m). In conclusion, C1CP enhances SOC entry, in part, via hyperpolarization of the E(m) and attenuation of the thapsigargin-invoked membrane depolarization, thus increasing the electrochemical gradient for Ca(2+) ions. Hence, C1CP may be a useful reagent for investigating the cellular effects of ceramide derivatives.

Acrylonitrile↗

PubMed2000

Different sensitivities of human and rat rho(1) GABA receptors to extracellular pH.

We have examined the sensitivity of human and rat homo-oligomeric rho(1) GABA receptors to variations in extracellular pH (pH(o)) using the whole-cell patch clamp technique. The GABA-induced conductance mediated by the rat rho(1) receptor (rho(1)-R) decreased with a decrease in pH(o) between 9.0 to 5.4. Below pH(o) 7.4 the effect of protons on the GABA-induced conductance was apparently competitive, but above pH(o) 7.4 the inhibitory effect of extracellular protons was almost independent on the GABA concentration. Titration of the GABA-induced conductance at 3 microM GABA revealed two protonation sites on rat rho(1)-R with pKa 6.4 and pKa 8.2. At 10 microM GABA the low pKa (6.4) was shifted to a clearly lower value (5.6), but the high pKa was only slightly decreased (from 8.2 to 7.9). Zn(2+) ions were capable of relieving the proton inhibition at low pH(o) indicating that Zn(2+) interacts with the low pKa site. Unlike the rat rho(1)-R, the human rho(1)-R was sensitive only to changes in pH(o) at acidic levels. Proton inhibition of human rho(1)-R was apparently competitive, as observed on rat-rho(1) at acidic pH(o). Titration of the human rho(1)-R gave a single H(+) binding site with a pKa of 6.3, similar to the value for the low pKa on rat rho(1)-R. The pKa value of human rho(1)-R was not dependent on the GABA concentration. A chimeric receptor, consisting of the N-terminal part of the rat rho(1)-R and C-terminal part of the human rho(1)-R, displayed pH(o) sensitivity similar to that observed for rat rho(1)-R. This indicates that the high pKa of rat rho(1)-R is attributable to the 11 amino acid differences between the rat and human rho(1)-R extracellular domains.

Amino Acid Sequence↗