Biophysical and pharmacological properties of the rapidly activating delayed rectifier current

The delayed rectifier current, IK, responsible for terminating the plateau phase of the cardiac action potential, is composed of two components; the slowly activating component, IKs, and the rapidly activating component, IKr. IKr is the primary target of Class III antiarrhythmic agents, inhibition of which prolongs cardiac action potential and reduces the probability of reentrant arrhythmia induction. However, Class III antiarrhythmic agents can also excessively prolong the QT interval and precipitate a potentially lethal arrhythmia called torsade de pointes (TdP). Furthermore, mutations at the gene coding for IKr can cause congenital long QT syndrome and TdP. Heterologous expression of the human ether-a-go-go -related gene (HERG) elicits currents that closely resemble native IKr. The work presented in this thesis explores in detail heterologously expressed HERG channels, in particular their biophysical and pharmacological properties. We first probed HERG channels with the divalent cation Ba 2+ and the potent Class III antiarrhythmic agent dofetilide to examine the voltage-dependent pore accessibility. Our experimental and mathematical modeling data indicate that Ba2+ blocked wild-type HERG channels in their closed and open states but channel inactivation relieved Ba 2+ block. In contrast to previous reports indicating that dofetilide only blocked the open state of HERG channels, our data clearly show that dofetilide interacted with both the open and inactivated states. We then examined the need for association between the recently cloned MiRP1 (minK-related peptide 1) subunit in imparting properties similar to that of native IKr to HERG. We demonstrated that the pharmacological characteristics of HERG channels expressed in CHO cells is very similar to IKr recorded from guinea pig myocytes. The minor discrepancies between the cloned and native currents (i.e. deactivation kinetics and voltage dependence of activation) were not rectified by MiRP1 co-expression. ThuIn summary, our findings demonstrate that HERG channels are accessible in a state-dependent manner from the extracellular side by Ba2+ ions and the intracellular side by dofetilide. The characteristics of HERG channels that govern its sensitivity to drug blockers do not appear to require an association with its putative MiRP1 beta-subunit. The latter finding has important implications for screening of novel drugs for an early determination of their potential to prolong the QT interval, in particular those agents that are not meant to possess IKr inhibitory properties. Inhibition of HERG channels expressed in a mammalian cell line will provide a very close approximation of the sensitivity of native IKr to these agents

A novel model of Quinidine induced torsade de pointes arrhythmias in the isolated rabbit heart /

Accumulating evidence suggest the underlying danger of Class IA and Class III antiarrhythmic agents in inducing potentially lethal torsade de pointes arrhythmias (TdP) if associated with hypokalemia and slow heart rates. The primary aim of the following study was to examine the proarrhythmic activity of the Class IA agent quinidine in isolated perfused rabbit hearts in an effort towards developing a reliable model of TdP.Thirty five rabbit hearts were perfused with therapeutic quinidine concentrations (2.5 or 5$mu$M) in the presence of hypokalemia (K$sp+$ - 2.7mM) and stimulated at cycle lengths (CLs) ranging from 0.5s-5.5s. Monophasic action potentials (MAPs) were recorded from left endocardial and right epicardial sites along with an ECG.We suggest that the described model of acquired TdP in isolated rabbit hearts can be adapted for the quantitative and qualitative assessment of the proarrhythmic potential of existing and novel antiarrhythmic agents, to test the efficacy of novel therapeutic strategies for the acquired long QT syndrome and/or to clarify the specific mechanism(s) of drug induced TdP. (Abstract shortened by UMI.

Abelson Tyrosine Kinase Links PDGFbeta Receptor Activation to Cytoskeletal Regulation of NMDA Receptors in CA1 Hippocampal Neurons

Background: We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA) currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl), control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results: Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDAevoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion: This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons

Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons

Abstract Background We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA) currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl), control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons.</p

Abelson tyrosine kinase links PDGFbeta receptor activation to cytoskeletal regulation of NMDA receptors in CA1 hippocampal neurons

Abstract Background We have previously demonstrated that PDGF receptor activation indirectly inhibits N-methyl-D-aspartate (NMDA) currents by modifying the cytoskeleton. PDGF receptor ligand is also neuroprotective in hippocampal slices and cultured neurons. PDGF receptors are tyrosine kinases that control a variety of signal transduction pathways including those mediated by PLCγ. In fibroblasts Src and another non-receptor tyrosine kinase, Abelson kinase (Abl), control PDGF receptor regulation of cytoskeletal dynamics. The mechanism whereby PDGF receptor regulates cytoskeletal dynamics in central neurons remains poorly understood. Results Intracellular applications of active Abl, but not heat-inactivated Abl, decreased NMDA-evoked currents in isolated hippocampal neurons. This mimics the effects of PDGF receptor activation in these neurons. The Abl kinase inhibitor, STI571, blocked the inhibition of NMDA currents by Abl. We demonstrate that PDGF receptors can activate Abl kinase in hippocampal neurons via mechanisms similar to those observed previously in fibroblasts. Furthermore, PDGFβ receptor activation alters the subcellular localization of Abl. Abl kinase is linked to actin cytoskeletal dynamics in many systems. We show that the inhibition of NMDA receptor currents by Abl kinase is blocked by the inclusion of the Rho kinase inhibitor, Y-27632, and that activation of Abl correlates with an increase in ROCK tyrosine phosphorylation. Conclusion This study demonstrates that PDGFβ receptors act via an interaction with Abl kinase and Rho kinase to regulated cytoskeletal regulation of NMDA receptor channels in CA1 pyramidal neurons

Control of excitatory synaptic transmission by C-terminal Src kinase

The induction of long-term potentiation at CA3-CA1 synapses is caused by an N-methyl-D-aspartate (NMDA) receptor-dependent accumulation of intracellular Ca2+, followed by Src family kinase activation and a positive feedback enhancement of NMDA receptors (NMDARs). Nevertheless, the amplitude of baseline transmission remains remarkably constant even though low frequency stimulation is also associated with an NMDAR-dependent influx of Ca2+ into dendritic spines. We show here that an interaction between C-terminal Src kinase (Csk) and NMDARs controls the Src-dependent regulation of NMDAR activity. Csk associates with the NMDAR signaling complex in the adult brain, inhibiting the Src-dependent potentiation of NMDARs in CA1 neurons and attenuating the Src-dependent induction of long-term potentiation. Csk associates directly with Src-phosphorylated NR2 subunits in vitro. An inhibitory antibody for Csk disrupts this physical association, potentiates NMDAR mediated excitatory postsynaptic currents, and induces long-term potentiation at CA3-CA1 synapses. Thus, Csk serves to maintain the constancy of baseline excitatory synaptic transmission by inhibiting Src kinase-dependent synaptic plasticity in the hippocampus. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc

A comparison of currents carried by HERG, with and without coexpression of MiRP1, and the native rapid delayed rectifier current. Is MiRP1 the missing link?

Although it has been suggested that coexpression of minK related peptide (MiRP1) is required for reconstitution of native rapid delayed-rectifier current (IKr) by human ether-a-go-go related gene (HERG), currents resulting from HERG (IHERG) and HERG plus MiRP1 expression have not been directly compared with native IKr. We compared the pharmacological and selected biophysical properties of IHERG with and without MiRP1 coexpression in Chinese hamster ovary (CHO) cells with those of guinea-pig IKr under comparable conditions. Comparisons were also made with HERG expressed in Xenopus oocytes. MiRP1 coexpression significantly accelerated IHERG deactivation at potentials negative to the reversal potential, but did not affect more physiologically relevant deactivation of outward IHERG, which remained slower than that of IKr. MiRP1 shifted IHERG activation voltage dependence in the hyperpolarizing direction, whereas IKr activated at voltages more positive than IHERG. There were major discrepancies between the sensitivity to quinidine, E-4031 and dofetilide of IHERG in Xenopus oocytes compared to IKr, which were not substantially affected by coexpression with MiRP1. On the other hand, the pharmacological sensitivity of IHERG in CHO cells was indistinguishable from that of IKr and was unaffected by MiRP1 coexpression. We conclude that the properties of IHERG in CHO cells are similar in many ways to those of native IKr under the same recording conditions, and that the discrepancies that remain are not reduced by coexpression with MiRP1. These results suggest that the physiological role of MiRP1 may not be to act as an essential consituent of the HERG channel complex carrying native IKr