T-type calcium channel blockers that attenuate thalamic burst firing and suppress absence seizures.

Absence seizures are a common seizure type in children with genetic generalized epilepsy and are characterized by a temporary loss of awareness, arrest of physical activity, and accompanying spike-and-wave discharges on an electroencephalogram. They arise from abnormal, hypersynchronous neuronal firing in brain thalamocortical circuits. Currently available therapeutic agents are only partially effective and act on multiple molecular targets, including γ-aminobutyric acid (GABA) transaminase, sodium channels, and calcium (Ca(2+)) channels. We sought to develop high-affinity T-type specific Ca(2+) channel antagonists and to assess their efficacy against absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model. Using a rational drug design strategy that used knowledge from a previous N-type Ca(2+) channel pharmacophore and a high-throughput fluorometric Ca(2+) influx assay, we identified the T-type Ca(2+) channel blockers Z941 and Z944 as candidate agents and showed in thalamic slices that they attenuated burst firing of thalamic reticular nucleus neurons in GAERS. Upon administration to GAERS animals, Z941 and Z944 potently suppressed absence seizures by 85 to 90% via a mechanism distinct from the effects of ethosuximide and valproate, two first-line clinical drugs for absence seizures. The ability of the T-type Ca(2+) channel antagonists to inhibit absence seizures and to reduce the duration and cycle frequency of spike-and-wave discharges suggests that these agents have a unique mechanism of action on pathological thalamocortical oscillatory activity distinct from current drugs used in clinical practice.

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 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, PDGFß 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 PDGFß 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) receptordependent accumulation of intracellular Ca(2+), 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 Ca(2+) 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.

Protein kinase C enhances glycine-insensitive desensitization of NMDA receptors independently of previously identified protein kinase C sites.

Protein kinase C (PKC) phosphorylates the NR1 and NR2A subunits of NMDARs at consensus sites located within their intracellular C-terminal tails. However, the functional consequences of these biochemical events are not well understood. In HEK293 cells expressing NR1/NR2A, activation of endogenous PKC by 4beta-phorbol 12-myristate 13-acetate (PMA) increased NMDAR desensitization as evidenced by a reduced steady-state current without any change in peak. The effects of PMA on NMDAR-mediated responses were prevented by specific PKC inhibitors and were not mimicked by an inactive enantiomer of PMA. The effects of PMA were preserved despite mutagenesis of the major PKC sites on the NR1 subunit (S889A, S890A, S896A and S897A) or removal of the entire NR1 C-terminal tail (NR1(stop838)). When co-expressing NR1(stop838)/NR2A the effects of PMA could only be observed with agonist concentrations sufficient to induce glycine-insensitive desensitization. Moreover, the effects of PMA were observed in receptors composed of NR1/NR2A and NR1/NR2B, but not NR1/NR2C, a subunit combination in which desensitization is absent. The NR2 subunit dependence suggested that the actions of PMA might require specific PKC sites previously identified within NR2A. However, a C-terminal truncated form of NR2A (NR2A(stop905)) remained responsive to PMA. We conclude that activation of PKC increases NMDAR glycine-insensitive desensitization independently of previously identified sites located within the NR1 C-terminus and distal segment of the NR2A C-terminus.

KvLQT1 modulates the distribution and biophysical properties of HERG. A novel alpha-subunit interaction between delayed rectifier currents.

Cardiac repolarization is under joint control of the slow (IKs) and rapid (IKr) delayed rectifier currents. Experimental and clinical evidence indicates important functional interactions between these components. We hypothesized that there might be more direct interactions between the KvLQT1 and HERG alpha-subunits of IKs and IKr and tested this notion with a combination of biophysical and biochemical techniques. Co-expression of KvLQT1 with HERG in a mammalian expression system significantly accelerated HERG current deactivation at physiologically relevant potentials by increasing the contribution of the fast component (e.g. upon repolarization from +20 mV to -50 mV: from 20 +/- 3 to 32 +/- 5%, p < 0.05), making HERG current more like native IKr. In addition, HERG current density was approximately doubled (e.g. tail current after a step to +10 mV: 18 +/- 3 versus 39 +/- 7 pA/picofarad, p < 0.01) by co-expression with KvLQT1. KvLQT1 co-expression also increased the membrane immunolocalization of HERG by approximately 2-fold (p < 0.05). HERG and KvLQT1 co-immunolocalized in canine ventricular myocytes and co-immunoprecipitated in cultured Chinese hamster ovary cells as well as in native cardiac tissue, indicating physical interactions between HERG and KvLQT1 proteins in vitro and in vivo. Protein interaction assays also demonstrated binding of KvLQT1 (but not another K+ channel alpha-subunit, Kv3.4) to a C-terminal HERG glutathione S-transferase fusion protein. Co-expression with HERG did not affect the membrane localization or ionic current properties of KvLQT1. This study shows that the alpha-subunit of IKs can interact with and modify the localization and current-carrying properties of the alpha-subunit of IKr, providing potentially novel insights into the molecular function of the delayed rectifier current system.

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 (I(Kr)) by human ether-a-go-go related gene (HERG), currents resulting from HERG (I(HERG)) and HERG plus MiRP1 expression have not been directly compared with native I(Kr). We compared the pharmacological and selected biophysical properties of I(HERG) with and without MiRP1 coexpression in Chinese hamster ovary (CHO) cells with those of guinea-pig I(Kr) under comparable conditions. Comparisons were also made with HERG expressed in Xenopus oocytes. MiRP1 coexpression significantly accelerated I(HERG) deactivation at potentials negative to the reversal potential, but did not affect more physiologically relevant deactivation of outward I(HERG), which remained slower than that of I(Kr). MiRP1 shifted I(HERG) activation voltage dependence in the hyperpolarizing direction, whereas I(Kr) activated at voltages more positive than I(HERG). There were major discrepancies between the sensitivity to quinidine, E-4031 and dofetilide of I(HERG) in Xenopus oocytes compared to I(Kr), which were not substantially affected by coexpression with MiRP1. On the other hand, the pharmacological sensitivity of I(HERG) in CHO cells was indistinguishable from that of I(Kr) and was unaffected by MiRP1 coexpression. We conclude that the properties of I(HERG) in CHO cells are similar in many ways to those of native I(Kr) 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 I(Kr).

Dofetilide block involves interactions with open and inactivated states of HERG channels.

Rapidly activating delayed rectifier current ( IKr) is the key target of class III antiarrhythmic drugs including dofetilide. Due to its complex gating properties, the precise channel state or states that interact with these agents remain poorly defined. We have undertaken a careful analysis of the state dependence of HERG channel block by dofetilide in Xenopus oocytes and Chinese Hamster Ovary (CHO) cells by devising a protocol in which brief sampling pulses were superimposed over a wide range of test potentials. The rate of block onset, maximal steady-state block and IC50 were similar for all test potentials over the activation range, demonstrating that the drug probably interacts with open and/or inactivated but not resting HERG channels with high affinity. Reducing the fraction of inactivated channels at 0 mV by augmenting the external potassium concentration did not alter the sensitivity to dofetilide. In contrast, the S631A and S620T HERG mutations both eliminated inward rectification and reduced dofetilide affinity by approximately 10- and approximately 100-fold respectively. We have also found a novel ultra-slow activation process which occurs in wild type HERG channels at threshold potentials. Overall, our data imply that dofetilide block occurs equally at all voltages positive to the activation threshold, and that the drug interacts with HERG channels in both the open and inactivated states.