A potassium leak channel silences hyperactive neurons and ameliorates status epilepticus.

OBJECTIVE: To develop a constitutively active K(+) leak channel using TREK-1 (TWIK-related potassium channel 1; TREK-M) that is resistant to compensatory down-regulation by second messenger cascades, and to validate the ability of TREK-M to silence hyperactive neurons using cultured hippocampal neurons. To test if adenoassociated viral (AAV) delivery of TREK-M could reduce the duration of status epilepticus and reduce neuronal death induced by lithium-pilocarpine administration. METHODS: Molecular cloning techniques were used to engineer novel vectors to deliver TREK-M via plasmids, lentivirus, and AAV using a cytomegalovirus (CMV)-enhanced GABRA4 promoter. Electrophysiology was used to characterize the activity and regulation of TREK-M in human embryonic kidney (HEK-293) cells, and the ability to reduce spontaneous activity in cultured hippocampal neurons. Adult male rats were injected bilaterally with self-complementary AAV particles composed of serotype 5 capsid into the hippocampus and entorhinal cortex. Lithium-pilocarpine was used to induce status epilepticus. Seizures were monitored using continuous video-electroencephalography (EEG) monitoring. Neuronal death was measured using Fluoro-Jade C staining of paraformaldehyde-fixed brain slices. RESULTS: TREK-M inhibited neuronal firing by hyperpolarizing the resting membrane potential and decreasing input resistance. AAV delivery of TREK-M decreased the duration of status epilepticus by 50%. Concomitantly it reduced neuronal death in areas targeted by the AAV injection. SIGNIFICANCE: These findings demonstrate that TREK-M can silence hyperexcitable neurons in the brain of epileptic rats and treat acute seizures. This study paves the way for an alternative gene therapy treatment of status epilepticus, and provides the rationale for studies of AAV-TREK-M's effect on spontaneous seizures in chronic models of temporal lobe epilepsy.

Validation of high throughput screening assays against three subtypes of Ca(v)3 T-type channels using molecular and pharmacologic approaches.

T-type Ca(2+) channels encoded by voltage-gated Ca(2+) channel (Ca(v)) 3.1, 3.2, and 3.3 genes play important physiological roles and serve as therapeutic targets for neurological and cardiovascular disorders. Currently there is no selective T-channel blocker. To screen for such a blocker, we developed three stable cell lines expressing human recombinant Ca(v)3.1, 3.2, or 3.3 channels and then examined their usefulness in high throughput screens. All three cell lines displayed an increase in intracellular Ca(2+) in response to changes in extracellular Ca(2+) as detected with Ca(2+)-sensitive dyes using a fluorometric imaging plate reader (FLIPR [Molecular Devices, Sunnyvale, CA] or FlexStation [Molecular Devices]). The signal-to-noise ratio was 2-4. Co-expression of Ca(v)3.2 with a mouse leak K(+) channel, which by virtue of being open at rest hyperpolarizes the cell membrane, blocked the fluorescent signal. Co-addition of KCl to these cells induced a Ca(2+) signal that was similar to that observed in the cell line expressing Ca(v)3.2 alone. These results confirm that the detection of intracellular Ca(2+) increase in cells expressing Ca(v)3.2 alone results from Ca(2+) entry through channels that are open at the resting membrane potential of each cell line (i.e., window currents). Testing known drugs on Ca(v)3 channels showed that block could be reliably detected using the FlexStation assay, FLIPR assay, or voltage clamp recordings using the IonWorks HT system (Molecular Devices). These results support the use of the FLIPR window current assay for primary drug screening and high throughput patch recordings for secondary screening of novel T-channel blockers.