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1.
Epilepsia ; 62(7): 1744-1758, 2021 07.
Article in English | MEDLINE | ID: mdl-34085706

ABSTRACT

OBJECTIVE: About one third of all patients with epilepsy have pharmacoresistant seizures. Thus there is a need for better pharmacological treatments. The human voltage-gated potassium (hKV ) channel hKV 7.2/7.3 is a validated antiseizure target for compounds that activate this channel. In a previous study we have shown that resin acid derivatives can activate the hKV 7.2/7.3 channel. In this study we investigated if these channel activators have the potential to be developed into a new type of antiseizure drug. Thus we examined their structure-activity relationships and the site of action on the hKV 7.2/7.3 channel, if they have unwanted cardiac and cardiovascular effects, and their potential antiseizure effect. METHODS: Ion channels were expressed in Xenopus oocytes or mammalian cell lines and explored with two-electrode voltage-clamp or automated patch-clamp techniques. Unwanted vascular side effects were investigated with isometric tension recordings. Antiseizure activity was studied in an electrophysiological zebrafish-larvae model. RESULTS: Fourteen resin acid derivatives were tested on hKV 7.2/7.3. The most efficient channel activators were halogenated and had a permanently negatively charged sulfonyl group. The compounds did not bind to the sites of other hKV 7.2/7.3 channel activators, retigabine, or ICA-069673. Instead, they interacted with the most extracellular gating charge of the S4 voltage-sensing helix, and the effects are consistent with an electrostatic mechanism. The compounds altered the voltage dependence of hKV 7.4, but in contrast to retigabine, there were no effects on the maximum conductance. Consistent with these data, the compounds had less smooth muscle-relaxing effect than retigabine. The compounds had almost no effect on the voltage dependence of hKV 11.1, hNaV 1.5, or hCaV 1.2, or on the amplitude of hKV 11.1. Finally, several resin acid derivatives had clear antiseizure effects in a zebrafish-larvae model. SIGNIFICANCE: The described resin acid derivatives hold promise for new antiseizure medications, with reduced risk for adverse effects compared with retigabine.


Subject(s)
Anticonvulsants/pharmacology , Epilepsy/prevention & control , KCNQ2 Potassium Channel/drug effects , KCNQ3 Potassium Channel/drug effects , Resins, Synthetic/pharmacology , Seizures/prevention & control , Animals , Carbamates/pharmacology , Humans , Ion Channel Gating/drug effects , Larva , Oocytes , Patch-Clamp Techniques , Phenylenediamines/pharmacology , Substrate Specificity , Xenopus laevis , Zebrafish
2.
Sci Rep ; 10(1): 18041, 2020 10 22.
Article in English | MEDLINE | ID: mdl-33093500

ABSTRACT

Classically, neurexins are thought to mediate synaptic connections through trans interactions with a number of different postsynaptic partners. Neurexins are cleaved by metalloproteases in an activity-dependent manner, releasing the soluble extracellular domain. Here, we report that in both immature (before synaptogenesis) and mature (after synaptogenesis) hippocampal neurons, the soluble neurexin-1ß ectodomain triggers acute Ca2+-influx at the dendritic/postsynaptic side. In both cases, neuroligin-1 expression was required. In immature neurons, calcium influx required N-type calcium channels and stimulated dendritic outgrowth and neuronal survival. In mature glutamatergic neurons the neurexin-1ß ectodomain stimulated calcium influx through NMDA-receptors, which increased presynaptic release probability. In contrast, prolonged exposure to the ectodomain led to inhibition of synaptic transmission. This secondary inhibition was activity- and neuroligin-1 dependent and caused by a reduction in the readily-releasable pool of vesicles. A synthetic peptide modeled after the neurexin-1ß:neuroligin-1 interaction site reproduced the cellular effects of the neurexin-1ß ectodomain. Collectively, our findings demonstrate that the soluble neurexin ectodomain stimulates growth of neurons and exerts acute and chronic effects on trans-synaptic signaling involved in setting synaptic strength.


Subject(s)
Calcium-Binding Proteins/pharmacology , Calcium/metabolism , Dendritic Cells/drug effects , Dendritic Cells/physiology , Neural Cell Adhesion Molecules/pharmacology , Synaptic Transmission/drug effects , Animals , Calcium-Binding Proteins/metabolism , Cell Adhesion Molecules, Neuronal/metabolism , Cells, Cultured , Hippocampus/metabolism , Mice, Inbred C57BL , Mice, Knockout , Neural Cell Adhesion Molecules/metabolism , Neurons/metabolism , Rats, Wistar , Receptors, N-Methyl-D-Aspartate/metabolism , Solubility , Stimulation, Chemical
3.
Curr Protoc Pharmacol ; 89(1): e75, 2020 06.
Article in English | MEDLINE | ID: mdl-32271492

ABSTRACT

Ligand-gated ion channels (LGICs) are a group of diverse ion channels that are gated by ligands and play important roles in normal physiological and pathological conditions. Many of them are drug targets that have been pursued, are being pursued, and will likely be pursued in the future by pharmaceutical companies and academic groups for a variety of diseases. One of those LGICs is the GABAA receptor, a heterooligomeric chloride channel that can be blocked and modulated at various sites. In order to study the receptor's functional response to compounds, the manual patch-clamp method provides a detailed but low-throughput electrophysiological characterization. QPatch II, a next-generation automated patch clamp machine that was recently developed by Sophion Bioscience, provides an automated electrophysiological study of ion channels. In this article, we use the GABAA receptor as an example for studying LGICs and describe two detailed protocols for using QPatch II to carry out pharmacological studies on the receptor. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Ligand concentration-response experiment (GABAA receptor) on QPatch II Alternate Protocol: Non-cumulative ligand concentration-response experiment (GABAA receptor) on QPatch II Support Protocol 1: Cell culture of HEK293-hGABAA (α5ß3γ2) Support Protocol 2: Data analysis for Basic Protocol 1 Support Protocol 3: Data analysis for Alternate Protocol Basic Protocol 2: Antagonist dose-response experiment (GABAA receptor) on QPatch II Support Protocol 3: Data analysis for Basic Protocol 2.


Subject(s)
Automation/instrumentation , Electrophysiology/instrumentation , Patch-Clamp Techniques/instrumentation , Receptors, GABA-A/metabolism , Animals , CHO Cells , Cell Culture Techniques , Cricetulus , HEK293 Cells , Humans , Ion Channels , Membrane Potentials/physiology
4.
J Neurosci ; 36(47): 11865-11880, 2016 11 23.
Article in English | MEDLINE | ID: mdl-27881774

ABSTRACT

Whether interactions between synaptotagmin-1 (syt-1) and the soluble NSF attachment protein receptors (SNAREs) are required during neurotransmission is debated. We examined five SNAP-25 mutations designed to interfere with syt-1 interactions. One mutation, D51/E52/E55A, targeted negative charges within region II of the primary interface (Zhou et al., 2015); two mutations targeted region I (D166A and D166/E170A) and one mutation targeted both (D51/E52/E55/D166A). The final mutation (D186/D193A) targeted C-terminal residues not expected to interact with syt-1. An in vitro assay showed that the region I, region II, and region I+II (D51/E52/E55/D166A) mutants markedly reduced the attachment between syt-1 and t-SNARE-carrying vesicles in the absence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. In the presence of PI(4,5)P2, vesicle attachment was unaffected by mutation. When expressed in Snap-25-null mouse autaptic neurons, region I mutations reduced the size of the readily releasable pool of vesicles, whereas the region II mutation reduced vesicular release probability. Combining both in the D51/E52/E55/D166A mutation abrogated evoked release. These data point to a division of labor between region I (vesicle priming) and region II (evoked release). Spontaneous release was disinhibited by region I mutations and found to correlate with defective complexin (Cpx) clamping in an in vitro fusion assay, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Mutation in region II (D51/E52/E55A) also unclamped release, but this effect could be overcome by synaptotagmin overexpression, arguing against an obligatory role in clamping. We conclude that three synaptic release functions of syt-1, vesicle priming, spontaneous release clamping, and evoked release triggering, depend on direct SNARE complex interaction. SIGNIFICANCE STATEMENT: The function of synaptotagmin-1 (syt-1):soluble NSF attachment protein receptor (SNARE) interactions during neurotransmission remains unclear. We mutated SNAP-25 within the recently identified region I and region II of the primary synaptotagmin:SNARE interface. Using in vitro assays and rescue experiments in autaptic neurons, we show that interactions within region II of the primary interface are necessary for synchronized calcium-triggered release, whereas region I is involved in vesicle priming. Spontaneous release was disinhibited by region I mutation and found to correlate with defective complexin (Cpx) clamping in vitro, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Therefore, vesicle priming, clamping spontaneous release, and eliciting evoked release are three different functions of syt-1 that involve different interaction modes with the SNARE complex.


Subject(s)
Action Potentials/physiology , Signal Transduction/physiology , Synaptic Transmission/physiology , Synaptic Vesicles/physiology , Synaptosomal-Associated Protein 25/metabolism , Synaptotagmin I/metabolism , Animals , Binding Sites , Calcium Signaling/physiology , Female , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Protein Binding , Structure-Activity Relationship , Synaptosomal-Associated Protein 25/genetics , Synaptotagmin I/genetics
5.
J Physiol ; 591(1): 185-201, 2013 Jan 01.
Article in English | MEDLINE | ID: mdl-23027824

ABSTRACT

The capacity to sense temperature is essential for the survival of all animals. At the molecular level, ion channels belonging to the transient receptor potential (TRP) family of channels function as temperature sensors in animals across several phyla. TRP channels are opened directly by changes in temperature and show pronounced sensitivity at their activation range. To determine how temperature activates these channels, we analysed channels belonging to the TRPA family, which detect heat in insects and cold in mammals. By constructing chimeric proteins consisting of human and Drosophila TRPA1 channels, we mapped regions that regulate thermal activation and identified residues in the pore helix that invert temperature sensitivity of TRPA1. From analysis of individual channels we defined the gating reaction of Drosophila TRPA1 and determined how mutagenesis alters the energy landscape for channel opening. Our results reveal specific molecular requirements for thermal activation of TRPA1 and provide mechanistic insight into this process.


Subject(s)
Calcium Channels/physiology , Drosophila Proteins/physiology , Nerve Tissue Proteins/physiology , TRPC Cation Channels/physiology , Transient Receptor Potential Channels/physiology , Animals , Calcium Channels/chemistry , Drosophila , Drosophila Proteins/chemistry , Hot Temperature , Humans , Ion Channels , Mutation , Nerve Tissue Proteins/chemistry , Porosity , TRPA1 Cation Channel , TRPC Cation Channels/chemistry , Transient Receptor Potential Channels/chemistry
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