N-methyl-D-aspartate receptors (NMDARs) play an essential role in excitatory neurotransmission in the mammalian brain, and their physiological importance is underscored by the large number of pathogenic mutations that have been identified in the receptor's GluN subunits and associated with a wide range of diseases and disorders. Here, we characterized the functional and pharmacological effects of the pathogenic N650K variant in the GluN1 subunit, which is associated with developmental delay and seizures. Our microscopy experiments showed that when expressed in HEK293 cells (from ATCC®), the GluN1-N650K subunit increases the surface expression of both GluN1/GluN2A and GluN1/GluN2B receptors, but not GluN1/GluN3A receptors, consistent with increased surface expression of the GluN1-N650K subunit expressed in hippocampal neurons (from embryonic day 18 of Wistar rats of both sexes). Using electrophysiology, we found that the GluN1-N650K variant increases the potency of GluN1/GluN2A receptors to both glutamate and glycine but decreases the receptor's conductance and open probability. In addition, the GluN1-N650K subunit does not form functional GluN1/GluN2B receptors but does form fully functional GluN1/GluN3A receptors. Moreover, in the presence of extracellular Mg2+, GluN1-N650K/GluN2A receptors have a similar and increased response to ketamine and memantine, respectively, while the effect of both drugs had markedly slower onset and offset compared to wild-type GluN1/GluN2A receptors. Finally, we found that expressing the GluN1-N650K subunit in hippocampal neurons reduces excitotoxicity, and memantine shows promising neuroprotective effects in neurons expressing either wild-type GluN1 or the GluN1-N650K subunit. This study provides the functional and pharmacological characterization of NMDARs containing the GluN1-N650K variant.
- Klíčová slova
- Glutamate receptor, Hippocampal neuron, Ion channel, Ketamine, Memantine, Neurodegeneration,
- MeSH
- HEK293 buňky MeSH
- krysa rodu Rattus MeSH
- kyselina glutamová MeSH
- lidé MeSH
- memantin * farmakologie MeSH
- potkani Wistar MeSH
- receptory N-methyl-D-aspartátu * genetika MeSH
- savci MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kyselina glutamová MeSH
- memantin * MeSH
- receptory N-methyl-D-aspartátu * MeSH
The transient receptor potential ankyrin 1 (TRPA1) channel is a polymodal sensor of environmental irritant compounds, endogenous proalgesic agents, and cold. Upon activation, TRPA1 channels increase cellular calcium levels via direct permeation and trigger signaling pathways that hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2 ) in the inner membrane leaflet. Our objective was to determine the extent to which a putative PIP2 -interaction site (Y1006-Q1031) is involved in TRPA1 regulation. The interactions of two specific peptides (L992-N1008 and T1003-P1034) with model lipid membranes were characterized by biophysical approaches to obtain information about affinity, peptide secondary structure, and peptide effect in the lipid organization. The results indicate that the two peptides interact with lipid membranes only if PIP2 is present and their affinities depend on the presence of calcium. Using whole-cell electrophysiology, we demonstrate that mutation at F1020 produced channels with faster activation kinetics and with a rightward shifted voltage-dependent activation curve by altering the allosteric constant that couples voltage sensing to pore opening. We assert that the presence of PIP2 is essential for the interaction of the two peptide sequences with the lipid membrane. The putative phosphoinositide-interacting domain comprising the highly conserved F1020 contributes to the stabilization of the TRPA1 channel gate.
- Klíčová slova
- TRP channel, ankyrin transient receptor potential, gating, peptide-lipid interaction, rectification,
- MeSH
- biofyzikální jevy MeSH
- fosfatidylinositol-4,5-difosfát chemie metabolismus MeSH
- fosfolipidy chemie metabolismus MeSH
- HEK293 buňky MeSH
- kationtový kanál TRPA1 chemie genetika MeSH
- kinetika MeSH
- lidé MeSH
- membránové potenciály genetika MeSH
- metabolismus lipidů genetika MeSH
- peptidy chemie MeSH
- sekundární struktura proteinů MeSH
- signální transdukce genetika MeSH
- vápník chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fosfatidylinositol-4,5-difosfát MeSH
- fosfolipidy MeSH
- kationtový kanál TRPA1 MeSH
- peptidy MeSH
- vápník MeSH
Exposure to repetitive low-frequency electromagnetic field (LF-EMF) shows promise as a non-invasive approach to treat various sensory and neurological disorders. Despite considerable progress in the development of modern stimulation devices, there is a limited understanding of the mechanisms underlying their biological effects and potential targets at the cellular level. A significant impact of electromagnetic field on voltage-gated calcium channels and downstream signalling pathways has been convincingly demonstrated in many distinct cell types. However, evidence for clear effects on primary sensory neurons that particularly may be responsible for the analgesic actions of LF-EMF is still lacking. Here, we used F11 cells derived from dorsal root ganglia neurons as an in vitro model of peripheral sensory neurons and three different protocols of high-induction magnetic stimulation to determine the effects on chemical responsiveness and spontaneous activity. We show that short-term (<180 sec.) exposure of F11 cells to LF-EMF reduces calcium transients in response to bradykinin, a potent pain-producing inflammatory agent formed at sites of injury. Moreover, we characterize an immediate and reversible potentiating effect of LF-EMF on neuronal spontaneous activity. Our results provide new evidence that electromagnetic field may directly modulate the activity of sensory neurons and highlight the potential of sensory neuron-derived cell line as a tool for studying the underlying mechanisms at the cellular and molecular level.
- Klíčová slova
- bradykinin receptor, electromagnetic field, ion channel, primary sensory neuron, transient receptor potential channel,
- MeSH
- bradykinin farmakologie MeSH
- buněčné linie MeSH
- elektromagnetická pole * MeSH
- kationtový kanál TRPA1 metabolismus MeSH
- lidé MeSH
- nervové receptory účinky léků metabolismus MeSH
- vápník metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bradykinin MeSH
- kationtový kanál TRPA1 MeSH
- TRPA1 protein, human MeSH Prohlížeč
- vápník MeSH
Transient receptor potential ankyrin 1 (TRPA1) is a temperature-sensitive ion channel activated by various pungent and irritant compounds that can produce pain in humans. Its activation involves an allosteric mechanism whereby electrophilic agonists evoke interactions within cytosolic domains and open the channel pore through an integrated nexus formed by intracellular membrane proximal regions that are densely packed beneath the lower segment of the S1-S4 sensor domain. Studies indicate that this part of the channel may contain residues that form a water-accessible cavity that undergoes changes in solvation during channel gating. We identified conserved polar residues facing the putative lower crevice of the sensor domain that were crucial determinants of the electrophilic, voltage, and calcium sensitivity of the TRPA1 channel. This part of the sensor may also comprise a domain capable of binding to membrane phosphoinositides through which gating of the channel is regulated in a state-dependent manner.
- MeSH
- alosterická regulace MeSH
- gating iontového kanálu * MeSH
- HEK293 buňky MeSH
- kationtový kanál TRPA1 chemie fyziologie MeSH
- konformace proteinů MeSH
- lidé MeSH
- membránové potenciály * MeSH
- molekulární modely MeSH
- mutace MeSH
- mutageneze cílená MeSH
- proteinové domény MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie MeSH
- vápník metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- kationtový kanál TRPA1 MeSH
- TRPA1 protein, human MeSH Prohlížeč
- vápník MeSH
