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.
- 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
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.
- 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
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.
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
Transient receptor potential A1 (TRPA1) is an excitatory ion channel that functions as a cellular sensor, detecting a wide range of proalgesic agents such as environmental irritants and endogenous products of inflammation and oxidative stress. Topical application of TRPA1 agonists produces an acute nociceptive response through peripheral release of neuropeptides, purines and other transmitters from activated sensory nerve endings. This, in turn, further regulates TRPA1 activity downstream of G-protein and phospholipase C-coupled signaling cascades. Despite the important physiological relevance of such regulation leading to nociceptor sensitization and consequent pain hypersensitivity, the specific domains through which TRPA1 undergoes post-translational modifications that affect its activation properties are yet to be determined at a molecular level. This review aims at providing an account of our current knowledge on molecular basis of regulation by neuronal inflammatory signaling pathways that converge on the TRPA1 channel protein and through modification of its specific residues influence the extent to which this channel may contribute to pain.
- MeSH
- bolest genetika metabolismus MeSH
- bradykinin genetika metabolismus MeSH
- kationtový kanál TRPA1 fyziologie MeSH
- lidé MeSH
- nociceptory fyziologie MeSH
- signální transdukce fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Gain-of-function (GOF) mutations in ion channels are rare events, which lead to increased agonist sensitivity or altered gating properties, and may render the channel constitutively active. Uncovering and following characterization of such mutants contribute substantially to the understanding of the molecular basis of ion channel functioning. Here we give an overview of some GOF mutants in polymodal ion channels specifically involved in transduction of painful stimuli--TRPV1 and TRPA1, which are scrutinized by scientists due to their important role in development of some pathological pain states. Remarkably, a substitution of single amino acid in the S4-S5 region of TRPA1 (N855S) has been recently associated with familial episodic pain syndrome. This mutation increases chemical sensitivity of TRPA1, but leaves the voltage sensitivity unchanged. On the other hand, mutations in the analogous region of TRPV1 (R557K and G563S) severely affect all aspects of channel activation and lead to spontaneous activity. Comparison of the effects induced by mutations in homologous positions in different TRP receptors (or more generally in other distantly related ion channels) may elucidate the gating mechanisms conserved during evolution.
- MeSH
- bolest patofyziologie MeSH
- kationtové kanály TRP chemie genetika metabolismus MeSH
- kationtové kanály TRPV chemie genetika metabolismus MeSH
- lidé MeSH
- mícha patofyziologie MeSH
- mutace genetika MeSH
- percepce bolesti MeSH
- proteiny nervové tkáně chemie genetika metabolismus MeSH
- vápníkové kanály chemie genetika metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
