Browsing by Author "Zamponi, Gerald W."
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Item Open Access Activity-dependent subcellular cotrafficking of the small GTPase Rem2 and Ca2+/CaM-dependent protein kinase IIα(PLOS one, 2012-07-18) Bernier, Nikolas; De Koninck, Paul; Flynn, Robyn E.; Labrie-Dion, Étienne; Colicos, Michael A.; Zamponi, Gerald W.Rem2 is a small monomeric GTP-binding protein of the RGK family, whose known functions are modulation of calcium channel currents and alterations of cytoskeletal architecture. Rem2 is the only RGK protein found predominantly in the brain, where it has been linked to synaptic development. We wished to determine the effect of neuronal activity on the subcellular distribution of Rem2 and its interacting partners.Item Open Access AKAP79 modulation of L-type channels involves disruption of intramolecular interactions in the CaV1.2 subunit(Landes Bioscience, 2012-05) Altier, Christophe; Dubel, Stefan J.; Barrère, Christian; Jarvis, Scott E.; Stotz, Stephanie C.; Scott, John D.; Nargeot, Joël; Zamponi, Gerald W.; Bourinet, EmmanuelL-type voltage gated calcium channels (VGCCs) interact with a variety of proteins that modulate both their function and localization. A-Kinase Anchoring Proteins (AKAPs) facilitate L-type calcium channel phosphorylation through β adrenergic stimulation. Our previous work indicated a role of neuronal AKAP79/150 in the membrane targeting of Ca(V)1.2 L-type calcium channels, which involved a proline rich domain (PRD) in the intracellular II-III loop of the channel.(1) Here, we show that mutation of proline 857 to alanine (P857A) into the PRD does not disrupt the AKAP79-induced increase in Ca(v)1.2 membrane expression. Furthermore, deletion of two other PRDs into the carboxy terminal domain of Ca(V)1.2 did not alter the targeting role of AKAP79. In contrast, the distal carboxy terminus region of the channel directly interacts with AKAP79. This protein-protein interaction competes with a direct association of the channel II-III linker on the carboxy terminal tail and modulates membrane targeting of Ca(V)1.2. Thus, our results suggest that the effects of AKAP79 occur through relief of an autoinhibitory mechanism mediated by intramolecular interactions of Ca(v)1.2 intracellular regions.Item Open Access Analgesia by intrathecal delta-9-tetrahydrocannabinol is dependent on Cav3.2 calcium channels(2023-05-25) de Maria Gadotti, Vinicius; Antunes, Flavia T. T.; Zamponi, Gerald W.Abstract Delta-9-tetrahydrocannabinol (Δ9-THC) is known to produce systemic analgesia that involves CB1 and CB2 cannabinoid receptors. However, there is compelling evidence that Δ9-THC can potently inhibit Cav3.2T-type calcium channels which are highly expressed in dorsal root ganglion neurons and in the dorsal horn of the spinal cord. Here, we investigated whether spinal analgesia produced by Δ9-THC involves Cav3.2 channels vis a vis cannabinoid receptors. We show that spinally delivered Δ9-THC produced dose-dependent and long-lasting mechanical anti-hyperalgesia in neuropathic mice, and showed potent analgesic effects in models of inflammatory pain induced by formalin or Complete Freund’s Adjuvant (CFA) injection into the hind paw, with the latter showing no overt sex differences. The Δ9-THC mediated reversal of thermal hyperalgesia in the CFA model was abolished in Cav3.2 null mice, but was unaltered in CB1 and CB2 null animals. Hence, the analgesic effects of spinally delivered Δ9-THC are due to an action on T-type calcium channels, rather than activation of spinal cannabinoid receptors.Item Open Access Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist(BioMed Central Ltd., 2013-07-01) You, Haitao; Gadotti, Vinícius Maria; Petrov, Ravil R.; Berger, N. Daniel; Diaz, Philippe J.; Zamponi, Gerald W.Cannabinoid receptors and T-type calcium channels are potential targets for treating pain. Here we report on the design, synthesis and analgesic properties of a new mixed cannabinoid/T-type channel ligand, NMP-181.Item Open Access Analysis of GPCR/ion channel interactions(Springer, 2011-01-01) Altier, Christophe; Zamponi, Gerald W.Voltage-gated calcium channels are key regulators of calcium homeostasis in excitable cells. A number of cellular signaling pathways serve to fine tune calcium channel activity, including the activation of G protein-coupled receptors. Besides regulating channel activity via second messengers, GPCRs can also physically associate with calcium channels to directly regulate their functions, as well as their trafficking to and from the plasma membrane. Here we provide some methods that can be used to examine channel-receptor interactions and co-trafficking. While we focus on voltage-gated calcium channels, the techniques described herein are broadly applicable to other types of channels.Item Open Access Antiallodynic effects of a confused α-conotoxin: Vc1.1 relieves neuropathic pain via off target actions on GABA(B) receptors and N-type channels(Elsevier, 2011-02-01) Zamponi, Gerald W.Item Open Access Antihyperalgesic effect of joint mobilization requires Cav3.2 calcium channels(2023-07-18) Martins, Daniel F.; Sorrentino, Victor; Mazzardo-Martins, Leidiane; Reed, William R.; Santos, Adair R. S.; Gadotti, Vinícius M.; Zamponi, Gerald W.Abstract The present study was undertaken to explore the relative contributions of Cav3.2 T-type channels to mediating the antihyperalgesic activity of joint manipulation (JM) therapy. We used the chronic constriction injury model (CCI) to induce peripheral neuropathy and chronic pain in male mice, followed by JM. We demonstrate that JM produces long-lasting mechanical anti-hyperalgesia that is abolished in Cav3.2 null mice. Moreover, we found that JM displays a similar analgesic profile as the fatty acid amide hydrolase inhibitor URB597, suggesting a possible converging mechanism of action involving endocannabinoids. Overall, our findings advance our understanding of the mechanisms through which JM produces analgesia.Item Open Access Biophysical characterization of cav1.4 l-type calcium channels(2007) Doering, Clinton John; Zamponi, Gerald W.Item Open Access Bipartite syntaxin 1A interactions mediate CaV2.2 calcium channel regulation(Elsevier, 2011-07-05) Davies, Jonathan N.; Jarvis, Scott E.; Zamponi, Gerald W.Functional interactions between syntaxin 1A and Ca(V)2 calcium channels are critical for fast neurotransmitter release in the mammalian brain, and coexpression of syntaxin 1A with these channels not only regulates channel availability, but also promotes G-protein inhibition. Both the syntaxin 1A C-terminal H3 domain, and N-terminal Ha domain have been shown to interact with the Ca(V)2.2 channel synprint region, suggesting a bipartite model of functional interaction, however the molecular determinants of this interaction have not been closely investigated. We used in vitro binding assays to assess interactions of syntaxin 1A truncation mutants with Ca(V)2.2 synprint and Ca(V)2.3 II-III linker regions. We identified two distinct interactions between the Ca(V)2.2 synprint region and syntaxin 1A: the first between C-terminal H3c domain of syntaxin 1A and residues 822-872 of Ca(V)2.2; and the second between the N-terminal 10 residues of the syntaxin 1A Ha region and residues 718-771 of Ca(V)2.2. The N-terminal syntaxin 1A fragment also interacted with the Ca(V)2.3 II-III linker. We then performed whole cell patch clamp recordings to test the effects of a putative interacting syntaxin 1A N-terminus peptide with Ca(V)2.2 and Ca(V)2.3 channels in a recombinant expression system. A YFP-tagged peptide corresponding to the N-terminal 10 residues of the syntaxin 1A Ha domain was sufficient to allosterically inhibit both Ca(V)2.2 and Ca(V)2.3 channel function but had no effect on G-protein mediated inhibition. Our results support a model of bipartite functional interactions between syntaxin 1A and Ca(V)2.2 channels and add accuracy to the two putative interacting domains, consistent with previous studies. Furthermore, we highlight the syntaxin 1A N-terminus as the minimal determinant for functional regulation of Ca(V)2.2 and Ca(V)2.3 channels.Item Open Access Block of T-type calcium channels by protoxins I and II(BioMed Central, 2014-05-09) Bladen, Chris; Hamid, Jawed; Souza, Ivana A.; Zamponi, Gerald W.Item Open Access The Brugada syndrome mutation A39V does not affect surface expression of neuronal rat Cav1.2 channels(BioMed Central, 2012-04-02) Simms, Brett A.; Zamponi, Gerald W.Item Open Access The Brugada syndrome mutation A39V does not affect surface expression of neuronal rat Cav1.2 channels(BioMed Central Ltd., 2012-03-02) Simms, Brent A.; Zamponi, Gerald W.A loss of function of the L-type calcium channel, Cav1.2, results in a cardiac specific disease known as Brugada syndrome. Although many Brugada syndrome channelopathies reduce channel function, one point mutation in the N-terminus of Cav1.2 (A39V) has been shown to elicit disease a phenotype because of a loss of surface trafficking of the channel. This lack of cell membrane expression could not be rescued by the trafficking chaperone Cavβ.Item Open Access Calcium channel inactivation(2003) Stotz, Stephanie Christine; Zamponi, Gerald W.Item Open Access Calcium Regulation of a Slow AHP in Hippocampal Pyramidal Cells(2018-04-12) Miclat, Jason; Turner, Raymond Joseph; Whelan, Patrick J.; Zamponi, Gerald W.A slow afterhyperpolarization (sAHP) in CA1 hippocampal pyramidal cells is calcium-dependent and involves activation of KCa3.1 and KCNQ potassium channels. Both KCa3.1 and KCNQ channels bind calmodulin (CaM) as a calcium sensor, with an additional role proposed for the protein hippocalcin. We measured the calcium sensitivity of the IsAHP and potassium channel associations with CaM or hipppocalcin. In recordings in hippocampal tissue slices in vitro the KCa3.1-mediated IsAHP steadily increased over 10 min equilibration to 0 µM calcium electrolyte, but decreased upon infusing 1 µM calcium, while the KCNQ-mediated IsAHP responded in an opposite manner. KCa3.1 channels contributed to the IsAHP in rat but not mouse pyramidal cells. Coimmunoprecipitation occurred between KCa3.1 and both CaM and hippocalcin, but only KCNQ and CaM. These data reveal a species-specific expression pattern for two potassium channels and in calcium sensor proteins present to generate the sAHP in the same class of hippocampal neuron.Item Open Access The Cavβ subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels(Nature, 2011-02) Altier, Christophe; You, Haitao; Chen, Lina; Walcher, Jan; Hermosilla, Tamara; García-Caballero, Agustín; Simms, Brent A.; Tedford, H. William; Zamponi, Gerald W.It is well established that the auxiliary Cavβ subunit regulates calcium channel density in the plasma membrane, but the cellular mechanism by which this occurs has remained unclear. We found that the Cavβ subunit increased membrane expression of Cav1.2 channels by preventing the entry of the channels into the endoplasmic reticulum-associated protein degradation (ERAD) complex. Without Cavβ, Cav1.2 channels underwent robust ubiquitination by the RFP2 ubiquitin ligase and interacted with the ERAD complex proteins derlin-1 and p97, culminating in targeting of the channels to the proteasome for degradation. On treatment with the proteasomal inhibitor MG132, Cavβ-free channels were rescued from degradation and trafficked to the plasma membrane. The coexpression of Cavβ interfered with ubiquitination and targeting of the channel to the ERAD complex, thereby facilitating export from the endoplasmic reticulum and promoting expression on the cell surface. Thus, Cavββ regulates the ubiquitination and stability of the calcium channel complex.Item Open Access CaVβ-subunit dependence of forward and reverse trafficking of CaV1.2 calcium channels(2022-05-09) Ferron, Laurent; Guderyan, Sydney D.; Smith, Ethan J.; Zamponi, Gerald W.Abstract Auxiliary CaVβ subunits interact with the pore forming CaVα1 subunit to promote the plasma membrane expression of high voltage-activated calcium channels and to modulate the biophysical properties of Ca2+ currents. However, the effect of CaVβ subunits on channel trafficking to and from the plasma membrane is still controversial. Here, we have investigated the impact of CaVβ1b and CaVβ2a subunits on plasma membrane trafficking of CaV1.2 using a live-labeling strategy. We show that the CaVβ1b subunit is more potent in increasing CaV1.2 expression at the plasma membrane than the CaVβ2a subunit and that this effect is not related to modification of intracellular trafficking of the channel (i.e. neither forward trafficking, nor recycling, nor endocytosis). We conclude that the differential effect of CaVβ subunit subtypes on CaV1.2 surface expression is likely due to their differential ability to protect CaV1.2 from degradation.Item Open Access CCR2 receptor ligands inhibit Cav3.2 T-type calcium channels(The American Society for Pharmacology and Experimental Therapeutics, 2010-02-01) You, Haitao; Altier, Christophe; Zamponi, Gerald W.Monocyte chemoattractant protein-1 (MCP-1) is a cytokine known to be involved in the recruitment of monocytes to sites of injury. MCP-1 activates the chemokine (C-C motif) receptor 2 (CCR2), a seven-transmembrane helix G protein-coupled receptor that has been implicated in inflammatory pain responses. Here we show that MCP-1 mediates activation of the CCR2 receptor and inhibits coexpressed N-type calcium channels in tsA-201 cells via a voltage-dependent pathway. Moreover, MCP-1 inhibits Ca(v)3.2 calcium channels, but not other members of the Cav3 calcium channel family, with nanomolar affinity. Unlike in N-type channels, this modulation does not require CCR2 receptor activation and seems to involve a direct action of the ligand on the channel. Whole-cell T-type calcium currents in acutely dissociated dorsal root ganglia neurons are effectively inhibited by MCP-1, consistent with the notion that these cells express Ca(v)3.2. The effects of MCP-1 were eliminated by heat denaturation. Furthermore, they were sensitive to the application of the divalent metal ion chelator diethylenetriaminepentaacetic acid, suggesting the possibility that metal ions may act as a cofactor. Finally, small organic CCR2 receptor antagonists inhibit Ca(v)3.2 and other members of the T-type channel family with micromolar affinity. Our findings provide novel avenues for the design of small organic inhibitors of T-type calcium channels for the treatment of pain and other T-type channel linked disorders.Item Open Access A cell-permeant peptide corresponding to the cUBP domain of USP5 reverses inflammatory and neuropathic pain(Sage Publishing, 2016-01-01) García-Caballero, Agustín; Gadotti, Vinícius Maria; Chen, Lina; Zamponi, Gerald W.Cav3.2 T-type calcium currents in primary afferents are enhanced in various painful pathological conditions, whereas inhibiting Cav3.2 activity or expression offers a strategy for combating the development of pain hypersensitivity. We have shown that Cav3.2 channel surface density is strongly regulated by the ubiquitination machinery and we identified the deubiquitinase USP5 as a Cav3.2 channel interacting protein and regulator of its cell surface expression. We also reported that USP5 is upregulated in chronic pain conditions. Conversely, preventing its binding to the channel in vivo mediates analgesia in inflammatory and neuropathic pain models.Item Open Access Cellular and Network Substrates of Neuronal Excitability in Relation to Epileptic Seizures(2007-07) Khosravani, Houman; Zamponi, Gerald W.; Federico, PaoloBrain function is, in part, maintained by an appropriate balance between excitatory and inhibitory elements. In relation to excitability, factors such as the complement and distribution of ion channels, properties and composition of synaptic proteins, and dynamics affecting network synchrony all interact to modulate neuronal firing and network activity. In this dissertation, I present a series of three focused studies at the level of ion channels (T-type calcium channels), synaptic transmission (prion protein), and network activity (high frequency oscillations) that affect neuronal excitability. With regards to Cav3.2 T-type voltage-gated calcium channels, I demonstrate that novel missense mutations, as identified in patients with idiopathic generalized epilepsies, can result in alteration of channel biophysical properties. The majority of mutants altered gating properties consistent with greater channel activity. However, most of these biophysical alterations were not large in magnitude suggesting that the role of these channels in relation to other cellular processes may be affected. At the level of synapses, I describe a novel interaction/modulation of NMDA receptor currents by the endogenous prion protein (PrP). Using PrP-null mice, I show that loss of PrP results in enhanced synaptic NMDA currents with greater amplitude and prolonged deactivation kinetics. These changes do not seem to be related to developmental effects and possibly involve an NMDA receptor subunit switch to functional receptors containing NR2D. At the network level, I show that high frequency oscillations in field recordings in vitro and in the EEG from patients with epilepsy are localized to the seizure onset zone and increase over time during the immediate pre-seizure period. This knowledge can be used to better localized seizures for surgical resection, thereby improving seizure control in intractable patients. These three topics and their relevance to hyperexcitable states are discussed in the context of epileptiform seizure activity and neurological disease.Item Open Access Cellular prion protein protects from inflammatory and neuropathic pain(BioMed Central Ltd., 2011-08-16) Gadotti, Vinícius Maria; Zamponi, Gerald W.Cellular prion protein (PrPC) inhibits N-Methyl-D-Aspartate (NMDA) receptors. Since NMDA receptors play an important role in the transmission of pain signals in the dorsal horn of spinal cord, we thus wanted to determine if PrPC null mice show a reduced threshold for various pain behaviours.We compared nociceptive thresholds between wild type and PrPC null mice in models of inflammatory and neuropathic pain, in the presence and the absence of a NMDA receptor antagonist. 2-3 months old male PrPC null mice exhibited an MK-801 sensitive decrease in the paw withdrawal threshold in response both mechanical and thermal stimuli. PrPC null mice also exhibited significantly longer licking/biting time during both the first and second phases of formalin-induced inflammation of the paw, which was again prevented by treatment of the mice with MK-801, and responded more strongly to glutamate injection into the paw. Compared to wild type animals, PrPC null mice also exhibited a significantly greater nociceptive response (licking/biting) after intrathecal injection of NMDA. Sciatic nerve ligation resulted in MK-801 sensitive neuropathic pain in wild-type mice, but did not further augment the basal increase in pain behaviour observed in the null mice, suggesting that mice lacking PrPC may already be in a state of tonic central sensitization. Altogether, our data indicate that PrPC exerts a critical role in modulating nociceptive transmission at the spinal cord level, and fit with the concept of NMDA receptor hyperfunction in the absence of PrPC.