Browsing by Author "Gandini, Maria A"

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    A CACNA1A variant associated with trigeminal neuralgia alters the gating of Cav2.1 channels
    (2021-01-07) Gambeta, Eder; Gandini, Maria A; Souza, Ivana A; Ferron, Laurent; Zamponi, Gerald W
    Abstract A novel missense mutation in the CACNA1A gene that encodes the pore forming α1 subunit of the CaV2.1 voltage-gated calcium channel was identified in a patient with trigeminal neuralgia. This mutation leads to a substitution of proline 2455 by histidine (P2455H) in the distal C-terminus region of the channel. Due to the well characterized role of this channel in neurotransmitter release, our aim was to characterize the biophysical properties of the P2455H variant in heterologously expressed CaV2.1 channels. Whole-cell patch clamp recordings of wild type and mutant CaV2.1 channels expressed in tsA-201 cells reveal that the mutation mediates a depolarizing shift in the voltage-dependence of activation and inactivation. Moreover, the P2455H mutant strongly reduced calcium-dependent inactivation of the channel that is consistent with an overall gain of function. Hence, the P2455H CaV2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in CaV2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.
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    Cav3.2 calcium channel interactions with the epithelial sodium channel ENaC
    (2019-02-08) Garcia-Caballero, Agustin; Gandini, Maria A; Huang, Shuo; Chen, Lina; Souza, Ivana A; Dang, Yan L; Stutts, M. J; Zamponi, Gerald W
    Abstract This study describes the functional interaction between Cav3.2 calcium channels and the Epithelial Sodium Channel (ENaC). β-ENaC subunits showed overlapping expression with endogenous Cav3.2 calcium channels in the thalamus and hypothalamus as detected by immunostaining. Moreover, β- and γ-ENaC subunits could be co-immunoprecipitated with Cav3.2 calcium channels from brain lysates, dorsal horn and lumbar dorsal root ganglia. Mutation of a cluster of lysines present in the intracellular N-terminus region of β-ENaC (K4R/ K5R/ K9R/ K16R/ K23R) reduced interactions with Cav3.2 calcium channels. Αβγ-ENaC channels enhanced Cav3.2 calcium channel trafficking to the plasma membrane in tsA-201 cells. This effect was reciprocal such that Cav3.2 channel expression also enhanced β-ENaC trafficking to the cell surface. T-type current density was increased when fully assembled αβγ-ENaC channels were transiently expressed in CAD cells, a neuronal derived cell line. Altogether, these findings reveal ENaC as an interactor and potential regulator of Cav3.2 calcium channels expressed in neuronal tissues.
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    Differential regulation of Cav2.2 channel exon 37 variants by alternatively spliced μ-opioid receptors
    (2019-11-27) Gandini, Maria A; Souza, Ivana A; Raval, Dvij; Xu, Jin; Pan, Ying-Xian; Zamponi, Gerald W
    Abstract We have examined the regulation of mutually exclusive Cav2.2 exon 37a and b variants by the mouse μ-opioid receptor (mMOR) C-terminal splice variants 1, 1C and 1O in tsA-201 cells. Electrophysiological analyses revealed that both channel isoforms exhibit DAMGO-induced voltage-dependent (Gβγ-mediated) inhibition and its recovery by voltage pre-pulses, as well as a voltage-independent component. However, the two channel isoforms differ in their relative extent of voltage-dependent and independent inhibition, with Cav2.2-37b showing significantly more voltage-dependent inhibition upon activation of the three mMOR receptors studied. In addition, coexpression of either mMOR1 or mMOR1C results in an agonist-independent reduction in the peak current density of Cav2.2-37a channels, whereas the peak current density of Cav2.2-37b does not appear to be affected. Interestingly, this decrease is not due to an effect on channel expression at the plasma membrane, as demonstrated by biotinylation experiments. We further examined the mechanism underlying the agonist-independent modulation of Cav2.2-37a by mMOR1C. Incubation of cells with pertussis toxin did not affect the mMOR1C mediated inhibition of Cav2.2-37a currents, indicating a lack of involvement of Gi/o signaling. However, when a Src tyrosine kinase inhibitor was applied, the effect of mMOR1C was lost. Moreover, when we recorded currents using a Cav2.2-37a mutant in which tyrosine 1747 was replaced with phenylalanine (Y1747F), the agonist independent effects of mMOR1C were abolished. Altogether our findings show that Cav2.2-37a and Cav2.2-37b isoforms are subject to differential regulation by C-terminal splice variants of mMORs, and that constitutive mMOR1C activity and downstream tyrosine kinase activity exert a selective inhibition of the Cav2.2-37a splice variant, an N-type channel isoform that is highly enriched in nociceptors. Our study provides new insights into the roles of the MOR full-length C-terminal variants in modulating Cav2.2 channel isoform activities.
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    Functional identification of potential non-canonical N-glycosylation sites within Cav3.2 T-type calcium channels
    (2020-11-11) Ficelova, Vendula; Souza, Ivana A; Cmarko, Leos; Gandini, Maria A; Stringer, Robin N; Zamponi, Gerald W; Weiss, Norbert
    Abstract Low-voltage-activated T-type calcium channels are important contributors to nervous system function. Post-translational modification of these channels has emerged as an important mechanism to control channel activity. Previous studies have documented the importance of asparagine (N)-linked glycosylation and identified several asparagine residues within the canonical consensus sequence N-X-S/T that is essential for the expression and function of Cav3.2 channels. Here, we explored the functional role of non-canonical N-glycosylation motifs in the conformation N-X-C based on site directed mutagenesis. Using a combination of electrophysiological recordings and surface biotinylation assays, we show that asparagines N345 and N1780 located in the motifs NVC and NPC, respectively, are essential for the expression of the human Cav3.2 channel in the plasma membrane. Therefore, these newly identified asparagine residues within non-canonical motifs add to those previously reported in canonical sites and suggest that N-glycosylation of Cav3.2 may also occur at non-canonical motifs to control expression of the channel in the plasma membrane. It is also the first study to report the functional importance of non-canonical N-glycosylation motifs in an ion channel.
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    Interactions of Rabconnectin-3 with Cav2 calcium channels
    (2019-06-28) Gandini, Maria A; Souza, Ivana A; Fan, Jing; Li, Katherine; Wang, Decheng; Zamponi, Gerald W
    Abstract This study describes the interaction between Cav2 calcium channels and Rabconnectin-3, a di-subunit protein that is associated with synaptic vesicles. Immunostaining reveals that both Rabconnectin-3α (RB-3α) and Rabconnectin-3β (RB-3β) are colocalized in mouse hippocampal neurons. Co-immunoprecipitations from brain tissue is consistent with the formation of a protein complex between RB-3α and RB-3β and both Cav2.2 and the related Cav2.1 calcium channel. The coexpression of either RB-3α or RB-3β with Cav2.2 calcium channels in tsA-201 cells led to a reduction in Cav2.2 current density without any effects on the voltage-dependence of activation or inactivation. Coexpression of both Rabconnectin-3 subunits did not cause an additive effect on current densities. Finally, the presence of Rabconnectin-3 did not interfere with μ-opioid receptor mediated Gβγ modulation of Cav2.2 channels. Altogether, our findings show that Rabconnectin-3 has the propensity to regulate calcium entry mediated by Cav2.2 channels.
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    Pathogenic Cav3.2 channel mutation in a child with primary generalized epilepsy
    (2019-10-24) Souza, Ivana A; Gandini, Maria A; Zhang, Fang-Xiong; Mitchell, Wendy G; Matsumoto, Joyce; Lerner, Jason; Pierson, Tyler M; Zamponi, Gerald W
    Abstract Two paternally-inherited missense variants in CACNA1H were identified and characterized in a 6-year-old child with generalized epilepsy. Febrile and unprovoked seizures were present in this child. Both variants were expressed in cis or isolation using human recombinant Cav3.2 calcium channels in tsA-201 cells. Whole-cell patch-clamp recordings indicated that one variant (c.3844C > T; p.R1282W) caused a significant increase in current density consistent with a pathogenic gain-of-function phenotype; while the other cis-related variant (c.5294C > T; p.A1765V) had a benign profile.
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    Rare functional missense variants in CACNA1H: What can we learn from Writer’s cramp?
    (2021-01-21) Huang, Miaozhen; Nibbeling, Esther A R; Lagrand, Tjerk J; Souza, Ivana A; Groen, Justus L; Gandini, Maria A; Zhang, Fang-Xiong; Koelman, Johannes H T M; Adir, Noam; Sinke, Richard J; Zamponi, Gerald W; Tijssen, Marina A J; Verbeek, Dineke S
    Abstract Writer’s cramp (WC) is a task-specific focal dystonia that occurs selectively in the hand and arm during writing. Previous studies have shown a role for genetics in the pathology of task-specific focal dystonia. However, to date, no causal gene has been reported for task-specific focal dystonia, including WC. In this study, we investigated the genetic background of a large Dutch family with autosomal dominant‒inherited WC that was negative for mutations in known dystonia genes. Whole exome sequencing identified 4 rare variants of unknown significance that segregated in the family. One candidate gene was selected for follow-up, Calcium Voltage-Gated Channel Subunit Alpha1 H, CACNA1H, due to its links with the known dystonia gene Potassium Channel Tetramerization Domain Containing 17, KCTD17, and with paroxysmal movement disorders. Targeted resequencing of CACNA1H in 82 WC cases identified another rare, putative damaging variant in a familial WC case that did not segregate. Using structural modelling and functional studies in vitro, we show that both the segregating p.Arg481Cys variant and the non-segregating p.Glu1881Lys variant very likely cause structural changes to the Cav3.2 protein and lead to similar gains of function, as seen in an accelerated recovery from inactivation. Both mutant channels are thus available for re-activation earlier, which may lead to an increase in intracellular calcium and increased neuronal excitability. Overall, we conclude that rare functional variants in CACNA1H need to be interpreted very carefully, and additional studies are needed to prove that the p.Arg481Cys variant is the cause of WC in the large Dutch family.
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    The de novo CACNA1A pathogenic variant Y1384C associated with hemiplegic migraine, early onset cerebellar atrophy and developmental delay leads to a loss of Cav2.1 channel function
    (2021-02-08) Gandini, Maria A; Souza, Ivana A; Ferron, Laurent; Innes, A. M; Zamponi, Gerald W
    Abstract CACNA1A pathogenic variants have been linked to several neurological disorders including familial hemiplegic migraine and cerebellar conditions. More recently, de novo variants have been associated with severe early onset developmental encephalopathies. CACNA1A is highly expressed in the central nervous system and encodes the pore-forming CaVα1 subunit of P/Q-type (Cav2.1) calcium channels. We have previously identified a patient with a de novo missense mutation in CACNA1A (p.Y1384C), characterized by hemiplegic migraine, cerebellar atrophy and developmental delay. The mutation is located at the transmembrane S5 segment of the third domain. Functional analysis in two predominant splice variants of the neuronal Cav2.1 channel showed a significant loss of function in current density and changes in gating properties. Moreover, Y1384 variants exhibit differential splice variant-specific effects on recovery from inactivation. Finally, structural analysis revealed structural damage caused by the tyrosine substitution and changes in electrostatic potentials.