Browsing by Author "Weiss, Norbert"

Now showing 1 - 9 of 9
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    A rare CACNA1H variant associated with amyotrophic lateral sclerosis causes complete loss of Cav3.2 T-type channel activity
    (2020-03-06) Stringer, Robin N; Jurkovicova-Tarabova, Bohumila; Huang, Sun; Haji-Ghassemi, Omid; Idoux, Romane; Liashenko, Anna; Souza, Ivana A; Rzhepetskyy, Yuriy; Lacinova, Lubica; Van Petegem, Filip; Zamponi, Gerald W; Pamphlett, Roger; Weiss, Norbert
    Abstract Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of cortical, brain stem and spinal motor neurons that leads to muscle weakness and death. A previous study implicated CACNA1H encoding for Cav3.2 calcium channels as a susceptibility gene in ALS. In the present study, two heterozygous CACNA1H variants were identified by whole genome sequencing in a small cohort of ALS patients. These variants were functionally characterized using patch clamp electrophysiology, biochemistry assays, and molecular modeling. A previously unreported c.454GTAC > G variant produced an inframe deletion of a highly conserved isoleucine residue in Cav3.2 (p.ΔI153) and caused a complete loss-of-function of the channel, with an additional dominant-negative effect on the wild-type channel when expressed in trans. In contrast, the c.3629C > T variant caused a missense substitution of a proline with a leucine (p.P1210L) and produced a comparatively mild alteration of Cav3.2 channel activity. The newly identified ΔI153 variant is the first to be reported to cause a complete loss of Cav3.2 channel function. These findings add to the notion that loss-of-function of Cav3.2 channels associated with rare CACNA1H variants may be risk factors in the complex etiology of ALS.
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    Control of low-threshold exocytosis by T-type calcium channels
    (Elsevier, 2013-07) Weiss, Norbert; Zamponi, Gerald W.
    Low-voltage-activated (LVA) T-type Ca²⁺ channels differ from their high-voltage-activated (HVA) homologues by unique biophysical properties. Hence, whereas HVA channels convert action potentials into intracellular Ca²⁺ elevations, T-type channels control Ca²⁺ entry during small depolarizations around the resting membrane potential. They play an important role in electrical activities by generating low-threshold burst discharges that occur during various physiological and pathological forms of neuronal rhythmogenesis. In addition, they mediate a previously unrecognized function in the control of synaptic transmission where they directly trigger the release of neurotransmitters at rest. In this review, we summarize our present knowledge of the role of T-type Ca²⁺ channels in vesicular exocytosis, and emphasize the critical importance of localizing the exocytosis machinery close to the Ca²⁺ source for reliable synaptic transmission. This article is part of a Special Issue entitled: Calcium channels.
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    De novo SCN8A and inherited rare CACNA1H variants associated with severe developmental and epileptic encephalopathy
    (2021-08-16) Stringer, Robin N.; Jurkovicova-Tarabova, Bohumila; Souza, Ivana A.; Ibrahim, Judy; Vacik, Tomas; Fathalla, Waseem M.; Hertecant, Jozef; Zamponi, Gerald W.; Lacinova, Lubica; Weiss, Norbert
    Abstract Developmental and epileptic encephalopathies (DEEs) are a group of severe epilepsies that are characterized by seizures and developmental delay. DEEs are primarily attributed to genetic causes and an increasing number of cases have been correlated with variants in ion channel genes. In this study, we report a child with an early severe DEE. Whole exome sequencing showed a de novo heterozygous variant (c.4873–4881 duplication) in the SCN8A gene and an inherited heterozygous variant (c.952G > A) in the CACNA1H gene encoding for Nav1.6 voltage-gated sodium and Cav3.2 voltage-gated calcium channels, respectively. In vitro functional analysis of human Nav1.6 and Cav3.2 channel variants revealed mild but significant alterations of their gating properties that were in general consistent with a gain- and loss-of-channel function, respectively. Although additional studies will be required to confirm the actual pathogenic involvement of SCN8A and CACNA1H, these findings add to the notion that rare ion channel variants may contribute to the etiology of DEEs.
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    Electrophysiological and computational analysis of Cav3.2 channel variants associated with familial trigeminal neuralgia
    (2022-11-17) Mustafá, Emilio R.; Gambeta, Eder; Stringer, Robin N.; Souza, Ivana A.; Zamponi, Gerald W.; Weiss, Norbert
    Abstract Trigeminal neuralgia (TN) is a rare form of chronic neuropathic pain characterized by spontaneous or elicited paroxysms of electric shock-like or stabbing pain in a region of the face. While most cases occur in a sporadic manner and are accompanied by intracranial vascular compression of the trigeminal nerve root, alteration of ion channels has emerged as a potential exacerbating factor. Recently, whole exome sequencing analysis of familial TN patients identified 19 rare variants in the gene CACNA1H encoding for Cav3.2T-type calcium channels. An initial analysis of 4 of these variants pointed to a pathogenic role. In this study, we assessed the electrophysiological properties of 13 additional TN-associated Cav3.2 variants expressed in tsA-201 cells. Our data indicate that 6 out of the 13 variants analyzed display alteration of their gating properties as evidenced by a hyperpolarizing shift of their voltage dependence of activation and/or inactivation resulting in an enhanced window current supported by Cav3.2 channels. An additional variant enhanced the recovery from inactivation. Simulation of neuronal electrical membrane potential using a computational model of reticular thalamic neuron suggests that TN-associated Cav3.2 variants could enhance neuronal excitability. Altogether, the present study adds to the notion that ion channel polymorphisms could contribute to the etiology of some cases of TN and further support a role for Cav3.2 channels.
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    Electrophysiological characterization of a Cav3.2 calcium channel missense variant associated with epilepsy and hearing loss
    (2023-09-21) Stringer, Robin N.; Cmarko, Leos; Zamponi, Gerald W.; De Waard, Michel; Weiss, Norbert
    Abstract T-type calcium channelopathies encompass a group of human disorders either caused or exacerbated by mutations in the genes encoding different T-type calcium channels. Recently, a new heterozygous missense mutation in the CACNA1H gene that encodes the Cav3.2 T-type calcium channel was reported in a patient presenting with epilepsy and hearing loss—apparently the first CACNA1H mutation to be associated with a sensorineural hearing condition. This mutation leads to the substitution of an arginine at position 132 with a histidine (R132H) in the proximal extracellular end of the second transmembrane helix of Cav3.2. In this study, we report the electrophysiological characterization of this new variant using whole-cell patch clamp recordings in tsA-201 cells. Our data reveal minor gating alterations of the channel evidenced by a mild increase of the T-type current density and slower recovery from inactivation, as well as an enhanced sensitivity of the channel to external pH change. To what extend these biophysical changes and pH sensitivity alterations induced by the R132H mutation contribute to the observed pathogenicity remains an open question that will necessitate the analysis of additional CACNA1H variants associated with the same pathologies.
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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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    How do T-type calcium channels control low-threshold exocytosis?
    (Landes Bioscience, 2012-03-12) Weiss, Norbert; De Waard, Michel; Zamponi, Gerald W.
    Low-voltage-activated T-type calcium channels act as a major pathway for calcium entry near the resting membrane potential in a wide range of neuronal cell types. Several reports have uncovered an unrecognized feature of T-type channels in the control of vesicular neurotransmitter and hormone release, a process so far thought to be mediated exclusively by high-voltage-activated calcium channels. However, the underlying molecular mechanisms linking T-type calcium channels to vesicular exocytosis have remained enigmatic. In a recent study, we have reported that Ca(v)3.2 T-type channel forms a signaling complex with the neuronal Q-SNARE syntaxin-1A and SNAP-25. This interaction that relies on specific Ca(v)3.2 molecular determinants, not only modulates T-type channel activity, but was also found essential to support low-threshold exocytosis upon Ca(v)3.2 channel expression in MPC 9/3L-AH chromaffin cells. Overall, we have indentified an unrecognized regulation pathway of T-type calcium channels by SNARE proteins, and proposed the first molecular mechanism by which T-type channels could mediate low-threshold exocytosis.
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    Identification of a molecular gating determinant within the carboxy terminal region of Cav3.3 T-type channels
    (2019-04-08) Jurkovicova-Tarabova, Bohumila; Cmarko, Leos; Rehak, Renata; Zamponi, Gerald W; Lacinova, Lubica; Weiss, Norbert
    Abstract The physiological functions controlled by T-type channels are intrinsically dependent on their gating properties, and alteration of T-type channel activity is linked to several human disorders. Therefore, it is essential to develop a clear understanding of the structural determinants responsible for the unique gating features of T-type channels. Here, we have investigated the specific role of the carboxy terminal region by creating a series a deletion constructs expressed in tsA-201 cells and analyzing them by patch clamp electrophysiology. Our data reveal that the proximal region of the carboxy terminus contains a structural determinant essential for shaping several gating aspects of Cav3.3 channels, including voltage-dependence of activation and inactivation, inactivation kinetics, and coupling between the voltage sensing and the pore opening of the channel. Altogether, our data are consistent with a model in which the carboxy terminus stabilizes the channel in a closed state.
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    Reciprocal Regulation of Neuronal Calcium Channels by Synaptic Proteins
    (Springer, 2013-06-01) Weiss, Norbert; Zamponi, Gerald W.
    Voltage-gated Ca2C channels represent one of the main pathways for Ca2C entry into nerve terminals where they play a critical role in the control of synaptic exocytosis. It is traditionally believed that the vesicle-docking/release machinery must be located in the vicinity of the calcium source in order to trigger fast, efficient and spatially delimited neurotransmitter release. This tight coupling is mostly achieved by a physical interaction of the presynaptic calcium channel with several actors of the synaptic vesicle release machinery. Conversely, the binding of synaptic proteins regulates calcium channel activity, providing for fine control of presynaptic Ca2C entry. Here, we review the current state of knowledge of the molecular mechanisms by which synaptic proteins regulates presynaptic Ca2C channel activity.