Missense mutations in the human secretary carrier-associated membrane protein 5 (SCAMP5) cause a variety of neurological disorders including neurodevelopmental delay, epilepsy, and Parkinson's disease. We recently documented the importance of SCAMP2 in the regulation of T-type calcium channel expression in the plasma membrane. Here, we show that similar to SCAMP2, the co-expression of SCAMP5 in tsA-201 cells expressing recombinant Cav3.1, Cav3.2, and Cav3.3 channels nearly abolished whole-cell T-type currents. Recording of intramembrane charge movements revealed that SCAMP5-induced inhibition of T-type currents is primarily caused by the reduced expression of functional channels in the plasma membrane. Moreover, we show that SCAMP5-mediated downregulation of Cav3.2 channels is essentially preserved with disease-causing SCAMP5 R91W and G180W mutations. Hence, this study extends our previous findings with SCAMP2 and indicates that SCAMP5 also contributes to repressing the expression of T-type channels in the plasma membrane.

• Keywords
• Calcium channels, Channelopathy, Ion channels, SCAMP5, Secretory carrier-associated membrane protein 5, T-type channels,
• MeSH
• Cell Membrane MeSH
• Down-Regulation MeSH
• Humans MeSH
• Membrane Proteins genetics   MeSH
• Mutation MeSH
• Calcium Channels, T-Type * genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH
• SCAMP5 protein, human MeSH Browser
• Calcium Channels, T-Type * MeSH

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.

• Keywords
• CACNA1H, Calcium channel, Cav3.2 channel, Channelopathy, Ion channel, Trigeminal neuralgia,
• MeSH
• Electrophysiological Phenomena MeSH
• Humans MeSH
• Membrane Potentials MeSH
• Trigeminal Neuralgia * genetics   MeSH
• Neurons MeSH
• Calcium Channels MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CACNA1H protein, human MeSH Browser
• Calcium Channels MeSH

Low-voltage-activated T-type Ca2+ channels are key regulators of neuronal excitability both in the central and peripheral nervous systems. Therefore, their recruitment at the plasma membrane is critical in determining firing activity patterns of nerve cells. In this study, we report the importance of secretory carrier-associated membrane proteins (SCAMPs) in the trafficking regulation of T-type channels. We identified SCAMP2 as a novel Cav3.2-interacting protein. In addition, we show that co-expression of SCAMP2 in mammalian cells expressing recombinant Cav3.2 channels caused an almost complete drop of the whole cell T-type current, an effect partly reversed by single amino acid mutations within the conserved cytoplasmic E peptide of SCAMP2. SCAMP2-induced downregulation of T-type currents was also observed in cells expressing Cav3.1 and Cav3.3 channel isoforms. Finally, we show that SCAMP2-mediated knockdown of the T-type conductance is caused by the lack of Cav3.2 expression at the cell surface as evidenced by the concomitant loss of intramembrane charge movement without decrease of total Cav3.2 protein level. Taken together, our results indicate that SCAMP2 plays an important role in the trafficking of Cav3.2 channels at the plasma membrane.

• Keywords
• Calcium channels, Cav3.2 channels, Ion channels, SCAMP2, Secretory carrier-associated membrane protein 2, T-type channels, Trafficking,
• MeSH
• Cell Membrane metabolism   MeSH
• Membrane Proteins metabolism   MeSH
• Neurons metabolism   MeSH
• Mammals metabolism   MeSH
• Carrier Proteins metabolism   MeSH
• Calcium metabolism   MeSH
• Calcium Channels, T-Type * metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Membrane Proteins MeSH
• Carrier Proteins MeSH
• Calcium MeSH
• Calcium Channels, T-Type * MeSH

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.

• Keywords
• Asparagine-linked glycosylation, Calcium channel, N-glycosylation, Non-canonical glycosylation, T-type channel, Trafficking, cav3.2 Channel,
• MeSH
• Amino Acid Motifs MeSH
• Asparagine metabolism   MeSH
• Glycosylation MeSH
• Humans MeSH
• Calcium Channels, T-Type chemistry   metabolism   MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Asparagine MeSH
• CACNA1H protein, human MeSH Browser
• Calcium Channels, T-Type MeSH

Low-voltage-activated Cav3 calcium channels (T-type) play an essential role in the functioning of the nervous system where they support oscillatory activities that relie on several channel molecular determinants that shape their unique gating properties. In a previous study, we documented the important role of the carboxy proximal region in the functioning of Cav3.3 channels. Here, we explore the ability of a TAT-based cell penetrating peptide containing this carboxy proximal region (TAT-C3P) to modulate the activity of Cav3 channels. We show that chronic application of TAT-C3P on tsA-201 cells expressing Cav3 channels selectively inhibits Cav3.3 channels without affecting Cav3.1 and Cav3.2 channels. Therefore, the TAT-C3P peptide described in this study represents a new tool to address the specific physiological role of Cav3.3 channels, and to potentially enhance our understanding of Cav3.3 in disease.

• Keywords
• Calcium channel, Cav3.3 channel, Inhibitor, T-type channel, TAT-peptide,
• MeSH
• Cell Line MeSH
• Humans MeSH
• Models, Molecular MeSH
• Neurons metabolism   MeSH
• Peptides metabolism   MeSH
• Amino Acid Sequence MeSH
• Calcium Channels, T-Type chemistry   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Peptides MeSH
• Calcium Channels, T-Type MeSH

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.

• Keywords
• ALS, Amyotrophic lateral sclerosis, Biophysics, CACNA1H, Calcium channel, Cav3.2 channel, Motor neuron disease, Mutation, T-type channel,
• MeSH
• Amyotrophic Lateral Sclerosis * genetics   MeSH
• Genes, Dominant MeSH
• Genetic Predisposition to Disease * MeSH
• Genetic Association Studies * MeSH
• Heterozygote MeSH
• Rats MeSH
• Humans MeSH
• Mutation * genetics   MeSH
• Amino Acid Sequence MeSH
• Whole Genome Sequencing MeSH
• Structural Homology, Protein MeSH
• Calcium Channels, T-Type * chemistry   genetics   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CACNA1H protein, human MeSH Browser
• Calcium Channels, T-Type * MeSH

Neuromuscular disorders encompass a wide range of conditions often associated with a genetic component. In the present study, we report a patient with severe infantile-onset amyotrophy in whom two compound heterozygous variants in the gene CACNA1H encoding for Cav3.2 T-type calcium channels were identified. Functional analysis of Cav3.2 variants revealed several alterations of the gating properties of the channel that were in general consistent with a loss-of-channel function. Taken together, these findings suggest that severe congenital amyoplasia may be related to CACNA1H and would represent a new phenotype associated with mutations in this gene.

• Keywords
• CACNA1H, Ca3.2 channel, Congenital amyotrophy, T-type channel, calcium channel, mutations,
• MeSH
• Brachial Plexus Neuritis genetics   physiopathology   MeSH
• Electrophysiology MeSH
• Phenotype MeSH
• Heterozygote MeSH
• Infant MeSH
• Humans MeSH
• Mutation, Missense * MeSH
• Exome Sequencing MeSH
• Calcium Channels, T-Type chemistry   genetics   physiology   MeSH
• Check Tag
• Infant MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CACNA1H protein, human MeSH Browser
• Calcium Channels, T-Type MeSH

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.

• Keywords
• Cav3.3 channel, Electrophysiology, Gating, T-type channels,
• MeSH
• Ion Channel Gating * MeSH
• HEK293 Cells MeSH
• Kinetics MeSH
• Humans MeSH
• Amino Acid Sequence MeSH
• Calcium Channels, T-Type metabolism   MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CACNA1I protein, human MeSH Browser
• Calcium Channels, T-Type MeSH