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Low-voltage-activated T-type Ca 2+ 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 Ca v 3.2-interacting protein. In addition, we show that co-expression of SCAMP2 in mammalian cells expressing recombinant Ca v 3.2 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 Ca v 3.1 and Ca v 3.3 channel isoforms. Finally, we show that SCAMP2-mediated knockdown of the T-type conductance is caused by the lack of Ca v 3.2 expression at the cell surface as evidenced by the concomitant loss of intramembrane charge movement without decrease of total Ca v 3.2 protein level. Taken together, our results indicate that SCAMP2 plays an important role in the trafficking of Ca v 3.2 channels at the plasma membrane., (© 2021. The Author(s).)

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 Na v 1.6 voltage-gated sodium and Ca v 3.2 voltage-gated calcium channels, respectively. In vitro functional analysis of human Na v 1.6 and Ca v 3.2 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., (© 2021. The Author(s).)

Mutations in the voltage-gated sodium channel Na v 1.1 (SCN1A) are linked to various epileptic phenotypes with different severities, however, the consequences of newly identified SCN1A variants on patient phenotype is uncertain so far. The functional impact of nine SCN1A variants, including five novel variants identified in this study, was studied using whole-cell patch-clamp recordings measurement of mutant Na v 1.1 channels expressed in HEK293T mammalian cells. E78X, W384X, E1587K, and R1596C channels failed to produce measurable sodium currents, indicating complete loss of channel function. E788K and M909K variants resulted in partial loss of function by exhibiting reduced current density, depolarizing shifts of the activation and hyperpolarizing shifts of the inactivation curves, and slower recovery from inactivation. Hyperpolarizing shifts of the activation and inactivation curves were observed in D249E channels along with slower recovery from inactivation. Slower recovery from inactivation was observed in E78D and T1934I with reduced current density in T1934I channels. Various functional effects were observed with the lack of sodium current being mainly associated with severe phenotypes and milder symptoms with less damaging channel alteration. In vitro functional analysis is thus fundamental for elucidation of the molecular mechanisms of epilepsy, to guide patients' treatment, and finally indicate misdiagnosis of SCN1A related epilepsies.

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 Ca v 3.2 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 Ca v 3.2 (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 Ca v 3.2 channel activity. The newly identified ΔI153 variant is the first to be reported to cause a complete loss of Ca v 3.2 channel function. These findings add to the notion that loss-of-function of Ca v 3.2 channels associated with rare CACNA1H variants may be risk factors in the complex etiology of ALS.

Voltage-gated Ca 2+ channels are embedded in a network of protein interactions that are fundamental for channel function and modulation. Different strategies such as high-resolution quantitative MS analyses and yeast-two hybrid screens have been used to uncover these Ca 2+ channel nanodomains. We applied the yeast split-ubiquitin system with its specific advantages to search for interaction partners of the Ca V 2.2 Ca 2+ channel and identified four proteins: reticulon 1 (RTN1), member 1 of solute carrier family 38 (SLC38), prostaglandin D2 synthase (PTGDS) and transmembrane protein 223 (TMEM223). Interactions were verified using the yeast split-ubiquitin system and narrowed down to Ca V 2.2 domain IV. Colocalization studies using fluorescent constructs demonstrated defined regions of subcellular localization. Detailed electrophysiological studies revealed that coexpression of RTN1 modulated Ca V 2.2 channels only to a minor extent. SLC38 accelerated the cumulative current inactivation during a high-frequency train of brief depolarizing pulses. As neurons expressing Ca V 2.2 channels were exposed to high-frequency bursts under physiological conditions, observed regulation may have a negative modulatory effect on transmitter release. Coexpression of PTGDS significantly lowered the average current density and slowed the kinetics of cumulative current inactivation. Since the latter effect was not significant, it may only partly compensate the first one under physiological conditions. Expression of TMEM223 lowered the average current density, accelerated the kinetics of cumulative current inactivation and slowed the kinetics of recovery from inactivation. Therefore, TMEM223 and, to a lesser extent, PTGDS, may negatively modulate Ca 2+ entry required for transmitter release and/or for dendritic plasticity under physiological conditions.

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 Ca v 3.3 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.

N-type or Ca V 2.2 high-voltage activated calcium channels are distinguished by exclusively neuronal tissue distribution, sensitivity to ω-conotoxins, prominent inhibition by G-proteins, and a unique role in nociception. Most investigated modulatory pathway regulating the Ca V 2.2 channels is G-protein-coupled receptor-activated pathway leading to current inhibition by Gβγ subunit of G-protein. Binding of Gβγ dimer to α 1 subunit of the Ca V 2.2 channel transfers the channel form "willing" to "reluctant" gating state. Channel phosphorylation by protein kinase C potentiates N-type calcium current. Ca V 2.2 channels could be functionally regulated also by a number of protein-protein interactions. Ca V 2.2 null mice are hyposensitive to inflammatory and neuropathic pain, otherwise they have a mild phenotype. Consistent with the mild phenotype of the Ca V 2.2-/- mice, reports on mutations linked to a disease phenotype are scarce. Only one mutation related to human heritable diseases was identified until now. Pharmaceutical inhibition of Ca V 2.2 channels either by direct inhibition of the channel, by an activation of G-protein coupled receptors, or by inhibition of membrane targeting of the channel protein are promising strategies for treatment of severe chronic and/or neuropathic pain.

Contributions of voltage sensing S4 segments in domains I - IV of Ca V 3.1 channel to channel activation were analyzed. Neutralization of the uppermost charge in individual S4 segments by exchange of arginine for cysteine was employed. Mutant channels with single exchange in domains I - IV, in two adjacent domains, and in all four domains were constructed and expressed in HEK 293 cells. Changes in maximal gating charge Q max and the relation between Q max and maximal conductance G max were evaluated. Q max was the most affected by single mutation in domain I and by double mutations in domains I + II and I + IV. The ratio G max /Q max proportional to opening probability of the channel was significantly decreased by the mutation in domain III and increased by mutations in domains I and II. In channels containing double mutations G max /Q max ratio increased significantly when the mutation in domain I was included. Mutations in domains II and III zeroed each other. Mutation in domain IV prevented the decrease caused by the mutation in domain III. Neither ion current nor gating current was observed when channels with quadruple mutations were expressed. Immunocytochemistry analysis did not reveal the presence of channel protein in the cell membrane. Likely, quadruple mutation results in a structural change that affects the channel's trafficking mechanism. Altogether, S4 segments in domains I-IV of the Ca V 3.1 channel unequally contribute to channel gating by voltage. We suggest the most important role of the voltage sensor in the domain I and lesser roles of voltage sensors in domains II and III.