Molecular cloning of the gene promoter encoding the human CaVγ2/Stargazin divergent transcript (CACNG2-DT): characterization and regulation by the cAMP-PKA/CREB signaling pathway.

CaVγ2 (Stargazin or TARPγ2) is a protein expressed in various types of neurons whose function was initially associated with a decrease in the functional expression of voltage-gated presynaptic Ca2+ channels (CaV) and which is now known to promote the trafficking of the postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) towards the cell membrane. Alterations in CaVγ2 expression has been associated with several neurological disorders, such as absence epilepsy. However, its regulation at the transcriptional level has not been intensively addressed. It has been reported that the promoter of the Cacng2 gene, encoding the rat CaVγ2, is bidirectional and regulates the transcription of a long non-coding RNA (lncRNA) in the antisense direction. Here, we investigate the proximal promoter region of the human CACNG2 gene in the antisense direction and show that this region includes two functional cAMP response elements that regulate the expression of a lncRNA called CACNG2-DT. The activity of these sites is significantly enhanced by forskolin, an adenylate cyclase activator, and inhibited by H89, a protein kinase A (PKA) antagonist. Therefore, this regulatory mechanism implies the activation of G protein-coupled receptors and downstream phosphorylation. Interestingly, we also found that the expression of CACNG2-DT may increase the levels of the CaVγ2 subunit. Together, these data provide novel information on the organization of the human CACNG2-DT gene promoter, describe modulatory domains and mechanisms that can mediate various regulatory inputs, and provide initial information on the molecular mechanisms that regulate the functional expression of the CaVγ2 protein.

Molecular Mechanisms of Nuclear Transport of the Neuronal Voltage-gated Ca2+ Channel ß3 Auxiliary Subunit.

Previous studies have shown that in addition to its role within the voltage-gated calcium channel complex in the plasma membrane, the neuronal CaVß subunit can translocate to the cell nucleus. However, little is known regarding the role this protein could play in the nucleus, nor the molecular mechanism used by CaVß to enter this cell compartment. This report shows evidence that CaVß3 has nuclear localization signals (NLS) that are not functional, suggesting that the protein does not use a classical nuclear import pathway. Instead, its entry into the nucleus could be associated with another protein that would function as a carrier, using a mechanism known as a piggyback. Mass spectrometry assays and bioinformatic analysis allowed the identification of proteins that could be participating in the entry of CaVß3 into the nucleus. Likewise, through proximity ligation assays (PLA), it was found that members of the heterogeneous nuclear ribonucleoproteins (hnRNPs) and B56δ, a regulatory subunit of the protein phosphatase 2A (PP2A), could function as proteins that regulate this piggyback mechanism. On the other hand, bioinformatics and site-directed mutagenesis assays allowed the identification of a functional nuclear export signal (NES) that controls the exit of CaVß3 from the nucleus, which would allow the completion of the nuclear transport cycle of the protein. These results reveal a novel mechanism for the nuclear transport cycle of the neuronal CaVß3 subunit.

Identification of Dp140 and α1-syntrophin as novel molecular interactors of the neuronal CaV2.1 channel.

The primary function of dystrophin is to form a link between the cytoskeleton and the extracellular matrix. In addition to this crucial structural function, dystrophin also plays an essential role in clustering and organizing several signaling proteins, including ion channels. Proteomic analysis of the whole rodent brain has stressed the role of some components of the dystrophin-associated glycoprotein complex (DGC) as potential interacting proteins of the voltage-gated Ca2+ channels of the CaV2 subfamily. The interaction of CaV2 with signaling and scaffolding proteins, such as the DGC components, may influence their function, stability, and location in neurons. This work aims to study the interaction between dystrophin and CaV2.1. Our immunoprecipitation data showed the presence of a complex formed by CaV2.1, CaVα2δ-1, CaVß4e, Dp140, and α1-syntrophin in the brain. Furthermore, proximity ligation assays (PLA) showed that CaV2.1 and CaVα2δ-1 interact with dystrophin in the hippocampus and cerebellum. Notably, Dp140 and α1-syntrophin increase CaV2.1 protein stability, half-life, permanence in the plasma membrane, and current density through recombinant CaV2.1 channels. Therefore, we have identified the Dp140 and α1-syntrophin as novel interaction partners of CaV2.1 channels in the mammalian brain. Consistent with previous findings, our work provides evidence of the role of DGC in anchoring and clustering CaV channels in a macromolecular complex.

Role of the Ca2+ channel α2δ-1 auxiliary subunit in proliferation and migration of human glioblastoma cells.

The overexpression of α2δ-1 is related to the development and degree of malignancy of diverse types of cancer. This protein is an auxiliary subunit of voltage-gated Ca2+ (CaV) channels, whose expression favors the trafficking of the main pore-forming subunit of the channel complex (α1) to the plasma membrane, thereby generating an increase in Ca2+ entry. Interestingly, TLR-4, a protein belonging to the family of toll-like receptors that participate in the inflammatory response and the transcription factor Sp1, have been linked to the progression of glioblastoma multiforme (GBM). Therefore, this report aimed to evaluate the role of the α2δ-1 subunit in the progression of GBM and investigate whether Sp1 regulates its expression after the activation of TLR-4. To this end, the expression of α2δ-1, TLR-4, and Sp1 was assessed in the U87 human glioblastoma cell line, and proliferation and migration assays were conducted using different agonists and antagonists. The actions of α2δ-1 were also investigated using overexpression and knockdown strategies. Initial luciferase assays and Western blot analyses showed that the activation of TLR-4 favors the transcription and expression of α2δ-1, which promoted the proliferation and migration of the U87 cells. Consistent with this, overexpression of α2δ-1, Sp1, and TLR-4 increased cell proliferation and migration, while their knockdown with specific siRNAs abrogated these actions. Our data also suggest that TLR-4-mediated regulation of α2δ-1 expression occurs through the NF-kB signaling pathway. Together, these findings strongly suggest that the activation of TLR-4 increases the expression of α2δ-1 in U87 cells, favoring their proliferative and migratory potential, which might eventually provide a theoretical basis to examine novel biomarkers and molecular targets for the diagnosis and treatment of GBM.

The role of voltage-gated calcium channels in the pathogenesis of Parkinson's disease.

Aim: Voltage-gated calcium (CaV) channels play an essential role in maintaining calcium homeostasis and regulating numerous physiological processes in neurons. Therefore, dysregulation of calcium signaling is relevant in many neurological disorders, including Parkinson's disease (PD). This review aims to introduce the role of CaV channels in PD and discuss some novel aspects of channel regulation and its impact on the molecular pathophysiology of the disease.Methods: an exhaustive search of the literature in the field was carried out using the PubMed database of The National Center for Biotechnology Information. Systematic searches were performed from the initial date of publication to May 2022.Results: Although α-synuclein aggregates are the main feature of PD, L-type calcium (CaV1) channels seem to play an essential role in the pathogenesis of PD. Changes in the functional expression of CaV1.3 channels alter Calcium homeostasis and contribute to the degeneration of dopaminergic neurons. Furthermore, recent studies suggest that CaV channel trafficking towards the cell membrane depends on the activity of the ubiquitin-proteasome system (UPS). In PD, there is an increase in the expression of L-type channels associated with a decrease in the expression of Parkin, an E3 enzyme of the UPS. Therefore, a link between Parkin and CaV channels could play a fundamental role in the pathogenesis of PD and, as such, could be a potentially attractive target for therapeutic intervention.Conclusion: The study of alterations in the functional expression of CaV channels will provide a framework to understand better the neurodegenerative processes that occur in PD and a possible path toward identifying new therapeutic targets to treat this condition.

Ion channel long non-coding RNAs in neuropathic pain.

Neuropathic pain is one of the primary forms of chronic pain and is the consequence of the somatosensory system's direct injury or disease. It is a relevant public health problem that affects about 10% of the world's general population. In neuropathic pain, alteration in neurotransmission occurs at various levels, including the dorsal root ganglia, the spinal cord, and the brain, resulting from the malfunction of diverse molecules such as receptors, ion channels, and elements of specific intracellular signaling pathways. In this context, there have been exciting advances in elucidating neuropathic pain's cellular and molecular mechanisms in the last decade, including the possible role that long non-coding RNAs (lncRNAs) may play, which open up new alternatives for the development of diagnostic and therapeutic strategies for this condition. This review focuses on recent studies associated with the possible relevance of lncRNAs in the development and maintenance of neuropathic pain through their actions on the functional expression of ion channels. Recognizing the changes in the function and spatio-temporal patterns of expression of these membrane proteins is crucial to understanding the control of neuronal excitability in chronic pain syndromes.