The Influence of Sex Steroid Hormone Fluctuations on Capsaicin-Induced Pain and TRPV1 Expression.

Sexual dimorphism among mammals includes variations in the pain threshold. These differences are influenced by hormonal fluctuations in females during the estrous and menstrual cycles of rodents and humans, respectively. These physiological conditions display various phases, including proestrus and diestrus in rodents and follicular and luteal phases in humans, distinctly characterized by varying estrogen levels. In this study, we evaluated the capsaicin responses in male and female mice at different estrous cycle phases, using two murine acute pain models. Our findings indicate that the capsaicin-induced pain threshold was lower in the proestrus phase than in the other three phases in both pain assays. We also found that male mice exhibited a higher pain threshold than females in the proestrus phase, although it was similar to females in the other cycle phases. We also assessed the mRNA and protein levels of TRPV1 in the dorsal root and trigeminal ganglia of mice. Our results showed higher TRPV1 protein levels during proestrus compared to diestrus and male mice. Unexpectedly, we observed that the diestrus phase was associated with higher TRPV1 mRNA levels than those in both proestrus and male mice. These results underscore the hormonal influence on TRPV1 expression regulation and highlight the role of sex steroids in capsaicin-induced pain.

Association Study of CACNA1D, KCNJ11, KCNQ1, and CACNA1E Single-Nucleotide Polymorphisms with Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a complex chronic disease characterized by decreased insulin secretion and the development of insulin resistance. Previous genome-wide association studies demonstrated that single-nucleotide polymorphisms (SNPs) present in genes coding for ion channels involved in insulin secretion increase the risk of developing this disease. We determined the association of 16 SNPs found in CACNA1D, KCNQ1, KCNJ11, and CACNA1E genes and the increased probability of developing T2DM. In this work, we performed a case-control study in 301 Mexican adults, including 201 cases with diabetes and 100 controls without diabetes. Our findings indicate a moderate association between T2DM and the C allele, and the C/C genotype of rs312480 within CACNA1D. The CAG haplotype surprisingly showed a protective effect, whereas the CAC and CGG haplotypes have a strong association with T2DM. The C allele and C/C genotype of rs5219 were significantly associated with diabetes. Also, an association was observed between diabetes and the A allele and the A/A genotype of rs3753737 and rs175338 in CACNA1E. The TGG and CGA haplotypes were also found to be significantly associated. The findings of this study indicate that the SNPs examined could serve as a potential diagnostic tool and contribute to the susceptibility of the Mexican population to this disease.

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.

LD block disorder-specific pleiotropic roles of novel CRHR1 in type 2 diabetes and depression disorder comorbidity.

Major depressive disorder (MDD) and type 2 diabetes (T2D) are complex disorders whose comorbidity can be due to hypercortisolism and may be explained by dysfunction of the corticotropin-releasing hormone receptor 1 (CRHR1) and cortisol feedback within the hypothalamic-pituitary-adrenal axis (HPA axis). To investigate the role of the CRHR1 gene in familial T2D, MDD, and MDD-T2D comorbidity, we tested 152 CRHR1 single-nucleotide-polymorphisms (SNPs), via 2-point parametric linkage and linkage disequilibrium (LD; i.e., association) analyses using 4 models, in 212 peninsular families with T2D and MDD. We detected linkage/LD/association to/with MDD and T2D with 122 (116 novel) SNPs. MDD and T2D had 4 and 3 disorder-specific novel risk LD blocks, respectively, whose risk variants reciprocally confirm one another. Comorbidity was conferred by 3 novel independent SNPs. In silico analyses reported novel functional changes, including the binding site of glucocorticoid receptor-alpha [GR-α] on CRHR1 for transcription regulation. This is the first report of CRHR1 pleiotropic linkage/LD/association with peninsular familial MDD and T2D. CRHR1 contribution to MDD is stronger than to T2D and may antecede T2D onset. Our findings suggest a new molecular-based clinical entity of MDD-T2D and should be replicated in other ethnic groups.

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.

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.

Cdk5-Dependent Phosphorylation of CaV3.2 T-Type Channels: Possible Role in Nerve Ligation-Induced Neuropathic Allodynia and the Compound Action Potential in Primary Afferent C Fibers.

Voltage-gated T-type Ca2+ (CaV3) channels regulate diverse physiological events, including neuronal excitability, and have been linked to several pathological conditions such as absence epilepsy, cardiovascular diseases, and neuropathic pain. It is also acknowledged that calcium/calmodulin-dependent protein kinase II and protein kinases A and C regulate the activity of T-type channels. Interestingly, peripheral nerve injury induces tactile allodynia and upregulates CaV3.2 channels and cyclin-dependent kinase 5 (Cdk5) in dorsal root ganglia (DRG) and spinal dorsal horn. Here, we report that recombinant CaV3.2 channels expressed in HEK293 cells are regulatory targets of Cdk5. Site-directed mutagenesis showed that the relevant sites for this regulation are residues S561 and S1987. We also found that Cdk5 may regulate CaV3.2 channel functional expression in rats with mechanical allodynia induced by spinal nerve ligation (SNL). Consequently, the Cdk5 inhibitor olomoucine affected the compound action potential recorded in the spinal nerves, as well as the paw withdrawal threshold. Likewise, Cdk5 expression was upregulated after SNL in the DRG. These findings unveil a novel mechanism for how phosphorylation may regulate CaV3.2 channels and suggest that increased channel activity by Cdk5-mediated phosphorylation after SNL contributes nerve injury-induced tactile allodynia.SIGNIFICANCE STATEMENT Neuropathic pain is a current public health challenge. It can develop as a result of injury or nerve illness. It is acknowledged that the expression of various ion channels can be altered in neuropathic pain, including T-type Ca2+ channels that are expressed in sensory neurons, where they play a role in the regulation of cellular excitability. The present work shows that the exacerbated expression of Cdk5 in a preclinical model of neuropathic pain increases the functional expression of CaV3.2 channels. This finding is relevant for the understanding of the molecular pathophysiology of the disease. Additionally, this work may have a substantial translational impact, since it describes a novel molecular pathway that could represent an interesting therapeutic alternative for neuropathic pain.

TRPV1 channels and the progesterone receptor Sig-1R interact to regulate pain.

The Transient Receptor Potential Vanilloid 1 (TRPV1) ion channel is expressed in nociceptors where, when activated by chemical or thermal stimuli, it functions as an important transducer of painful and itch-related stimuli. Although the interaction of TRPV1 with proteins that regulate its function has been previously explored, their modulation by chaperones has not been elucidated, as is the case for other mammalian TRP channels. Here we show that TRPV1 physically interacts with the Sigma 1 Receptor (Sig-1R), a chaperone that binds progesterone, an antagonist of Sig-1R and an important neurosteroid associated to the modulation of pain. Antagonism of Sig-1R by progesterone results in the down-regulation of TRPV1 expression in the plasma membrane of sensory neurons and, consequently, a decrease in capsaicin-induced nociceptive responses. This is observed both in males treated with a synthetic antagonist of Sig-1R and in pregnant females where progesterone levels are elevated. This constitutes a previously undescribed mechanism by which TRPV1-dependent nociception and pain can be regulated.

Regulation of the Ca2+ channel α2δ-1 subunit expression by epidermal growth factor via the ERK/ELK-1 signaling pathway.

Voltage-gated Ca2+ (CaV) channels are expressed in endocrine cells where they contribute to hormone secretion. Diverse chemical messengers, including epidermal growth factor (EGF), are known to affect the expression of CaV channels. Previous studies have shown that EGF increases Ca2+ currents in GH3 pituitary cells by increasing the number of high voltage-activated (HVA) CaV channels at the cell membrane, which results in enhanced prolactin (PRL) secretion. However, little is known regarding the mechanisms underlying this regulation. Here, we show that EGF actually increases the expression of the CaVα2δ-1 subunit, a key molecular component of HVA channels. The analysis of the gene promoter encoding CaVα2δ-1 (CACNA2D1) revealed binding sites for transcription factors activated by the Ras/Raf/MEK/ERK signaling cascade. Chromatin immunoprecipitation and site-directed mutagenesis showed that ELK-1 is crucial for the transcriptional regulation of CACNA2D1 in response to EGF. Furthermore, we found that EGF increases the membrane expression of CaVα2δ-1 and that ELK-1 overexpression increases HVA current density, whereas ELK-1 knockdown decreases the functional expression of the channels. Hormone release assays revealed that CaVα2δ-1 overexpression increases PRL secretion. These results suggest a mechanism for how EGF, by activating the Ras/Raf/MEK/ERK/ELK-1 pathway, may influence the expression of HVA channels and the secretory behavior of pituitary cells.

Cdk5 phosphorylates CaV1.3 channels and regulates GABAA-mediated miniature inhibitory post-synaptic currents in striato-nigral terminals.

Neurotransmission is one of the most important processes in neuronal communication and depends largely on Ca2+ entering synaptic terminals through voltage-gated Ca2+ (CaV) channels. Although the contribution of L-type CaV channels in neurotransmission has not been unambiguously established, increasing evidence suggests a role for these proteins in noradrenaline, dopamine, and GABA release. Here we report the regulation of L-type channels by Cdk5, and its possible effect on GABA release in the substantia nigra pars reticulata (SNpr). Using patch-clamp electrophysiology, we show that Cdk5 inhibition by Olomoucine significantly increases current density through CaV1.3 (L-type) channels heterologously expressed in HEK293 cells. Likewise, in vitro phosphorylation showed that Cdk5 phosphorylates residue S1947 in the C-terminal region of the pore-forming subunit of CaV1.3 channels. Consistent with this, the mutation of serine into alanine (S1947A) prevented the regulation of Cdk5 on CaV1.3 channel activity. Our data also revealed that the inhibition of Cdk5 increased the frequency of high K+-evoked miniature inhibitory postsynaptic currents in rat SNpr neurons, acting on L-type channels. These results unveil a novel regulatory mechanism of GABA release in the SNpr that involves a direct action of Cdk5 on L-type channels.

TRPV1 Channel: A Noxious Signal Transducer That Affects Mitochondrial Function.

The Transient Receptor Vanilloid 1 (TRPV1) or capsaicin receptor is a nonselective cation channel, which is abundantly expressed in nociceptors. This channel is an important transducer of several noxious stimuli, having a pivotal role in pain development. Several TRPV1 studies have focused on understanding its structure and function, as well as on the identification of compounds that regulate its activity. The intracellular roles of these channels have also been explored, highlighting TRPV1's actions in the homeostasis of Ca2+ in organelles such as the mitochondria. These studies have evidenced how the activation of TRPV1 affects mitochondrial functions and how this organelle can regulate TRPV1-mediated nociception. The close relationship between this channel and mitochondria has been determined in neuronal and non-neuronal cells, demonstrating that TRPV1 activation strongly impacts on cell physiology. This review focuses on describing experimental evidence showing that TRPV1 influences mitochondrial function.

Steroids and TRP Channels: A Close Relationship.

Transient receptor potential (TRP) channels are remarkable transmembrane protein complexes that are essential for the physiology of the tissues in which they are expressed. They function as non-selective cation channels allowing for the signal transduction of several chemical, physical and thermal stimuli and modifying cell function. These channels play pivotal roles in the nervous and reproductive systems, kidney, pancreas, lung, bone, intestine, among others. TRP channels are finely modulated by different mechanisms: regulation of their function and/or by control of their expression or cellular/subcellular localization. These mechanisms are subject to being affected by several endogenously-produced compounds, some of which are of a lipidic nature such as steroids. Fascinatingly, steroids and TRP channels closely interplay to modulate several physiological events. Certain TRP channels are affected by the typical genomic long-term effects of steroids but others are also targets for non-genomic actions of some steroids that act as direct ligands of these receptors, as will be reviewed here.

Epidermal Growth Factor Potentiates Migration of MDA-MB 231 Breast Cancer Cells by Increasing NaV1.5 Channel Expression.

INTRODUCTION: Breast cancer is one of the leading causes of death worldwide and is the result of dysregulation of various signaling pathways in mammary epithelial cells. The mortality rate in patients suffering from breast cancer is high because the tumor cells have a prominent invasive capacity towards the surrounding tissues. Previous studies carried out in tumor cell models show that voltage-gated ion channels may be important molecular actors that contribute to the migratory and invasive capacity of the tumor cells. METHODS: In this study, by using an experimental strategy that combines cell and molecular biology assays with electrophysiological recording, we sought to determine whether the voltage-dependent sodium channel NaV1.5 regulates the migratory capacity of the human breast cancer cell line MDA-MB 231, when cells are maintained in the presence of epidermal growth factor (EGF), as an inductor of the epithelial-mesenchymal transition. RESULTS: Our data show that EGF stimulates the migratory capacity of MDA-MB 231 cells, by regulating the functional expression of NaV1.5 channels. Consistent with this, the stimulatory actions of the growth factor were prevented by the use of tetrodotoxin, an Na+ channel selective blocker, as well as by resveratrol, an antioxidant that can also affect Na+ channel activity. DISCUSSION: The understanding of molecular mechanisms, such as the EGF pathway in the progression of breast cancer is fundamental for the design of more effective therapeutic strategies for the disease.

Biochemical and Functional Interplay Between Ion Channels and the Components of the Dystrophin-Associated Glycoprotein Complex.

Dystrophin is a cytoskeleton-linked membrane protein that binds to a larger multiprotein assembly called the dystrophin-associated glycoprotein complex (DGC). The deficiency of dystrophin or the components of the DGC results in the loss of connection between the cytoskeleton and the extracellular matrix with significant pathophysiological implications in skeletal and cardiac muscle as well as in the nervous system. Although the DGC plays an important role in maintaining membrane stability, it can also be considered as a versatile and flexible molecular complex that contribute to the cellular organization and dynamics of a variety of proteins at specific locations in the plasma membrane. This review deals with the role of the DGC in transmembrane signaling by forming supramolecular assemblies for regulating ion channel localization and activity. These interactions are relevant for cell homeostasis, and its alterations may play a significant role in the etiology and pathogenesis of various disorders affecting muscle and nerve function.

Leptin regulation of inward membrane currents, electrical activity and LH release in isolated bovine gonadotropes.

Leptin, a peptide hormone produced by adipocytes, is recognized as one of the signals involved in the onset of reproductive activity. The leptin receptor has been found in hypothalamic neurons and pituitary gonadotropes, suggesting that the hormone may act at both sites to stimulate the secretion of GnRH and consequently, FSH and LH. In response to a stimulus such as a hypothalamic secretagogue, gonadotropes respond with changes in electrical activity, intracellular Ca2+ and hormone release. The main aim of this report was to investigate whether leptin promotes a change in the electrical and secretory activities of bovine gonadotropes. After 48 h of treatment with leptin (10 nM) significant changes in the action potential properties were observed in gonadotropes, which included an increase in amplitude, time-to-pike and post-hyperpolarization, as well as a decrease in firing threshold. Likewise, leptin induced a significant (∼1.3-fold) up-regulation of voltage-gated Na+ channel current density, and a selective increase (∼2.1-fold) in Ca2+ current density through high voltage-activated channels. Consistent with this, leptin enhanced GnRH-induced secretion of LH measured by ELISA. We suggest that leptin enhances membrane expression of voltage-gated Na+ and Ca2+ channels, which results in a modulation of the action potential properties and an increase in hormone release from gonadotropes.

Structural determinants of the transient receptor potential 1 (TRPV1) channel activation by phospholipid analogs.

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1.

A role for ß-dystroglycan in the organization and structure of the nucleus in myoblasts.

We recently characterized a nuclear import pathway for ß-dystroglycan; however, its nuclear role remains unknown. In this study, we demonstrate for the first time, the interaction of ß-dystroglycan with distinct proteins from different nuclear compartments, including the nuclear envelope (NE) (emerin and lamins A/C and B1), splicing speckles (SC35), Cajal bodies (p80-coilin), and nucleoli (Nopp140). Electron microscopy analysis revealed that ß-dystroglycan localized in the inner nuclear membrane, nucleoplasm, and nucleoli. Interestingly, downregulation of ß-dystroglycan resulted in both mislocalization and decreased expression of emerin and lamin B1, but not lamin A/C, as well in disorganization of nucleoli, Cajal bodies, and splicing speckles with the concomitant decrease in the levels of Nopp140, and p80-coilin, but not SC35. Quantitative reverse transcription PCR and cycloheximide-mediated protein arrest assays revealed that ß-dystroglycan deficiency did not change mRNA expression of NE proteins emerin and lamin B1 bud did alter their stability, accelerating protein turnover. Furthermore, knockdown of ß-dystroglycan disrupted NE-mediated processes including nuclear morphology and centrosome-nucleus linkage, which provides evidence that ß-dystroglycan association with NE proteins is biologically relevant. Unexpectedly, ß-dystroglycan-depleted cells exhibited multiple centrosomes, a characteristic of cancerous cells. Overall, these findings imply that ß-dystroglycan is a nuclear scaffolding protein involved in nuclear organization and NE structure and function, and that might be a contributor to the biogenesis of nuclear envelopathies.

Transcription factor Sp1 regulates T-type Ca(2+) channel CaV 3.1 gene expression.

Voltage-gated T-type Ca(2+) (CaV 3) channels mediate a number of physiological events in developing and mature cells, and are implicated in neurological and cardiovascular diseases. In mammals, there are three distinct T-channel genes (CACNA1G, CACNA1H, and CACNA1I) encoding proteins (CaV 3.1-CaV 3.3) that differ in their localization as well as in molecular, biophysical, and pharmacological properties. The CACNA1G is a large gene that contains 38 exons and is localized in chromosome 17q22. Only basic characteristics of the CACNA1G gene promoter region have been investigated classifying it as a TATA-less sequence containing several potential transcription factor-binding motifs. Here, we cloned and characterized a proximal promoter region and initiated the analysis of transcription factors that control CaV 3.1 channel expression using the murine Cacna1g gene as a model. We isolated a ∼1.5 kb 5'-upstream region of Cacna1g and verified its transcriptional activity in the mouse neuroblastoma N1E-115 cell line. In silico analysis revealed that this region possesses a TATA-less minimal promoter that includes two potential transcription start sites and four binding sites for the transcription factor Sp1. The ability of one of these sites to interact with the transcription factor was confirmed by electrophoretic mobility shift assays. Consistent with this, Sp1 over-expression enhanced promoter activity while siRNA-mediated Sp1 silencing significantly decreased the level of CaV 3.1 protein and reduced the amplitude of whole-cell T-type Ca(2+) currents expressed in the N1E-115 cells. These results provide new insights into the molecular mechanisms that control CaV 3.1 channel expression.

Familial hemiplegic migraine type 1 mutations W1684R and V1696I alter G protein-mediated regulation of Ca(V)2.1 voltage-gated calcium channels.

Familial hemiplegic migraine type 1 (FHM-1) is a monogenic form of migraine with aura that is characterized by recurrent attacks of a typical migraine headache with transient hemiparesis during the aura phase. In a subset of patients, additional symptoms such as epilepsy and cerebellar ataxia are part of the clinical phenotype. FHM-1 is caused by missense mutations in the CACNA1A gene that encodes the pore-forming subunit of Ca(V)2.1 voltage-gated Ca(2+) channels. Although the functional effects of an increasing number of FHM-1 mutations have been characterized, knowledge on the influence of most of these mutations on G protein regulation of channel function is lacking. Here, we explored the effects of G protein-dependent modulation on mutations W1684R and V1696I which cause FHM-1 with and without cerebellar ataxia, respectively. Both mutations were introduced into the human Ca(V)2.1α(1) subunit and their functional consequences investigated after heterologous expression in human embryonic kidney 293 (HEK-293) cells using patch-clamp recordings. When co-expressed along with the human µ-opioid receptor, application of the agonist [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) inhibited currents through both wild-type (WT) and mutant Ca(V)2.1 channels, which is consistent with the known modulation of these channels by G protein-coupled receptors. Prepulse facilitation, which is a way to characterize the relief of direct voltage-dependent G protein regulation, was reduced by both FHM-1 mutations. Moreover, the kinetic analysis of the onset and decay of facilitation showed that the W1684R and V1696I mutations affect the apparent dissociation and reassociation rates of the Gßγ dimer from the channel complex, suggesting that the G protein-Ca(2+) channel affinity may be altered by the mutations. These biophysical studies may shed new light on the pathophysiology underlying FHM-1.

Isolation and characterization of the 5´-upstream region of the human voltage-gated Ca(2+) channel α 2δ-1 auxiliary subunit gene: promoter analysis and regulation by transcription factor Sp1.

The α2δ proteins are auxiliary subunits of high-voltage-activated Ca(2+) channels associated with alterations of surface expression, kinetics, and voltage-dependent properties of the channel complex. Four mammalian genes and several splice α2δ subunit variants have been cloned and described, though very little information concerning the transcriptional mechanisms that regulate their expression is available. Here, we report the identification and characterization of the human α2δ-1 subunit gene promoter and its regulation by specific transcription factor 1 (Sp1). Transient transfection of human neuroblastoma SH-SY5Y cells with a promoter/luciferase reporter construct revealed a ~1.5 kb 5´-UTR fragment of the CACNA2D1 gene that produced high levels of luciferase activity. Deletional analysis of this sequence showed that the minimal promoter was located within a 413-bp region (nt -326 to +98) with respect to the transcription start site. In this region, no canonical TATA box was present, but a high GC content and five potential Sp1 binding sites were found. The ability of two of these sites to interact with the transcription factor was confirmed by electrophoretic mobility shift assays. Likewise, Sp1 overexpression enhanced promoter activity while siRNA-mediated Sp1 silencing significantly decreased the level of α2δ protein expressed in the SH-SY5Y cells, as well as reduced the amplitude of whole-cell patch clamp Ca(2+) currents in dorsal root ganglion neurons. This study thus represents the first identification of the transcriptional control region in the gene encoding the Ca(2+) channel α2δ-1 auxiliary subunit.