The voltage-gated calcium channel functions as the molecular switch of synaptic transmission.

Transmitter release is a fast Ca(2+)-dependent process triggered in response to membrane depolarization. It involves two major calcium-binding proteins, the voltage-gated calcium channel (VGCC) and the vesicular protein synaptotagmin (syt1). Ca(2+) binding triggers transmitter release with a time response of conformational changes that are too fast to be accounted for by Ca(2+) binding to syt1. In contrast, conformation-triggered release, which engages Ca(2+) binding to VGCC, better accounts for the fast rate of the release process. Here, we summarize findings obtained from heterologous expression systems, neuroendocrine cells, and reconstituted systems, which reveal the molecular mechanism by which Ca(2+) binding to VGCC triggers exocytosis prior to Ca(2+) entry into the cell. This review highlights the molecular aspects of an intramembrane signaling mechanism in which a signal is propagated from the channel transmembrane (TM) domain to the TM domain of syntaxin 1A to trigger transmitter release. It discusses fundamental problems of triggering transmitter release by syt1 and suggests a classification of docked vesicles that might explain synchronous transmitter release, spontaneous release, and facilitation of transmitter release.

Cysteine induces mitochondrial reductive stress in glioblastoma through hydrogen peroxide production.

Glucose and amino acid metabolism are critical for glioblastoma (GBM) growth, but little is known about the specific metabolic alterations in GBM that are targetable with FDA-approved compounds. To investigate tumor metabolism signatures unique to GBM, we interrogated The Cancer Genome Atlas for alterations in glucose and amino acid signatures in GBM relative to other human cancers and found that GBM exhibits the highest levels of cysteine and methionine pathway gene expression of 32 human cancers. Treatment of patient-derived GBM cells with the FDA-approved single cysteine compound N-acetylcysteine (NAC) reduced GBM cell growth and mitochondrial oxygen consumption, which was worsened by glucose starvation. Normal brain cells and other cancer cells showed no response to NAC. Mechanistic experiments revealed that cysteine compounds induce rapid mitochondrial H2O2 production and reductive stress in GBM cells, an effect blocked by oxidized glutathione, thioredoxin, and redox enzyme overexpression. From analysis of the clinical proteomic tumor analysis consortium (CPTAC) database, we found that GBM cells exhibit lower expression of mitochondrial redox enzymes than four other cancers whose proteomic data are available in CPTAC. Knockdown of mitochondrial thioredoxin-2 in lung cancer cells induced NAC susceptibility, indicating the importance of mitochondrial redox enzyme expression in mitigating reductive stress. Intraperitoneal treatment of mice bearing orthotopic GBM xenografts with a two-cysteine peptide induced H2O2 in brain tumors in vivo. These findings indicate that GBM is uniquely susceptible to NAC-driven reductive stress and could synergize with glucose-lowering treatments for GBM.

ß-Subunit of the voltage-gated Ca2+ channel Cav1.2 drives signaling to the nucleus via H-Ras.

Depolarization-induced signaling to the nucleus by the L-type voltage-gated calcium channel Cav1.2 is widely assumed to proceed by elevating intracellular calcium. The apparent lack of quantitative correlation between Ca2+ influx and gene activation suggests an alternative activation pathway. Here, we demonstrate that membrane depolarization of HEK293 cells transfected with α11.2/ß2b/α2δ subunits (Cav1.2) triggers c-Fos and MeCP2 activation via the Ras/ERK/CREB pathway. Nuclear signaling is lost either by absence of the intracellular ß2 subunit or by transfecting the cells with the channel mutant α11.2W440A/ß2b/α2δ, a mutation that disrupts the interaction between α11.2 and ß2 subunits. Pulldown assays in neuronal SH-SY5Y cells and in vitro binding of recombinant H-Ras and ß2 confirmed the importance of the intracellular ß2 subunit for depolarization-induced gene activation. Using a Ca2+-impermeable mutant channel α11.2L745P/ß2b/α2δ or disrupting Ca2+/calmodulin binding to the channel using the channel mutant α11.2I1624A/ß2b/α2δ, we demonstrate that depolarization-induced c-Fos and MeCP2 activation does not depend on Ca2+ transport by the channel. Thus, in contrast to the paradigm that elevated intracellular Ca2+ drives nuclear signaling, we show that Cav1.2-triggered c-Fos or MeCP2 is dependent on extracellular Ca2+ and Ca2+ occupancy of the open channel pore, but is Ca2+-influx independent. An indispensable ß-subunit interaction with H-Ras, which is triggered by conformational changes at α11.2 independently of Ca2+ flux, brings to light a master regulatory role of ß2 in transcriptional activation via the ERK/CREB pathway. This mode of H-Ras activation could have broad implications for understanding the coupling of membrane depolarization to the rapid induction of gene transcription.

Voltage-gated calcium channels function as Ca2+-activated signaling receptors.

Voltage-gated calcium channels (VGCCs) are transmembrane cell surface proteins responsible for multifunctional signals. In response to voltage, VGCCs trigger synaptic transmission, drive muscle contraction, and regulate gene expression. Voltage perturbations open VGCCs enabling Ca(2+) binding to the low affinity Ca(2+) binding site of the channel pore. Subsequent to permeation, Ca(2+) targets selective proteins to activate diverse signaling pathways. It is becoming apparent that the Ca(2+)-bound channel triggers secretion in excitable cells and drives contraction in cardiomyocytes prior to Ca(2+) permeation. Here, I highlight recent data implicating receptor-like function of the Ca(2+)-bound channel in converting external Ca(2+) into an intracellular signal. The two sequential mechanistic perspectives of VGCC function are discussed in the context of the prevailing and long-standing current models of depolarization-evoked secretion and cardiac contraction.

Non-ionotropic voltage-gated calcium channel signaling.

Voltage-gated calcium channels (VGCCs) are the major conduits for calcium ions (Ca2+) within excitable cells. Recent studies have highlighted the non-ionotropic functionality of VGCCs, revealing their capacity to activate intracellular pathways independently of ion flow. This non-ionotropic signaling mode plays a pivotal role in excitation-coupling processes, including gene transcription through excitation-transcription (ET), synaptic transmission via excitation-secretion (ES), and cardiac contraction through excitation-contraction (EC). However, it is noteworthy that these excitation-coupling processes require extracellular calcium (Ca2+) and Ca2+ occupancy of the channel ion pore. Analogous to the "non-canonical" characterization of the non-ionotropic signaling exhibited by the N-methyl-D-aspartate receptor (NMDA), which requires extracellular Ca2+ without the influx of ions, VGCC activation requires depolarization-triggered conformational change(s) concomitant with Ca2+ binding to the open channel. Here, we discuss the contributions of VGCCs to ES, ET, and EC coupling as Ca2+ binding macromolecules that transduces external stimuli to intracellular input prior to elevating intracellular Ca2+. We emphasize the recognition of calcium ion occupancy within the open ion-pore and its contribution to the excitation coupling processes that precede the influx of calcium. The non-ionotropic activation of VGCCs, triggered by the upstroke of an action potential, provides a conceptual framework to elucidate the mechanistic aspects underlying the microseconds nature of synaptic transmission, cardiac contractility, and the rapid induction of first-wave genes.

N-Acetylcysteine, N-Acetylcysteine Amide, and Thioredoxin Mimetic Peptides Regenerate Mercaptoalbumin and Exhibit Antioxidant Activity.

Albumin (HSA) is the most abundant circulating protein and plays a pivotal role in maintaining the redox state of the plasma. Three HSA proteoforms have been identified based on the redox state of cysteine 34. These proteoforms comprise of the reduced state (HSA-SH) referred to as mercaptoalbumin, non-mercaptoalbumin-1, containing a disulfide with small thiols such as cysteine (HSA-Cys), and non-mercaptoalbumin-2, representing the higher oxidized proteoform. Several clinical studies have shown a relationship between an individual's serum HSA redox status and the severity of diseases such as heart failure, diabetes mellitus, and liver disease. Furthermore, when HSA undergoes oxidation, it can worsen certain health conditions and contribute to their advancement. This study aimed to evaluate the ability of the redox compounds AD4/NACA and the thioredoxin mimetic (TXM) peptides TXM-CB3, TXM-CB13, and TXM-CB30 to regenerate HSA-SH and to enhance its redox activity. The HSA proteoforms were quantified by LC-MS, and the antioxidant activity was determined using dichlorofluorescin. Each of the compounds exhibited a significant increase in HSA-SH and a reduction in HSA-Cys levels. The increase in HSA-SH was associated with a recovery of its antioxidant activity. In this work, we unveil a novel mechanistic facet of the antioxidant activity of AD4/NACA and TXM peptides. These results suggest an additional therapeutic approach for addressing oxidative stress-related conditions.

TXM peptides inhibit SARS-CoV-2 infection, syncytia formation, and lower inflammatory consequences.

After three years of the SARS-CoV-2 pandemic, the search and availability of relatively low-cost benchtop therapeutics for people not at high risk for a severe disease are still ongoing. Although vaccines and new SARS-CoV-2 variants reduce the death toll, the long COVID-19 along with neurologic symptoms can develop and persist even after a mild initial infection. Reinfections, which further increase the risk of sequelae in multiple organ systems as well as the risk of death, continue to require caution. The spike protein of SARS-CoV-2 is an important target for both vaccines and therapeutics. The presence of disulfide bonds in the receptor binding domain (RBD) of the spike protein is essential for its binding to the human ACE2 receptor and cell entry. Here, we demonstrate that thiol-reducing peptides based on the active site of oxidoreductase thioredoxin 1, called thioredoxin mimetic (TXM) peptides, can prevent syncytia formation, SARS-CoV-2 entry into cells, and infection in a mouse model. We also show that TXM peptides inhibit the redox-sensitive HIV pseudotyped viral cell entry. These results support disulfide targeting as a common therapeutic strategy for treating infections caused by viruses using redox-sensitive fusion. Furthermore, TXM peptides exert anti-inflammatory properties by lowering the activation of NF-κB and IRF signaling pathways, mitogen-activated protein kinases (MAPKs) and lipopolysaccharide (LPS)-induced cytokines in mice. The antioxidant and anti-inflammatory effects of the TXM peptides, which also cross the blood-brain barrier, in combination with prevention of viral infections, may provide a beneficial clinical strategy to lower viral infections and mitigate severe consequences of COVID-19.

N-acetylcysteine Amide AD4/NACA and Thioredoxin Mimetic Peptides Inhibit Platelet Aggregation and Protect against Oxidative Stress.

In the present study, we tested the effect of small-molecular-weight redox molecules on collagen-induced platelet aggregation. We used N-acetylcysteine amide (AD4/NACA), the amide form of N-acetylcysteine (NAC), a thiol antioxidant with improved lipophilicity and bioavailability compared to NAC, and the thioredoxin-mimetic (TXM) peptides, TXM-CB3, TXM-CB13, and TXM-CB30. All compounds significantly inhibited platelet aggregation induced by collagen, with TXM-peptides and AD4 being more effective than NAC. The levels of TxB2 and 12-HETE, the main metabolites derived from the cyclooxygenase and lipoxygenase pathways following platelet activation, were significantly reduced in the presence of AD4, TXM peptides, or NAC, when tested at the highest concentration (0.6 mM). The effects of AD4, TXM-peptides, and NAC were also tested on the clotting time (CT) of whole blood. TXM-CB3 and TXM-CB30 showed the greatest increase in CT. Furthermore, two representative compounds, TXM-CB3 and NAC, showed an increase in the anti-oxidant free sulfhydryl groups of plasma detected via Ellman's method, suggesting a contribution of plasma factors to the antiaggregating effects. Our results suggest that these small-molecular-weight redox peptides might become useful for the prevention and/or treatment of oxidative stress conditions associated with platelet activation.

Voltage-driven Ca(2+) binding at the L-type Ca(2+) channel triggers cardiac excitation-contraction coupling prior to Ca(2+) influx.

The activation of the ryanodine Ca(2+) release channels (RyR2) by the entry of Ca(2+) through the L-type Ca(2+) channels (Cav1.2) is believed to be the primary mechanism of excitation-contraction (EC) coupling in cardiac cells. This proposed mechanism of Ca(2+)-induced Ca(2+) release (CICR) cannot fully account for the lack of a termination signal for this positive feedback process. Using Cav1.2 channel mutants, we demonstrate that the Ca(2+)-impermeable α(1)1.2/L775P/T1066Y mutant introduced through lentiviral infection into neonate cardiomyocytes triggers Ca(2+) transients in a manner independent of Ca(2+) influx. In contrast, the α(1)1.2/L775P/T1066Y/4A mutant, in which the Ca(2+)-binding site of the channel was destroyed, supports neither the spontaneous nor the electrically evoked contractions. Ca(2+) bound at the channel selectivity filter appears to initiate a signal that is conveyed directly from the channel pore to RyR2, triggering contraction of cardiomyocytes prior to Ca(2+) influx. Thus, RyR2 is activated in response to a conformational change in the L-type channel during membrane depolarization and not through interaction with Ca(2+) ions diffusing in the junctional gap space. Accordingly, termination of the RyR2 activity is achieved when the signal stops upon the return of the L-channel to the resting state. We propose a new model in which the physical link between Cav1.2 and RyR2 allows propagation of a conformational change induced at the open pore of the channel to directly activate RyR2. These results highlight Cav1.2 as a signaling protein and provide a mechanism for terminating the release of Ca(2+) from RyR2 through protein-protein interactions. In this model, the L-type channel is a master regulator of both initiation and termination of EC coupling in neonate cardiomyocytes.

A novel dithiol amide CB3 attenuates allergic airway disease through negative regulation of p38 mitogen-activated protein kinase.

RATIONALE: Cellular redox homeostasis altered by excessive production of reactive oxygen species (ROS) and weakening of the antioxidant defense leads to oxidative stress. Oxidative stress is characterized as a decrease in glutathione/glutathione disulfide (GSH/GSSG) and the triggering of a number of the redox-sensitive signaling cascades. Recent studies have demonstrated that ROS play an important role in the pathogenesis of airway inflammation and hyperresponsiveness. OBJECTIVES: Here we characterized for the first time the protective properties of a new hydrophobic thiol compound, N-acetyl cysteine proline cysteine amide (CB3), in allergic airway diseases. METHODS: We used ovalbumin (OVA)-inhaled mice to evaluate the role of CB3 as an antiinflammatory reagent and to determine its molecular signaling activity in allergic airways. MEASUREMENTS AND MAIN RESULTS: The administration of CB3 (1-50 mg/kg) to OVA-inhaled mice restored the decreased GSH levels, enhanced IL-10 expression, and significantly reduced the increase of Th2 cytokines and OVA-specific IgE. CB3 decreased the number of inflammatory cells and airway hyperresponsiveness in the lungs. We also found that the administration of CB3 dramatically decreased the nuclear translocation of the nuclear factor-κB (NF-κB) and the phosphorylation of p38 mitogen-activated protein kinases (MAPKs) in lungs after OVA inhalation. In addition, allergen-induced airway inflammation and hyperresponsiveness were substantially reduced by the administration of inhibitors of NF-κB and p38 MAPK, BAY 11-7085, and SB 239063, respectively. CONCLUSIONS: These results suggest that CB3 attenuates allergic airway disease by up-regulation of GSH levels as well as inhibition of NF-κB and p38 MAPK activity.

Revisiting the molecular basis of synaptic transmission.

Measurements of the time elapsed during synaptic transmission has shown that synaptic vesicle (SV) fusion lags behind Ca2+-influx by approximately 60 microseconds (µsec). The conventional model cannot explain this extreme rapidity of the release event. Synaptic transmission occurs at the active zone (AZ), which comprises of two pools of SV, non-releasable "tethered" vesicles, and a readily-releasable pool of channel-associated Ca2+-primed vesicles, "RRP". A recent TIRF study at cerebellar-mossy fiber-terminal, showed that subsequent to an action potential, newly "tethered" vesicles, became fusion-competent in a Ca2+-dependent manner, 300-400 ms after tethering, but were not fused. This time resolution may correspond to priming of tethered vesicles through Ca2+-binding to Syt1/Munc13-1/complexin. It confirms that Ca2+-priming and Ca2+-influx-independent fusion, are two distinct events. Notably, we have established that Ca2+ channel signals evoked-release in an ion flux-independent manner, demonstrated by Ca2+-impermeable channel, or by substitution of Ca2+ with channel -impermeable La3+. Thus, conformational changes in a channel coupled to RRP appear to directly activate the release machinery and account for a µsec Ca2+-influx-independent vesicle fusion. Rapid vesicle fusion driven by non-ionotropic channel signaling strengthens a conformational-coupling mechanism of synaptic transmission, and contributes to better understanding of neuronal communication vital for brain function.

Novel anti-apoptotic L-DOPA precursors SuperDopa and SuperDopamide as potential neuroprotective agents for halting/delaying progression of Parkinson's disease.

Parkinson's disease (PD) is characterized by a gradual degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNpC). Levodopa, the standard PD treatment, provides the missing dopamine in SNpC, but ultimately after a honeymoon with levodopa treatment the neurodegenerative process and the progression of the disease continue. Aimed at prolonging the life of dopaminergic cells, we prepared the levodopa precursors SuperDopa (SD) and SueprDopamide (SDA), in which levodopa is merged with the antioxidant N-acetylcysteine (NAC) into a single molecule. Rotenone is a mitochondrial complex inhibitor often used as experimental model of PD. In vivo, SD and SDA treatment show a significant relief of motor disabilities in rotenone-injected rats. SD and SDA also lower rotenone-induced-α-synuclein (α-syn) expression in human SH-SY5Y cells, and α-syn oligomerization in α-syn-overexpressing-HEK293 cells. In the neuronal SH-SY5Y cells, SD and SDA reverse oxidative stress-induced phosphorylation of cJun-N-terminal kinase (JNK) and p38-mitogen-activated kinase (p38MAPK). Attenuation of the MAPK-inflammatory/apoptotic pathway in SH-SY5Y cells concurrent with protection of rotenone-triggered motor impairment in rats, is a manifestation of the combined antioxidant/anti-inflammatory activity of SD and SDA together with levodopa release. The concept of joined therapies into a single molecule, where levodopa precursors confer antioxidant activity by enabling NAC delivery across the BBB, provides a potential disease-modifying treatment for slowing PD progression.

Autism associated mutations in ß2 subunit of voltage-gated calcium channels constitutively activate gene expression.

Membrane depolarization triggers gene expression through voltage-gated calcium channels (VGCC) in a process called Excitation-transcription (ET) coupling. Mutations in the channel subunits α11.2, or ß2d, are associated with neurodevelopmental disorders such as ASD. Here, we found that two mutations S143F and G113S within the rat Cavß2a corresponding to autistic related mutations Cavß2dS197F and Cavß2dG167S in the human Cavß2d, activate ET-coupling via the RAS/ERK/CREB pathway. Membrane depolarization of HEK293 cells co-expressing α11.2 and α2δ with Cavß2aS143F or Cavß2aG113S triggers constitutive transcriptional activation, which is correlated with facilitated channel activity. Similar to the Timothy-associated autistic mutation α11.2G406R, constitutive gene activation is attributed to a hyperpolarizing shift in the activation kinetics of Cav1.2. Pulldown of RasGRF2 and RhoGEF by wt and the Cavß2a autistic mutants is consistent with Cavß2/Ras activation in ET coupling and implicates Rho signaling as yet another molecular pathway activated by Cavα11.2/Cavß2 . Facilitated spontaneous channel activity preceding enhanced gene activation via the Ras/ERK/CREB pathway, appears a general molecular mechanism for Ca2+ channel mediated ASD and other neurodevelopmental disorders.

Conformational changes induced in voltage-gated calcium channel Cav1.2 by BayK 8644 or FPL64176 modify the kinetics of secretion independently of Ca2+ influx.

The role of the L-type calcium channel (Cav1.2) as a molecular switch that triggers secretion prior to Ca(2+) transport has previously been demonstrated in bovine chromaffin cells and rat pancreatic beta cells. Here, we examined the effect of specific Cav1.2 allosteric modulators, BayK 8644 (BayK) and FPL64176 (FPL), on the kinetics of catecholamine release, as monitored by amperometry in single bovine chromaffin cells. We show that 2 microm BayK or 0.5 microm FPL accelerates the rate of catecholamine secretion to a similar extent in the presence either of the permeable Ca(2+) and Ba(2+) or the impermeable charge carrier La(3+). These results suggest that structural rearrangements generated through the binding of BayK or FPL, by altering the channel activity, could affect depolarization-evoked secretion prior to cation transport. FPL also accelerated the rate of secretion mediated by a Ca(2+)-impermeable channel made by replacing the wild type alpha(1)1.2 subunit was replaced with the mutant alpha(1)1.2/L775P. Furthermore, BayK and FPL modified the kinetic parameters of the fusion pore formation, which represent the initial contact between the vesicle lumen and the extracellular medium. A direct link between the channel activity and evoked secretion lends additional support to the view that the voltage-gated Ca(2+) channels act as a signaling molecular switch, triggering secretion upstream to ion transport into the cell.

Emerging therapeutic opportunities of novel thiol-amides, NAC-amide (AD4/NACA) and thioredoxin mimetics (TXM-Peptides) for neurodegenerative-related disorders.

Understanding neurodegenerative diseases have challenged scientists for decades. It has become apparent that a decrease in life span is often correlated with the development of neurodegenerative disorders. Oxidative stress and the subsequent inflammatory damages appear to contribute to the different molecular and biochemical mechanisms associated with neurodegeneration. In this review, I examine the protective properties of novel amino acid based compounds, comprising the AD series (AD1-AD7) in particular N-acetylcysteine amide, AD4, also called NACA, and the series of thioredoxin mimetic (TXM) peptides, TXM-CB3-TXM-CB16. Designed to cross the blood-brain-barrier (BBB) and permeate the cell membrane, these antioxidant/anti-inflammatory compounds may enable effective treatment of neurodegenerative related disorders. The review addresses the molecular mechanism of cellular protection exhibited by these new reagents, focusing on the reversal of oxidative stress, mitochondrial stress, inflammatory damages, and prevention of premature cell death. In addition, it will cover the outlook of the clinical prospects of AD4/NACA and the thioredoxin-mimetic peptides, which are currently in development.

Elevated basal transcription can underlie timothy channel association with autism related disorders.

Timothy syndrome (TS) is a neurodevelopmental disorder caused by mutations in the pore-forming subunit α11.2 of the L-type voltage-gated Ca2+-channel Cav1.2, at positions G406R or G402S. Although both mutations cause cardiac arrhythmias, only Cav1.2G406R is associated with the autism-spectrum-disorder (ASD). We show that transcriptional activation by Cav1.2G406R and Cav1.2G402S is driven by membrane depolarization through the Ras/ERK/CREB pathway in a process called excitation-transcription (ET) coupling, as previously shown for wt Cav1.2. This process requires the presence of the intracellular ß-subunit of the channel. We found that only the autism-associated mutant Cav1.2G406R, as opposed to the non-autistic mutated channel Cav1.2G402S, exhibits a depolarization-independent CREB phosphorylation, and spontaneous transcription of cFos and MeCP2. A leftward voltage-shift typical of Cav1.2G406R activation, increases channel opening at subthreshold potentials, resulting in an enhanced channel activity, as opposed to a rightward shift in Cav1.2G402S. We suggest that the enhanced spontaneous Cav1.2G406R activity accounts for the increase in basal transcriptional activation. This uncontroled transcriptional activation may result in the manifestation of long-term dysregulations such as autism. Thus, gating changes provide a mechanistic framework for understanding the molecular events underlying the autistic phenomena caused by the G406R Timothy mutation. They might clarify whether a constitutive transcriptional activation accompanies other VGCC that exhibit a leftward voltage-shift of activation and are also associated with long-term cognitive disorders.

N-acetylcysteine amide (AD4) attenuates oxidative stress in beta-thalassemia blood cells.

Many aspects of the pathology in beta-hemoglobinopathies (beta-thalassemia and sickle cell anemia) are mediated by oxidative stress. In the present study we tested a novel thiol compound, N-acetylcysteine amide (AD4), the amide form of N-acetyl cysteine (NAC) for its antioxidant effects. Using flow-cytometry, we showed that in vitro treatment of blood cells from beta-thalassemic patients with AD4 elevated the reduced glutathione (GSH) content of red blood cells (RBC), platelets and polymorphonuclear (PMN) leukocytes, and reduced their ROS. These effects resulted in a significant reduced sensitivity of thalassemic RBC to hemolysis and phagocytosis by macrophages. Intra-peritoneal injection of AD4 to beta-thalassemic mice (150 mg/kg) reduced the parameters of oxidative stress (p<0.001). Our results show the superiority of AD4, compared to NAC, in reducing oxidative stress markers in thalassemic cells both in vitro and in vivo.

Ion occupancy of the channel pore is critical for triggering excitation-transcription (ET) coupling.

During membrane depolarization the voltage-gated calcium channel (VGCC) activates gene expression in excitable cells by means of a signal-transduction pathway termed excitation transcription (ET) coupling. The L-type calcium channel Cav1.2 can drive nuclear activity by either the ERK-CREB pathway, the Ca2+/calmodulin-dependent protein kinase II (CaMKII) cascade, or via the Ca2+-dependent protein phosphatase calcineurin. The ERK-CREB pathway mediates nuclear activity via a direct interaction of the intracellular ß subunit of VGCC with the Ras/GRF1 complex. Here we show that ET coupling in HEK293 cells transfected with wt Cav1.2 or the Timothy mutant Cav1.2G406R is mediated by substituting Ca2+ with the impermeable lanthanum (La3+). In the absence of extracellular Ca2+ or La3+, ET coupling was not triggered. This implies that cation occupancy of the selectivity filter, as opposed to calcium influx, plays an essential role in depolarization triggered signaling to the nucleus. ET coupling triggered by membrane depolarization in Cav1.2 transfected HEK293 cells and neuroendocrine PC12 cells was also supported by substituting Ba2+ for Ca2+ as the charge carrier. Since Ba2+ ions do not bind to calmodulin this implies activation of ET coupling via a Ca2+/calmodulin-independent pathway. Together, these results suggest a model whereby nuclear signaling through the ERK-CREB pathway is driven by voltage-dependent conformational change that requires channel pore occupancy and is Ca2+ influx-independent. This model is also consistent with the previous observation that ET coupling can be driven by the Ca2+-impermeable Cav1.2L745P mutant. Thus, the conversion of synaptic stimuli to transcriptional activation is mediated by the metabotropic function (Ca2+-inflow independent) of Cav1.2, similar to the ion-influx independent depolarization-triggered transmitter release and transcription activation mediated by the NMDA receptors.

The voltage-gated Ca(2+) channel is the Ca(2+) sensor protein of secretion.

Neurotransmitter release involves two consecutive Ca(2+)-dependent steps, an initial Ca(2+) binding to the selectivity filter of voltage-gated Ca(2+) channels (VGCC) followed by Ca(2+) binding to synaptic vesicle protein. The unique Ca(2+)-binding site of the VGCC is located within the alpha(1) subunit of the Ca(2+) channel. The structure of the selectivity filter allows for the binding of Ca(2+), Sr(2+), Ba(2+), and La(3+). Despite its cell impermeability, La(3+) supports secretion, which is in contradistinction to the commonly accepted mechanism in which elevation of cytosolic ion concentrations ([Ca(2+)](i)) and binding to synaptotagmin(s) trigger release. Here we show that a Cav1.2-mutated alpha(1)1.2/L775P subunit which does not conduct Ca(2+) currents supports depolarization-evoked release by means of Ca(2+) binding to the pore. Bovine chromaffin cells, which secrete catecholamine almost exclusively via nifedipine-sensitive Cav1.2, were infected with the Semliki Forest Virus, pSFV alpha(1)1.2/L775P. This construct also harbored a second mutation that rendered the channel insensitive to nifedipine. Depolarization of cells infected with alpha(1)1.2/L775P triggered release in the presence of nifedipine. Thus, the initial Ca(2+) binding at the pore of the channel appeared to be sufficient to trigger secretion, indicating that the VGCC could be the primary Ca(2+) sensor protein. The 25% lower efficiency, however, implied that additional ancillary effects of elevated [Ca(2+)](i) were essential for optimizing the overall release process. Our findings suggest that the rearrangement of Ca(2+) ions within the pore of the channel during membrane depolarization triggers secretion prior to Ca(2+) entry. This allows for a tight temporal coupling between the depolarization event and exocytosis of vesicles tethered to the channel.

Reconstitution of depolarization and Ca2+-evoked secretion in Xenopus oocytes monitored by membrane capacitance.

The identity of the proteins that constitute the "minimal molecular machinery" required for depolarization-evoked neurotransmitter release at synapses is still not fully disclosed. Using capacitance monitoring combined with heterologous protein expression in Xenopus oocytes, we were able to reconstitute a fast (<.5 s) secretion that was triggered directly by membrane depolarization. The functional assembly of voltage-gated Ca2+ channel (Cav1.2 or Cav2.2) coexpressed with syntaxin 1A, synaptosome-associated protein of 25 kDa (SNAP-25), and synaptotagmin led to the reconstitution of depolarization-evoked secretion. Botulinum C1, botulinum A, and tetanus toxin were used to establish that this minimal set of proteins, named the excitosome complex, was necessary and sufficient for reconstituting depolarization-induced exocytosis. Similar to synaptic transmission, the capacitance changes were sensitive to neurotoxins, modulated by divalent cations (Ca2+, Ba2+, and Sr2+) or channels (Lc or N type; ionotropic glutamate GLUR3), and depended nonlinearly on extracellular divalent cation concentration. Expression of a recombinant intracellular domain of the calcium channel (Lc753-893) abolished evoked release in the reconstituted assay. Also, mutations at the synaptotagmin C2A polylysine motif, a channel interaction site, abolished depolarization-evoked capacitance transients, consistent with release studies in PC12 cells. Because of its improved speed, native trigger, and great experimental versatility, this reconstitution assay provides a novel, promising tool to study synaptic and nonsynaptic exocytosis and examine the role of other proteins implicated in these processes.