Channel Cav1 Research Articles

MEPIRAPIM-derived synthetic cannabinoids inhibit T-type calcium channels with divergent effects on seizures in rodent models of epilepsy.

Background: T-type Ca2+ channels (Cav3) represent emerging therapeutic targets for a range of neurological disorders, including epilepsy and pain. To aid the development and optimisation of new therapeutics, there is a need to identify novel chemical entities which act at these ion channels. A number of synthetic cannabinoid receptor agonists (SCRAs) have been found to exhibit activity at T-type channels, suggesting that cannabinoids may provide convenient chemical scaffolds on which to design novel Cav3 inhibitors. However, activity at cannabinoid type 1 (CB1) receptors can be problematic because of central and peripheral toxicities associated with potent SCRAs. The putative SCRA MEPIRAPIM and its analogues were recently identified as Cav3 inhibitors with only minimal activity at CB1 receptors, opening the possibility that this scaffold may be exploited to develop novel, selective Cav3 inhibitors. Here we present the pharmacological characterisation of SB2193 and SB2193F, two novel Cav3 inhibitors derived from MEPIRAPIM. Methods: The potency of SB2193 and SB2193F was evaluated in vitro using a fluorometric Ca2+ flux assay and confirmed using whole-cell patch-clamp electrophysiology. In silico docking to the cryo-EM structure of Cav3.1 was also performed to elucidate structural insights into T-type channel inhibition. Next, in vivo pharmacokinetic parameters in mouse brain and plasma were determined using liquid chromatography-mass spectroscopy. Finally, anticonvulsant activity was assayed in established genetic and electrically-induced rodent seizure models. Results: Both MEPIRAPIM derivatives produced potent inhibition of Cav3 channels and were brain penetrant, with SB2193 exhibiting a brain/plasma ratio of 2.7. SB2193 was further examined in mouse seizure models where it acutely protected against 6Hz-induced seizures. However, SB2193 did not reduce spontaneous seizures in the Scn1a +/- mouse model of Dravet syndrome, nor absence seizures in the Genetic Absence Epilepsy Rat from Strasbourg (GAERS). Surprisingly, SB2193 appeared to increase the incidence and duration of spike-and-wave discharges in GAERS animals over a 4h recording period. Conclusion: These results show that MEPIRAPIM analogues provide novel chemical scaffolds to advance Cav3 inhibitors against certain seizure types.

Self-Assembly Nanostructure of Myristoylated ω-Conotoxin MVIIA Increases the Duration of Efficacy and Reduces Side Effects

Chronic pain is one of the most prevalent health problems worldwide. An alternative to suppress or alleviate chronic pain is the use of peptide drugs that block N-type Ca2+ channels (Cav2.2), such as ω-conotoxin MVIIA. Nevertheless, the narrow therapeutic window, severe neurological side effects and low stability associated with peptide MVIIA have restricted its widespread use. Fortunately, self-assembly endows the peptide with high stability and multiple functions, which can effectively control its release to prolong its duration of action. Inspired by this, MVIIA was modified with appropriate fatty acid chains to render it amphiphilic and easier to self-assemble. In this paper, an N-terminal myristoylated MVIIA (Myr-MVIIA, medium carbon chain length) was designed and prepared to undergo self-assembly. The present results indicated that Myr-MVIIA can self-assemble into micelles. Self-assembled micelles formed by Myr-MVIIA at higher concentrations than MVIIA can prolong the duration of the analgesic effect and significantly reduce or even eliminate the side effects of tremor and coordinated motor dysfunction in mice.

Distinct pools of synaptic vesicles are released by different calcium channels

Mueller et al. [1] uncover distinct roles for CaV1 and CaV2 channels in neurotransmitter release at the C. elegans neuromuscular junction. Although nanodomain coupling occurs via clustered CaV2 channels, evidence is also presented that release of a separate vesicular pool is mediated by more peripheral, dispersed CaV1 channels, requiring obligatory coupling with RYR to amplify the Ca2+ signal.

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, CaVβ4e, 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.

Molecular Modeling and Potential Ca2+ Channel Blocker Activity of Diphenylmethoxypiperidine Derivatives

Molecular interactions of 4-diphenylmethoxy-1-methylpiperidine derivatives with the calcium channel CaV1.1 (pdb:6JP5) are described. All the compounds tested, previously shown to inhibit adrenergic vascular contractions, display similar binding energetics and interactions with the trans-membrane domain of 6JP5 on the opposite side relative to the channel pore, where nifedipine, a known dihydropyridine Ca2+ channel blocker binds. Additionally, the compounds tested inhibit Ca2+-dependent contractions in isolated mouse mesenteric arteries. Thus, diphenylpyraline analogs may exert their anticontractile effects, at least partially, by blocking vascular Ca2+ channels.

Nanomaterial's interfacial stimulation of vascular endothelial cells and divergent guidances for nanomedicine treating vasculature-associated diseases

Blood vasculature plays an essential role in transporting nanomaterials in vivo. However, the interaction between vascular endothelial cells (VECs) and nanomaterials lacked deep understanding, impeding the development of safe and efficient nanomedicine. Here, we discover that VECs can be significantly stimulated by two-dimensional PEGylated lamellar materials (PLMs) without being internalized. Additional experiments and computational simulations suggest that the unique VEC stimulation can be ascribed to the interaction between the PLMs and the membrane lipids, which induces ion-transport-related conformational change of L-type calcium ion channel Cav1.3. Additionally, the in vivo studies demonstrate that PLMs-stimulated VECs have divergent impacts on two types of vasculature-associated diseases: exacerbating the development of atherosclerosis while magnifying the accumulation of nanomedicine within tumors. Thus, the current study not only advances our understanding of how specific proteins on VEC membranes could be stimulated by PLMs but also guides the rational use of promising PLMs in nanomedicine.

In Vivo CaV3 Channel Inhibition Promotes Maturation of Glucose-Dependent Ca2+ Signaling in Human iPSC-Islets

CaV3 channels are ontogenetically downregulated with the maturation of certain electrically excitable cells, including pancreatic β cells. Abnormally exaggerated CaV3 channels drive the dedifferentiation of mature β cells. This led us to question whether excessive CaV3 channels, retained mistakenly in engineered human-induced pluripotent stem cell-derived islet (hiPSC-islet) cells, act as an obstacle to hiPSC-islet maturation. We addressed this question by using the anterior chamber of the eye (ACE) of immunodeficient mice as a site for recapitulation of in vivo hiPSC-islet maturation in combination with intravitreal drug infusion, intravital microimaging, measurements of cytoplasmic-free Ca2+ concentration ([Ca2+]i) and patch clamp analysis. We observed that the ACE is well suited for recapitulation, observation and intervention of hiPSC-islet maturation. Intriguingly, intraocular hiPSC-islet grafts, retrieved intact following intravitreal infusion of the CaV3 channel blocker NNC55-0396, exhibited decreased basal [Ca2+]i levels and increased glucose-stimulated [Ca2+]i responses. Insulin-expressing cells of these islet grafts indeed expressed the NNC55-0396 target CaV3 channels. Intraocular hiPSC-islets underwent satisfactory engraftment, vascularization and light scattering without being influenced by the intravitreally infused NNC55-0396. These data demonstrate that inhibiting CaV3 channels facilitates the maturation of glucose-activated Ca2+ signaling in hiPSC-islets, supporting the notion that excessive CaV3 channels as a developmental error impede the maturation of engineered hiPSC-islet insulin-expressing cells.

Non-prophylactic resveratrol-mediated protection of neurite integrity under chronic hypoxia is associated with reduction of Cav1.2 channel expression and calcium overloading.

Cellular hypoxia is a major cause of oxidative stress, culminating in neuronal damage in neurodegenerative diseases. Numerous ex vivo studies have implicated that hypoxia episodes leading to disruption of Ca2+ homeostasis and redox status contribute to the progression of various neuropathologies and cell death. Isolation and maintenance of primary cell culture being cost-intensive, the details of the time course relationship between Ca2+ overload, L-type Ca2+ channel function, and neurite retraction under chronic and long-term hypoxia remain undefined. In order to explore the effect of oxidative stress and Ca2+ overload on neurite length, first, we developed a 5-day-long neurite outgrowth model using N2a cell line. Second, we propose a chronic hypoxia model to investigate the modulation of the L-type Ca2+ channel (Cav1.2) and oxidative resistance gene (OXR1) expression level during the process of neurite retraction and neuronal damage over 32h. Thirdly, we developed a framework for quantitative analysis of cytosolic Ca2+, superoxide formation, neurite length, and constriction formation in individual cells using live imaging that provides an understanding of molecular targets. Our findings suggest that an increase in cytosolic Ca2+ is a feature of an early phase of hypoxic stress. Further, we demonstrate that augmentation in the L-type channel leads to amplification in Ca2+ overload, ROS accumulation, and a reduction in neurite length during the late phase of hypoxic stress. Next, we demonstrated that non-prophylactic treatment of resveratrol leads to the reduction of calcium overloading under chronic hypoxia via lowering of L-type channel expression. Finally, we demonstrate that resveratrol-mediated reduction of Cav1.2 channel and STAT3 expression are associated with retention of neurite integrity. The proposed in vitro model assumes significance in the context of drug designing and testing that demands monitoring of neurite length and constriction formations by imaging before animal testing.

CaV1 and CaV2 calcium channels mediate the release of distinct pools of synaptic vesicles.

Activation of voltage-gated calcium channels at presynaptic terminals leads to local increases in calcium and the fusion of synaptic vesicles containing neurotransmitter. Presynaptic output is a function of the density of calcium channels, the dynamic properties of the channel, the distance to docked vesicles, and the release probability at the docking site. We demonstrate that at Caenorhabditis elegans neuromuscular junctions two different classes of voltage-gated calcium channels, CaV2 and CaV1, mediate the release of distinct pools of synaptic vesicles. CaV2 channels are concentrated in densely packed clusters ~250 nm in diameter with the active zone proteins Neurexin, α-Liprin, SYDE, ELKS/CAST, RIM-BP, α-Catulin, and MAGI1. CaV2 channels are colocalized with the priming protein UNC-13L and mediate the fusion of vesicles docked within 33 nm of the dense projection. CaV2 activity is amplified by ryanodine receptor release of calcium from internal stores, triggering fusion up to 165 nm from the dense projection. By contrast, CaV1 channels are dispersed in the synaptic varicosity, and are colocalized with UNC-13S. CaV1 and ryanodine receptors are separated by just 40 nm, and vesicle fusion mediated by CaV1 is completely dependent on the ryanodine receptor. Distinct synaptic vesicle pools, released by different calcium channels, could be used to tune the speed, voltage-dependence, and quantal content of neurotransmitter release.

Activation of Endothelial Large Conductance Potassium Channels Protects against TNF-α-Induced Inflammation.

Elevated TNF-α levels in serum and broncho-alveolar lavage fluid of acute lung injury patients correlate with mortality rates. We hypothesized that pharmacological plasma membrane potential (Em) hyperpolarization protects against TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells through inhibition of inflammatory Ca2+-dependent MAPK pathways. Since the role of Ca2+ influx in TNF-α-mediated inflammation remains poorly understood, we explored the role of L-type voltage-gated Ca2+ (CaV) channels in TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells. The CaV channel blocker, Nifedipine, decreased both CCL-2 and IL-6 secretion, suggesting that a fraction of CaV channels is open at the significantly depolarized resting Em of human microvascular pulmonary endothelial cells (-6 ± 1.9 mV), as shown by whole-cell patch-clamp measurements. To further explore the role of CaV channels in cytokine secretion, we demonstrated that the beneficial effects of Nifedipine could also be achieved by Em hyperpolarization via the pharmacological activation of large conductance K+ (BK) channels with NS1619, which elicited a similar decrease in CCL-2 but not IL-6 secretion. Using functional gene enrichment analysis tools, we predicted and validated that known Ca2+-dependent kinases, JNK-1/2 and p38, are the most likely pathways to mediate the decrease in CCL-2 secretion.

Caldendrin represses neurite regeneration and growth in dorsal root ganglion neurons

Caldendrin is a Ca2+ binding protein that interacts with multiple effectors, such as the Cav1 L-type Ca2+ channel, which play a prominent role in regulating the outgrowth of dendrites and axons (i.e., neurites) during development and in response to injury. Here, we investigated the role of caldendrin in Cav1-dependent pathways that impinge upon neurite growth in dorsal root ganglion neurons (DRGNs). By immunofluorescence, caldendrin was localized in medium- and large- diameter DRGNs. Compared to DRGNs cultured from WT mice, DRGNs of caldendrin knockout (KO) mice exhibited enhanced neurite regeneration and outgrowth. Strong depolarization, which normally represses neurite growth through activation of Cav1 channels, had no effect on neurite growth in DRGN cultures from female caldendrin KO mice. Remarkably, DRGNs from caldendrin KO males were no different from those of WT males in terms of depolarization-dependent neurite growth repression. We conclude that caldendrin opposes neurite regeneration and growth, and this involves coupling of Cav1 channels to growth-inhibitory pathways in DRGNs of females but not males.

Structure-Function Studies of Sponge-Derived Compounds on the Cardiac CaV3.1 Channel.

T-type calcium (CaV3) channels are involved in cardiac automaticity, development, and excitation-contraction coupling in normal cardiac myocytes. Their functional role becomes more pronounced in the process of pathological cardiac hypertrophy and heart failure. Currently, no CaV3 channel inhibitors are used in clinical settings. To identify novel T-type calcium channel ligands, purpurealidin analogs were electrophysiologically investigated. These compounds are alkaloids produced as secondary metabolites by marine sponges, and they exhibit a broad range of biological activities. In this study, we identified the inhibitory effect of purpurealidin I (1) on the rat CaV3.1 channel and conducted structure-activity relationship studies by characterizing the interaction of 119 purpurealidin analogs. Next, the mechanism of action of the four most potent analogs was investigated. Analogs 74, 76, 79, and 99 showed a potent inhibition on the CaV3.1 channel with IC50's at approximately 3 μM. No shift of the activation curve could be observed, suggesting that these compounds act like a pore blocker obstructing the ion flow by binding in the pore region of the CaV3.1 channel. A selectivity screening showed that these analogs are also active on hERG channels. Collectively, a new class of CaV3 channel inhibitors has been discovered and the structure-function studies provide new insights into the synthetic design of drugs and the mechanism of interaction with T-type CaV channels.

Impairment of β-adrenergic regulation and exacerbation of pressure-induced heart failure in mice with mutations in phosphoregulatory sites in the cardiac CaV1.2 calcium channel.

The cardiac calcium channel CaV1.2 conducts L-type calcium currents that initiate excitation-contraction coupling and serves as a crucial mediator of β-adrenergic regulation of the heart. We evaluated the inotropic response of mice with mutations in C-terminal phosphoregulatory sites under physiological levels of β-adrenergic stimulation in vivo, and we assessed the impact of combining mutations of C-terminal phosphoregulatory sites with chronic pressure-overload stress. Mice with Ser1700Ala (S1700A), Ser1700Ala/Thr1704Ala (STAA), and Ser1928Ala (S1928A) mutations had impaired baseline regulation of ventricular contractility and exhibited decreased inotropic response to low doses of β-adrenergic agonist. In contrast, treatment with supraphysiogical doses of agonist revealed substantial inotropic reserve that compensated for these deficits. Hypertrophy and heart failure in response to transverse aortic constriction (TAC) were exacerbated in S1700A, STAA, and S1928A mice whose β-adrenergic regulation of CaV1.2 channels was blunted. These findings further elucidate the role of phosphorylation of CaV1.2 at regulatory sites in the C-terminal domain for maintaining normal cardiac homeostasis, responding to physiological levels of β-adrenergic stimulation in the fight-or-flight response, and adapting to pressure-overload stress.

Differential regulation of Cav 3.2 and Cav 2.2 calcium channels by CB1 receptors and cannabidiol.

Cannabinoids are a promising therapeutic avenue for chronic pain. However, clinical trials often fail to report analgesic efficacy of cannabinoids. Inhibition of voltage gate calcium (Cav ) channels is one mechanism through which cannabinoids may produce analgesia. We hypothesized that cannabinoids and cannabinoid receptor agonists target different types of Cav channels through distinct mechanisms. Electrophysiological recordings from tsA-201 cells expressing either Cav 3.2 or Cav 2.2 were used to assess inhibition by HU-210 or cannabidiol (CBD) in the absence and presence of the CB1 receptor. Homology modelling assessed potential interaction sites for CBD in both Cav 2.2 and Cav 3.2. Analgesic effects of CBD were assessed in mouse models of inflammatory and neuropathic pain. HU-210 (1μM) inhibited Cav 2.2 function in the presence of CB1 receptor but had no effect on Cav 3.2 regardless of co-expression of CB1 receptor. By contrast, CBD (3μM) produced no inhibition of Cav 2.2 and instead inhibited Cav 3.2 independently of CB1 receptors. Homology modelling supported these findings, indicating that CBD binds to and occludes the pore of Cav 3.2, but not Cav 2.2. Intrathecal CBD alleviated thermal and mechanical hypersensitivity in both male and female mice, and this effect was absent in Cav 3.2 null mice. Our findings reveal differential modulation of Cav 2.2 and Cav 3.2 channels by CB1 receptors and CBD. This advances our understanding of how different cannabinoids produce analgesia through action at different voltage-gated calcium channels and could influence the development of novel cannabinoid-based therapeutics for treatment of chronic pain.

Catspermasome: The matrimony of CatSper and SLCO6C1.

CatSper is a highly specialized Ca2+ channel mediating Ca2+ entry in spermatozoa, which is essential for sperm motility and fertility. CatSper dysfunction would lead to male infertility, and it has been an attractive target for male infertility treatment as well as development of novel non-hormonal contraceptives. CatSper is the most complex ion channel known, consisting of at least ten identified subunits. It is very different from classic Cav channels in many aspects. For instance, the ion conducting pore of CatSper consists of four separate chains CatSper1-4 instead of a single chain in Cav channels. The auxiliary subunits CatSperβ, γ, δ, ε contain large extracellular domains while none of them are homologous to the α2δ subunit of Cav channels, indicating unique assembly of CatSper. To provide structural insights into CatSper composition, assembly, and regulation mechanisms, we purified the native CatSper channel from mouse testicular and epididymal tissues and resolved its structure at an overall resolution of 2.9 Å by cryo-EM. In the extracellular view, CatSper1-4 heterotetramer conform to the conventional domain-swapped voltage-gated ion channel fold, following a counterclockwise arrangement. CatSperβ, γ, δ and ε—each of which contains a single transmembrane segment and a large extracellular domain—constitute a pavilion-like structure that stabilizes the entire complex through interactions with CatSper4, 1, 3 and 2, respectively. Besides, the high-resolution EM map reveals several previously uncharacterized components, including TMEM249, TMEM262 (also named CatSperη) and an organic anion transporter SLCO6C1. Thus, CatSper is actually a channel-transporter ultracomplex that we termed the CatSpermasome. The structure and assembly details of the CatSpermasome revealed by our study help to lay the foundation for the development of CatSpermasome-related treatments for male infertility and non-hormonal contraceptives.

FacePalm: Probing dynamic S-palmitoylation of CaV channel complexes using engineered depalmitoylases.

S-palmitoylation is a powerful post-translational modification of ion channels that tunes its function and localization. Unlike other lipid modifications, palmitoylation is a reversible process orchestrated by zDHHC family of protein palmitoyl-transferases and acyl-protein thioesterases that attach and excise palmitic groups, respectively. Even so, dissecting spatial and temporal dynamics of palmitoylation of large channel complexes remains challenging, owing to limitations in molecular approaches to acutely manipulate palmitoylation of specific targets. Here, we develop a new approach to rapidly and inducible depalmitoylate target proteins by engineering the ABHD17 (α/β-hydrolase domain containing protein 17) family of protein depalmitoylases with an FKBP/FRBP chemically inducible dimerization system. This approach is termed FKBP/FRB-activated chemogenetic excision of Palmitoylation or FacePalm. Application of FacePalm revealed multifaced regulation of CaV1/2 channel complexes by palmitoylation. First, time-lapse confocal imaging revealed dynamic redistribution of the CaVβ2A subunit from the plasma membrane to the cytoplasm. Second, whole-cell and single-channel electrophysiology show differences in both inactivation and activation of CaV2 channels depending on palmitoylation of distinct sites on either the β2A or the pore-forming α-subunit. Third, targeting FacePalm to distinct subcellular compartments revealed intrinsic differences in the rate of depalmitoylation of CaVβ2A subunit in different organelles. To further demonstrate generality, we applied FacePalm to various cellular proteins and found substrate-specific differences in palmitoylation dynamics. In all, these studies provide critical insights into the exquisite molecular and cellular mechanisms regulating CaV channel function. Furthermore, FacePalm opens new frontiers to dissect dynamic palmitoylation of ion channel complexes in distinct physiological settings.

Inhibition of Cav1.4 channels by protons depends on the identity of the α2δ subunit.

In retinal photoreceptors, CaV1.4 channels coassemble with the β2 and α2δ-4 subunits and regulate the release of neurotransmitter. Like other CaV channels, CaV1.4 undergoes a prominent inhibition by extracellular protons with low pH decreasing the maximal current amplitude (Imax) and depolarizing the half maximal activation voltage (Vh). However, the mechanisms underlying this form of regulation remain controversial. Here, we analyzed the role of subunit identity on proton-mediated inhibition of CaV1.4 (coexpressed with β2x13) in transfected HEK 293T cells. In cells cotransfected with α2δ-4, changing the extracellular pH from 8.0 to 6.25 caused a significantly greater inhibition of Imax (ΔIMax) and shift in Vh (ΔVh) (ΔIMax = 89.8 ± 1.1 %, ΔVh = 22.7 ± 1.7 mV) than in cells cotransfected with α2δ-1 (ΔIMax = 64.6 ± 4.9 %, ΔVh = 9.0 ± 1.4 mV) or expressing CaV1.4 alone (ΔIMax = 48.3 ± 3.0 %, ΔVh = 9.4 ± 2.1 mV). Notably, this effect of α2δ-4 was selectively diminished for a C-terminal variant of CaV1.4 channels with hyperpolarized voltage-dependence of activation due to a disease-causing mutation (K1591X) but not alternative splicing (CaV1.4 Δ47). The increase in ΔIMax due to α2δ-4 was significantly lower in cells expressing K1591X (11.0 ± 9.9 %) as compared to CaV1.4 (46.2 ± 0.7 %) and CaV1.4 Δ47 (28.7 ± 2.4 %). We conclude that α2δ-4 potentiates proton-mediated inhibition of CaV1.4 in a manner that is modified by pathological variations in the C-terminal domain of CaV1.4.

Restoration of β-adrenergic responsivity in the aging heart.

Progressive cardiac hypo-responsivity to β-adrenergic receptor (βAR)-stimulation occurs with aging, diminishing capacity to manage acute stress, and rendering the heart more vulnerable to systemic stressors including hypertension and diabetes. Disruption of this crucial regulatory mechanism reduces inotropic reserve and lowers the threshold for heart failure. Yet the causal links between aging and β-AR hypo-responsivity are unclear. Here, using electrophysiology, advanced imaging approaches and biochemistry, we propose a mechanism that explains this age-dependent hypo-responsivity, and a restorative measure to rejuvenate the aging heart. Our results indicate that cardiac Ca2+ channels CaV1.2 and type 2 ryanodine receptors display altered nanoscale distribution, and impaired βAR-stimulated reorganization and recycling with aging that limits their functional augmentation. We find that age-associated overexpression of bridging integrator 1 (BIN1) contributes to dysregulated endosomal recycling and disrupted trafficking dynamics of these channels. BIN1 knockdown restored βAR-signaling responsivity and inotropic reserve to youthful levels. Paired echocardiograms of old mice before and after BIN1 knockdown revealed a restoration in cardiac contractility in vivo. In conclusion, we find that βAR hypo-responsivity in the aging heart occurs in part due to expressional upregulation of BIN1 that contributes to deficits in the endosomal recycling pathway, resulting in enlarged endosomes, altered trafficking dynamics of cardiac Ca2+ channels, and limiting the plasticity of excitation-contraction coupling. We propose targeted BIN1 knockdown as a novel therapeutic strategy to rejuvenate the aging myocardium.

The inhibitory effect of escitalopram on mouse detrusor contractility: The role of L-type calcium channels

Selective serotonin reuptake inhibitors (SSRIs) are associated with urinary problems attributed to their central effects. ESC is a preferred SSRI and several case reports described that ESC is related to urinary retention. However, the direct effect of ESC on detrusor contractility is still not completely elucidated. Thus, we investigated the effect of ESC on detrusor contractility and mechanism(s) of its action in isolated mouse detrusor strips. Molecular docking and measurement of intracellular calcium were performed to determine the possible calcium channel blocking effect of ESC. The contractile responses to carbachol (CCh), KCl and electrical field stimulation of detrusor strips were significantly abolished by ESC (10 or 100 μM). ESC relaxed KCl-precontracted detrusor strips concentration-dependently, which was not affected by tetraethylammonium, glibenclamide, 4-aminopyridine, propranolol, L-NAME or methylene blue. ESC (10 or 100 μM) reduced both the CaCl2- and CCh-induced contractions under calcium-free conditions, indicating the role of calcium-involved mechanisms in ESC-mediated relaxation. Furthermore, ESC significantly decreased Bay K8644-induced contraction and the cytosolic calcium level in fura-2-loaded A7r5 cells. Molecular docking study also revealed the potential of ESC to bind L-type calcium (Cav1) channels. Our results demonstrate that ESC inhibits detrusor contractility via blocking Cav1 channels, which provides evidence for the direct effect of ESC on detrusor contractility and its mechanism.

Towards next-generation optogenetic actuators of voltage-gated Ca2+ channels.

CaV1 channels (CaV1.2/1.3) are fundamentally involved in the normal function and pathophysiology of the heart and the brain. These channels convey Ca2+ influx that sculpts action potentials, triggers muscle contraction, and mediates excitation-transcription coupling. To orchestrate these diverse functions, CaV1 activity is fine-tuned by multiple regulatory processes, including Ca2+-dependent inactivation (CDI), which is mediated by the Ca2+-binding protein, calmodulin (CaM). Disruption of CaM regulation is linked to cardiac arrhythmias and neurodevelopmental disorders. However, the precise role of this modulation in various physiological settings is not fully understood. In part, this gap in understanding stems from a lack of approaches to dynamically alter CDI. Here, we engineer a novel optogenetic tool to acutely prevent CDI. To do so, we undertook a biomimetic approach by leveraging naturally occurring CaV1 regulatory mechanisms that modify CDI. Specifically, SH3 and cysteine-rich-domain (stac) protein was recently found to selectively prevent CaV1 CDI. Our previous work identified a short amino acid segment of stac called the U-domain that strongly inhibits CDI. Here, we attached the stac3 U-domain to a light-sensitive Lov2 domain to optically switch its accessibility for modulating CaV1 channels, yielding an actuator known as LovU-CaV1. Whole-cell electrophysiological recordings showed that co-expression of LovU-CaV1 resulted in low basal inhibition of CDI of CaV1.2. However, upon light activation of LovU-CaV1, we observed a rapid reduction in CDI that was then reversible. LovU-CaV1 provides a new avenue to optically manipulate endogenous Ca channels in both the heart and the brain. Future studies will employ this tool to dissect the importance of L-type channel inactivation in both cardiomyocytes and in neurons.