Regulation Of Cav1 Research Articles

Exploration of the Molecular Mechanism by Which Caveolin-1 Regulates Changes in Blood-Brain Barrier Permeability Leading to Eosinophilic Meningoencephalitis.

Angiostrongylus cantonensis, a zoonotic parasite, can invade the human central nervous system (CNS) and cause acute eosinophilic meningitis or eosinophilic meningoencephalitis. Mice infected with A. cantonensis show elevated levels of pro-inflammatory cytokines, plasminogen activators, and matrix metalloproteinase-9, resulting in disruption of the blood-brain barrier (BBB) and immune cell infiltration into the CNS. Caveolin-1 (Cav-1) regulates the permeability of the BBB, which affects immune cells and cerebrospinal fluid. This intricate interaction ultimately fuels the progression of brain damage and edema. This study aims to investigate the regulatory role of Cav-1 in the pathogenesis of meningoencephalitis induced by A. cantonensis infection. We investigated pathological alterations by triphenyl-tetrazolium chloride, brain water content, BBB permeability, Western blot analysis, and gelatin zymography in BALB/c mice after A. cantonensis. The study evaluates the critical role of Cav-1 regulation through the TLR4/MyD88 signaling pathway, modulates tight junction proteins, influences BBB permeability, and contributes to brain damage in A. cantonensis-induced meningoencephalitis.

Abstract 7080: Regulation of caveolin-1 by RAS/RAF oncogenic signaling in cancer cells

Abstract Background and Purpose: Caveolin-1 (Cav-1), a scaffolding protein implicated in cellular signaling and membrane dynamics, undergoes intricate regulation at multiple levels. Recent evidence has suggested a potential association between oncogenic mutations and alterations in Cav-1 expression. Our study aimed to comprehensively explore how RAS/RAF mutations influence Cav-1 expression in cancer cells, as well as the underlying molecular mechanisms, using tetracycline (tet)-inducible cell lines and cancer cell lines harboring endogenous BRAF or KRAS mutations. Experimental Design: We assessed the regulation of Cav-1 expression using tet-inducible cell lines with BRAFV600E, KRASG12D, or KRASG12C mutations. We measured the half-life of Cav-1 at protein levels by using cycloheximide. We monitored Cav-1 localization and degradation dynamics by SNAP-based pulse-chase assay. Additionally, we performed protein and gene expression analyses by immunoblotting and qRT-PCR, luciferase reporter assay, and immunofluorescence to dissect the impact of BRAF and KRAS mutations on Cav-1 regulation at various stages of its life cycle. Results: We found a significant downregulation of Cav-1 at both mRNA and protein levels upon induction of BRAF or KRAS mutations. The use of KRASG12C, BRAFV600E, ERK, and/or MEK inhibitors in tet-inducible cell lines rescued the effects observed upon induction. The rescue effects have also been observed in H23, MiaPaCa-2 and 8505-C cancer cell lines bearing either BRAF or KRAS mutations. Notably, BRAFV600E exhibited a more pronounced reduction in Cav-1 expression than other mutations. We observed shortened half-life of Cav-1 at protein levels after induction of RAS/RAF mutations by using cycloheximide, and SNAP-based pulse-chase. These mutations accelerated lysosomal-mediated degradation of Cav-1 as determined by immunoblotting and immunofluorescence, which was reversed by lysosomal inhibitors. Conclusions: Our study establishes a novel regulatory paradigm wherein RAS/RAF mutations exert a multi-faceted influence on Cav-1 dynamics in cancer cells. The transcriptional and translational downregulation, coupled with accelerated lysosomal degradation, collectively contributes to the decreased half-life of Cav-1 following the induction of BRAF or KRAS mutations. These findings provide insights into the intricate molecular mechanisms underpinning the dysregulation of Cav-1 in the context of RAS/RAF mutations, offering potential avenues for therapeutic interventions. Citation Format: Veronica Castro Aceituno, Tiantian Cui, Haihua Feng, Linlin Yang, Sindhu Nair, Terence M. Williams. Regulation of caveolin-1 by RAS/RAF oncogenic signaling in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7080.

Spatiotemporal organization and regulation of CaV1.2 in adult mouse cardiomyocytes

Spatiotemporal organization and regulation of CaV1.2 in adult mouse cardiomyocytes

AKAPs contribute to the indirect pathway for regulation of CaV1.2 channels by RAD and PKA

AKAPs contribute to the indirect pathway for regulation of CaV1.2 channels by RAD and PKA

MiRNA-26a-5p inhibits preterm labor initiation by targeting and regulating TRPC3 ion channel protein expression.

The incidence of preterm birth (PTB) is increasing annually worldwide, leading to various health problems or even fetal deaths. Our previous work demonstrated the activation of transient receptor potential cation channel subfamily C 3 (TRPC3) in mice with PTB, and its activation could promote inward flow of calcium ions and uterine smooth muscle (USM) contraction via regulation of Cav3.2, Cav3.1, and Cav1.2. However, the upstream regulators of TRPC3 and its mechanisms remain unknown. In the present study, the binding of miR-26a-5p to the 3' untranslated region of TRPC3 was predicted by bioinformatics databases (TargetScanHuman and starBase v3.0) and confirmed by a dual-luciferase assay. MiR-26a-5p was downregulated, while TRPC3 was upregulated in the USM tissues of patients with PTB compared to people without PTB. The results showed that miR-26a-5p mimic transfection markedly reduced TRPC3 expression in LPS-stimulated USM cells. Additionally, miR-26a-5p regulated intracellular Ca2+ levels in USM cells by targeting TRPC3. Furthermore, miR-26a-5p inhibited the CPI17/PKC/PLCγ signaling pathway and reduced the expression of Cav3.2, Cav3.1, and Cav1.2. In conclusion, miR-26a-5p regulated the initiation of PTB by targeting TRPC3 and regulating intracellular Ca2+ levels. This study provides a promising diagnostic biomarker and therapeutic target for PTB.

MicroRNA-510 mediated negative regulation of Caveolin-1 in fibroblasts promotes aggressive tumor growth.

In the US, despite the recent decline in breast cancer deaths, a persistent mortality disparity exists between black and white women with breast cancer, with black women having a 41% higher death rate. Several studies are now reporting that racial disparities can exist independent of socioeconomic and standard of care issues, suggesting that biological factors may be involved. Caveolin-1 (Cav1) loss in the tumor stromal compartment is a novel clinical biomarker for predicting poor outcome in breast cancer including triple negative subtype, however the mechanism of Cav1 loss is unknown. We previously identified miR-510-5p as a novel oncomir and propose here that the high levels observed in patients is a novel mechanism leading to stromal Cav1 loss and worse outcomes. Cav1 was identified as a direct target of miR-510-5p through luciferase, western blot and qPCR assays. Stromal cross talk between epithelial cells and fibroblasts was assessed in vitro using transwell co-culture assays and in vivo using xenograft assays. We found that Cav1 is a direct target of miR-510-5p and that expression in fibroblasts results in an 'activated' phenotype. We propose that this could be important in the context of cancer disparities as we also observed increased levels of circulating miR-510-5p and reduced levels of stromal Cav1 in black women compared to white women with breast cancer. Finally, we observed a significant increase in tumor growth when tumor cells were co-injected with miR-510-5p expressing cancer associated fibroblasts in vivo. We propose that miR-510-5p mediated negative regulation of Cav1 in fibroblasts is a novel mechanism of aggressive tumor growth and may be a driver of breast cancer disparity.

Yue-bi-tang attenuates adriamycin-induced nephropathy edema through decreasing renal microvascular permeability via inhibition of the Cav-1/ eNOS pathway.

Edema is one of the most typical symptoms of nephrotic syndrome. Increased vascular permeability makes a significant contribution to the progression of edema. Yue-bi-tang (YBT) is a traditional formula with excellent clinical efficacy in the treatment of edema. This study investigated the effect of YBT on renal microvascular hyperpermeability-induced edema in nephrotic syndrome and its mechanism. In our study, the content of target chemical components of YBT was identified using UHPLC-Q-Orbitrap HRMS analysis. A nephrotic syndrome model was replicated based on male Sprague-Dawley rats with Adriamycin (6.5mg/kg) by tail vein injection. The rats were randomly divided into control, model, prednisone, and YBT (22.2g/kg, 11.1g/kg, and 6.6g/kg) groups. After 14d of treatment, the severity of renal microvascular permeability, edema, the degree of renal injury, and changes in the Cav-1/eNOS pathway were assessed. We found that YBT could regulate renal microvascular permeability, alleviate edema, and reduce renal function impairment. In the model group, the protein expression of Cav-1 was upregulated, whereas VE-cadherin was downregulated, accompanied by the suppression of p-eNOS expression and activation of the PI3K pathway. Meanwhile, an increased NO level in both serum and kidney tissues was observed, and the above situations were improved with YBT intervention. It thus indicates YBT exerts therapeutic effects on the edema of nephrotic syndrome, as it improves the hyperpermeability of renal microvasculature, and that YBT is engaged in the regulation of Cav-1/eNOS pathway-mediated endothelial function.

Regulation of Cardiac Cav1.2 Channels by Calmodulin

Cav1.2 Ca2+ channels, a type of voltage-gated L-type Ca2+ channel, are ubiquitously expressed, and the predominant Ca2+ channel type, in working cardiac myocytes. Cav1.2 channels are regulated by the direct interactions with calmodulin (CaM), a Ca2+-binding protein that causes Ca2+-dependent facilitation (CDF) and inactivation (CDI). Ca2+-free CaM (apoCaM) also contributes to the regulation of Cav1.2 channels. Furthermore, CaM indirectly affects channel activity by activating CaM-dependent enzymes, such as CaM-dependent protein kinase II and calcineurin (a CaM-dependent protein phosphatase). In this article, we review the recent progress in identifying the role of apoCaM in the channel 'rundown' phenomena and related repriming of channels, and CDF, as well as the role of Ca2+/CaM in CDI. In addition, the role of CaM in channel clustering is reviewed.

Pathophysiology of Cav1.3 L-type calcium channels in the heart.

Ca2+ plays a crucial role in excitation-contraction coupling in cardiac myocytes. Dysfunctional Ca2+ regulation alters the force of contraction and causes cardiac arrhythmias. Ca2+ entry into cardiomyocytes is mediated mainly through L-type Ca2+ channels, leading to the subsequent Ca2+ release from the sarcoplasmic reticulum. L-type Ca2+ channels are composed of the conventional Cav1.2, ubiquitously expressed in all heart chambers, and the developmentally regulated Cav1.3, exclusively expressed in the atria, sinoatrial node, and atrioventricular node in the adult heart. As such, Cav1.3 is implicated in the pathogenesis of sinoatrial and atrioventricular node dysfunction as well as atrial fibrillation. More recently, Cav1.3 de novo expression was suggested in heart failure. Here, we review the functional role, expression levels, and regulation of Cav1.3 in the heart, including in the context of cardiac diseases. We believe that the elucidation of the functional and molecular pathways regulating Cav1.3 in the heart will assist in developing novel targeted therapeutic interventions for the aforementioned arrhythmias.

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.

Convergent regulation of Cav1.2 channels by direct phosphorylation and by the small GTPase rad in the cardiac fight-or-flight response.

The cardiac CaV1.2 calcium channel serves as a key regulator of heart rate, rhythm, and force of contraction. CaV1.2 is regulated by direct β-adrenergic/PKA-mediated phosphorylation of its auto-inhibitory C-terminal domain (CT). The small GTPase RAD (Ras associated with diabetes) has also emerged as a potent inhibitor of CaV1.2 with a key role in mediating β-adrenergic/PKA up-regulation of channel activity. However, the relative roles of direct phosphorylation of CaV1.2 channels and phosphorylation of RAD in channel regulation remain uncertain. In a tsA-201 human embryonic kidney cell heterologous expression system, both RAD and the proteolytically processed distal CT (dCT) significantly reduced peak current conducted by the C-terminal truncated CaV1.2α1800 isoform. Activation of PKA by forskolin reversed this inhibition and increased CaV1.2 currents synergistically through PKA phosphorylation of RAD and PKA phosphorylation of Ser 1700 in the proximal CT (pCT). In contrast, alanine substitution for serine at CaV1.2 PKA site Ser1700 and at RAD PKA sites (Ser25, Ser38, Ser272, Ser300) both eliminated PKA-mediated up-regulation in the presence of dCT and RAD. Our findings reveal that the proteolytically processed form of CaV1.2 undergoes convergent regulation by direct PKA phosphorylation of the CT and by PKA phosphorylation of RAD. These parallel regulatory pathways provide a flexible mechanism for up-regulation of the activity of CaV1.2 channels in the fight-or-flight response. Supported by NIH Research Grant R01HL112808.

BIN1 is essential for β-adrenergic regulation of cardiac function.

The number and activity of voltage gated L-type CaV1.2 Ca2+ channels in the sarcolemma of cardiomyocytes tunes the magnitude of Ca2+-induced Ca2+ release and subsequent contraction. Previous studies from our group revealed that β-adrenergic receptor stimulation in mouse ventricular myocytes triggers mobilization of CaV1.2 channels from early and recycling endosomal reservoirs to augment sarcolemmal expression and clustering and enhancing excitation-contraction coupling. In the heart, the membrane curvature-mediating protein bridging integrator 1 (BIN1) mediates t-tubule micro-folding, and targeted delivery of CaV1.2 channels to t-tubules via anchored microtubules. BIN1 deficits are associated with heart failure. In neurons, BIN1 is known to play an essential role in tubulation of endosomes to permit cargo exit and recycling. We used myocytes isolated from cardiac-specific BIN1 knockout mice, to test the hypothesis that expressional deficits in BIN1 impairs isoproterenol-stimulated endosomal recycling of CaV1.2, reducing the ability to respond to acute stress. Super-resolution Airyscan imaging of immunostained ventricular myocytes revealed that CaV1.2 pools on early and recycling endosomes are mobilized to the sarcolemma to bolster channel expression in response to β-adrenergic stimulation with isoproterenol in WT but not BIN1-/- cells. Moreover, single molecule localization microscopy revealed that isoproterenol-stimulated CaV1.2 upregulation and super-clustering at the t-tubule sarcolemma occurs in wild-type myocytes but is reduced in BIN1+/- and absent in BIN1-/- myocytes. To assess the functional implications on β-adrenergic regulation of CaV1.2 and excitation-contraction coupling, we performed voltage-clamp electrophysiological recordings of whole-cell Ca2+ currents and measured Ca2+transients from paced myocytes finding impaired responses to isoproterenol in BIN1 knockout cells versus wild-type controls. Based on these data, we propose that tight regulation of BIN1 expression is required to support β-adrenergic receptor stimulated CaV1.2 channel trafficking and recycling to increase Ca2+ influx and inotropy in times of acute stress.

Mechanisms of Vascular CaV1.2 Channel Regulation During Diabetic Hyperglycemia.

Diabetes is a leading cause of disability and mortality worldwide. A major underlying factor in diabetes is the excessive glucose levels in the bloodstream (e.g., hyperglycemia). Vascular complications directly result from this metabolic abnormality, leading to disabling and life-threatening conditions. Dysfunction of vascular smooth muscle cells is a well-recognized factor mediating vascular complications during diabetic hyperglycemia. The function of vascular smooth muscle cells is exquisitely controlled by different ion channels. Among the ion channels, the L-type CaV1.2 channel plays a key role as it is the main Ca2+ entry pathway regulating vascular smooth muscle contractile state. The activity of CaV1.2 channels in vascular smooth muscle is altered by diabetic hyperglycemia, which may contribute to vascular complications. In this chapter, we summarize the current understanding of the regulation of CaV1.2 channels in vascular smooth muscle by different signaling pathways. We place special attention on the regulation of CaV1.2 channel activity in vascular smooth muscle by a newly uncovered AKAP5/P2Y11/AC5/PKA/CaV1.2 axis that is engaged during diabetic hyperglycemia. We further describe the pathophysiological implications of activation of this axis as it relates to myogenic tone and vascular reactivity and propose that this complex may be targeted for developing therapies to treat diabetic vascular complications.

Rad regulation of CaV1.2 channels controls cardiac fight-or-flight response

Fight-or-flight responses involve β-adrenergic-induced increases in heart rate and contractile force. In the present study, we uncover the primary mechanism underlying the heart’s innate contractile reserve. We show that four protein kinase A (PKA)-phosphorylated residues in Rad, a calcium channel inhibitor, are crucial for controlling basal calcium current and essential for β-adrenergic augmentation of calcium influx in cardiomyocytes. Even with intact PKA signaling to other proteins modulating calcium handling, preventing adrenergic activation of calcium channels in Rad-phosphosite-mutant mice (4SA-Rad) has profound physiological effects: reduced heart rate with increased pauses, reduced basal contractility, near-complete attenuation of β-adrenergic contractile response and diminished exercise capacity. Conversely, expression of mutant calcium-channel β-subunits that cannot bind 4SA-Rad is sufficient to enhance basal calcium influx and contractility to adrenergically augmented levels of wild-type mice, rescuing the failing heart phenotype of 4SA-Rad mice. Hence, disruption of interactions between Rad and calcium channels constitutes the foundation toward next-generation therapeutics specifically enhancing cardiac contractility.

Convergent regulation of CaV1.2 channels by direct phosphorylation and by the small GTPase RAD in the cardiac fight-or-flight response

The L-type calcium currents conducted by the cardiac CaV1.2 calcium channel initiate excitation-contraction coupling and serve as a key regulator of heart rate, rhythm, and force of contraction. CaV1.2 is regulated by β-adrenergic/protein kinase A (PKA)-mediated protein phosphorylation, proteolytic processing, and autoinhibition by its carboxyl-terminal domain (CT). The small guanosine triphosphatase (GTPase) RAD (Ras associated with diabetes) has emerged as a potent inhibitor of CaV1.2, and accumulating evidence suggests a key role for RAD in mediating β-adrenergic/PKA upregulation of channel activity. However, the relative roles of direct phosphorylation of CaV1.2 channels and phosphorylation of RAD in channel regulation remain uncertain. Here, we investigated the hypothesis that these two mechanisms converge to regulate CaV1.2 channels. Both RAD and the proteolytically processed distal CT (dCT) strongly reduced CaV1.2 activity. PKA phosphorylation of RAD and phosphorylation of Ser-1700 in the proximal CT (pCT) synergistically reversed this inhibition and increased CaV1.2 currents. Our findings reveal that the proteolytically processed form of CaV1.2 undergoes convergent regulation by direct phosphorylation of the CT and by phosphorylation of RAD. These parallel regulatory pathways provide a flexible mechanism for upregulation of the activity of CaV1.2 channels in the fight-or-flight response.

Storax Inhibits Caveolae-Mediated Transcytosis at Blood-Brain Barrier After Ischemic Stroke in Rats.

Background and Purpose: Blood-brain barrier (BBB) disruption following ischemic stroke (IS) contributes to hemorrhagic transformation, brain edema, increased neural dysfunction, secondary injury, and mortality. The prevailing view attributes the destruction of tight junction proteins (TJs) to the resulting BBB damage following IS. However, recent studies define a stepwise impairment of the transcellular barrier followed by the paracellular barrier which accounts for the BBB leakage in IS. The increased endothelial transcytosis that has been proven to be caveolae-mediated, preceding and independent of TJs disintegration. Emerging experimental investigations suggested Storax attenuates BBB damage after stroke. This study aimed to test our hypothesis that Storax inhibits caveolae-mediated transcytosis at BBB after ischemic stroke in rats. Methods: Male Wistar rats (250–300 g) were subjected to transient middle cerebral artery occlusion (t-MCAO). Brain water content and the cerebral infarction size were assessed by brain tissue drying-wet method and 2,3,5-triphenyltetrazolium chloride (TTC) staining. BBB permeability was detected by the leakage of Evans blue and Albumin-Alexa594. The ultrastructure of BBB was examined by transmission electron microscopy (TEM). Cav-1 and Mfsd2a were quantified by western blotting and immunofluorescence staining, AQP4, PDGFR-β, ZO-1 and Occludin were quantified by western blotting. Results: Storax treatment of 0.1 g/kg had no significant effects on brain lesions. Storax treatment of 0.2, 0.4, and 0.8 g/kg led to a significant decrease in infarction size, and the Storax 0.4, 0.8 g/kg groups displayed a significant reduction in brain water content. Storax treatment of 0.8 g/kg showed mild toxic reactions. Thus, 0.4 g/kg Storax was selected as the optimal dose for subsequent studies. Storax significantly inhibited the fluorescent albumin intensity in the brain parenchyma and the number of caveolae in ECs, alongside attenuating the ultrastructural disruption of BBB at 6 h after stroke. Meanwhile, Storax significantly increased the expression of Mfsd2a and PDGFR-β, and decrease the expression of Cav-1 and AQP4, corresponding to the significantly decreased Cav-1 positive cells and increased Mfsd2a positive cells. However, Storax has no significant effects on Evan blue leakage or the expression ZO-1, Occludin. Conclusion: Our experimental findings demonstrate Storax treatment inhibits caveolae-mediated transcytosis at BBB in the focal stroke model of rats. We also speculate that regulation of Cav-1, Mfsd2a, AQP4, and PDGFR-β expressions might be associated with its beneficial pharmacological effect, but remain to define and elucidate in future investigation.

Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels.

Voltage-gated calcium channel Cav2.1 undergoes Ca2+-dependent facilitation and inactivation, which are important in short-term synaptic plasticity. In presynaptic terminals, Cav2.1 forms large protein complexes that include synaptotagmins. Synaptotagmin-7 (Syt-7) is essential to mediate short-term synaptic plasticity in many synapses. Here, based on evidence that Cav2.1 and Syt-7 are both required for short-term synaptic facilitation, we investigated the direct interaction of Syt-7 with Cav2.1 and probed its regulation of Cav2.1 function. We found that Syt-7 binds specifically to the α1A subunit of Cav2.1 through interaction with the synaptic-protein interaction (synprint) site. Surprisingly, this interaction enhances facilitation in paired-pulse protocols and accelerates the onset of facilitation. Syt-7α induces a depolarizing shift in the voltage dependence of activation of Cav2.1 and slows Ca2+-dependent inactivation, whereas Syt-7β and Syt-7γ have smaller effects. Our results identify an unexpected, isoform-specific interaction between Cav2.1 and Syt-7 through the synprint site, which enhances Cav2.1 facilitation and modulates its inactivation.

A Role of Caveolae in Trabecular Meshwork Mechanosensing and Contractile Tone.

Polymorphisms in the CAV1/2 gene loci impart increased risk for primary open-angle glaucoma (POAG). CAV1 encodes caveolin-1 (Cav1), which is required for biosynthesis of plasma membrane invaginations called caveolae. Cav1 knockout mice exhibit elevated intraocular pressure (IOP) and decreased outflow facility, but the mechanistic role of Cav1 in IOP homeostasis is unknown. We hypothesized that caveolae sequester/inhibit RhoA, to regulate trabecular meshwork (TM) mechanosensing and contractile tone. Using phosphorylated myosin light chain (pMLC) as a surrogate indicator for Rho/ROCK activity and contractile tone, we found that pMLC was elevated in Cav1-deficient TM cells compared to control (131 ± 10%, n = 10, p = 0.016). Elevation of pMLC levels following Cav1 knockdown occurred in cells on a soft surface (137 ± 7%, n = 24, p < 0.0001), but not on a hard surface (122 ± 17%, n = 12, p = 0.22). In Cav1-deficient TM cells where pMLC was elevated, Rho activity was also increased (123 ± 7%, n = 6, p = 0.017), suggesting activation of the Rho/ROCK pathway. Cyclic stretch reduced pMLC/MLC levels in TM cells (69 ± 7% n = 9, p = 0.002) and in Cav1-deficient TM cells, although not significantly (77 ± 11% n = 10, p = 0.059). Treatment with the Cav1 scaffolding domain mimetic, cavtratin (1 μM) caused a reduction in pMLC (70 ± 5% n = 7, p = 0.001), as did treatment with the scaffolding domain mutant cavnoxin (1 μM) (82 ± 7% n = 7, p = 0.04). Data suggest that caveolae differentially regulate RhoA signaling, and that caveolae participate in TM mechanotransduction. Cav1 regulation of these key TM functions provide evidence for underlying mechanisms linking polymorphisms in the Cav1/2 gene loci with increased POAG risk.

Divergent Ca2+/calmodulin feedback regulation of CaV1 and CaV2 voltage-gated calcium channels evolved in the common ancestor of Placozoa and Bilateria

CaV1 and CaV2 voltage-gated calcium channels evolved from an ancestral CaV1/2 channel via gene duplication somewhere near the stem animal lineage. The divergence of these channel types led to distinguishing functional properties that are conserved among vertebrates and bilaterian invertebrates and contribute to their unique cellular roles. One key difference pertains to their regulation by calmodulin (CaM), wherein bilaterian CaV1 channels are uniquely subject to pronounced, buffer-resistant Ca2+/CaM-dependent inactivation, permitting negative feedback regulation of calcium influx in response to local cytoplasmic Ca2+ rises. Early diverging, nonbilaterian invertebrates also possess CaV1 and CaV2 channels, but it is unclear whether they share these conserved functional features. The most divergent animals to possess both CaV1 and CaV2 channels are placozoans such as Trichoplax adhaerens, which separated from other animals over 600 million years ago shortly after their emergence. Hence, placozoans can provide important insights into the early evolution of CaV1 and CaV2 channels. Here, we build upon previous characterization of Trichoplax CaV channels by determining the cellular expression and ion-conducting properties of the CaV1 channel orthologue, TCaV1. We show that TCaV1 is expressed in neuroendocrine-like gland cells and contractile dorsal epithelial cells. In vitro, this channel conducts dihydropyridine-insensitive, high-voltage–activated Ca2+ currents with kinetics resembling those of rat CaV1.2 but with left-shifted voltage sensitivity for activation and inactivation. Interestingly, TCaV1, but not TCaV2, exhibits buffer-resistant Ca2+/CaM-dependent inactivation, indicating that this functional divergence evolved prior to the emergence of bilaterian animals and may have contributed to their unique adaptation for cytoplasmic Ca2+ signaling within various cellular contexts.

Evaluating the impact of Cav1.2 mutations on neuronal function

Timothy Syndrome (TS) is a multisystem disorder characterized by severe cardiac and neurological deficits including autism spectrum disorder (ASD). The cause of TS is a single point mutation in the CaV1.2 L-type Ca2+ channel (LTCC), and this mutation has been shown to disrupt channel regulation. The original TS mutation, G406R, imparts impaired voltage dependent inactivation, calcium dependent inactivation and voltage dependent activation, leading to this excess Ca2+ entry. Since the discovery of G406R, the number of known mutations in CaV1.2 has grown considerably, and biophysical studies have demonstrated varying effects of each mutation on the regulation of CaV1.2.