Regulation Of Calcium Channels Research Articles

Abstract 4475: SMYD2 inhibitor BAY-598 reveals the giant scaffold AHNAK as a novel multi-methylated substrate

Abstract The SET and MYND domain-containing protein 2 (SMYD2) has been reported to mono-methylate several lysine residues on histone and non-histone proteins. Overexpression of SMYD2 has been reported in cancer cell lines as well as in esophageal squamous cell carcinoma (ESCC), bladder carcinoma, gastric cancer and pediatric acute lymphoblastic leukemia patients and correlates with lower survival rates. Although several studies uncovered important roles of SMYD2 in promoting tumorgenesis by protein methylation of key proteins (e.g. p53, PTEN, Rb, PARP), the biology of SMYD2 and its possible role in cancer biology is still far from being fully understood. Therefore, we employed the SMYD2 inhibitor BAY-598 (AACR-2015 abstract 2829, publication in preparation), and used proteomics to find novel substrates of SMYD2 in cancer cells. We confirmed with both BAY-598 and by knockdown experiments that the giant scaffold protein AHNAK is a novel major substrate of SMYD2. Different studies have tried to decipher the function of AHNAK in normal and diseased tissues. AHNAK forms multi-protein complexes acting as a structural scaffold involved in different processes, including calcium channel regulation, cell contact signaling, migration, and tumor metastasis. Interestingly, we found AHNAK to be mono-methylated by SMYD2 on multiple sites in its central repeated domain as well as on its C-terminal domain. BAY-598 potently inhibited methylation at these sites on AHNAK. To further determine the prevalence of AHNAK methylation, we analyzed different cancer cell lines and tissues from mice. AHNAK methylation correlated with SMYD2 expression, but not with AHNAK expression, suggesting tissue-specific regulation of AHNAK methylation by SMYD2. In summary, the novel finding of AHNAK regulation by methylation further complements the understanding of the roles of SMYD2 in cancer biology and underlines the utility of using probe inhibitors like BAY-598 to explore potential therapeutic targets in cancer. Citation Format: Erik Eggert, Timo Stellfeld, Daniel Korr, Carlo Stresemann. SMYD2 inhibitor BAY-598 reveals the giant scaffold AHNAK as a novel multi-methylated substrate. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4475.

The T-type calcium channel antagonist Z944 rescues impairments in crossmodal and visual recognition memory in Genetic Absence Epilepsy Rats from Strasbourg

Childhood absence epilepsy (CAE) is often comorbid with behavioral and cognitive symptoms, including impaired visual memory. Genetic Absence Epilepsy Rats from Strasbourg (GAERS) is an animal model closely resembling CAE; however, cognition in GAERS is poorly understood. Crossmodal object recognition (CMOR) is a recently developed memory task that examines not only purely visual and tactile memory, but also requires rodents to integrate sensory information about objects gained from tactile exploration to enable visual recognition. Both the visual and crossmodal variations of the CMOR task rely on the perirhinal cortex, an area with dense expression of T-type calcium channels. GAERS express a gain-in-function missense mutation in the Cav3.2 T-type calcium channel gene. Therefore, we tested whether the T-type calcium channel blocker Z944 dose dependently (1, 3, 10mg/kg; i.p.) altered CMOR memory in GAERS compared to the non-epileptic control (NEC) strain. GAERS demonstrated recognition memory deficits in the visual and crossmodal variations of the CMOR task that were reversed by the highest dose of Z944. Electroencephalogram recordings determined that deficits in CMOR memory in GAERS were not the result of seizures during task performance. In contrast, NEC showed a decrease in CMOR memory following Z944 treatment. These findings suggest that T-type calcium channels mediate CMOR in both the GAERS and NEC strains. Future research into the therapeutic potential of T-type calcium channel regulation may be particularly fruitful for the treatment of CAE and other disorders characterized by visual memory deficits.

Regulation of the Cardiac L-Type Calcium Channel by the Cyclic Nucleotide Cross-Talk Signaling Network

Regulation of L-type Calcium (Ca2+) channel (LCC) gating is critical to shaping the cardiac action potential (AP) and excitation-contraction coupling (ECC), the process by which electrical excitation leads to mechanical contraction of cardiac myocytes. The cyclic nucleotide (cN) cross-talk signaling network, which encompasses the β-adrenergic and the Nitric Oxide (NO)/cGMP/Protein Kinase G (PKG) pathways and their interaction through distinctively-regulated phosphodiesterase isoenzymes (PDEs), regulates LCC current via Protein Kinase A (PKA)- and PKG-mediated phosphorylation. Due to tightly-coupled biochemical reactions, it remains to be clarified how LCC gating is ultimately regulated by this signaling network. In addition, the large number of potential ECC-related phosphorylation targets of PKA and PKG makes it difficult to quantify and isolate the effect of LCC regulation. We have developed a multi-scale, biophysically-detailed computational model of LCC regulation by the cN signaling network based on a wide range of experimental data. Changes in LCC current properties arising from interaction with PKA and PKG are modeled by redistributing the number of channels among four gating modes, corresponding to the four channel phosphorylation states: non-, PKA-, PKG-, and PKA-and-PKG- phosphorylated. The model exhibits experimentally observed single LCC gating characteristics, as well as whole-cell LCC current. Simulations predict how LCC gating modes are redistributed in response to a variety of simulation protocols. Results distinguish LCC regulation exerted between two mechanisms which are difficult to resolve by experimentation alone: 1) LCC interaction with activated PKA and PKG and 2) regulation of PKA and PKG activation as a result of cN cross-talk. Consequently, the model reveals underlying mechanisms that explain LCC current observations under various stimulation scenarios of the signaling network. These results provide insights into how cN signals are integrated via LCC regulation.

Microdomain-Specific Remodelling of Autonomic Regulation of L-Type Calcium Channels Revealed by Super-Resolution Scanning Patch Clamp in Rat Atrial Myocytes in Heart Failure

It has recently become evident that discrete clusters of L-type Ca2+ channels (LTCCs) exist along the cardiomyocyte sarcolemma in association with distinct membrane structures and receptors forming macromolecular signalling complexes. Disruption in subcellular targeting of Ca2+ signaling complexes secondary to changes in cardiomyocyte structure, may contribute to the pathophysiology of heart failure (HF) altering cardiomyocyte function. Here, we used Ca2+ imaging and super-resolution scanning patch-clamp to examine microdomain-specific regulation of LTCCs by β1 adrenergic (β1ARs) and adenosine A1 receptors (AdoA1Rs) in atrial myocytes isolated from control and 16-weeks post-MI HF rats. In control, β1ARs stimulation (ISO 100 nM and β2AR antagonist ICI 50 nM) enhanced spontaneous Ca2+ activity which was abolished by AdoA1Rs stimulation (2′-MeCCPA 200 nM). Accordingly, activation of single LTCCs in both t-tubules and on the crest of sarcolemma from non-localized β1ARs stimulation (ISO 2 µM and ICI 50 nM in external solution) was also abolished by local AdoA1R stimulation (10 µM). In HF, both β1ARs and AdoA1R effects on LTCCs were preserved in t-tubules and lost on the crest. Similar, the effect of β1AR stimulation on spontaneous Ca2+ activities was significantly reduced (28% vs 134% in HF vs control, P<0.01) and the anti-adrenergic effect of AdoA1R was lost (8% vs 84% in HF vs control, P<0.01). In addition, the effects of both β1ARs and AdoA1Rs on normalized cell shortening were significantly reduced in HF. Our findings provide the first direct evidence for microdomain-specific remodeling of LTCC regulation by adrenergic and adenosine receptors in HF which could be linked to local disruption in the interactions between LTCCs and their cellular microenvironment.

Redefining the role of syndecans in C. elegans biology.

Cytosolic calcium is an important factor during fertilization, development and differentiation. Hence, the control of cytosolic calcium levels has been studied extensively for several decades. Numerous calcium channels have been identified and their mechanism of action elucidated. However, the mode of calcium channel regulation remains elusive. Here we discuss our recent findings regarding the role of syndecans in the regulation of cytosolic calcium levels. Syndecans are transmembrane proteoglycans present in both vertebrates and invertebrates that interact with extracellular ligands resulting in the activation of several downstream signaling pathways. We identified a previously unappreciated role of syndecans in cytosolic calcium regulation in mammals that is conserved in C. elegans. We concluded that calcium regulation is the basic, evolutionarily conserved role for syndecans, which enables them to be integral for multiple cellular functions.

Klotho-Dependent Cellular Transport Regulation.

Klotho is a transmembrane protein that in humans is encoded by the hKL gene. This protein is known to have aging suppressor effects and is predominantly expressed in the distal convoluted tubule of the kidney, parathyroid glands, and choroid plexus of the brain. The Klotho protein exists in both full-length membrane form and a soluble secreted form, which exerts numerous distinct functions. The extracellular domain of Klotho can be enzymatically cleaved off and released into the systemic circulation where it functions as β-glucuronidase and a hormone. Soluble Klotho is a multifunction protein present in the biological fluids including blood, urine, and cerebrospinal fluid of mammals. Klotho deficiency leads to multiple organ failure accompanied by early appearance of multiple age-related disorders and early death, whereas overexpression of Klotho results in the opposite effects. Klotho, an enzyme and hormone, has been reported to participate in the regulation of cellular transport processes across the plasma membrane either indirectly through inhibiting calcitriol (1,25(OH)2D3) formation or other mechanism, or by directly affecting transporter proteins, including ion channels, cellular carriers, and Na(+)/K(+)-ATPase. Accordingly, Klotho protein serves as a powerful regulator of cellular transport across the plasma membrane. Importantly, Klotho-dependent cellular transport regulation implies stimulatory or inhibitory effects. Klotho has been shown to play a key role in the regulation of multiple calcium and potassium ion channels, and various cellular carriers including the Na(+)-coupled cotransporters such as NaPi-IIa, NaPi-IIb, EAAT3, and EAAT4, CreaT1 as well as Na(+)/K(+)-ATPase. These regulations are parts of the antiaging function of Klotho, which will be discussing throughout this chapter. Clearly, further experimental efforts are required to investigate the effect of Klotho on other transport proteins and underlying molecular mechanisms by which Klotho exerts its effect.

Conformational Dynamics in FKBP Domains: Relevance to Molecular Signaling and Drug Design.

Among the 22 FKBP domains in the human genome, FKBP12.6 and the first FKBP domains (FK1) of FKBP51 and FKBP52 are evolutionarily and structurally most similar to the archetypical FKBP12. As such, the development of inhibitors with selectivity among these four FKBP domains poses a significant challenge for structure-based design. The pleiotropic effects of these FKBP domains in a range of signaling processes such as the regulation of ryanodine receptor calcium channels by FKBP12 and FKBP12.6 and steroid receptor regulation by the FK1 domains of FKBP51 and FKBP52 amply justify the efforts to develop selective therapies. In contrast to their close structural similarities, these four FKBP domains exhibit a substantial diversity in their conformational flexibility. A number of distinct conformational transitions have been characterized for FKBP12 spanning timeframes from 20 s to 10 ns and in each case these dynamics have been shown to markedly differ from the conformational behavior for one or more of the other three FKBP domains. Protein flexibilitybased inhibitor design could draw upon the transitions that are significantly populated in only one of the targeted proteins. Both the similarities and differences among these four proteins valuably inform the understanding of how dynamical effects propagate across the FKBP domains as well as potentially how such intramolecular transitions might couple to the larger scale transitions that are central to the signaling complexes in which these FKBP domains function.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Both Orai1 and TRPC1 are Involved in Excessive Store-Operated Calcium Entry in Striatal Neurons Expressing Mutant Huntingtin Exon 1.

It has been previously reported that N-terminus of mutant huntingtin (product of the 1st exon) is sufficient to cause a Huntington's disease (HD) pathological phenotype. In view of recent data suggesting that improper regulation of store-operated calcium (SOC) channels is involved in neurodegenerative processes, we investigated influence of expression of the mutant huntingtin N-terminal fragment (Htt138Q-1exon) on SOC entry (SOCE) in mouse neuroblastoma cells (Neuro-2a) and in primary culture of medium spiny neurons (MSNs) isolated from mice. The results show that SOCE in these cells is enhanced upon lentiviral expression of the Htt138Q-1exon. Moreover, we demonstrated that RNAi-mediated knockdown of TRPC1, Orai1, or STIM1 proteins leads to dramatic reduction of abnormal SOCE in both Neuro-2a and MSNs, expressing Htt138Q-1exon. Thus, we concluded that abnormal SOCE in these cells is maintained by both TRPC1- and Orai1-containing channels and required STIM1 for its activation. Furthermore, EVP4593 compound previously tested as a potential anti-HD drug in a Drosophila screening system has proved to be capable of reducing SOCE to the normal level in MSNs expressing the Htt138Q-1exon.

Abstract 11905: Loss of Adrenergic and Adenosine Regulation of Extradyadic L-type Calcium Channels in Rat Atrial Myocytes in Heart Failure

It has recently become evident that discrete clusters of L-type Ca2+ channels (LTCCs) exist along the cardiomyocyte sarcolemma in association with distinct membrane structures. Such microdomain-specific localization impact channel function and regulation by a variety of neurohormonal pathways, including adrenergic and adenosine. Disruption in their subcellular targeting may contribute to the pathophysiology of cardiac diseases, including heart failure (HF). We used Ca2+ imaging and super-resolution scanning patch clamp to examine microdomain-specific regulation of LTCCs in atrial myocytes isolated from control and 16-weeks post-MI HF rats. In control, β1 adrenergic receptors (β1ARs) stimulation (ISO 100 nM and β2AR antagonist ICI 50 nM) enhanced spontaneous Ca2+ release events while the following adenosine A1 receptors (AdoA1Rs) stimulation (2'-MeCCPA 200 nM) abolished β1ARs effects. Non-localized β1ARs stimulation (ISO 2 μM and ICI 50 nM in external solution) activated single LTCCs in both t-tubules and on the crest of sarcolemma, increasing LTCC occurrence and enhancing their open probability and amplitude. Local AdoA1R stimulation through the pipette (10 μM) on β1ARs-pretreated cells completely abolished β1ARs effects. In HF, both β1ARs and AdoA1R effects on LTCCs were preserved in t-tubules and lost on the crest. Similar, the effect of β1AR stimulation on spontaneous Ca2+ release events was significantly reduced in HF (28% vs 134% in HF vs control, P&lt;0.01) and the anti-adrenergic effect of AdoA1R was lost (8% vs 84% in HF vs control, P&lt;0.01), despite up-regulation of β1 and AdoA1Rs mRNA. No changes in caveolae density were observed in HF (4.6±0.4 caveolae/μm vs 3.7±0.5 caveolae/μm for HF vs control, P=0.167). Our findings provide the first direct evidence for microdomain-specific remodeling of LTCC regulation by adrenergic and adenosine receptors in HF which could be linked to local disruption in the interactions between LTCCs and their cellular microenvironment. This extends beyond the classical concept of electrical remodeling, stressing the involvement of alteration of spatial compartmentation of ion channels and receptors in addition to classical concepts of changes in protein expression and post-translational modifications.

Myocardial expression profiles of candidate molecules in patients with arrhythmogenic right ventricular cardiomyopathy/dysplasia compared to those with dilated cardiomyopathy and healthy controls

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is mainly an autosomal dominant disease characterized by fibrofatty infiltration of the right ventricle, leading to ventricular arrhythmias. Mutations in desmosomal proteins can be identified in about half of the patients. The pathogenic mechanisms leading to disease expression remain unclear. The purpose of this study was to investigate myocardial expression profiles of candidate molecules involved in the pathogenesis of ARVC/D. Myocardial messenger RNA (mRNA) expression of 62 junctional molecules, 5 cardiac ion channel molecules, 8 structural molecules, 4 apoptotic molecules, and 6 adipogenic molecules was studied. The averaged expression of candidate mRNAs was compared between ARVC/D samples (n = 10), nonfamilial dilated cardiomyopathy (DCM) samples (n = 10), and healthy control samples (n = 8). Immunohistochemistry and quantitative protein expression analysis were performed. Genetic analysis using next generation sequencing was performed in all patients with ARVC/D. Following mRNA levels were significantly increased in patients with ARVC/D compared to those with DCM and healthy controls: phospholamban (P ≤ .001 vs DCM; P ≤ .001 vs controls), healthy tumor protein 53 apoptosis effector (P = .001 vs DCM; P ≤ .001 vs controls), and carnitine palmitoyltransferase 1β (P ≤ .001 vs DCM; P = 0.008 vs controls). Plakophillin-2 (PKP-2) mRNA was downregulated in patients with ARVC/D with PKP-2 mutations compared with patients with ARVC/D without PKP-2 mutations (P = .04). Immunohistochemistry revealed significantly increased protein expression of phospholamban, tumor protein 53 apoptosis effector, and carnitine palmitoyltransferase 1β in patients with ARVC/D and decreased PKP-2 expression in patients with ARVC/D carrying a PKP-2 mutation. Changes in the expression profiles of sarcolemmal calcium channel regulation, apoptosis, and adipogenesis suggest that these molecular pathways may play a critical role in the pathogenesis of ARVC/D, independent of the underlying genetic mutations.

T-type Calcium Channel Regulation of Neural Tube Closure and EphrinA/EPHA Expression.

A major class of human birth defects arise from aberrations during neural tube closure (NTC). We report on a NTC signaling pathway requiring T-type calcium channels (TTCCs) that is conserved between primitive chordates (Ciona) and Xenopus. With loss of TTCCs, there is a failure to seal the anterior neural folds. Accompanying loss of TTCCs is an upregulation of EphrinA effectors. Ephrin signaling is known to be important in NTC, and ephrins can affect both cell adhesion and repulsion. In Ciona, ephrinA-d expression is downregulated at the end of neurulation, whereas, with loss of TTCC, ephrinA-d remains elevated. Accordingly, overexpression of ephrinA-d phenocopied TTCC loss of function, while overexpression of a dominant-negative Ephrin receptor was able to rescue NTC in a Ciona TTCC mutant. We hypothesize that signaling through TTCCs is necessary for proper anterior NTC through downregulation of ephrins, and possibly elimination of a repulsive signal.

A new role for AMP-activated protein kinase in the circadian regulation of L-type voltage-gated calcium channels in late-stage embryonic retinal photoreceptors.

AMP-activated protein kinase (AMPK) is a cellular energy sensor, which is activated when the intracellular ATP production decreases. The activities of AMPK display circadian rhythms in various organs and tissues, indicating that AMPK is involved in the circadian regulation of cellular metabolism. In vertebrate retina, the circadian clocks regulate many aspects of retinal function and physiology, including light/dark adaption, but whether and how AMPK was involved in the retinal circadian rhythm was not known. We hypothesized that the activation of AMPK (measured as phosphorylated AMPK) in the retina was under circadian control, and AMPK might interact with other intracellular signaling molecules to regulate photoreceptor physiology. We combined ATP assays, western blots, immunostaining, patch-clamp recordings, and pharmacological treatments to decipher the role of AMPK in the circadian regulation of photoreceptor physiology. We found that the overall retinal ATP content displayed a diurnal rhythm that peaked at early night, which was nearly anti-phase to the diurnal and circadian rhythms of AMPK phosphorylation. AMPK was also involved in the circadian phase-dependent regulation of photoreceptor L-type voltage-gated calcium channels (L-VGCCs), the ion channel essential for sustained neurotransmitter release. The activation of AMPK dampened the L-VGCC currents at night with a corresponding decrease in protein expression of the L-VGCCα1 pore-forming subunit, while inhibition of AMPK increased the L-VGCC current during the day. AMPK appeared to be upstream of extracellular-signal-regulated kinase and mammalian/mechanistic target of rapamycin complex 1 (mTORC1) but downstream of adenylyl cyclase in regulating the circadian rhythm of L-VGCCs. Hence, as a cellular energy sensor, AMPK integrates into the cell signaling network to regulate the circadian rhythm of photoreceptor physiology. We found that in chicken embryonic retina, the activation of AMP-activated protein kinase (AMPK) is under circadian control and anti-phase to the retinal ATP rhythm. While ATP content is higher at night, phosphorylated AMPK (pAMPK) is higher during the day. AMPK appears to be upstream of extracellular signal-regulated kinase (ERK), protein kinase B (AKT), and mammalian target of rapamycin complex 1 (mTORC1) but downstream of adenylyl cyclase in regulating the circadian rhythm of L-VGCCs. Therefore, as a cellular energy sensor, AMPK integrates into the cell signaling network to regulate the circadian rhythm of photoreceptor physiology.

Arterial Smooth Muscle Mitochondria Amplify Hydrogen Peroxide Microdomains Functionally Coupled to L-Type Calcium Channels.

Mitochondria are key integrators of convergent intracellular signaling pathways. Two important second messengers modulated by mitochondria are calcium and reactive oxygen species. To date, coherent mechanisms describing mitochondrial integration of calcium and oxidative signaling in arterial smooth muscle are incomplete. To address and add clarity to this issue, we tested the hypothesis that mitochondria regulate subplasmalemmal calcium and hydrogen peroxide microdomain signaling in cerebral arterial smooth muscle. Using an image-based approach, we investigated the impact of mitochondrial regulation of L-type calcium channels on subcellular calcium and reactive oxygen species signaling microdomains in isolated arterial smooth muscle cells. Our single-cell observations were then related experimentally to intact arterial segments and to living animals. We found that subplasmalemmal mitochondrial amplification of hydrogen peroxide microdomain signaling stimulates L-type calcium channels, and that this mechanism strongly impacts the functional capacity of the vasoconstrictor angiotensin II. Importantly, we also found that disrupting this mitochondrial amplification mechanism in vivo normalized arterial function and attenuated the hypertensive response to systemic endothelial dysfunction. From these observations, we conclude that mitochondrial amplification of subplasmalemmal calcium and hydrogen peroxide microdomain signaling is a fundamental mechanism regulating arterial smooth muscle function. As the principle components involved are fairly ubiquitous and positioning of mitochondria near the plasma membrane is not restricted to arterial smooth muscle, this mechanism could occur in many cell types and contribute to pathological elevations of intracellular calcium and increased oxidative stress associated with many diseases.

Towards a Unified Theory of Calmodulin Regulation (Calmodulation) of Voltage-Gated Calcium and Sodium Channels

Voltage-gated Na and Ca(2+) channels represent two major ion channel families that enable myriad biological functions including the generation of action potentials and the coupling of electrical and chemical signaling in cells. Calmodulin regulation (calmodulation) of these ion channels comprises a vital feedback mechanism with distinct physiological implications. Though long-sought, a shared understanding of the channel families remained elusive for two decades as the functional manifestations and the structural underpinnings of this modulation often appeared to diverge. Here, we review recent advancements in the understanding of calmodulation of Ca(2+) and Na channels that suggest a remarkable similarity in their regulatory scheme. This interrelation between the two channel families now paves the way towards a unified mechanistic framework to understand vital calmodulin-dependent feedback and offers shared principles to approach related channelopathic diseases. An exciting era of synergistic study now looms.

Regulation of Cardiac Calcium Channels in the Fight-or-Flight Response.

Intracellular calcium transients generated by activation of voltage-gated calcium (CaV) channels generate local signals, which initiate physiological processes such as secretion, synaptic transmission, and excitation-contraction coupling. Regulation of calcium entry through CaV channels is crucial for control of these physiological processes. In this article, I review experimental results that have emerged over several years showing that cardiac CaV1.2 channels form a local signaling complex, in which their proteolytically processed distal C-terminal domain, an A-Kinase Anchoring Protein, and cyclic AMP-dependent protein kinase (PKA) interact directly with the transmembrane core of the ion channel through the proximal C-terminal domain. This signaling complex is the substrate for β-adrenergic up-regulation of the CaV1.2 channel in the heart during the fight-or-flight response. Protein phosphorylation of two sites at the interface between the distal and proximal C-terminal domains contributes importantly to control of basal CaV1.2 channel activity, and phosphorylation of Ser1700 by PKA at that interface up-regulates CaV1.2 activity in response to β-adrenergic signaling. Thus, the intracellular C-terminal domain of CaV1.2 channels serves as a signaling platform, mediating beat-to-beat physiological regulation of channel activity and up-regulation by β-adrenergic signaling in the fight-or-flight response.

Novel approaches to examine the regulation of voltage-gated calcium channels in the heart.

The cardiac L-type Ca(2+) channel plays a key role in cardiac excitation-contraction coupling, action potential duration, and gene expression. Abnormalities in CaV1.2 function, including increased long-opening-mode gating and blunted adrenergic responsiveness, are associated with heart failure and hypertrophy. The increased activation of CaV1.2, in turn, triggers Ca(2+) -responsive signaling pathways, which contribute to the pathogenesis of heart failure and hypertrophy. CaV1.2 in heart is associated with large supramolecular complexes that impact on channel trafficking, localization, turnover, and function. Much of the prevailing dogma relating to mechanisms underlying CaV1.2 trafficking and modulation is derived from studies using recombinant channels reconstituted in heterologous expression systems. However, recent results using knock-in mice indicate that several long-standing "facts" about CaV1.2 regulation derived from heterologous expression studies are not replicated in native heart, emphasizing the critical need for mechanistic studies in the context of actual cardiomyocytes. In this review, we discuss the use of the use of knockin and knockout mice as well as new tools, including doxycycline-induced expression of informative α1C mutants within cardiomyocytes to probe adrenergic regulation of CaV1.2.

Redox and trace metal regulation of ion channels in the pain pathway.

Given the clinical significance of pain disorders and the relative ineffectiveness of current therapeutics, it is important to identify alternative means of modulating nociception. The most obvious pharmacological targets are the ion channels that facilitate nervous transmission from pain sensors in the periphery to the processing regions within the brain and spinal cord. In order to design effective pharmacological tools for this purpose, however, it is first necessary to understand how these channels are regulated. A growing area of research involves the investigation of the role that trace metals and endogenous redox agents play in modulating the activity of a diverse group of ion channels within the pain pathway. In the present review, the most recent literature concerning trace metal and redox regulation of T-type calcium channels, NMDA (N-methyl-D-aspartate) receptors, GABAA (γ-aminobutyric acid A) receptors and TRP (transient receptor potential) channels are described to gain a comprehensive understanding of the current state of the field as well as to provide a basis for future thought and experimentation.

Molecular Basis of the Membrane Interaction of the β2e Subunit of Voltage-Gated Ca2+ Channels

The auxiliary β subunit plays an important role in the regulation of voltage-gated calcium (CaV) channels. Recently, it was revealed that β2e associates with the plasma membrane through an electrostatic interaction between N-terminal basic residues and anionic phospholipids. However, a molecular-level understanding of β-subunit membrane recruitment in structural detail has remained elusive. In this study, using a combination of site-directed mutagenesis, liposome-binding assays, and multiscale molecular-dynamics (MD) simulation, we developed a physical model of how the β2e subunit is recruited electrostatically to the plasma membrane. In a fluorescence resonance energy transfer assay with liposomes, binding of the N-terminal peptide (23 residues) to liposome was significantly increased in the presence of phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). A mutagenesis analysis suggested that two basic residues proximal to Met-1, Lys-2 (K2) and Trp-5 (W5), are more important for membrane binding of the β2e subunit than distal residues from the N-terminus. Our MD simulations revealed that a stretched binding mode of the N-terminus to PS is required for stable membrane attachment through polar and nonpolar interactions. This mode obtained from MD simulations is consistent with experimental results showing that K2A, W5A, and K2A/W5A mutants failed to be targeted to the plasma membrane. We also investigated the effects of a mutated β2e subunit on inactivation kinetics and regulation of CaV channels by PIP2. In experiments with voltage-sensing phosphatase (VSP), a double mutation in the N-terminus of β2e (K2A/W5A) increased the PIP2 sensitivity of CaV2.2 and CaV1.3 channels by ∼3-fold compared with wild-type β2e subunit. Together, our results suggest that membrane targeting of the β2e subunit is initiated from the nonspecific electrostatic insertion of N-terminal K2 and W5 residues into the membrane. The PS-β2e interaction observed here provides a molecular insight into general principles for protein binding to the plasma membrane, as well as the regulatory roles of phospholipids in transporters and ion channels.

Regulation of L-type calcium channel by phospholemman in cardiac myocytes

We evaluated whether phospholemman (PLM) regulates L-type Ca2+ current (ICa) in mouse ventricular myocytes. Expression of α1-subunit of L-type Ca2+ channels between wild-type (WT) and PLM knockout (KO) hearts was similar. Compared to WT myocytes, peak ICa (at −10mV) from KO myocytes was ~41% larger, the inactivation time constant (τinact) of ICa was ~39% longer, but deactivation time constant (τdeact) was similar. In the presence of isoproterenol (1μM), peak ICa was ~48% larger and τinact was ~144% higher in KO myocytes. With Ba2+ as the permeant ion, PLM enhanced voltage-dependent inactivation but had no effect on τdeact. To dissect the molecular determinants by which PLM regulated ICa, we expressed PLM mutants by adenovirus-mediated gene transfer in cultured KO myocytes. After 24h in culture, KO myocytes expressing green fluorescent protein (GFP) had significantly larger peak ICa and longer τinact than KO myocytes expressing WT PLM; thereby independently confirming the observations in freshly isolated myocytes. Compared to KO myocytes expressing GFP, KO myocytes expressing the cytoplasmic domain truncation mutant (TM43), the non-phosphorylatable S68A mutant, the phosphomimetic S68E mutant, and the signature PFXYD to alanine (ALL5) mutant all resulted in lower peak ICa. Expressing PLM mutants did not alter expression of α1-subunit of L-type Ca2+ channels in cultured KO myocytes. Our results suggested that both the extracellular PFXYD motif and the transmembrane domain of PLM but not the cytoplasmic tail were necessary for regulation of peak ICa amplitude. We conclude that PLM limits Ca2+ influx in cardiac myocytes by reducing maximal ICa and accelerating voltage-dependent inactivation.