Calcium Entry Channels Research Articles

TRPM3 channel expression and block in vascular smooth muscle cells

TRPM3 is a calcium‐permeable channel activated by sphingolipids, including D‐erythro‐sphingosine (Grimm et al 2005, Mol Pharmacol 67, 798–805). Its cellular functions are largely unknown. Using methods to develop isoform‐specific channel blockers (Xu et al 2005, Nat Biotechnol 23, 1289–93) we produced a TRPM3‐specific blocking agent (TM3E3). Preincubation of cells with TM3E3 produced about 40% block of TRPM3‐mediated calcium‐entry or ionic current. TM3E3 had no effect on the closely related channel TRPM2, or other TRP family members TRPC5 and TRPV4, confirming its specificity for TRPM3. Recently, novel activators for TRPM3 have been suggested. In conjunction with TM3E3, these activators have been used to study a potential role of TRPM3 in human vascular smooth muscle cells. The cells express mRNA encoding TRPM3 and the presence of TRPM3 is indicated by labelling with anti‐TRPM3 antibodies. Sphingosine and its analogues evoked calcium responses that could be inhibited by gadolinium, 2‐APB, TM3E3 or siRNA targeting TRPM3. The data suggest TRPM3 is a novel calcium‐entry channel of vascular smooth muscle cells.The research was supported by a BBSRC Industrial Co‐operative Award in Science and Engineering, the Wellcome Trust and the British Heart Foundation.

Orai1 expression and function in vascular smooth muscle cells

Recent studies suggest TRPC5 is a component of calcium‐entry channels that help drive vascular smooth muscle cell migration (Xu et al 2006, Circ Res 98, 1381–9). Here we investigated another type of channel protein (Orai1) which is distinct from the TRP proteins and has been suggested to have pivotal roles in calcium‐entry of lymphocytes (Hogan & Rao 2007, Trends Biochem Sci 32, 235–45). Our experiments focused on proliferating vascular smooth muscle cells of the human saphenous vein. Quantitative RT‐PCR revealed high levels of mRNA encoding Orai1 and its homologues Orai2 and Orai3. Calcium‐entry was measured in the cells after store‐depletion with thapsigargin and using fura‐2 indicator dye on a real‐time 96‐well plate reader. RNAi knock‐down of Orai1 suppressed calcium‐entry, while knock‐down of Orai2 or Orai3 had no effect. Functional relevance was studied using modified Boyden chamber assays to quantify cell migration (chemotaxis) and invasion (through basement membrane matrix) in the absence of store‐depletion. Knock‐down of Orai1 markedly suppressed migration and invasion. Invasion is facilitated by secretion of matrix metalloproteinases (eg MMP‐2) but knock‐down of Orai1 had no significant effect on secretion of MMP‐2. The data suggest Orai1 is a novel calcium channel protein of vascular smooth muscle cells with important roles in cell motility.Supported by the British Heart Foundation and Wellcome Trust.

Gain-of-function haplotype in the epithelial calcium channel TRPV6 is a risk factor for renal calcium stone formation

The rate-limiting step of dietary calcium absorption in the intestine requires the brush border calcium entry channel TRPV6. The TRPV6 gene was completely sequenced in 170 renal calcium stone patients. The frequency of an ancestral TRPV6 haplotype consisting of three non-synonymous polymorphisms (C157R, M378V, M681T) was significantly higher (P = 0.039) in calcium stone formers (8.4%; derived = 502, ancestral = 46) compared to non-stone-forming individuals (5.4%; derived = 645, ancestral = 37). Mineral metabolism was investigated on four different calcium regimens: (i) free-choice diet, (ii) low calcium diet, (iii) fasting and (iv) after a 1 g oral calcium load. When patients homozygous for the derived haplotype were compared with heterozygous patients, no differences were found with respect to the plasma concentrations of 1,25-vitamin D, PTH and calcium, and the urinary excretion of calcium. In one stone-forming patient, the ancestral haplotype was found to be homozygous. This patient had absorptive hypercalciuria. We therefore expressed the ancestral protein (157R+378V+681T) in Xenopus oocytes and found a significantly enhanced calcium permeability when tested by a (45)Ca(2+) uptake assay (7.11 +/- 1.93 versus 3.61 +/- 1.01 pmol/min/oocyte for ancestral versus derived haplotype, P < 0.01). These results suggest that the ancestral gain-of-function haplotype in TRPV6 plays a role in calcium stone formation in certain forms of absorptive hypercalciuria.

The role of TRPV6 in breast carcinogenesis

TRPV6 is an endothelial calcium entry channel that is strongly expressed in breast adenocarcinoma tissue. In this study, we further confirmed this observation by analysis of breast cancer tissues, which indicated that TRPV6 mRNA expression was up-regulated between 2-fold and 15-fold compared with the average in normal breast tissue. Whereas TRPV6 is expressed in the cancer tissue, its role as a calcium channel in breast carcinogenesis is poorly understood. Therefore, we investigated how TRPV6 affects the viability, apoptosis, and calcium transport in the breast cancer cell line T47D. Hormones can also affect the tumor development; hence, we determined the effects of estradiol, progesterone, and 1,25-vitamin D on TRPV6 transcription. Interestingly, the estrogen receptor antagonist tamoxifen reduced expression of TRPV6 and is able to inhibit its calcium transport activity (IC(50), 7.5 micromol/L). The in vitro model showed that TRPV6 can be regulated by estrogen, progesterone, tamoxifen, and 1,25-vitamin D and has a large influence on breast cancer cell proliferation. Moreover, the effect of tamoxifen on cell viability was enhanced when TRPV6 expression was silenced with small interfering RNA. TRPV6 may be a novel target for the development of calcium channel inhibitors to treat breast adenocarcinoma expressing TRPV6.

Methacholine and PDGF activate store-operated calcium entry in neuronal precursor cells via distinct calcium entry channels

Neurons are a diverse cell type exhibiting hugely different morphologies and neurotransmitter specifications. Their distinctive phenotypes are established during differentiation from pluripotent precursor cells. The signalling pathways that specify the lineage down which neuronal precursor cells differentiate remain to be fully elucidated. Among the many signals that impinge on the differentiation of neuronal cells, cytosolic calcium (Ca2+) has an important role. However, little is known about the nature of the Ca2+ signals involved in fate choice in neuronal precursor cells, or their sources. In this study, we show that activation of either muscarinic or platelet-derived growth factor (PDGF) receptors induces a biphasic increase in cytosolic Ca2+ that consists of release from intracellular stores followed by sustained entry across the plasma membrane. For both agonists, the prolonged Ca2+ entry occurred via a store-operated pathway that was pharmacologically indistinguishable from Ca2+ entry initiated by thapsigargin. However, muscarinic receptor-activated Ca2+ entry was inhibited by siRNA-mediated knockdown of TRPC6, whereas Ca2+ entry evoked by PDGF was not. These data provide evidence for agonist-specific activation of molecularly distinct store-operated Ca2+ entry pathways, and raise the possibility of privileged communication between these Ca2+ entry pathways and downstream processes.

Transport-dependent calcium signaling in spatially segregated cellular caveolar domains

We developed a two-dimensional model of transport-dependent intracellular calcium signaling in endothelial cells (ECs). Our purpose was to evaluate the effects of spatial colocalization of endothelial nitric oxide synthase (eNOS) and capacitative calcium entry (CCE) channels in caveolae on eNOS activation in response to ATP. Caveolae are specialized microdomains of the plasma membrane that contain a variety of signaling molecules to optimize their interactions and regulate their activity. In ECs, these molecules include CCE channels and eNOS. To achieve a quantitative understanding of the mechanisms of microdomain calcium signaling and the preferential sensitivity of eNOS to calcium entering the cell through CCE channels, we constructed a mathematical model incorporating the cell morphology and cellular physiological processes. The model predicts that the spatial segregation of calcium channels in ECs can create transport-dependent sharp gradients in calcium concentration within the cell. The calcium concentration gradient is affected by channel density and cell geometry. This transport-dependent calcium signaling specificity effect is enhanced in ECs by increasing the spatial segregation of the caveolar signaling domains. Our simulation significantly advances the understanding of how Ca2+, despite its many potential actions, can mediate selective activation of signaling pathways. We show that diffusion-limited calcium transport allows functional compartmentalization of signaling pathways based on the spatial arrangements of Ca2+ sources and targets.

The determination of the absolute configurations of chiral molecules using vibrational circular dichroism (VCD) spectroscopy

The vibrational circular dichroism (VCD) spectra of the two enantiomers of a chiral molecule are of equal magnitude and opposite sign: i.e. mirror-image enantiomers give mirror-image VCD spectra. In principle, the absolute configuration (AC) of a chiral molecule can therefore be determined from its VCD spectrum. In practice, the determination of the AC of a chiral molecule from its experimental VCD spectrum requires a methodology which reliably predicts the VCD spectra of its enantiomers. The only reliable methodology developed to date uses the Stephens quantum-mechanical theory of the rotational strengths of fundamental vibrational transitions, developed in the early 1980s, implemented using ab initio density functional theory in the GAUSSIAN program in the mid 1990s. This methodology has by now been widely used in determining ACs from experimental VCD spectra. In this article we discuss the protocol for determining the ACs of chiral molecules with optimum reliability and its implementation for a variety of molecules, including the D3 symmetry perhydrotriphenylene, a thiazino-oxadiazolone recently shown to be a highly active calcium entry channel blocker, the alkaloid natural products schizozygine, iso-schizogaline, and iso-schizogamine, and the iridoid natural products plumericin, iso-plumericin, and prismatomerin. The power of VCD spectroscopy in determining ACs, even for large organic molecules and for substantially conformationally-flexible organic molecules is clearly documented.

Calcium Inhibition and Calcium Potentiation of Orai1, Orai2, and Orai3 Calcium Release-activated Calcium Channels

The recent discoveries of Stim1 and Orai proteins have shed light on the molecular makeup of both the endoplasmic reticulum Ca(2+) sensor and the calcium release-activated calcium (CRAC) channel, respectively. In this study, we investigated the regulation of CRAC channel function by extracellular Ca(2+) for channels composed primarily of Orai1, Orai2, and Orai3, by co-expressing these proteins together with Stim1, as well as the endogenous channels in HEK293 cells. As reported previously, Orai1 or Orai2 resulted in a substantial increase in CRAC current (I(crac)), but Orai3 failed to produce any detectable Ca(2+)-selective currents. However, sodium currents measured in the Orai3-expressing HEK293 cells were significantly larger in current density than Stim1-expressing cells. Moreover, upon switching to divalent free external solutions, Orai3 currents were considerably more stable than Orai1 or Orai2, indicating that Orai3 channels undergo a lesser degree of depotentiation. Additionally, the difference between depotentiation from Ca(2+) and Ba(2+) or Mg(2+) solutions was significantly less for Orai3 than for Orai1 or -2. Nonetheless, the Na(+) currents through Orai1, Orai2, and Orai3, as well as the endogenous store-operated Na(+) currents in HEK293 cells, were all inhibited by extracellular Ca(2+) with a half-maximal concentration of approximately 20 mum. We conclude that Orai1, -2, and -3 channels are similarly inhibited by extracellular Ca(2+), indicating similar affinities for Ca(2+) within the selectivity filter. Orai3 channels appeared to differ from Orai1 and -2 in being somewhat resistant to the process of Ca(2+) depotentiation.

Determination of the Absolute Configuration of a Chiral Oxadiazol-3-one Calcium Channel Blocker, Resolved Using Chiral Chromatography, via Concerted Density Functional Theory Calculations of Its Vibrational Circular Dichroism, Electronic Circular Dichroism, and Optical Rotation

The chiral oxadiazol-3-one 2 has recently been shown to exhibit myocardial calcium entry channel blocking activity, substantially higher than that of diltiazem. To determine the enantioselectivity of this activity, the enantiomers of 2 have been resolved using chiral chromatography. The absolute configuration (AC) of 2 has been determined by comparison of density functional theory (DFT) calculations of its vibrational circular dichroism (VCD) spectrum, electronic circular dichroism (ECD) spectrum, and optical rotation (OR) to experimental VCD, ECD, and OR data. All three chiroptical properties yield identical ACs; the AC of 2 is unambiguously determined to be S(+)/R(-).

Organization and function of TRPC channelosomes

TRPC proteins constitute a family of conserved Ca2+-permeable cation channels which are activated in response to agonist-stimulated PIP2 hydrolysis. These channels were initially proposed to be components of the store-operated calcium entry channel (SOC). Subsequent studies have provided substantial evidence that some TRPCs contribute to SOC activity. TRPC proteins have also been shown to form agonist-stimulated calcium entry channels that are not store-operated but are likely regulated by PIP2 or diacylglycerol. Further, and consistent with the presently available data, selective homomeric or heteromeric interactions between TRPC monomers generate distinct agonist-stimulated cation permeable channels. We suggest that interaction between TRPC monomers, as well as the association of these channels with accessory proteins, determines their mode of regulation as well as their cellular localization and function. Currently identified accessory proteins include key Ca2+ signaling proteins as well as proteins involved in vesicle trafficking, cytoskeletal interactions, and scaffolding. Studies reported until now demonstrate that TRPC proteins are segregated into specific Ca2+ signaling complexes which can generate spatially and temporally controlled [Ca2+]i signals. Thus, the functional organization of TRPC channelosomes dictates not only their regulation by extracellular stimuli but also serves as a platform to coordinate specific downstream cellular functions that are regulated as a consequence of Ca2+ entry. This review will focus on the accessory proteins of TRPC channels and discuss the functional implications of TRPC channelosomes and their assembly in microdomains.

Diltiazem Analogues: The Last Ten Years on Structure Activity Relationships

Cardiovascular diseases as hypertension, angina and/or supraventricular arrhythmias are among the most important death causes in the world. For the treatment of heart pathologies, calcium channel entry blockers are very important drugs, owing to their therapeutic versatility. Although few calcium antagonists described until today are structurally related to diltiazem and to the benzothiazepine class, the still high pharmaceutical interest on diltiazem analogues justifies this review. Diltiazem and its first analogues developed in the early '70s became popular in the '80s, and were pharmacologically characterized for a long time. It is in the '90s that several research groups carried out structural variations identifying novel scaffolds for diltiazem-related compounds, with significant calcium antagonist behaviour. Recently, a series of thiazino-oxadiazolone derivatives were identified as potent and selective antagonists for calcium influx into cardiac cells, and they were subsequently used to search for novel chemotypes by means of virtual screening techniques. The resulting hits could open interesting perspectives for the development of drugs to treat cardiovascular diseases. In the present review, an updated collection of diltiazem analogues is reported over the last ten years. The chemical structure and the structure activity relationships will be given, with additional mention to the potential therapeutic applications.

Regulation of capacitative calcium entries by α1‐syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of α1‐syntrophin

Calcium mishandling in Duchenne dystrophic muscle suggested that dystrophin, a membrane-associated cytoskeleton protein, might regulate calcium signaling cascade such as calcium influx pathway. It was previously shown that abnormal calcium entries involve uncontrolled stretch-activated currents and store-operated Ca2+ currents supported by TRPC1 channels. Moreover, our recent work demonstrated that reintroduction of minidystrophin in dystrophic myotubes restores normal capacitative calcium entries (CCEs). However, until now, no molecular link between the dystrophin complex and calcium entry channels has been described. This study is the first to show by coimmunoprecipitation assays the molecular association of TRPC1 with dystrophin and alpha1-syntrophin in muscle cells. TRPC1 was also associated with alpha1-syntrophin in dystrophic muscle cells independently of dystrophin. Furthermore, glutathione S-transferase (GST) pull-down assays showed that TRPC1 binds to the alpha1-syntrophin PDZ domain. Transfected recombinant alpha1-syntrophin formed a complex with TRPC1 channels and restored normal CCEs in dystrophic muscle cells. We suggest that normal regulation of CCEs in skeletal muscle depends on the association between TRPC1 channels and alpha1-syntrophin that may anchor the store-operated channels to the dystrophin-associated protein complex (DAPC). The loss of this molecular association could participate in the calcium alterations observed in dystrophic muscle cells. This study provides a new model for the regulation of calcium influx by interaction with the scaffold of the DAPC in muscle cells.

Compartmentalization of TRPC1‐STIM1 interactions into lipid raft domains

Lipid rafts are cholesterol enriched plasma membrane microdomains that serve as cellular signaling platforms. Numerous physiological signals are relayed to the cells interior via these distinctly compartmentalized plasma membrane microdomains. Here we demonstrate the association of Transient Receptor Potential Cannonical-1 (TRPC1, a store operated calcium entry (SOCE) channel) with lipid raft/caveolar compartments of the plasma membrane. Depletion of endoplasmic reticulum(ER) calcium stores by thapsigargin (Tg, a inhibitor for sarco/endoplasmic calcium ATPase) enhanced the recruitment of TRPC1 protein to the plasma membrane rafts. This was associated with the calcium influx property of TRPC1 that was sensitive to specific SOCE blockers. We also identify Stromal Interacting Molecule-1(STIM1 an ER calcium sensor) as a candidate SOCE element involved in ER store dependant activation of TRPC1. STIM1 communicates the ER calcium store depletion status by translocating to the plasma membrane and interacting with TRPC1. Down-regulation of STIM1 by siRNA reduced SOCE which was similar in cells expressing TRPC1-siRNA. Alternatively, sequestration of membrane cholesterol by MβCD or Fillipin-III altered raft-associated TRPC1 localization and its calcium influx property. Altogether, our findings demonstrate activation of TRPC1 relies on its association with STIM1 and lipid raft integrity.

Adenylyl cyclase 5/6 colocalizes with TRPC4 and cell adhesion molecules to caveolin‐enriched fractions of pulmonary microvascular endothelial cells (PMVECs)

Strongly adherent PMVECs interact to form the pulmonary capillary barrier, necessary to maintain efficient gas exchange in the lung. Tight cell-cell junctions between adjacent pulmonary capillary endothelial cells contribute to the integrity of this barrier and are dynamically regulated by transitions in cAMP. Within PMVECs calcium sensitive adenylyl cyclase activity has been resolved in a phosphodiesterase 4 regulated membrane fraction. However, compartmentation of adenylyl cyclase and other signaling molecules involved in barrier regulation within the PMVEC membrane has not been described. Here we utilize a detergent free discontinuous sucrose density gradient centrifugation technique to separate caveolae/lipid raft fractions from the bulk plasma membrane. AC5/6 immunoreactivity was detected in the caveolin rich light-density fractions while beta adaptin immunoreactivity was detected in the high-density fraction of non-caveolae regions within the plasma membrane. TRPC4 a putative molecular component of the store operated calcium entry channel also resides in the caveolin rich fraction. In addition, cell adhesion molecules N-cadherin and ALCAM and the scaffolding molecule Dlg localize to caveolae. Therefore, it appears that AC6 compartmentalizes to caveolae with other signaling components that dynamically regulate endothelial barrier. Supported by the Welcome Trust, HL66299 and HL60024

Inhibitory Effect of the Phosphoinositide 3-Kinase Inhibitor LY294002 on Muscarinic Acetylcholine Receptor-Induced Calcium Entry in PC12h Cells

Phosphoinositide-3 kinase (PI3K) and phospholipase C (PLC) utilize the same phosphoinositides as substrates to produce different signaling molecules. These enzymes are activated by a similar set of cell signaling mechanisms, i.e., tyrosine kinases and G proteins, and affect common cell functions, including proliferation, motility, and intracellular trafficking. Despite these similarities, the interplay between these enzymes is not well understood. To address this issue, the effects of the PI3K inhibitor LY294002 on carbachol-induced calcium increase in PC12h cells were examined. As carbachol stimulates both Gq- and Gi-coupled muscarinic acetylcholine receptors (mAChRs), PI3K and PLC are activated simultaneously in this protocol. LY294002 was found to reduce the carbachol-induced calcium increase, and the reduction was attributed to suppression of calcium entry. As LY294002 did not affect either carbachol-induced calcium release or calcium entry induced by calcium store depletion, this agent was found to suppress calcium entry directly activated by mAChRs. Although PI3K was supposed to compete for substrates with PLC, the PI3K inhibitor did not enhance PLC-dependent cellular responses. As LY294002 was still effective by treating cells after carbachol stimulation, it is likely that this agent blocks the calcium entry channels directly.

STIM1 carboxyl-terminus activates native SOC, Icrac and TRPC1 channels

Receptor-evoked Ca2+ signalling involves Ca2+ release from the endoplasmic reticulum, followed by Ca2+ influx across the plasma membrane. Ca2+ influx is essential for many cellular functions, from secretion to transcription, and is mediated by Ca2+-release activated Ca2+ (I(crac)) channels and store-operated calcium entry (SOC) channels. Although the molecular identity and regulation of I(crac) and SOC channels have not been precisely determined, notable recent findings are the identification of STIM1, which has been indicated to regulate SOC and I(crac) channels by functioning as an endoplasmic reticulum Ca2+ sensor, and ORAI1 (ref. 7) or CRACM1 (ref. 8)--both of which may function as I(crac) channels or as an I(crac) subunit. How STIM1 activates the Ca2+ influx channels and whether STIM1 contributes to the channel pore remains unknown. Here, we identify the structural features that are essential for STIM1-dependent activation of SOC and I(crac) channels, and demonstrate that they are identical to those involved in the binding and activation of TRPC1. Notably, the cytosolic carboxyl terminus of STIM1 is sufficient to activate SOC, I(crac) and TRPC1 channels even when native STIM1 is depleted by small interfering RNA. Activity of STIM1 requires an ERM domain, which mediates the selective binding of STIM1 to TRPC1, 2 and 4, but not to TRPC3, 6 or 7, and a cationic lysine-rich region, which is essential for gating of TRPC1. Deletion of either region in the constitutively active STIM1(D76A) yields dominant-negative mutants that block native SOC channels, expressed TRPC1 in HEK293 cells and I(crac) in Jurkat cells. These observations implicate STIM1 as a key regulator of activity rather than a channel component, and reveal similar regulation of SOC, I(crac) and TRPC channel activation by STIM1.

On the activation mechanism of store-operated calcium channels

The development of the patch clamp technique has revolutionised our understanding of the life sciences. One area in which it has made an enormous contribution is cellular signalling. In many cell types, calcium influx across the plasma membrane is essential for the regulation of a wide range of critical physiological responses including secretion, gene transcription and cell growth. For many years the calcium influx pathways in non-excitable cells remained unknown, despite their importance in physiological and pathophysiological states. Very careful and insightful work by James Putney led to the formulation of the capacitative calcium entry (store-operated calcium influx) model, in which the process of emptying intracellular calcium stores resulted in the activation of calcium entry channels. Unequivocal evidence for this revolutionary model was provided by patch clamp studies carried out by Markus Hoth and Reinhold Penner, who demonstrated that store depletion activated a novel class of calcium channel called the CRAC channel. This review provides a historical perspective on the development of store-operated calcium influx and how patch clamping resolved a long-standing controversy in cell physiology. The review also discusses current ideas relating to how store emptying opens channels in the plasma membrane.

Movement of calcium signals and calcium-binding proteins: firewalls, traps and tunnels

In the board game 'Snakes and Ladders', placed on the image of a pancreatic acinar cell, calcium ions have to move from release sites in the secretory region to the nucleus. There is another important contraflow - from calcium entry channels in the basal part of the cell to ER (endoplasmic reticulum) terminals in the secretory granule region. Both transport routes are perilous as the messenger can disappear in any place on the game board. It can be grabbed by calcium ATPases of the ER (masquerading as a snake but functioning like a ladder) and tunnelled through its low buffering environment, it can be lured into the whirlpools of mitochondria uniporters and forced to regulate the tricarboxylic acid cycle, and it can be permanently placed inside the matrix of secretory granules and released only outside the cell. The organelles could trade calcium (e.g. from the ER to mitochondria and vice versa) almost depriving this ion the light of the cytosol and noble company of cytosolic calcium buffers. Altogether it is a rich and colourful story.

Functional Coupling between TRPC3 and RyR1 Regulates the Expressions of Key Triadic Proteins

We have shown that TRPC3 (transient receptor potential channel canonical type 3) is sharply up-regulated during the early part of myotube differentiation and remains elevated in mature myotubes compared with myoblasts. To examine its functional roles in muscle, TRPC3 was "knocked down" in mouse primary skeletal myoblasts using retroviral-delivered small interference RNAs and single cell cloning. TRPC3 knockdown myoblasts (97.6 +/- 1.9% reduction in mRNA) were differentiated into myotubes (TRPC3 KD) and subjected to functional and biochemical assays. By measuring rates of Mn(2+) influx with Fura-2 and Ca(2+) transients with Fluo-4, we found that neither excitation-coupled Ca(2+) entry nor thapsigargin-induced store-operated Ca(2+) entry was significantly altered in TRPC3 KD, indicating that expression of TRPC3 is not required for engaging either Ca(2+) entry mechanism. In Ca(2+) imaging experiments, the gain of excitation-contraction coupling and the amplitude of the Ca(2+) release seen after direct RyR1 activation with caffeine was significantly reduced in TRPC3 KD. The decreased gain appears to be due to a decrease in RyR1 Ca(2+) release channel activity, because sarcoplasmic reticulum (SR) Ca(2+) content was not different between TRPC3 KD and wild-type myotubes. Immunoblot analysis demonstrated that TRPC1, calsequestrin, triadin, and junctophilin 1 were up-regulated (1.46 +/- 1.91-, 1.42 +/- 0.08-, 2.99 +/- 0.32-, and 1.91 +/- 0.26-fold, respectively) in TRPC3 KD. Based on these data, we conclude that expression of TRPC3 is tightly regulated during muscle cell differentiation and propose that functional interaction between TRPC3 and RyR1 may regulate the gain of SR Ca(2+) release independent of SR Ca(2+) load.

Regulation of endothelial Isoc function by calcium phosphate complexation

The endothelial ISOC channel is a calcium selective store-operated calcium (SOC) entry channel whose activation is an important step leading to intercellular gap formation and increased permeability. The transient receptor potential canonical (TRPC) homologues 1 and 4 contribute to ISOC channel structure. Protein 4.1 physically and functionally links the ISOC channel to the membrane skeleton, and activation of the ISOC channel requires a proline rich region/protein 4.1 binding domain on the carboxy terminus of the TRPC4 subunit. This region contains both serine and threonine as potential phosphorylation targets. We observed that the TRPC4 subunit is phosphorylated upon thapsigargin-induced activation of SOC entry. Calcium entry through SOC entry channels leads to high calcium concentration near the pore of the channel. We hypothesized that free calcium coming through the channel pore complexes with the phosphate group(s) to regulate the channel open/closed state. As a proof of concept, calcium (Ca2+) and lanthanum (La3+) complex formation of phosphoserine was evaluated using nuclear magnetic resonance (NMR) spectroscopy. The change in 2JC-P and 3JC-P coupling constants in carbon-13 and phosphorus-31 NMR following Ca2+ (or La3+) addition to phosphoserine indicated that Ca2+ (or La3+) had readily complexed with the phosphate group. This observation is compatible with the idea that the formation of a calcium phosphate complex is important in ISOC channel function. Supported by HL60024 and HL66299.