Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca²⁺ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca²⁺ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP₃Rs. Blockade of Ca²⁺-induced Ca²⁺ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca²⁺ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca²⁺ overload whereas IP₃R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca²⁺ release through RyRs contributes to cytosolic Ca²⁺ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes.     

The role of cAMP dependent protein kinase in modulating spontaneous intracellular Ca waves in interstitial cells of Cajal from the rabbit urethra

Interstitial cells of Cajal (ICC) serve as electrical pacemakers in the rabbit urethra. Pacemaking activity in ICC results from spontaneous intracellular Ca²⁺ waves that rely on Ca²⁺ release from endoplasmic reticulum (ER) stores. The purpose of this study was to investigate if the action of protein kinase A (PKA) affected the generation of Ca²⁺ waves in ICC. Intracellular [Ca²⁺] was measured in fluo-4 loaded ICC, freshly isolated from the rabbit urethra using a Nipkow spinning disc confocal microscope. Application of the PKA inhibitor H-89 (10 μM) significantly inhibited the generation of spontaneous Ca²⁺ waves in ICC and this was associated with a significant decrease in the ER Ca²⁺ load, measured with 10 mM caffeine responses. Ca²⁺ waves could be rescued in the presence of H-89 by stimulating ryanodine receptors (RyRs) with 1 mM caffeine but not by activation of inositol 1,4,5 tri-phosphate receptors (IP₃Rs) with 10 μM phenylephrine. Increasing intracellular PKA with the cAMP agonists forskolin and 8-bromo-cAMP failed to yield an increase in Ca²⁺ wave activity. We conclude that PKA may be maximally active under basal conditions in ICC and that inhibition of PKA with H-89 leads to a decreased ER Ca²⁺ load sufficient to inactivate IP₃Rs but not RyRs.     

Fluvoxamine rescues mitochondrial Ca transport and ATP production through σ1-receptor in hypertrophic cardiomyocytes

Aims

We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor with high affinity for the σ₁-receptor (σ₁R), ameliorates cardiac hypertrophy and dysfunction via σ₁R stimulation. Although σ₁R on non-cardiomyocytes interacts with the IP₃ receptor (IP₃R) to promote mitochondrial Ca^2 + transport, little is known about its physiological and pathological relevance in cardiomyocytes.

Main methods

Here we performed Ca^2 + imaging and measured ATP production to define the role of σ₁Rs in regulating sarcoplasmic reticulum (SR)-mitochondrial Ca^2 + transport in neonatal rat ventricular cardiomyocytes treated with angiotensin II to promote hypertrophy.

Key finding

These cardiomyocytes exhibited imbalances in expression levels of σ₁R and IP₃R and impairments in both phenylephrine-induced mitochondrial Ca^2 + mobilization from the SR and ATP production. Interestingly, σ₁R stimulation with fluvoxamine rescued impaired mitochondrial Ca^2 + mobilization and ATP production, an effect abolished by treatment of cells with the σ₁R antagonist, NE-100. Under physiological conditions, fluvoxamine stimulation of σ₁Rs suppressed intracellular Ca^2 + mobilization through IP₃Rs and ryanodine receptors (RyRs). In vivo, chronic administration of fluvoxamine to TAC mice also rescued impaired ATP production.

Significance

These results suggest that σ₁R stimulation with fluvoxamine promotes SR-mitochondrial Ca^2 + transport and mitochondrial ATP production, whereas σ₁R stimulation suppresses intracellular Ca^2 + overload through IP₃Rs and RyRs. These mechanisms likely underlie in part the anti-hypertrophic and cardioprotective action of the σ₁R agonists including fluvoxamine.     

IP3 receptors in cell survival and apoptosis: Ca2+ release and beyond

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) serve to discharge Ca²⁺ from ER stores in response to agonist stimulation. The present review summarizes the role of these receptors in models of Ca²⁺-dependent apoptosis. In particular we focus on the regulation of IP₃Rs by caspase-3 cleavage, cytochrome c, anti-apoptotic proteins and Akt kinase. We also address the evidence that some of the effects of IP₃Rs in apoptosis may be independent of their ion-channel function. The role of IP₃Rs in delivering Ca²⁺ to the mitochondria is discussed from the perspective of the factors determining inter-organellar dynamics and the spatial proximity of mitochondria and ER membranes.     

Re-evaluation of the Role of Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) in Cellular Calcium Signaling

Changes in cytoplasmic Ca²⁺ concentration, resulting from activation of intracellular Ca²⁺ channels within the endoplasmic reticulum, regulate several aspects of cellular growth and differentiation. Ca²⁺ homeostasis endoplasmic reticulum protein (CHERP) is a ubiquitously expressed protein that has been proposed as a regulator of both major families of endoplasmic reticulum Ca²⁺ channels, inositol 1,4,5-trisphosphate receptors (IP₃Rs) and ryanodine receptors (RyRs), with resulting effects on mitotic cycling. However, the manner by which CHERP regulates intracellular Ca²⁺ channels to impact cellular growth is unknown. Here, we challenge previous findings that CHERP acts as a direct cytoplasmic regulator of IP₃Rs and RyRs and propose that CHERP acts in the nucleus to impact cellular proliferation by regulating the function of the U2 snRNA spliceosomal complex. The previously reported effects of CHERP on cellular growth therefore are likely indirect effects of altered spliceosomal function, consistent with prior data showing that loss of function of U2 snRNP components can interfere with cell growth and induce cell cycle arrest.     

Mechanisms of calcium signaling in smooth muscle cells explored with fluorescence confocal imaging

A rise in the intracellular concentration of ionized calcium ([Ca²⁺]_i) is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques, with special accent on fluorescence confocal microscopy, and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for studying the relationship between the structural organization of living smooth muscle cells (SMCs) and features of calcium signaling at the subcellular level. Applying fluorescent confocal imaging, patch-clamp recording, immunostaining, and flash photolysis techniques to freshly isolated SMCs, we have demonstrated that: (i) Ca²⁺ sparks are mediated by spontaneous clustered opening of ryanodine receptors (RyRs) and occur at the highest rate at preferred sites (frequent discharge sites, FDSs), the number of which depends on SMC type; (ii) FDSs are associated with sub-plasmalemmal sarcoplasmic reticulum (SR) elements, but not with polarized mitochondria; (iii) Ca²⁺ spark frequency increases with membrane depolarization in voltage-clamped SMCs or following neurotransmitter application to SMCs, in which the membrane potential was not controlled, leading to spark summation and resulting in a cell-wide increase in [Ca²⁺]_i and myocyte contraction; (iv) cross-talk between RyRs and inositol trisphosphate receptors (IP₃Rs) is an important determinant of the [Ca²⁺]_i dynamics and recruits neighboring Ca²⁺-release sites to generate [Ca²⁺]_i waves; (v) [Ca²⁺]_i waves induced by depolarization of the plasma membrane or by noradrenaline or caffeine, but not by carbachol (CCh), originate at FDSs; (vi) Ca²⁺-dependent K⁺ and Cl^- channels sense the local changes in [Ca²⁺]_i during a Ca²⁺ spark and thereby may couple changes in [Ca²⁺]_i within a microdomain to changes in the membrane potential, thus affecting the cell excitability; (vii) the muscarinic cation current (mI _cat) does not mirror changes in [Ca²⁺]_i, thus reflecting the complexity of [Ca²⁺]_i — muscarinic cationic channel coupling; (viii) RyR-mediated Ca²⁺ release, either spontaneous or caffeine-induced, does not augment mI _cat; (ix) intracellular flash release of Ca²⁺ is less effective in augmentation of mI _cat than flash release of IP₃, suggesting that IP₃ may sensitize muscarinic cationic channels to Ca²⁺; (x) intracellular flash release of IP₃ fails to augment mI _cat in SMCs, in which [Ca²⁺]_i was strongly buffered, suggesting that IP₃ exerts no direct effect on muscarinic cationic channel gating, and that these channels sense an increase in [Ca²⁺]_i rather than depletion of the IP₃-dependent Ca²⁺ store; and (xi) predominant expression of IP₃R type 1 in the peripheral SR provides a structural basis for a tight functional coupling between IP₃R-mediated Ca²⁺ release and muscarinic cationic channel opening.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 455–465, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

Atrial local Ca signaling and inositol 1,4,5-trisphosphate receptors

In atrial myocytes lacking t-tubules, action potential triggers junctional Ca²⁺ releases in the cell periphery, which propagates into the cell interior. The present article describes growing evidence on atrial local Ca²⁺ signaling and on the functions of inositol 1,4,5-trisphosphate receptors (IP₃Rs) in atrial myocytes, and show our new findings on the role of IP₃R subtype in the regulation of spontaneous focal Ca²⁺ releases in the compartmentalized areas of atrial myocytes. The Ca²⁺ sparks, representing focal Ca²⁺ releases from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR) clusters, occur most frequently at the peripheral junctions in isolated resting atrial cells. The Ca²⁺ sparks that were darker and longer lasting than peripheral and non-junctional (central) sparks, were found at peri-nuclear sites in rat atrial myocytes. Peri-nuclear sparks occurred more frequently than central sparks. Atrial cells express larger amounts of IP₃Rs compared with ventricular cells and possess significant levels of type 1 IP₃R (IP₃R1) and type 2 IP₃R (IP₃R2). Over the last decade the roles of atrial IP₃R on the enhancement of Ca²⁺-induced Ca²⁺ release and arrhythmic Ca²⁺ releases under hormonal stimulations have been well documented. Using protein knock-down method and confocal Ca²⁺ imaging in conjunction with immunocytochemistry in the adult atrial cell line HL-1, we could demonstrate a role of IP₃R1 in the maintenance of peri-nuclear and non-junctional Ca²⁺ sparks via stimulating a posttranslational organization of RyR clusters.     

Mitochondrial Calcium Signaling Driven by the IP3 Receptor

Many agonists bring about their effects on cellular functions through a rise incytosolic [Ca²⁺]([Ca²⁺]_c) mediated by the second messenger inositol 1,4,5-trisphosphate (IP₃). Imaging studiesof single cells have demonstrated that [Ca²⁺]_c signals display cell specific spatiotemporalorganization that is established by coordinated activation of IP₃ receptor Ca²⁺ channels.Evidence emerges that cytosolic calcium signals elicited by activation of the IP₃ receptors areefficiently transmitted to the mitochondria. An important function of mitochondrial calciumsignals is to activate the Ca²⁺-sensitive mitochondrial dehydrogenases, and thereby to meetdemands for increased energy in stimulated cells. Activation of the permeability transitionpore (PTP) by mitochondrial calcium signals may also be involved in the control of cell death.Furthermore, mitochondrial Ca²⁺ transport appears to modulate the spatiotemporal organizationof [Ca²⁺]_c responses evoked by IP₃ and so mitochondria may be important in cytosolic calciumsignaling as well. This paper summarizes recent research to elucidate the mechanisms andsignificance of IP₃-dependent mitochondrial calcium signaling.     

Comparison of Ca2+ puffs evoked by extracellular agonists and photoreleased IP3

The inositol trisphosphate (IP₃) signaling pathway evokes local Ca²⁺ signals (Ca²⁺ puffs) that arise from the concerted openings of clustered IP₃ receptor/channels in the ER membrane. Physiological activation is triggered by binding of agonists to G-protein-coupled receptors (GPCRs) on the cell surface, leading to cleavage of phosphatidyl inositol bisphosphate and release of IP₃ into the cytosol. Photorelease of IP₃ from a caged precursor provides a convenient and widely employed means to study the final stage of IP₃-mediated Ca²⁺ liberation, bypassing upstream signaling events to enable more precise control of the timing and relative concentration of cytosolic IP₃. Here, we address whether Ca²⁺ puffs evoked by photoreleased IP₃ fully replicate those arising from physiological agonist stimulation. We imaged puffs in individual SH-SY5Y neuroblastoma cells that were sequentially stimulated by picospritzing extracellular agonist (carbachol, CCH or bradykinin, BK) followed by photorelease of a poorly-metabolized IP₃ analog, i-IP₃. The centroid localizations of fluorescence signals during puffs evoked in the same cells by agonists and photorelease substantially overlapped (within ∼1 μm), suggesting that IP₃ from both sources accesses the same, or closely co-localized clusters of IP₃Rs. Moreover, the time course and spatial spread of puffs evoked by agonists and photorelease matched closely. Because photolysis generates IP₃ uniformly throughout the cytoplasm, our results imply that IP₃ generated in SH-SY5Y cells by activation of receptors to CCH and BK also exerts broadly distributed actions, rather than specifically activating a subpopulation of IP₃Rs that are scaffolded in close proximity to cell surface receptors to form a signaling nanodomain.     

Inositol 1,4,5-trisphosphate receptor-isoform diversity in cell death and survival

Cell-death and -survival decisions are critically controlled by intracellular Ca^2 + homeostasis and dynamics at the level of the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) play a pivotal role in these processes by mediating Ca^2 + flux from the ER into the cytosol and mitochondria. Hence, it is clear that many pro-survival and pro-death signaling pathways and proteins affect Ca^2 + signaling by directly targeting IP₃R channels, which can happen in an IP₃R-isoform-dependent manner. In this review, we will focus on how the different IP₃R isoforms (IP₃R1, IP₃R2 and IP₃R3) control cell death and survival. First, we will present an overview of the isoform-specific regulation of IP₃Rs by cellular factors like IP₃, Ca^2 +, Ca^2 +-binding proteins, adenosine triphosphate (ATP), thiol modification, phosphorylation and interacting proteins, and of IP₃R-isoform specific expression patterns. Second, we will discuss the role of the ER as a Ca^2 + store in cell death and survival and how IP₃Rs and pro-survival/pro-death proteins can modulate the basal ER Ca^2 + leak. Third, we will review the regulation of the Ca^2 +-flux properties of the IP₃R isoforms by the ER-resident and by the cytoplasmic proteins involved in cell death and survival as well as by redox regulation. Hence, we aim to highlight the specific roles of the various IP₃R isoforms in cell-death and -survival signaling. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

IP3 receptors and Ca2+ entry

Inositol 1,4,5-trisphosphate receptors (IP₃R) are the most widely expressed intracellular Ca²⁺ release channels. Their activation by IP₃ and Ca²⁺ allows Ca²⁺ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca²⁺] may directly regulate cytosolic effectors or fuel Ca²⁺ uptake by other organelles, while the decrease in ER luminal [Ca²⁺] stimulates store-operated Ca²⁺ entry (SOCE). We are close to understanding the structural basis of both IP₃R activation, and the interactions between the ER Ca²⁺-sensor, STIM, and the plasma membrane Ca²⁺ channel, Orai, that lead to SOCE. IP₃Rs are the usual means through which extracellular stimuli, through ER Ca²⁺ release, stimulate SOCE. Here, we review evidence that the IP₃Rs most likely to respond to IP₃ are optimally placed to allow regulation of SOCE. We also consider evidence that IP₃Rs may regulate SOCE downstream of their ability to deplete ER Ca²⁺ stores. Finally, we review evidence that IP₃Rs in the plasma membrane can also directly mediate Ca²⁺ entry in some cells.     

Variable luminal sarcoplasmic reticulum Ca buffer capacity in smooth muscle cells

Sarcoplasmic reticulum contains the internal Ca²⁺ store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca²⁺ level is the same all throughout the SR; however, whether the Ca²⁺ buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca²⁺ buffer capacity of the SR by comparing the reduction in SR Ca²⁺ levels with the corresponding increase in [Ca²⁺]_i during activation of either IP₃Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca²⁺ buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca²⁺ buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca²⁺ buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca²⁺ replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca²⁺ influx, indicate the relevance of luminal SR Ca²⁺ buffer capacity in the [Ca²⁺]_i response. To further study the importance of luminal SR Ca²⁺ buffer capacity in the release process we used low levels of heparin to partially inhibit IP₃Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca²⁺ levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca²⁺-binding proteins in the high-capacity, low-affinity conformation, particularly for IP₃R-mediated Ca²⁺ release.     

Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle

Ca²⁺-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca²⁺ inhibited cardiac RyRs with an average K _m = 15 mm, skeletal RyRs with K _m = 0.7 mm and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca²⁺ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [³H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca²⁺-inhibition in cardiac RyRs (Chu, Fill, Stefani &; Entman (1993) J. Membrane Biol. 135, 49–59). Ryanodine prevented Ca²⁺ from inhibiting either cardiac or skeletal RyRs. Ca²⁺-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mm Cs⁺, but not by 500 mm K⁺, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca²⁺-activation. Ca²⁺-inhibition in skeletal RyRs was retained in the presence of 500 mm Cs⁺. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mm Cs⁺ in the solutions fail to demonstrate Ca²⁺-inhibition, while Ca²⁺-inhibition of Ca²⁺ release is observed in vesicle studies where K⁺ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca²⁺-inhibition in skeletal RyRs and cardiac RyRs, with different Ca²⁺ affinities for inhibition. We conclude that differences in the Ca²⁺-inhibition in cardiac and skeletal channels depends on their Ca²⁺ binding properties.     

The IP3 receptor-mitochondria connection in apoptosis and autophagy

The amount of Ca²⁺ taken up in the mitochondrial matrix is a crucial determinant of cell fate; it plays a decisive role in the choice of the cell between life and death. The Ca²⁺ ions mainly originate from the inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores of the endoplasmic reticulum (ER). The uptake of these Ca²⁺ ions in the mitochondria depends on the functional properties and the subcellular localization of the IP₃ receptor (IP₃R) in discrete domains near the mitochondria. To allow for an efficient transfer of the Ca²⁺ ions from the ER to the mitochondria, structural interactions between IP₃Rs and mitochondria are needed. This review will focus on the key proteins involved in these interactions, how they are regulated, and what are their physiological roles in apoptosis, necrosis and autophagy. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Tracing the evolutionary history of Ca2+-signaling modulation by human Bcl-2: Insights from the Capsaspora owczarzaki IP3 receptor ortholog

Recently, a functional IP₃R ortholog (CO.IP₃R-A) capable of IP₃-induced Ca²⁺ release has been discovered in Capsaspora owczarzaki, a close unicellular relative to Metazoa. In contrast to mammalian IP₃Rs, CO.IP₃R-A is not modulated by Ca²⁺, ATP or PKA. Protein-sequence analysis revealed that CO.IP₃R-A contained a putative binding site for anti-apoptotic Bcl-2, although Bcl-2 was not detected in Capsaspora owczarzaki and only appeared in Metazoa. Here, we examined whether human Bcl-2 could form a complex with CO.IP₃R-A channels and modulate their Ca²⁺-flux properties using ectopic expression approaches in a HEK293 cell model in which all three IP₃R isoforms were knocked out. We demonstrate that human Bcl-2 via its BH4 domain could functionally interact with CO.IP₃R-A, thereby suppressing Ca²⁺ flux through CO.IP₃R-A channels. The BH4 domain of Bcl-2 was sufficient for interaction with CO.IP₃R-A channels. Moreover, mutating the Lys17 of Bcl-2's BH4 domain, the residue critical for Bcl-2-dependent modulation of mammalian IP₃Rs, abrogated Bcl-2's ability to bind and inhibit CO.IP₃R-A channels. Hence, this raises the possibility that a unicellular ancestor of animals already had an IP₃R that harbored a Bcl-2-binding site. Bcl-2 proteins may have evolved as controllers of IP₃R function by exploiting this pre-existing site, thereby counteracting Ca²⁺-dependent apoptosis.     

The actin cytoskeleton differentially regulates NG115-401L cell ryanodine receptor and inositol 1,4,5-trisphosphate receptor induced calcium signaling pathways

Regulation of bi-directional communication between intracellular Ca²⁺ pools and surface Ca²⁺ channels remains incompletely characterized. We report Ca²⁺ release mediated by inositol 1,4,5-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR) pathways is diminished under actin cytoskeleton disruption in NG115-401L (401L) neuronal cells, yet despite truncated Ca²⁺ release, Ca²⁺ influx was not significantly altered in these experiments. However, disruption of cortical actin networks completely abolished IP₃R induced Ca²⁺ release, whereas RyR-mediated Ca²⁺ release was preserved, albeit attenuated. Moreover, cortical actin disruption completely abolished IP₃R and RyR linked Ca²⁺ influx even though Ca²⁺ pool sensitivities were different. These findings suggest discrete Ca²⁺ store/Ca²⁺ channel coupling mechanisms in the IP₃R and RyR pathways as revealed by the differential sensitivity to actin perturbation.     

Regulation of transient receptor potential melastatin 4 channel by sarcoplasmic reticulum inositol trisphosphate receptors: Role in human detrusor smooth muscle function

We recently reported key physiologic roles for Ca²⁺-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca²⁺-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca²⁺, inositol 1,4,5-trisphosphate receptor (IP₃R)-mediated Ca²⁺ release from the sarcoplasmic reticulum represents a potential Ca²⁺ source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP₃Rs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IP₃Rs and are activated by IP₃R-mediated Ca²⁺ release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IP₃Rs in human DSM cells. As the TRPM4 channels and IP₃Rs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca²⁺-mediated TRPM4-IP₃R regulatory mechanism. To investigate IP₃R regulation of TRPM4 channel activity, we sought to determine the consequences of IP₃R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, blocking the IP₃Rs with the selective IP₃R inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IP₃Rs have a key role in mediating the Ca²⁺-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IP₃Rs are spatially and functionally coupled in human DSM.     

Properties of the charibdotoxin-sensitive component of Ca2+-dependent K+ current in smooth muscle cells of the guinea pigTaenia Coli

Using the voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea pigtaenia coli. In our study, we identified and studied a charibdotoxin-sensitive component of Ca²⁺-dependent K⁺ current carried through the channels of high conductance (in most publications called “big conductance,”I _BK(Ca)). This component was completely blocked by 100 nM charibdotoxin and by tetraethylammonium in concentrations as low as 1 mM.I _BK(Ca) demonstrated fast kinetics of inactivation, which nearly coincided with that of Ca²⁺ current. In addition to the dependence on Ca²⁺ concentration, this current also showed voltage-dependent properties: with a rise in the level of depolarization its amplitude increased. In many cells, depolarizing shifts in the membrane potential evoke spontaneous outward currents. Such currents probably represent the secondary effect of cyclic Ca²⁺ release from the caffeine-sensitive intracellular stores that result in short-term activation of charibdotoxin-sensitive Ca²⁺-dependent K⁺ channels.     

Single-Molecule Tracking of Inositol Trisphosphate Receptors Reveals Different Motilities and Distributions

Puffs are local Ca²⁺ signals that arise by Ca²⁺ liberation from the endoplasmic reticulum through the concerted opening of tightly clustered inositol trisphosphate receptors/channels (IP₃Rs). The locations of puff sites observed by Ca²⁺ imaging remain static over several minutes, whereas fluorescence recovery after photobleaching (FRAP) experiments employing overexpression of fluorescently tagged IP₃Rs have shown that the majority of IP₃Rs are freely motile. To address this discrepancy, we applied single-molecule imaging to locate and track type 1 IP₃Rs tagged with a photoswitchable fluorescent protein and expressed in COS-7 cells. We found that ∼70% of the IP₃R1 molecules were freely motile, undergoing random walk motility with an apparent diffusion coefficient of ∼0.095 μm s⁻¹, whereas the remaining molecules were essentially immotile. A fraction of the immotile IP₃Rs were organized in clusters, with dimensions (a few hundred nanometers across) comparable to those previously estimated for the IP₃R clusters underlying functional puff sites. No short-term (seconds) changes in overall motility or in clustering of immotile IP₃Rs were apparent following activation of IP₃/Ca²⁺ signaling. We conclude that stable clusters of small numbers of immotile IP₃Rs may underlie local Ca²⁺ release sites, whereas the more numerous motile IP₃Rs appear to be functionally silent.     

Functional role of the calmodulin- and inositol 1,4,5-trisphosphate receptor-binding (CIRB) site of TRPC6 in human platelet activation

Background

All identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP₃Rs)-binding (CIRB) site involved in the regulation of TRPC channel function.

Objectives

To assess the basis of CaM/IP₃Rs binding to the CIRB site of TRPC6 and its role in platelet physiology.

Methods

Protein association was detected by co-immunoprecipitation and Western blotting, Ca²⁺ mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry.

Results

Co-immunoprecipitation of TRPC6 with CaM or the IP₃Rs at different cytosolic free Ca²⁺ concentrations ([Ca²⁺]_c) indicates that the association between these proteins is finely regulated by cytosolic Ca²⁺ via association of CaM and displacement of the IP₃Rs at high [Ca²⁺]_c. Thrombin-stimulated association of TRPC6 with CaM or the IP₃Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca²⁺-dependent and -independent mechanisms. Incorporation of an anti-TRPC6 C-terminal antibody, whose epitope overlaps the CIRB region, impaired the dynamics of the association of TRPC6 with CaM and the IP₃Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca²⁺ entry in the presence of low or high, respectively, extracellular Ca²⁺ concentrations, as well as altered thrombin-evoked platelet aggregation.

Conclusions

Our results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca²⁺ entry and aggregation through its interaction with CaM and IP₃Rs.