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Statins, inhibitors of HMG-CoA reductase, are mainstay treatment for hypercholesterolaemia. However, muscle pain and weakness prevent many patients from benefiting from their cardioprotective effects. We previously demonstrated that simvastatin activates skeletal ryanodine receptors (RyR1), an effect that could be important in initiating myopathy. Using a range of structurally diverse statin analogues, we examined structural features associated with RyR1 activation, aiming to identify statins lacking this property.Compounds were screened for RyR1 activity utilising [All UK-prescribed statins activated RyR1 at nanomolar concentrations. Cerivastatin, withdrawn from the market due to life-threatening muscle-related side effects, was more effective than currently-prescribed statins and possessed the unique ability to open RyR1 channels independently of cytosolic CaThat cerivastatin activates RyR1 most strongly supports the hypothesis that RyR1 activation is implicated in statin-induced myopathy. Demonstrating that statin regulation of RyR1 and HMG-CoA reductase are separable effects will allow the role of RyR1 in statin-induced myopathy to be further elucidated by the tool compounds we have identified, allowing development of effective cardioprotective statins with improved patient tolerance.

Key points RyR2 mutations can cause type-1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a lethal, autosomal-dominant arrhythmic disease. However, the changes in RyR2 ion-channel function that result from the many different patient mutations are rarely investigated in detail and often only recombinant RyR2, homozygous for the mutation, are studied. As CPVT1 is a heterozygous disease and the tetrameric RyR2 channels expressed in the heart will contain varying numbers of mutated monomers, we have investigated the range of RyR2 single-channel abnormalities found in the hearts of mice heterozygous for the CPVT1 mutation, V2475F(+/-)-RyR2. Specific alterations to ligand regulation of V2475F(+/-)-RyR2 were observed. Multiple sub-populations of channels exhibited varying degrees of abnormality. In particular, an increased sensitivity to activating and inactivating cytosolic [Ca2+ ], and reduced sensitivity to Mg2+ inhibition were evident. Our results provide mechanistic insight into the changes to RyR2 gating that destabilise SR Ca2+ -release causing life-threatening arrhythmias in V2475F(+/-)-CPVT1 patients. Abstract Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is a lethal genetic disease causing arrhythmias and sudden cardiac death in children and young adults and is linked to mutations in the cardiac ryanodine receptor (RyR2). The effects of CPVT1 mutations on RyR2 ion-channel function are often investigated using purified recombinant RyR2 channels homozygous for the mutation. However, CPVT1 patients are heterozygous for the disease, so this approach does not reveal the true changes to RyR2 function across the entire RyR2 population of channels in the heart. We therefore investigated the native cardiac RyR2 single-channel abnormalities in mice heterozygous for the CPVT1 mutation, V2475F(+/-)-RyR2, and applied molecular modelling techniques to investigate the possible structural changes that could initiate any altered function. We observed that increased sensitivity of cardiac V2475F(+/-)-RyR2 channels to both activating and inactivating levels of cytosolic Ca2+ , plus attenuation of Mg2+ inhibition, were the most marked changes. Severity of abnormality was not uniform across all channels, giving rise to multiple sub-populations with differing functional characteristics. For example, 46% of V2475F(+/-)-RyR2 channels exhibited reduced Mg2+ inhibition and 23% were actually activated by Mg2+ . Using homology modelling, we discovered that V2475 is situated at a hinge between two regions of the RyR2 helical-domain-1 (HD1). Our model proposes that detrimental functional changes to RyR2 arise because mutation at this critical site reduces the angle between these regions. Our results demonstrate the necessity of characterising the total heterozygous population of CPVT1-mutated channels in order to understand CPVT1 phenotypes in patients. Abstract figure: (Left) RyR2 Helical Domain-1 with the V2475F mutation site indicated by the red circle. (Top right) Typical RyR2 single-channel recordings highlighting the increased opening burst durations of RyR2 isolated from V2475F(+/-) hearts compared to wild type hearts. (Bottom right) Possible RyR2 tetramer formations in V2475F(+/-) hearts. White (V) and green (F) circles represent wild type and mutated monomers, respectively. This article is protected by copyright. All rights reserved.

Ca2+ signaling is altered substantially in many cancers. The ryanodine receptors (RYRs) are among the key ion channels in Ca2+ signaling. This study aimed to establish the mutational profile of RYR in cancers and investigate the correlation between RYR alterations and cancer phenotypes. The somatic mutation and clinical data of 11,000 cancer patients across 33 cancer types was downloaded from The Cancer Genome Atlas (TCGA) database. Subsequent data processing was performed with corresponding packages of the R software. Mutational profile was analyzed and its correlation with tumor mutational burden (TMB), patient prognosis, age and smoking status was analyzed and compared. All three RYR isoforms exhibited random mutational distribution without hotspot mutations when all cancers were analyzed together. The number of mutations in RYR2 (2388 mutations) far overweight that of RYR1 (1439 mutations) and RYR3 (1573 mutations). Linear correlation was observed between cumulative TMB and cumulative number of mutations for all RYR isoforms. Patients with RYR mutations exhibited significantly higher TMB than those without RYR mutations for most cancer types. Strong correlation was also revealed in the average number of mutations per person between pairs of RYR isoforms. No stratification of patient overall survival (OS) by mutational status was found for all three RYR isoforms when all cancers were analyzed together, however, significant stratification of OS by RYR mutations was revealed in several individual cancers, most strikingly in LUAD (P = 0.0067, RYR1), BLCA (P = 0.00071, RYR2), LUSC (P = 0.036, RYR2) and KIRC (P = 0.0042, RYR3). Furthermore, RYR mutations were correlated with higher age, higher smoking history grading and higher number of pack years. Characteristic mutation profile of RYRs in cancers has been revealed for the first time. RYR mutations were correlated with TMB, age, smoking status and capable of stratifying the prognosis of patients in several cancer types.

Trimeric intracellular cation channels (TRIC-A and TRIC-B), found in the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes, are thought to provide countercurrents to balance Ca2+-movements across the SR, but there is also evidence that they physically interact with ryanodine receptors (RYR). We therefore investigated if TRIC channels could modulate the single-channel function of RYR2 after incorporation of vesicles isolated from HEK293 cells expressing TRIC-A or TRIC-B with RYR2 into artificial membranes under voltage clamp. We also examined the gating and conductance properties of TRIC channels. Co-expression of RYR2 with either TRIC-A or TRIC-B significantly altered the gating behavior of RYR2; however, co-expression with TRIC-A was particularly effective at potentiating the activating effects of cytosolic Ca2+. Fusing membrane vesicles containing TRIC-A or TRIC-B together with RYR2 into bilayers produced large currents of rapidly gating current fluctuations of multiple amplitudes. In 740 cytosolic/210 luminal mM KCl gradient, current-voltage relationships of macroscopic currents revealed average reversal potentials (Erev) of −13.67 ± 9.02 (n = 7), −2.11 ± 3.84 (n = 11), and 13.19 ± 3.23 (n = 13, **, P = 0.0025) from vesicles from RYR2 only, RyR2 + TRIC-A, or RyR2 + TRIC-B cells, respectively. Thus, with the incorporation of TRIC channels, the Erevs depart further from the calculated Erev for ideally selective cation channels than occurs when vesicles from RYR2-only cells are incorporated, suggesting that TRIC channels are permeable to both K+ and Cl−. In conclusion, our results indicate that both TRIC-A and TRIC-B regulate the gating of RYR2, but that TRIC-A has greater capacity to stimulate the RYR2 opening. The results also suggest that TRIC channels may be relatively nonselective ion channels being permeable to both cations and anions. This property would enable TRIC channels to be versatile providers of counter-ion current throughout the SR of many cell types.

Recessive ryanodine receptor 1 (RYR1) mutations cause congenital myopathies including multiminicore disease (MmD), congenital fiber-type disproportion and centronuclear myopathy. We created a mouse model knocked-in for the Q1970fsX16+A4329D RYR1 mutations, which are isogenic with those identified in a severely affected child with MmD. During the first 20 weeks after birth the body weight and the spontaneous running distance of the mutant mice were 20% and 50% lower compared to wild-type littermates. Skeletal muscles from mutant mice contained ‘cores’ characterized by severe myofibrillar disorganization associated with misplacement of mitochondria. Furthermore, their muscles developed less force and had smaller electrically evoked calcium transients. Mutant RyR1 channels incorporated into lipid bilayers were less sensitive to calcium and caffeine, but no change in single-channel conductance was observed. Our results demonstrate that the phenotype of the RyR1Q1970fsX16+A4329D compound heterozygous mice recapitulates the clinical picture of multiminicore patients and provide evidence of the molecular mechanisms responsible for skeletal muscle defects.

Background and purpose Statins, inhibitors of HMG-CoA reductase, are mainstay treatment for hypercholesterolemia. However, muscle pain and weakness prevent many patients from benefiting from their cardioprotective effects. We previously demonstrated that simvastatin activates skeletal ryanodine receptors (RyR1), an effect that could be important in initiating myopathy. We therefore investigated if RyR1 activation is a standard property of commonly-prescribed statins. Using a range of structurally-diverse statin analogues we examined structural features associated with RyR1 activation, aiming to identify statins lacking this property. Experimental Approach Compounds were screened for RyR1 activity utilising [H]ryanodine binding. Mechanistic insight into RyR1 activity was studied by incorporating RyR1 channels from sheep, mouse or rabbit skeletal muscle into bilayers. Key Results All UK-prescribed statins activated RyR1 at nanomolar concentrations. Cerivastatin, withdrawn from the market due to life-threatening muscle-related side effects, was more effective than currently-prescribed statins and possessed the unique ability to open RyR1 channels independently of cytosolic Ca. We synthesised the statin pharmacophore and it did not activate RyR1. We also identified five analogues retaining potent HMG-CoA reductase inhibition that inhibited RyR1 and four analogues that lacked the ability to activate RyR1. Conclusion and Implications That cervistatin activates RyR1 most strongly supports the hypothesis that RyR1 activation is implicated in statin-induced myopathy. Demonstrating that statin-regulation of RyR1 and HMG-CoA reductase are separable effects allows the role of RyR1 in statin-induced myopathy to be further elucidated by the tool compounds identified, thus paving the way for the development of effective cardioprotective statins with improved patient tolerance.

Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is a lethal genetic disease causing arrhythmias and sudden cardiac death in children and young adults and is linked to mutations in the cardiac ryanodine receptor (RyR2). The effects of CPVT1 mutations on RyR2 ion-channel function are often investigated using purified recombinant RyR2 channels homozygous for the mutation. However, CPVT1 patients are heterozygous for the disease, so this approach does not reveal the true changes to RyR2 function across the entire RyR2 population of channels in the heart. We therefore investigated the native cardiac RyR2 single-channel abnormalities in mice heterozygous for the CPVT1 mutation, V2475F(+/−)-RyR2, and applied molecular modelling techniques to investigate the possible structural changes that could initiate any altered function. We observed that increased sensitivity of cardiac V2475F(+/−)-RyR2 channels to both activating and inactivating levels of cytosolic Ca(2+), plus attenuation of Mg(2+) inhibition, were the most marked changes. Severity of abnormality was not uniform across all channels, giving rise to multiple sub-populations with differing functional characteristics. For example, 46% of V2475F(+/−)-RyR2 channels exhibited reduced Mg(2+) inhibition and 23% were actually activated by Mg(2+). Using homology modelling, we discovered that V2475 is situated at a hinge between two regions of the RyR2 helical-domain-1 (HD1). Our model proposes that detrimental functional changes to RyR2 arise because mutation at this critical site reduces the angle between these regions. Our results demonstrate the necessity of characterising the total heterozygous population of CPVT1-mutated channels in order to understand CPVT1 phenotypes in patients.

Nicotinic acid adenine dinucleotide phosphate (NAADP) potently releases Ca(2+) from acidic intracellular endolysosomal Ca(2+) stores. It is widely accepted that two types of two-pore channels, termed TPC1 and TPC2, are responsible for the NAADP-mediated Ca(2+) release but the underlying mechanisms regulating their gating appear to be different. For example, although both TPC1 and TPC2 are activated by NAADP, TPC1 appears to be additionally regulated by cytosolic Ca(2+) . Ion conduction and permeability also differ markedly. TPC1 and TPC2 are permeable to a range of cations although biophysical experiments suggest that TPC2 is slightly more selective for Ca(2+) over K(+) than TPC1 and hence capable of releasing greater quantities of Ca(2+) from acidic stores. TPC1 is also permeable to H(+) and therefore may play a role in regulating lysosomal and cytosolic pH, possibly creating localised acidic domains. The significantly different gating and ion conducting properties of TPC1 and TPC2 suggest that these two ion channels may play complementary physiological roles as Ca(2+) -release channels of the endolysosomal system.

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase and ion channel activity, or specific chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident calcium-regulated lipid scramblase. Our crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional structures solved by cryo-EM reveal extensive conformational changes extending from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity. Our results suggest mechanisms by which missense variants of TMEM16K could cause SCAR10 ataxia, providing new hypotheses to explore for therapy.

What is the topic of this review? Excitation-contraction coupling in fish hearts is maintained over a range of temperatures that would be cardioplegic to most mammals. Here, we review what is known about the fish cardiac ryanodine receptor, and consider how it may be regulated in a different manner from the mammalian cardiac isoforms of this channel. What advances does it highlight? We highlight how a better understanding of the basic gating and conducting properties of fish cardiac ryanodine receptors could provide considerable insight into mechanisms underlying sarcoplasmic reticulum calcium release in fish hearts and the role of the sarcoplasmic reticulum in the evolution of the heart. The fish cardiac sarcoplasmic reticulum (SR) holds large quantities of Ca(2+), but calcium-induced calcium release (CICR) is weak in these myocytes, and contraction and relaxation are largely determined by transsarcolemmal Ca(2+) flux. Many fish species live in a cold and seasonally variable thermal habitat, which could provide challenges to regulation of excitation-contraction coupling. Here, we focus on the cardiac SR Ca(2+)-release channel (RyR2) in fish and ask whether it may be regulated in a different manner from the mammalian RyR2. We review data indicating that fish cardiac RyR are present at lower density, are more spatially separated within the SR membrane and are less responsive to cytosolic Ca(2+) than mammalian RyR2 channels. All of these features would contribute to the weak CICR evident from functional studies. We also consider how CICR can be enhanced in fish myocytes following β-adrenergic stimulation and application of low levels of caffeine, and how acute and chronic temperature change may affect the gating properties of fish RyR2s. It is clear that a lack of insight into the fundamental gating and conductance properties of fish RyR2 channels is hindering our understanding of the role of the SR in fish cardiac excitation-contraction coupling. We conclude by reflecting on how studies that probe the biophysical properties of fish RyR2 channel gating in response to various ligands and temperatures would be very instructive for our understanding of the role of the SR in the evolution of the heart.

Once opened, ryanodine receptors (RyR) are efficient pathways for the release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR). The precise nature of the Ca2+-release event, however, requires fine-tuning for the specific process and type of cell involved. For example, the spatial organization of RyRs, the luminal [Ca2+] and the influence of soluble regulators that fluctuate under physiological and pathophysiological control mechanisms, all affect the amplitude and duration of RyR Ca2+ fluxes. Various proteins are docked tightly to the huge bulky structure of RyR and there is growing evidence that, together, they provide a sophisticated and integrated system for regulating RyR channel gating. This review focuses on those proteins that are relevant to phosphorylation of RyR channels with particular reference to the cardiac isoform of RyR (RyR2). How phosphorylation of RyR affects channel activity and whether proteins such as the FK-506 binding proteins (FKBP12 and FKBP12.6) are involved, have been highly controversial subjects for more than a decade. But that is expected given the large number of participating proteins, the relevance of phosphorylation in heart failure and inherited arrhythmic diseases, and the frustrations of predicting relationships between structure and function before the advent of a high resolution structure of RyR. Abbreviations: CaM, calmodulin; CaMKII, Ca2+/calmodulin-dependent protein kinase II; CPVT, catecholaminergic polymorphic ventricular tachycardia; FKBP, FK-506 binding proteins; MH, malignant hyperthermia; mKAP, muscle A kinase anchoring protein; pDE4D, cAMP-specific enzyme 3′,5′-cyclic phosphodiesterase 4D; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PKI, protein kinase A inhibitor; PP1, protein phosphatase 1; PP2A, protein phosphatase 2; Po, open probability; RyR, ryanodine receptor

Two-pore channels (TPCs) are ubiquitously expressed in animal and plant cells but there is argument over the ions that they conduct under physiological conditions and, hence, their cellular roles are disputed. In animals, there are three isoforms, TPC1, TPC2 and TPC3 but only TPC1 and TPC2 are present in all animals and these are located on lysosomal and endolysosomal acidic Ca2+ stores. A plant version of TPC1 is found on vacuolar membranes and the crystal structure of the channel from Arabidopsis thaliana, AtTPC1, has recently been determined. AtTPC1 has been identified as a voltage- and Ca2+-regulated non-selective cation channel and is involved in many cellular processes including the regulation of Ca2+ movements. In contrast, it is argued over whether the mammalian isoforms of TPC1 and TPC2 behave as Na+ channels or as Ca2+-release channels. We here review the structure and function of TPCs from a single-channel perspective with particular emphasis on their permeability properties.

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are Ca2+-mobilizing messengers important for modulating cardiac excitation–contraction coupling and pathophysiology. CD38, which belongs to the ADP-ribosyl cyclase family, catalyzes synthesis of both NAADP and cADPR in vitro. However, it remains unclear whether this is the main enzyme for their production under physiological conditions. Here we show that membrane fractions from WT but not CD38−/− mouse hearts supported NAADP and cADPR synthesis. Membrane permeabilization of cardiac myocytes with saponin and/or Triton X-100 increased NAADP synthesis, indicating that intracellular CD38 contributes to NAADP production. The permeabilization also permitted immunostaining of CD38, with a striated pattern in WT myocytes, whereas CD38−/− myocytes and nonpermeabilized WT myocytes showed little or no staining, without striation. A component of β-adrenoreceptor signaling in the heart involves NAADP and lysosomes. Accordingly, in the presence of isoproterenol, Ca2+ transients and contraction amplitudes were smaller in CD38−/− myocytes than in the WT. In addition, suppressing lysosomal function with bafilomycin A1 reduced the isoproterenol-induced increase in Ca2+ transients in cardiac myocytes from WT but not CD38−/− mice. Whole hearts isolated from CD38−/− mice and exposed to isoproterenol showed reduced arrhythmias. SAN4825, an ADP-ribosyl cyclase inhibitor that reduces cADPR and NAADP synthesis in mouse membrane fractions, was shown to bind to CD38 in docking simulations and reduced the isoproterenol-induced arrhythmias in WT hearts. These observations support generation of NAADP and cADPR by intracellular CD38, which contributes to effects of β-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm.

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are Ca2+-mobilizing messengers important for modulating cardiac excitation–contraction coupling and pathophysiology. CD38, which belongs to the ADP-ribosyl cyclase family, catalyzes synthesis of both NAADP and cADPR in vitro. However, it remains unclear whether this is the main enzyme for their production under physiological conditions. Here we show that membrane fractions from WT but not CD38−/− mouse hearts supported NAADP and cADPR synthesis. Membrane permeabilization of cardiac myocytes with saponin and/or Triton X-100 increased NAADP synthesis, indicating that intracellular CD38 contributes to NAADP production. The permeabilization also permitted immunostaining of CD38, with a striated pattern in WT myocytes, whereas CD38−/− myocytes and nonpermeabilized WT myocytes showed little or no staining, without striation. A component of β-adrenoreceptor signaling in the heart involves NAADP and lysosomes. Accordingly, in the presence of isoproterenol, Ca2+ transients and contraction amplitudes were smaller in CD38−/− myocytes than in the WT. In addition, suppressing lysosomal function with bafilomycin A1 reduced the isoproterenol-induced increase in Ca2+ transients in cardiac myocytes from WT but not CD38−/− mice. Whole hearts isolated from CD38−/− mice and exposed to isoproterenol showed reduced arrhythmias. SAN4825, an ADP-ribosyl cyclase inhibitor that reduces cADPR and NAADP synthesis in mouse membrane fractions, was shown to bind to CD38 in docking simulations and reduced the isoproterenol-induced arrhythmias in WT hearts. These observations support generation of NAADP and cADPR by intracellular CD38, which contributes to effects of β-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm.

Intracellular Ca2+ release through ryanodine receptor (RyR) and inositol trisphosphate receptor (IP3R) channels is supported by a complex network of additional proteins that are located in or near the Ca2+ release sites. In this review, we focus, not on RyR/IP3R, but on other ion-channels that are known to be present in the sarcoplasmic/endoplasmic reticulum (ER/SR) membranes. We review their putative physiological roles and the evidence suggesting that they may support the process of intracellular Ca2+ release, either indirectly by manipulating ionic fluxes across the ER/SR membrane or by directly interacting with a Ca2+-release channel. These channels rarely receive scientific attention because of the general lack of information regarding their biochemical and/or electrophysiological characteristics makes it difficult to predict their physiological roles and their impact on SR Ca2+ fluxes. We discuss the possible role of SR K+ channels and, in parallel, detail the known biochemical and biophysical properties of the trimeric intracellular cation (TRIC) proteins and their possible biological and pathophysiological roles in ER/SR Ca2+ release. We summarise what is known regarding Cl− channels in the ER/SR and the non-selective cation channels or putative ‘Ca2+ leak channels’, including mitsugumin23 (MG23), pannexins, presenilins and the transient receptor potential (TRP) channels that are distributed across ER/SR membranes but which have not yet been fully characterised functionally.

Funding: British Heart Foundation We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12(E31Q/D32N/W59F)) where the residues Glu(31), Asp(32), and Trp(59) were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12(E31Q/D32N/W59F) mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12(E31Q/D32N/W59F) activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu31, Asp(32), and Trp(59) in FKBP12 and Gln(31), Asn(32), and Phe(59) in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues. Publisher PDF

Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli.

Key points The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric‐a gene knockout (KO) alters the single‐channel function of skeletal RyR (RyR1).We find that RyR1 from Tric‐a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+, ATP, caffeine and luminal Ca2+.In the presence of Mg2+, ATP cannot effectively activate RyR1 from Tric‐a KO mice.Additionally, RyR1 from Tric‐a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of β‐adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+‐release.The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+‐release observed in skeletal muscle from Tric‐a KO mice, further highlighting the importance of TRIC‐A for normal physiological regulation of SR Ca2+‐release in skeletal muscle. Abstract The type A trimeric intracellular cation channel (TRIC‐A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric‐a knockout (KO) mice is characterized by Ca2+ overloaded and swollen SR and by changes in the properties of SR Ca2+ release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric‐a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage‐clamp conditions. We find that RyR1 channels from Tric‐a KO mice respond normally to cytosolic Ca2+, ATP, adenine, caffeine and to luminal Ca2+. However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg2+, ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC‐A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC‐A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K+ fluxes across the SR via TRIC‐A., Key points The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric‐a gene knockout (KO) alters the single‐channel function of skeletal RyR (RyR1).We find that RyR1 from Tric‐a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+, ATP, caffeine and luminal Ca2+.In the presence of Mg2+, ATP cannot effectively activate RyR1 from Tric‐a KO mice.Additionally, RyR1 from Tric‐a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of β‐adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+‐release.The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+‐release observed in skeletal muscle from Tric‐a KO mice, further highlighting the importance of TRIC‐A for normal physiological regulation of SR Ca2+‐release in skeletal muscle.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca2+ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca2+ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca2+ release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca2+ that will enable it to act as a Ca2+ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca2+] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca2+ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca2+ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μM but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.

The ryanodine receptor (RyR) is the pathway for release of the sarcoplasmic reticulum (SR) Ca2+ required for cardiac and skeletal muscle contraction. The opening of SR K+ channels is thought to support this process. Movements of the RyR and SR K+ channel gates required for channel opening and closing will be dependent on the biophysical properties of the SR membrane which, in turn, will be affected by factors such as temperature and membrane composition. Cardiac RyR channel opening is hypersensitive to temperature changes (Sitsapesan et al., 1991, J Physiol., 434:469-488) although single-channel recordings are invariably performed at room temperature. We have therefore incorporated mammalian cardiac and skeletal RyR and SR K+ channels into bilayers under voltage-clamp conditions to investigate the effects of changing temperature and membrane lipid composition on single-channel behaviour.Changing bilayer lipid composition from phosphatidylethanolamine (PE) only, to a more physiological composition containing a 5:4:1 ratio of PE: phosphatidylserine (PS): phosphatidylcholine (PC) at 23oC, significantly increased the open probability (Po) and single channel conductance of both channels. For example, RyR Po increased from 0.06±0.03 to 0.26±0.08 (n=8; SEM; P

The primary reason for cessation of statin therapy is skeletal myopathy but the underlying mechanism and apparent absence of detrimental effects of statins on cardiac muscle are not fully understood. We have reported that statin treatment in vivo causes a profound leak of Ca2+ from the sarcoplasmic reticulum (SR) of intact skeletal myocytes, a common myopathic mechanism. Here we compare the effect of simvastatin on Ca2+ sparks in rat skeletal and cardiac myocytes. Simvastatin treatment in vivo (40 mg/kg/day for 4 weeks) markedly increased Ca2+ spark frequency (280%) and duration (140%) in fluo4-loaded flexor digitorum brevis (FDB)fibres (P

Trimeric intracellular cation-selective (TRIC) channel subtypes, namely TRIC-A and TRIC-B, are derived from distinct genes and distributed throughout the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes. TRIC-A is preferentially expressed at high levels in excitable tissues, while TRIC-B is ubiquitously detected at relatively low levels in various tissues. TRIC channels are composed of ~300 amino acid residues and contain three putative membrane-spanning segments to form a bullet-shaped homo-trimeric assembly. Both native and purified recombinant TRIC subtypes form functional monovalent cation-selective channels in a lipid bilayer reconstitution system. The electrophysiological data indicate that TRIC channels behave as K(+) channels under intracellular conditions, although the detailed channel characteristics remain to be investigated. The pathophysiological defects detected in knockout mice suggest that TRIC channels support SR/ER Ca(2+) release mediated by ryanodine (RyR) and inositol trisphosphate receptor (IP(3)R) channels. For example, Tric-a-knockout mice develop hypertension resulting from vascular hypertonicity, and the mutant vascular smooth muscle cells exhibit insufficient RyR-mediated Ca(2+) release for inducing hyperpolarization. Tric-b-knockout mice show respiratory failure at birth, and IP(3)R-mediated Ca(2+) release essential for surfactant handling is impaired in the mutant alveolar epithelial cells. Moreover, double-knockout mice lacking both TRIC subtypes show embryonic heart failure, and SR Ca(2+) handling is deranged in the mutant cardiomyocytes. Current evidence strongly suggests that TRIC channels mediate counter-K(+) movements, in part, to facilitate physiological Ca(2+) release from intracellular stores.

Mitsugumin 23 (MG23) is a 23 kDa transmembrane protein localized to the sarcoplasmic/endoplasmic reticulum and nuclear membranes in a wide variety of cells. Although the characteristics imply the participation in a fundamental function in intracellular membrane systems, the physiological role of MG23 is unknown. Here we report the biochemical and biophysical characterization of MG23. Hydropathicity profile and limited proteolytic analysis proposed three transmembrane segments in the MG23 primary structure. Chemical cross-linking analysis suggested a homo-oligomeric assembly of MG23. Ultrastructural observations detected a large symmetrical particle as the predominant component and a small asymmetric assembly as the second major component in highly purified MG23 preparations. Single-particle three-dimensional reconstruction revealed that MG23 forms a large bowl-shaped complex equipped with a putative central pore, which is considered an assembly of the small asymmetric subunit. After reconstitution into planar phospholipid bilayers, purified MG23 behaved as a voltage-dependent, cation-conducting channel, permeable to both K+ and Ca2+. A feature of MG23 gating was that multiple channels always appeared to be gating together in the bilayer. Our observations suggest that the bowl-shaped MG23 can transiently assemble and disassemble. These building transitions may underlie the unusual channel gating behavior of MG23 and allow rapid cationic flux across intracellular membrane systems.

SUMMARY It was first proposed that cyclic ADP-ribose (cADPR) could activate ryanodine receptors (RyR) in 1991. Following a subsequent report that cADPR could activate cardiac RyR (RyR2) reconstituted into artificial membranes and stimulate Ca2+-release from isolated cardiac SR, there has been a steadily mounting stockpile of publications proclaiming the physiological and pathophysiological importance of cADPR in the cardiovascular system. It was only 2 years earlier, in 1989, that cADPR was first identified as the active metabolite of nicotinamide adenine dinucleotide (NAD), responsible for triggering the release of Ca2+ from crude homogenates of sea urchin eggs. Twenty years later, can we boast of being any closer to unraveling the mechanisms by which cADPR modulates intracellular Ca2+-release? This review sets out to examine the mechanisms underlying the effects of cADPR and ask whether cADPR is an important signaling molecule in the heart.

Background and purpose: Diadenosine polyphosphates are normally present in cells at low levels, but significant increases in concentrations can occur during cellular stress. The aim of this study was to investigate the effects of diadenosine pentaphosphate (Ap5A) and an oxidized analogue, oAp5A on the gating of sheep cardiac ryanodine receptors (RyR2). Experimental approach: RyR2 channel function was monitored after incorporation into planar bilayers under voltage-clamp conditions. Key results: With10 µmol·L−1 cytosolic Ca2+, a significant ‘hump’ or plateau at the base of the dose–response relationship to Ap5A was revealed. Open probability (Po) was significantly increased to a plateau of approximately 0.2 in the concentration range 100 pmol·L−1–10 µmol·L−1. High Po values were observed at >10 µmol·L−1 Ap5A, and Po values close to 1 could be achieved. Nanomolar levels of ATP and adenosine also revealed a hump at the base of the dose–response relationships, although GTP did not activate at any concentration, indicating a common, high-affinity binding site on RyR2 for adenine-based compounds. The oxidized analogue, oAp5A, did not significantly activate RyR2 via the high-affinity binding site; however, it could fully open the channel with an EC50 of 16 µmol·L−1 (Ap5A EC50 = 140 µmol·L−1). Perfusion experiments suggest that oAp5A and Ap5A dissociate slowly from their binding sites on RyR2. Conclusions and implications: The ability of Ap5A compounds to increase Po even in the presence of ATP and their slow dissociation from the channel may enable these compounds to act as physiological regulators of RyR2, particularly under conditions of cellular stress.

Background and purpose: Ca(2+)-calmodulin (Ca(2+)CaM) is widely accepted as an inhibitor of cardiac ryanodine receptors (RyR2); however, the effects of physiologically relevant CaM concentrations have not been fully investigated. Experimental approach: We investigated the effects of low concentrations of Ca(2+)CaM (50-100 nmol.L(-1)) on the gating of native sheep RyR2, reconstituted into bilayers. Suramin displaces CaM from RyR2 and we have used a gel-shift assay to provide evidence of the mechanism underlying this effect. Finally, using suramin to displace endogenous CaM from RyR2 in permeabilized cardiac cells, we have investigated the effects of 50 nmol.L(-1) CaM on sarcoplasmic reticulum (SR) Ca(2+)-release. Key results: Ca(2+)CaM activated or inhibited single RyR2, but activation was much more likely at low (50-100 nmol.L(-1)) concentrations. Also, suramin displaced CaM from a peptide of the CaM binding domain of RyR2, indicating that, like the skeletal isoform (RyR1), suramin directly competes with CaM for its binding site on the channel. Pre-treatment of rat permeabilized ventricular myocytes with suramin to displace CaM, followed by addition of 50 nmol.L(-1) CaM to the mock cytoplasmic solution caused an increase in the frequency of spontaneous Ca(2+)-release events. Application of caffeine demonstrated that 50 nmol.L(-1) CaM reduced SR Ca(2+) content. Conclusions and implications: We describe for the first time how Ca(2+)CaM is capable, not only of inactivating, but also of activating RyR2 channels in bilayers in a CaM kinase II-independent manner. Similarly, in cardiac cells, CaM stimulates SR Ca(2+)-release and the use of caffeine suggests that this is a RyR2-mediated effect.

What is the topic of this review? Excitation-contraction coupling in fish hearts is maintained over a range of temperatures that would be cardioplegic to most mammals. Here, we review what is known about the fish cardiac ryanodine receptor, and consider how it may be regulated in a different manner from the mammalian cardiac isoforms of this channel. What advances does it highlight? We highlight how a better understanding of the basic gating and conducting properties of fish cardiac ryanodine receptors could provide considerable insight into mechanisms underlying sarcoplasmic reticulum calcium release in fish hearts and the role of the sarcoplasmic reticulum in the evolution of the heart. The fish cardiac sarcoplasmic reticulum (SR) holds large quantities of Ca(2+), but calcium-induced calcium release (CICR) is weak in these myocytes, and contraction and relaxation are largely determined by transsarcolemmal Ca(2+) flux. Many fish species live in a cold and seasonally variable thermal habitat, which could provide challenges to regulation of excitation-contraction coupling. Here, we focus on the cardiac SR Ca(2+)-release channel (RyR2) in fish and ask whether it may be regulated in a different manner from the mammalian RyR2. We review data indicating that fish cardiac RyR are present at lower density, are more spatially separated within the SR membrane and are less responsive to cytosolic Ca(2+) than mammalian RyR2 channels. All of these features would contribute to the weak CICR evident from functional studies. We also consider how CICR can be enhanced in fish myocytes following β-adrenergic stimulation and application of low levels of caffeine, and how acute and chronic temperature change may affect the gating properties of fish RyR2s. It is clear that a lack of insight into the fundamental gating and conductance properties of fish RyR2 channels is hindering our understanding of the role of the SR in fish cardiac excitation-contraction coupling. We conclude by reflecting on how studies that probe the biophysical properties of fish RyR2 channel gating in response to various ligands and temperatures would be very instructive for our understanding of the role of the SR in the evolution of the heart.

Ion channels that are located on intracellular organelles have always posed challenges for biophysicists seeking to measure their ion conduction, selectivity, and gating kinetics. Unlike cell surface ion channels, intracellular ion channels cannot be accessed for biophysical single-channel recordings using the patch-clamp technique while remaining in a physiological setting. Disruption of the cell is always necessary and hence experiments inevitably have a certain "artificial" nature about them. This drawback is turned to considerable advantage if the internal membranes containing the channels of interest can be isolated or if the channels can be purified because they can then be incorporated into artificial membranes of controlled composition. This approach guarantees a tight but flexible control over the biophysical and biochemical environment of the ion channel molecules. This includes the lipid composition of the membrane and the ionic solutions on both sides of the channel, thus allowing the conductance properties of the channel to be accurately measured. Since the influence of multiple unknown regulators of channel function (that could be present within the physiological membrane or in cytosolic, or intraorganelle compartments) is removed, the identification and characterization of physiological and pharmacological regulators that directly affect channel gating can also be achieved. This cannot be performed in a cellular environment. These techniques have typically been used to study the properties of channels located on endoplasmic/sarcoplasmic reticulum (ER/SR) membranes but in this chapter we describe how the techniques are also suited for ion channels of the acidic lysosomal and endolysosomal Ca(2+) stores.

The effects of the covalent modifier of amino groups, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) on the single-channel properties of purified sheep cardiac ryanodine receptors (RyR) incorporated into planar phospholipid bilayers were investigated. DIDS increased single-channel conductance and open probability (Po) and induced unique modifications to the voltage-dependence of gating. The effects of DIDS on conduction and gating were irreversible within the time scale of the experiments, and both effects were dependent on the permeant ion. DIDS induced a greater increase in conductance with Ca2+ (20%) compared with K+ (8%) as the permeant ion. After modification by DIDS, all channels could be rapidly inactivated in a voltage-dependent manner. The open probability of the DIDS-modified channel decreased with increasing positive or negative transmembrane potentials; however, inactivation was only observed at negative potentials. Our results demonstrate that inactivation of RyR channels is dependent on the ligand activating the channel, and this will have consequences for the control and termination of sarcoplasmic reticulum Ca2+ release in cardiac cells.

NAADP potently triggers Ca(2+) release from acidic lysosomal and endolysosomal Ca(2+) stores. Human two-pore channels (TPC1 and TPC2), which are located on these stores, are involved in this process but there is controversy over whether TPC1 and TPC2 constitute the Ca(2+) release channels. We, therefore, examined the single-channel properties of human TPC1 after reconstitution into bilayers of controlled composition. We found TPC1 was permeable not only to Ca(2+) but also to monovalent cations and that permeability to protons was the highest (relative permeability sequence H(+)>>K(+)>Na(+)≥Ca(2+)). NAADP or Ca(2+) activated TPC1 and the presence of one of these ligands was required for channel activation. The endolysosome-located lipid phosphatidylinositol 3,5 bisphosphate, [PI(3,5)P(2)] had no effect on TPC1 open probability but significantly increased the relative permeability of Na(+) to Ca(2+) and of H(+) to Ca(2+). Furthermore, our data showed that, although both TPC1 and TPC2 are stimulated by NAADP, these channels differ in ion selectivity and modulation by Ca(2+) and pH. We propose that NAADP triggers H(+) release from lysosomes and endolysomes through activation of TPC1 but that the Ca(2+)-releasing ability of TPC1 will depend on the ionic composition of the acidic stores and may be influenced by other regulators that affect TPC1 ion permeation.

The effects of adenosine and adenine on the gating of native sheep cardiac ryanodine receptor (RyR) channels were investigated. By examining the mechanisms underlying channel activation and by using comparative molecular field analysis (CoMFA) we have investigated the structural features of adenine-based ligands involved in channel activation. In the presence of 10 μM cytosolic Ca2+, adenosine and adenine both activate the channel but only to a level approximately 10 and 20% respectively of that of ATP indicating that both are partial agonists of low efficacy. Adenosine was able to antagonize the ATP-induced increase in open probability (Po) as expected for a partial agonist of low efficacy at the ATP sites on the cardiac RyR. GTP (100 μM–10 mM) had no effect on channel gating indicating that the adenine ring structure is important for agonist activity at the ATP-sites on RyR. CoMFA revealed an extremely strong correlation between the structural features of the five ATP analogues and the ability to increase (Po). Our model indicates that the high efficacy of ATP results primarily from the large electrostatic field established by the ionized phosphate groups. Reducing the number of phosphate groups lowers the strength of this field, leading to ligands with lower efficacy. In addition, steric interactions between the α-phosphate and ribose moieties and the RyR are correlated with low Po. British Journal of Pharmacology (2000) 130, 1618–1626; doi:10.1038/sj.bjp.0703459

We have investigated the ability of AMP to modulate the native sheep cardiac ryanodine receptor (RyR) channel at various cytosolic [Ca2+]. Channels were incorporated into planar phospholipid bilayers and current fluctuations through the bilayer were monitored under voltage clamp conditions. We demonstrate that AMP only exhibits agonist activity if the cytosolic [Ca2+] is sufficiently high. Even in the presence of a high cytosolic [Ca2+] (65 μM), AMP cannot fully open the channel and the maximum open probability (Po) observed is approximately 0.3 at 2 mM AMP. Concentrations of AMP above the maximally activating level cause inactivation of the channel. Our experiments indicate that AMP is an agonist with such low efficacy at the ATP sites on the cardiac RyR that it is effectively an antagonist of ATP-induced increases in Po. Our study demonstrates that the number of phosphates attached to the 5′-carbon of the ribose ring of adenine-based compounds determines the efficacy of the ligand to increase the Po of the cardiac RyR. Substitution of groups at this position may lead to the identification of potent antagonists at ATP sites on RyR. British Journal of Pharmacology (1999) 127, 161–171; doi:10.1038/sj.bjp.0702491

The mechanisms involved in 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS)- and 4,4'-dibenzamidostilbene-2, 2'-disulfonic acid (DBDS)- modification of sheep cardiac ryanodine receptor (RyR) channel function have been investigated. DIDS (50-500 microm) exerts at least three effects on single channel function. With Ca2+ as the permeant ion, DIDS increases both channel open probability (Po) and single channel conductance in a similar manner to the effects observed with suramin. Both effects occur immediately and are fully reversible. Similar effects were observed with DBDS (10 microm-2 mm), a compound with the 4,4'-NCS groups of DIDS replaced with NHCOC6H5. DIDS (500 microm) also caused irreversible modification to the fully open channel level in 74% of the channels. This effect was not observed with suramin or DBDS (10 microm-1 mm). Competition studies with DBDS and suramin coupled with the close similarities in the effects of DIDS, DBDS and suramin on gating and conduction suggest that these ligands may all bind to the same sites on RyR. The DIDS-induced irreversible modification to the fully open state may result from the binding of the isothiocyanate groups to positively charged amino acids at or near the suramin binding sites although it is possible that this modification is unrelated to its other effects on channel function.

1. The effects of Ca2+, ATP and caffeine on the gating of lobster skeletal muscle ryanodine receptors (RyR) was investigated after reconstitution of the channels into planar phospholipid bilayers and by using [3H]-ryanodine binding studies. 2. The single channel studies reveal that the EC50 (60 microM) for activation of the lobster skeletal RyR by Ca2+ as the sole ligand is higher than for any other isoform of RyR studied. 3. Inactivation of the channel by Ca2+ (EC50 = 1 mM) occurs at concentrations slightly higher than those required to inactivate mammalian skeletal RyR (RyR1) but lower than those required to inactivate mammalian cardiac RyR (RyR2). 4. Lifetime analysis demonstrates that cytosolic Ca2+, as the sole activating ligand, cannot fully open the lobster skeletal RyR (maximum Po approximately 0.2). The mechanism for the increase in open probability (Po) is an increase in both the frequency and the duration of the open events. 5. ATP is a very effective activator of the lobster RyR and can almost fully open the channel in the presence of activating cytosolic [Ca2+]. In the presence of 700 microM Ca2+, 1 mM ATP increased Po to approximately 0.8. 6. Caffeine, often used as a tool to identify the presence of RyR channels, is relatively ineffective and cannot increase Po above the level that can be attained with Ca2+ alone. 7. The results reveal that caffeine increases Po by a different mechanism to that of cytosolic Ca2+ demonstrating that the mechanism for channel activation by caffeine is not 'sensitization' to cytosolic Ca2+. 8. By studying the mechanisms involved in the activation of the lobster RyR we have demonstrated that the channel responds in a unique manner to Ca2+ and to caffeine. The results strongly indicate that these ligand binding sites on the channel are different to those on mammalian isoforms of RyR.

Intracellular Ca2+ release from the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential for many cellular processes and it is well recognised that ryanodine receptors (RyRs) or inositol trisphosphate receptors (IP3Rs) provide the main pathways for that release of Ca2+. It is increasingly clear, however, that normal, physiological Ca2+release is carefully orchestrated by a consortium of regulatory proteins that are located in the ER/SR. Some proteins may be specific for a particular cell type or interact only with one isoform of RyR or IP3R. Some proteins can influence RyR or IP3R channel opening directly; other proteins exhibit indirect regulation. In the symposium we did not attempt to be comprehensive in covering all the ER/SR proteins that support the process of SR Ca2+release but instead focused on a few recent discoveries to highlight novel proteins or control mechanisms. Figure 1 visually summarises the proteins that were discussed. Figure 1 Representation of the T-tubule/sarcoplasmic reticulum membrane system of striated muscle to show key proteins discussed at the symposium Hiroshi Takeshima highlighted the role of two proteins: junctophilins, which form complexes between the plasma and SR membranes in excitable cells; and TRIC proteins, thought to behave as K+ channels on intracellular membrane systems (Yamazaki et al. 2009). He discussed mutations and polymorphisms found in human junctophilin and TRIC genes that are associated with hypertension risk and genetic diseases including cardiac hypertrophy and osteogenesis imperfecta. The next speaker, Elisa Venturi, described the single-channel properties of SR K+ channels derived from Tric-a null mice and discussed the evidence for their putative but controversial physiological role in providing counter-ion K+ fluxes during Ca2+ movements across the SR (Venturi et al. 2013). Michael Ibrahim then explained the changes to the T-tubule system that occur in the cardiac cells of heart failure patients. He discussed how changes in mechanical load can reverse both the structural architecture of the T-tubules and the loss in cardiac contractility, and the implications that these discoveries have for the treatment of heart failure (Ibrahim & Terracciano, 2013). Moving to skeletal muscle function, Francesco Zorzato described how decreases of muscle strength that occur in ageing and certain clinical diseases can be debilitating and life threatening. His presentation focused on a novel protein, JP45, and its interactions with both the skeletal muscle dihydropyridine receptor (Cav1.1) and the SR Ca2+ binding protein calsequestrin (CSQ) (Mosca et al. 2013). He presented data suggesting that JP45 and CSQ1 can modulate Cav1.1 activity and strengthen skeletal muscle contraction and, as such, are novel therapeutic targets. Finally, Isabelle Marty summarised new insights into the functional roles of triadin in cardiac and skeletal muscle, providing evidence that different isoforms of triadin are crucial not only for maintaining the structural integrity of the junctional RyR complex of proteins, but also in regulating Ca2+release via IP3Rs. The identification of triadin as a new gene responsible for the inherited arrhythmic disease catecholaminergic polymorphic ventricular tachycardia was discussed (Roux-Buisson et al. 2012).

The effects of ATP, ADP, and inorganic phosphate (Pi) on the gating of native sheep cardiac ryanodine receptor channels incorporated into planar phospholipid bilayers were investigated. We demonstrate that ATP and ADP can activate the channel by Ca2+-dependent and Ca2+-independent mechanisms. ATP and ADP appear to compete for the same site/s on the cardiac ryanodine receptor, and in the presence of cytosolic Ca2+ both agents tend to inactivate the channel at supramaximal concentrations. Our results reveal that ATP not only has a greater affinity for the adenine nucleotide site/s than ADP, but also has a greater efficacy. The EC50 value for channel activation is approximately 0.2 mM for ATP compared to 1.2 mM for ADP. Most interesting is the fact that, even in the presence of cytosolic Ca2+, ADP cannot activate the channel much above an open probability (Po) of 0.5, and therefore acts as a partial agonist at the adenine nucleotide binding site on the channel. We demonstrate that Pi also increases Po in a concentration and Ca2+-dependent manner, but unlike ATP and ADP, has no effect in the absence of activating cytosolic [Ca2+]. We demonstrate that Pi does not interact with the adenine nucleotide site/s but binds to a distinct domain on the channel to produce an increase in Po.

Trimeric intracellular cation channels (TRIC-A/TRIC-B) are found in the endo/sarcoplasmic reticulum (ER/SR) and nuclear membranes. TRIC-B knockout mice die in respiratory failure following birth but there are additional diverse, severe pathological symptoms (Yamazaki D. et al. (2009)). The physiological roles of TRIC-B are not fully understood and few regulators of channel activity are known. We here investigate if TRIC-B function is sensitive to pH by incorporating light SR membrane vesicles from TRIC-A knockout skeletal muscle into artificial membranes under voltage-clamp conditions in symmetrical 210 mM K-PIPES, pH 7.2. Open probability was determined at ±30 mV when ≤2 channels were gating in the bilayer. With ≥3 channels present, noise analysis was performed because the complex sub-conductance state gating prevented accurate measurements. As previously observed (Venturi E. et al. (2009)), TRIC-B was more open at positive potentials but exhibited variable gating behaviour. For example, at pH 7.2, at +30 mV, noise analysis yielded a mean current of 1.25±0.25 pA (SEM; n=23) but ranged from 0.025 to 3.36 pA. We observed that TRIC-B became more open as cytosolic pH was increased. At +30 mV, as cytosolic pH was raised from 7.2 to 9.2, we observed a 3.34 fold increase in mean current (SEM; n=7; p

Suramin, a polysulfonated napthylurea, increases the open probability and the single-channel conductance of rabbit skeletal and sheep cardiac ryanodine receptor channels. The main mechanism for the increase in Po is an increase in the duration of open lifetimes. The effects on conduction and gating are completely reversible and involve an interaction with the cytosolic side of the channel. 10 mM dithiothreitol had no effect on the suramin-induced increase in conductance or Po. Therefore oxidation of sulfhydryl groups on the channels does not appear to be involved. Suramin has been used as an antagonist of ATP at P2 purinoceptors, however, we find that suramin does not antagonize the effect of ATP at skeletal or cardiac ryanodine receptor channels. The unusual gating kinetics induced by suramin suggest that it does not interact with the adenine nucleotide binding site on the ryanodine receptor but rather binds at a novel site(s). The suramin-induced changes to channel gating and conduction do not prevent the characteristic modification of single-channel properties by micromolar ryanodine.

Trimeric intracellular cation channels (TRIC-A and TRIC-B) are located in the sarcoplasmic/endoplasmic reticulum (SR/ER) of most cells. Identifying the distinct biophysical properties of TRIC-A and TRIC-B is difficult because both channels are present in most tissues, yet this is crucial for delineating their individual physiological roles. Skeletal muscle SR vesicles (LSR) from TRIC-A knockout mice were incorporated into artificial membranes under voltage-clamp conditions as previously described [Pitt et al., 2010, Biophys. J. 99, 417-426] and single-channel recordings of native TRIC-B were obtained in symmetrical solutions of 210 mM K-PIPES, pH 7.2. The maximum single-channel conductance of TRIC-B was 197 ± 2 pS (n=32; SEM). TRIC-B channels always exhibited sub-conductance gating states and while these were of a variable nature, the predominant sub-conductance levels were found at 156 ± 3 pS (n=17; SEM), 125 ± 2 pS (n=19; SEM), 96 ± 2 pS (n=19; SEM) and 62 ± 2 pS (n=27; SEM). TRIC-B channel gating was voltage-dependent and channels were inhibited at negative holding potentials. For example, the probability of dwelling in the full open channel level was 0.0478±0.0194 at +30 mV but only 0.0010±0.0008 at −30 mV (n=6; SEM; ∗p