Caffeine Response Research Articles

Two Gr Genes Underlie Sugar Reception in Drosophila

We have analyzed the molecular basis of sugar reception in Drosophila. We define the response spectrum, concentration dependence, and temporal dynamics of sugar-sensing neurons. Using in situ hybridization and reporter gene expression, we identify members of the Gr5a-related taste receptor subfamily that are coexpressed in sugar neurons. Neurons expressing reporters of different Gr5a-related genes send overlapping but distinct projections to the brain and thoracic ganglia. Genetic analysis of receptor genes shows that Gr5a is required for response to one subset of sugars and Gr64a for response to a complementary subset. A Gr5a;Gr64a double mutant shows no physiological or behavioral responses to any tested sugar. The simplest interpretation of our results is that Gr5a and Gr64a are each capable of functioning independently of each other within individual sugar neurons and that they are the primary receptors used in the labellum to detect sugars.

Comparison of the effects of inorganic phosphate on caffeine-induced Ca2+ release in fast- and slow-twitch mammalian skeletal muscle

We compared the effects of 50 mM P(i) on caffeine-induced Ca(2+) release in mechanically skinned fast-twitch (FT) and slow-twitch (ST) skeletal muscle fibers of the rat. The time integral (area) of the caffeine response was reduced by approximately 57% (FT) and approximately 27% (ST) after 30 s of exposure to 50 mM P(i) in either the presence or absence of creatine phosphate (to buffer ADP). Differences in the sarcoplasmic reticulum (SR) Ca(2+) content between FT and ST fibers [ approximately 40% vs. 100% SR Ca(2+) content (pCa 6.7), respectively] did not contribute to the different effects of P(i) observed; underloading the SR of ST fibers so that the SR Ca(2+) content approximated that of FT fibers resulted in an even smaller ( approximately 21%), but not significant, reduction in caffeine-induced Ca(2+) release by P(i). These observed differences between FT and ST fibers could arise from fiber-type differences in the ability of the SR to accumulate Ca(2+)-P(i) precipitate. To test this, fibers were Ca(2+) loaded in the presence of 50 mM P(i). In FT fibers, the maximum SR Ca(2+) content (pCa 6.7) was subsequently increased by up to 13 times of that achieved when loading for 2 min in the absence of P(i). In ST fibers, the SR Ca(2+) content was only doubled. These data show that Ca(2+) release in ST fibers was less affected by P(i) than FT fibers, and this may be due to a reduced capacity of ST SR to accumulate Ca(2+)-P(i) precipitate. This may account, in part, for the fatigue-resistant nature of ST fibers.

Sugar Receptors in Drosophila

The detection and discrimination of chemical compounds in potential foods are essential sensory processes when animals feed. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of mostly nonvolatile chemicals that include sugars, a diverse group of toxic compounds present in many inedible plants and spoiled foods, and pheromones [1-6]. With the exception of a trehalose (GR5a) and a caffeine (GR66a) receptor [7-9], the functions of GRs involved in feeding are unknown. Here, we show that the Gr64 genes encode receptors for numerous sugars. We generated a fly strain that contained a deletion for all six Gr64 genes (DeltaGr64) and showed that these flies exhibit no or a significantly diminished proboscis extension reflex (PER) response when stimulated with glucose, maltose, sucrose, and several other sugars. The only considerable response was detected when Gr64 mutant flies were stimulated with fructose. Interestingly, response to trehalose is also abolished in these flies, even though they contain a functional Gr5a gene, which has been previously shown to encode a receptor for this sugar [8, 9]. This observation indicates that two or more Gr genes are necessary for trehalose detection, suggesting that GRs function as multimeric receptor complexes. Finally, we present evidence that some members of the Gr64 gene family are transcribed as a polycistronic mRNA, providing a mechanism for the coexpression of multiple sugar receptors in the same taste neurons.

Depressing effects of caffeine at crayfish neuromuscular synapses I. Dosage response and Ca++ gradient effects.

The response of crayfish synaptic terminals to drugs began to be studied to characterize the terminal's physiological characteristics. Caffeine, the first drug to be studied, was selected to enhance synaptic transmission because of its ability to increase calcium release from internal stores.1. The largest excitor neuron to the superficial flexor muscle system of Procambarus clarkii was stimulated at 10 Hz while recording junction potentials from several lateral muscle fibers.2. Caffeine unexpectedly decreased synaptic transmission in this system in a dosage-dependent manner. The depressing effect of caffeine was observed at 5 mM caffeine and junction potentials disappeared completely at 50 mM. Washing the preparation in fresh control Ringers did not restore the amplitudes of the junction potentials.3. Changes in extracellular calcium concentrations delayed or depressed the caffeine effect depending on the calcium gradient across the membrane or the caffeine dosage. The data suggest that calcium is involved in caffeine's response in this system in a way yet to be determined.

Fluvastatin and Atorvastatin Affect Calcium Homeostasis of Rat Skeletal Muscle Fibers in Vivo and in Vitro by Impairing the Sarcoplasmic Reticulum/Mitochondria Ca2+-Release System

The mechanism by which the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) induce skeletal muscle injury is still under debate. By using fura-2 cytofluorimetry on intact extensor digitorum longus muscle fibers, here we provided the first evidence that 2 months in vivo chronic treatment of rats with fluvastatin (5 and 20 mg kg-1) and atorvastatin (5 and 10 mg kg-1) caused an alteration of calcium homeostasis. All treated animals showed a significant increase of resting cytosolic calcium [Ca2+]i, up to 60% with the higher fluvastatin dose and up to 20% with the other treatments. The [Ca2+]i rise induced by statin administration was not due to an increase of sarcolemmal permeability to calcium. Furthermore, the treatments reduced caffeine responsiveness. In vitro application of fluvastatin caused changes of [Ca2+]i, resembling the effect obtained after the in vivo administration. Indeed, fluvastatin produced a shift of mechanical threshold for contraction toward negative potentials and an increase of resting [Ca2+]i. By using ruthenium red and cyclosporine A, we determined the sequence of the statin-induced Ca2+ release mechanism. Mitochondria appeared as the cellular structure responsible for the earlier event leading to a subsequent large sarcoplasmic reticulum Ca2+ release. In conclusion, we suggest that calcium homeostasis alteration may be a crucial event for myotoxicity induced by this widely used class of hypolipidemic drugs.

Occurrence of atypical Ca 2+ transients in triadin-binding deficient-RYR1 mutants

Triadin in the junctional sarcoplasmic reticulum (SR) of skeletal muscle cells has been suggested to interact with ryanodine receptor 1 (RYR1) via its KEKE motifs. Recently, we showed that amino acid residues D4878, D4907, and E4908 in RYR1 are critical for triadin-binding in vitro [J.M. Lee, S.H. Rho, D.W. Shin, C. Cho, W.J. Park, S.H. Eom, J. Ma, D.H. Kim, Negatively charged amino acids within the intraluminal loop of ryanodine receptor are involved in the interaction with triadin, J. Biol. Chem. 279 (2004) 6994–7000]. In order to test whether a disruption of the triadin-binding site(s) in RYR1 affects SR Ca 2+ release, alanine-substituted single (D4878A, D4907A, and E4908A) and triple (RYR1-TM) mutants of D4878, D4907, and E4908 were expressed in RYR1-null myotubes. Co-immunoprecipitation experiments showed a 50–60% decrease of triadin brought down in the D4907A and RYR1-TM complexes compared to the triadin–wtRYR1 complex. Ca 2+ imaging experiments using Fluo-4-AM showed atypical caffeine responses in myotubes expressing D4907A and RYR1-TM characterized by either a lack of or slower activation and faster inactivation of Ca 2+ transients. The results suggest that disruption of interaction between triadin and RYR1 impairs RYR1 function and SR Ca 2+ release.

Aldolase potentiates DIDS activation of the ryanodine receptor in rabbit skeletal sarcoplasmic reticulum

DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate), an anion channel blocker, triggers Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). The present study characterized the effects of DIDS on rabbit skeletal single Ca2+-release channel/RyR1 (ryanodine receptor type 1) incorporated into a planar lipid bilayer. When junctional SR vesicles were used for channel incorporation (native RyR1), DIDS increased the mean P(o) (open probability) of RyR1 without affecting unitary conductance when Cs+ was used as the charge carrier. Lifetime analysis of single RyR1 activities showed that 10 microM DIDS induced reversible long-lived open events (P(o)=0.451+/-0.038) in the presence of 10 microM Ca2+, due mainly to a new third component for both open and closed time constants. However, when purified RyR1 was examined in the same condition, 10 microM DIDS became considerably less potent (P(o)=0.206+/-0.025), although the caffeine response was similar between native and purified RyR1. Hence we postulated that a DIDS-binding protein, essential for the DIDS sensitivity of RyR1, was lost during RyR1 purification. DIDS-affinity column chromatography of solubilized junctional SR, and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis of the affinity-column-associated proteins, identified four major DIDS-binding proteins in the SR fraction. Among them, aldolase was the only protein that greatly potentiated DIDS sensitivity. The association between RyR1 and aldolase was further confirmed by co-immunoprecipitation and aldolase-affinity batch-column chromatography. Taken together, we conclude that aldolase is physically associated with RyR1 and could confer a considerable potentiation of the DIDS effect on RyR1.

A Taste Receptor Required for the Caffeine Response In Vivo

Caffeine is a methylxanthine present in the coffee tree, tea plant, and other naturally occurring sources and is among the most commonly consumed drugs worldwide. Whereas the pharmacological action of caffeine has been studied extensively, relatively little is known concerning the molecular mechanism through which this substance is detected as a bitter compound. Unlike most tastants, which are detected through cell-surface G protein-coupled receptors, it has been proposed that caffeine and related methylxanthines activate taste-receptor cells through inhibition of a cyclic nucleotide phosphodiesterase (PDE) . Here, we show that the gustatory receptor Gr66a is expressed in the dendrites of Drosophila gustatory receptor neurons and is essential for the caffeine response. In a behavioral assay, the aversion to caffeine was specifically disrupted in flies missing Gr66a. Caffeine-induced action potentials were also eliminated, as was the response to theophylline, the methylxanthine in tea. The Gr66a mutant exhibited normal tastant-induced action potentials upon presentation of theobromine, a methylxanthine in cocoa. Given that theobromine and caffeine inhibit PDEs with equal potencies , these data further support the role of Gr66a rather than a PDE in mediating the caffeine response. Gr66a is the first gustatory receptor shown to be essential for caffeine-induced behavior and activity of gustatory receptor cells in vivo.

Adenosine affects the release of Ca2+ from the sarcoplasmic reticulum via A2A receptors in ferret skinned cardiac fibres

In this study, it was shown that adenosine potentiates caffeine-induced Ca2+ release. It was then proposed that the enhancement of the caffeine-induced Ca2+ release might occur by a direct effect on the ryanodine Ca2+ release channel or on other Ca2+ regulation mechanisms. Furthermore, A2A receptors may be functional on the ferret cardiac sarcoplasmic reticulum. Using chemically skinned fibres, experiments were conducted on ferret cardiac muscle to find out whether adenosine and the A1 and A2A adenosine receptor agonists (CCPA and CGS 21680) and antagonists (DPCPX and ZM 241385) affected caffeine-induced Ca2+ release and the Ca2+ sensitivity of contractile proteins. Changes in the caffeine-induced contracture brought about by adenosine and by adenosine-receptor agonists and antagonists were recorded in saponin-skinned fibres (50 microg ml(-1)). Tension-pCa relationships were then obtained by exposing Triton X-100-skinned fibres (1% v/v) sequentially to solutions of decreasing pCa. Adenosine (1-100 nm) and the specific A2A receptor agonist CGS 21680 (1-50 nm) produced a concentration-dependant potentiation of the caffeine-induced Ca2+ release from saponin-skinned fibres. The data plotted versus adenosine and CGS 21680 concentrations displayed sigmoid relationships (Hill relationship), with potentiation of Ca2+ release by 22.2 +/- 1.6 (n = 6) and 10.9 +/- 0.4% (n = 6), respectively. In addition, the potentiation of caffeine-induced Ca2+ release by adenosine (50 nm; 15.3 +/- 1.0%; n = 6) and by CGS 21680 (50 nm; 11.2 +/- 0.4%; n = 6) was reduced by the specific A2A receptor antagonist ZM 241385 (50 nm) to 8.0 +/- 1.4 (n = 4) and 5.4 +/- 1.2% (n = 4), respectively. The A1 receptor agonist CCPA (1-50 nm) and antagonist DPCPX (50 nm) had no significant effects on caffeine responses. In Triton X-100-skinned fibres, the maximal Ca(2+)-activated tension of the contractile proteins (41.3 +/- 4.1 mN mm(-2); n = 8), the Hill coefficient (nH = 2.2 +/- 0.1; n = 8) and the pCa50 (6.15 +/- 0.05; n = 8) were not significantly modified by adenosine (100 nm) or by CGS 21680 (50 nm).

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

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

RNA Interference Decreases PAR-2 Expression and Function in Human Airway Smooth Muscle Cells

Asthma is characterized by bronchial inflammation and hyperresponsiveness that involves mast cell tryptase and potentially its specific receptor protease activated receptor 2 (PAR-2). Tryptase increases free intracellular calcium concentration ([Ca2+]i), a key step in activation of human airway smooth muscle cells (HASMC). The aim of this study was to analyze the effect of PAR-2 gene silencing on HASMC, in terms of calcium response, since no antagonist is available for this receptor. Five siRNA against PAR-2 were synthesized and transfected in HASMC using lipid agents, and PAR-2 expression was examined using Western blot, fluorescence-activated cell sorter, immunocytochemistry and RT-PCR. [Ca2+]i was measured using microspectrofluorimetry in response to tryptase, the activating peptide SLIGKV, trypsin, or caffeine. Two siRNA significantly inhibited PAR-2 expression in terms of both total and surface protein expression, as well as mRNA levels. Tryptase- and SLIGKV-induced transient increase in [Ca2+]i was significantly inhibited after transfection with the most appropriate siRNA, whereas neither trypsin nor caffeine response was altered. Two control siRNA had no effect in terms of both PAR-2 expression and calcium response. Transfection efficiency was maximal after 24 h and disappeared after 48 h. Gene silencing using siRNA can thus be used in vitro to assess the function of PAR-2 in HASMC.

Release and sequestration of Ca 2+ by a caffeine- and ryanodine-sensitive store in a sub-population of human SH-SY5Y neuroblastoma cells

We have used single cell fluorescence imaging techniques to examine the role that ryanodine receptors play in the stimulus-induced Ca 2+ responses of SH-SY5Y cells. The muscarinic agonist methacholine (1 mM) resulted in a Ca 2+ signal in 95% of all cells. Caffeine (30 mM) however stimulated a Ca 2+ signal in only 1–7% of N-type (neuroblastic) cells within any given field. The caffeine response was independent of extracellular Ca 2+, regenerative in nature, and abolished in a use-dependent fashion by ryanodine. In caffeine-responsive cells, the magnitude of the methacholine-induced Ca 2+ signal was inhibited by 75.07 ± 5.51% by pretreatment with caffeine and ryanodine, suggesting that the caffeine-sensitive store may act as a Ca 2+ source after muscarinic stimulation. When these data were combined with equivalent data from non-caffeine-responsive cells, the degree of apparent inhibition was significantly reduced. In contrast, after store depletion by caffeine, the Ca 2+ signal induced by 55 mM K + was potentiated 2.5-fold in the presence of ryanodine, suggesting that the store may act a Ca 2+ sink after depolarisation. We conclude that a caffeine- and ryanodine-sensitive store can act as a Ca 2+ source and sink in SH-SY5Y cells, and that effects of the store can become obscured if data from caffeine-insensitive cells are not excluded.

Inhibition of sarcoplasmic reticular function by chronic interleukin‐6 exposure via iNOS in adult ventricular myocytes

Interleukin (IL)-6 has been shown to decrease cardiac contractility via a nitric oxide synthase (NOS)-dependent pathway during acute exposure. We previously reported that IL-6 decreases contractility and increases inducible NOS (iNOS) in adult rat ventricular myocytes (ARVM) after 2 h exposure. The goal of this study was to investigate the cellular mechanism underlying this chronic IL-6-induced negative inotropy and the role of iNOS. Pretreatment for 2 h with 10 ng ml-1 IL-6 decreased the kinetics of cell shortening (CS) and contractile responsiveness to Ca2+o ([Ca2+]o from(0) to 2 mM) in ARVM. We first examined whether IL-6 reduced Ca2+ influx via L-type Ca2+ -channel current (ICa,L). Whole-cell ICa,L in ARVM was measured under conditions similar to those used for CS measurements, and it was found to be unaltered by IL-6. The sarcoplasmic reticular (SR) function was then assessed by examining postrest potentiation (PRP) and caffeine responsiveness of CS. Results showed that treatment with IL-6 for 2 h significantly decreased PRP, which was concomitant with a decrease in the phosphorylation of phospholamban. Following removal of IL-6, PRP and responsiveness to 10 mM caffeine were also reduced. Meanwhile, the IL-6-induced increase in nitric oxide (NO) production after 2 h (but not 1 h) was abolished by NG-monomethyl-l-arginine (l-NMMA) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; a selective inhibitor of iNOS). Furthermore, IL-6-elicited suppressions of PRP and responsiveness to caffeine and Ca2+o were abolished by L-NMMA and AMT. Thus, these results suggest that activation of iNOS mediates IL-6-induced inhibition of SR function in ARVM during chronic exposure.

Reversal of caffeine-induced anxiety by neurosteroid 3-alpha-hydroxy-5-alpha-pregnane-20-one in rats

Caffeine has been shown to increase brain and plasma content of neurosteroid 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) that allosterically modulates GABA A receptors. The present study evaluated the role of neurosteroid 3α,5α-THP in the caffeine-induced anxiogenic-like effect using the elevated plus-maze (EPM) test in rats. Acute administration of caffeine (50 or 100 mg/kg, i.p.) produced anxiogenic-like activity that was reversed by pretreatment with the neurosteroid 3α,5α-THP or progesterone, the GABA A agonist muscimol, or the benzodiazepine receptor agonist diazepam. On the contrary, caffeine produced higher anxiety in animals previously treated with the GABA A receptor antagonist, bicuculline or either of the various neurosteroid biosynthesis enzyme inhibitors viz. trilostane, finasteride or indomethacin. Furthermore, pretreatment with DHEAS, a neurosteroid that negatively modulates GABA A receptors also enhanced the caffeine-induced anxiety. Moreover, adrenalectomy potentiated the anxiogenic-like response of caffeine indicating the contributory role of peripheral steroidogenesis. Thus, it is speculated that neurosteroid 3α,5α-THP through positive modulation of GABA A receptor activity may serve as a counter-regulatory mechanism against caffeine-induced anxiety.

Mild dystrophic damage in the androgen-sensitive levator ani muscle of the mdx mouse

The time course of muscular dystrophy on the androgen-sensitive levator ani muscle was compared to that of the diaphragm of dystrophic (mdx) mice aged 1–20 months. Muscle growth, isometric contractile properties and caffeine-induced contractures were determined to assess the hormone myotrophic effect, muscle strength and sarcoplasmic reticulum function, respectively, of both control and dystrophic muscles. Histological analysis of mdx muscles showed variable fiber size, centronucleated cells, infiltration of connective tissue, and necrosis which was less severe in the levator ani than in the diaphragm muscle. Tetanic tension per unit weight in the mdx levator ani was reduced (29%) after aging, while the contraction time remained unchanged. The tetanic tension of the mdx diaphragm muscle decreased with age from 3 to 20 months (20–64%), and the relaxation time was prolonged after aging (22%). Gonadectomy of young adult mdx mice caused atrophy of the levator ani muscle, accelerated muscle wasting, reduced the tetanic force (31%), but it did not affect the contraction time and caffeine responses. The results showed that testosterone does not prevent the progress of muscle disease in the mdx levator ani, but androgen withdrawal accelerates muscle wasting suggesting a normonal beneficial effect.

Effects of volatile anesthetics on store-operated Ca(2+) influx in airway smooth muscle.

In airway smooth muscle (ASM), volatile anesthetics deplete sarcoplasmic reticulum (SR) Ca(2+) stores by increasing Ca(2+) "leak." Accordingly, SR replenishment becomes dependent on Ca(2+) influx. Depletion of SR Ca(2+) stores triggers Ca(2+) influx via specific plasma membrane channels, store-operated Ca(2+) channels (SOCC). We hypothesized that anesthetics inhibit SOCC triggered by increased SR Ca(2+) "leak," preventing SR replenishment and enhancing ASM relaxation. In porcine ASM cells, SR Ca was depleted by cyclopiazonic acid or caffeine in 0 extracellular Ca(2+), nifedipine and KCl (preventing Ca(2+) influx through L-type and SOCC channels). Extracellular Ca(2+) was rapidly introduced to selectively activate SOCC. After SOCC activation, SR was replenished and the protocol repeated in the presence of 1 or 2 minimum alveolar concentration halothane, isoflurane, or sevoflurane. In other cells, characteristics of SOCC and interactions between acetylcholine (Ach) and volatile anesthetics were examined. Cyclopiazonic acid produced slow SR leak, whereas the caffeine response was transient in ASM cells. Reintroduction of extracellular Ca(2+) rapidly increased [Ca(2+)]i. This influx was insensitive to nifedipine, SKF-96365, and KBR-7943, inhibited by Ni and blockade of inositol 1,4,5-triphosphate-induced SR Ca(2+) release, and enhanced by ACh. Preexposure to 1 or 2 minimum alveolar concentration halothane completely inhibited Ca(2+) influx when extracellular Ca(2+) was reintroduced, whereas isoflurane and sevoflurane produced less inhibition. Only halothane and isoflurane inhibited ACh-induced augmentation of Ca(2+) influx. Volatile anesthetics inhibit a Ni/La-sensitive store-operated Ca(2+) influx mechanism in porcine ASM cells, which likely helps maintain anesthetic-induced bronchodilation.

The Predicted TM10 Transmembrane Sequence of the Cardiac Ca2+ Release Channel (Ryanodine Receptor) Is Crucial for Channel Activation and Gating

The predicted TM10 transmembrane sequence, (4844)IIFDITFFFFVIVILLAIIQGLII(4867), has been proposed to be the pore inner helix of the ryanodine receptor (RyR) and to play a crucial role in channel activation and gating, as with the inner helix of bacterial potassium channels. However, experimental evidence for the involvement of the TM10 sequence in RyR channel activation and gating is lacking. In the present study, we have systematically investigated the effects of mutations of each residue within the 24-amino acid TM10 sequence of the mouse cardiac ryanodine receptor (RyR2) on channel activation by caffeine and Ca(2+). Intracellular Ca(2+) release measurements in human embryonic kidney 293 cells expressing the RyR2 wild type and TM10 mutants revealed that several mutations in the TM10 sequence either abolished caffeine response or markedly reduced the sensitivity of the RyR2 channel to activation by caffeine. By assessing the Ca(2+) dependence of [(3)H]ryanodine binding to RyR2 wild type and TM10 mutants we also found that mutations in the TM10 sequence altered the sensitivity of the channel to activation by Ca(2+) and enhanced the basal activity of [(3)H]ryanodine binding. Furthermore, single I4862A mutant channels exhibited considerable channel openings and altered gating at very low concentrations of Ca(2+). Our data indicate that the TM10 sequence constitutes an essential determinant for channel activation and gating, in keeping with the proposed role of TM10 as an inner helix of RyR. Our results also shed insight into the orientation of the TM10 helix within the RyR channel pore.

Characterization of Recombinant Skeletal Muscle (Ser-2843) and Cardiac Muscle (Ser-2809) Ryanodine Receptor Phosphorylation Mutants

Phosphorylation of the skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptors has been reported to modulate channel activity. Abnormally high phosphorylation levels (hyperphosphorylation) at Ser-2843 in RyR1 and Ser-2809 in RyR2 and dissociation of FK506-binding proteins from the receptors have been implicated as one of the causes of altered calcium homeostasis observed during human heart failure. Using site-directed mutagenesis, we prepared recombinant RyR1 and RyR2 mutant receptors mimicking constitutively phosphorylated and dephosphorylated channels carrying a Ser/Asp (RyR1-S2843D and RyR2-S2809D) and Ser/Ala (RyR1-S2843A and RyR2-S2809A) substitution, respectively. Following transient expression in human embryonic kidney 293 cells, the effects of Ca2+, Mg2+, and ATP on channel function were determined using single channel and [3H]ryanodine binding measurements. In both assays, neither the skeletal nor cardiac mutants showed significant differences compared with wild type. Similarly essentially identical caffeine responses were observed in Ca2+ imaging measurements. Co-immunoprecipitation and Western blot analysis showed comparable binding of FK506-binding proteins to wild type and mutant receptors. Finally metabolic labeling experiments showed that the cardiac ryanodine receptor was phosphorylated at additional sites. Taken together, the results did not support the view that phosphorylation of a single site (RyR1-Ser-2843 and RyR2-Ser-2809) substantially changes RyR1 and RyR2 channel function.

Cyclic ADP-ribose increases Ca2+ removal in smooth muscle.

Ca2+ release via ryanodine receptors (RyRs) is vital in cell signalling and regulates diverse activities such as gene expression and excitation-contraction coupling. Cyclic ADP ribose (cADPR), a proposed modulator of RyR activity, releases Ca2+ from the intracellular store in sea urchin eggs but its mechanism of action in other cell types is controversial. In this study, caged cADPR was used to examine the effect of cADPR on Ca2+ signalling in single voltage-clamped smooth muscle cells that have RyR but lack FKBP12.6, a proposed target for cADPR. Although cADPR released Ca2+ in sea urchin eggs (a positive control), it failed to alter global or subsarcolemma [Ca2+]c, to cause Ca2+-induced Ca2+ release or to enhance caffeine responses in colonic myocytes. By contrast, caffeine (an accepted modulator of RyR) was effective in these respects. The lack of cADPR activity on Ca2+ release was unaffected by the introduction of recombinant FKBP12.6 into the myocytes. Indeed in western blots, using brain membrane preparations as a source of FKBP12.6, cADPR did not bind to FKBPs, although FK506 was effective. However, cADPR increased and its antagonist 8-bromo-cADPR slowed the rate of Ca2+ removal from the cytoplasm. The evidence indicates that cADPR modulates [Ca2+]c but not via RyR; the mechanism may involve the sarcolemma Ca2+ pump.

Decrease in resting calcium and calcium entry associated with slow-to-fast transition in unloaded rat soleus muscle.

Using fura-2 and the manganese quenching technique, we show here that sarcolemmal permeability to cations (SP-Ca) of slow-twitch muscles is greater than that of fast-twitch ones. This appears to be related to a higher expression and/or activity of stretch-activated channels, whereas leak channel activities are similar. During hindlimb suspension (HU), we found highly correlated decreases in SPCa and resting calcium of soleus muscle toward values of extensor digitorum longus (EDL) muscle. This was significant as soon as 3 days of suspension, contrary to soleus muscle caffeine sensitivity and responsiveness that were not modified after this HU period. After 14 days of HU, SP-Ca, resting calcium, and caffeine response of soleus muscle became similar to that normally observed in EDL muscle. These results demonstrate that the correlated decreases in SP-Ca and resting calcium precede most functional changes due to HU. Given the known shortening of HU soleus muscle, we proposed that this could induce a decrease of SP-Ca and a consequent reduction of resting calcium. According to the crucial role of resting cytosolic free calcium in the maintenance and the adaptation of muscle phenotype, our results suggest that slow-to-fast transition of HU soleus muscle is calcium dependent.