Biomedical subjects

F Anthony Lai

Publications and source records attributed to F Anthony Lai.

32 records · Page 2Linked to original sources

PubMed2004

Functional heterogeneity of ryanodine receptor mutations associated with sudden cardiac death.

OBJECTIVES: Point mutations in the cardiac ryanodine receptor (RyR2) mediate abnormal intracellular Ca(2+) release and are associated with stress-induced ventricular tachycardia (VT), leading to sudden cardiac death (SCD). Although the precise molecular basis of RyR2 dysfunction in SCD remains controversial, there is consensus that the mutations characterised to date all exhibit gain-of-function Ca(2+) release properties following cell stimulation. We investigated the functional impact of a distinct set of SCD-linked RyR2 mutations (L(433)P, N(2386)I, R(176)Q/T(2504)M) on intracellular Ca(2+) handling. METHODS: We expressed full-length recombinant human wild-type (WT) and SCD-linked RyR2 mutations in human embryonic kidney (HEK) cells, and profiled the spatial and amplitude characteristics of caffeine-evoked Ca(2+) release through homo-tetrameric channels in living cells using rapid confocal laser scanning microscopy. RESULTS: Analysis of the precise mode of Ca(2+) release in HEK cells expressing RyR2 mutants demonstrated profound differences when compared with WT channels. The SCD-linked RyR2 mutations characterised in this study exhibited heterogeneous Ca(2+) release profiles, including the novel observation that one of the mutants, (L(433)P), exhibited a marked reduction in sensitivity to channel activation. However, all SCD-linked RyR2 mutations characterised in this study resulted in an increased duration of elevated cytoplasmic Ca(2+) levels following channel activation. CONCLUSIONS: Our live cell-based data demonstrates functional heterogeneity of Ca(2+) release through SCD-linked RyR2 mutants, suggesting that the mechanistic basis of RyR2 dysfunction in SCD may be more complex than previously anticipated. These findings may have profound consequences for the therapeutic modulation of RyR2 in stress-induced VT and SCD.

Arrhythmias, Cardiac↗

PubMed2004

Cell cycle-dependent Ca2+ oscillations in mouse embryos are regulated by nuclear targeting of PLCzeta.

During the first cell cycle Ca2+ oscillations are regulated in a cell cycle-dependent manner, such that the oscillations are unique to M phase. How the Ca2+ oscillations are regulated with such cell cycle stage-dependency is unknown, despite their importance for egg activation and embryo development. We recently identified a novel, sperm-specific phospholipase C (PLCzeta; PLCzeta) that triggers Ca2+ oscillations similar to those caused by sperm. We show that PLCzeta-induced Ca2+ oscillations also occur exclusively during M phase. The cell cycle-dependency can be explained by PLCzeta's localisation to the pronuclei, which depends specifically upon a nuclear localisation signal sequence. Preventing pronuclear localisation of PLCzeta by mutation of the nuclear localisation signal, or by inhibiting pronuclear formation/import, can prolong Ca2+ oscillations or allow them to occur during interphase. These data suggest a novel mechanism for regulating a PLC through nuclear sequestration and may explain the cell cycle-dependent regulation of Ca2+ oscillations following fertilisation.

Amino Acid Substitution↗

PubMed2004

Ryanodine receptor regulation by intramolecular interaction between cytoplasmic and transmembrane domains.

Ryanodine receptors (RyR) function as Ca(2+) channels that regulate Ca(2+) release from intracellular stores to control a diverse array of cellular processes. The massive cytoplasmic domain of RyR is believed to be responsible for regulating channel function. We investigated interaction between the transmembrane Ca(2+)-releasing pore and a panel of cytoplasmic domains of the human cardiac RyR in living cells. Expression of eGFP-tagged RyR constructs encoding distinct transmembrane topological models profoundly altered intracellular Ca(2+) handling and was refractory to modulation by ryanodine, FKBP12.6 and caffeine. The impact of coexpressing dsRed-tagged cytoplasmic domains of RyR2 on intracellular Ca(2+) phenotype was assessed using confocal microscopy coupled with parallel determination of in situ protein: protein interaction using fluorescence resonance energy transfer (FRET). Dynamic interactions between RyR cytoplasmic and transmembrane domains were mediated by amino acids 3722-4610 (Interacting or "I"-domain) which critically modulated intracellular Ca(2+) handling and restored RyR sensitivity to caffeine activation. These results provide compelling evidence that specific interaction between cytoplasmic and transmembrane domains is an important mechanism in the intrinsic modulation of RyR Ca(2+) release channels.

Animals↗

PubMed2004

Ca2+ syntillas, miniature Ca2+ release events in terminals of hypothalamic neurons, are increased in frequency by depolarization in the absence of Ca2+ influx.

Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of approximately 250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of -80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals.

Animals↗

PubMed2004

Ryanodine receptor oligomeric interaction: identification of a putative binding region.

Specific interactions between adjacent ryanodine receptor (RyR) molecules to form ordered two-dimensional arrays in the membrane have been demonstrated using electron microscopy both in situ, in tissues and cells, and in vitro, with the purified protein. RyR interoligomeric association has also been inferred from observations of simultaneous channel gating during multi-RyR channel recordings in lipid bilayers. In this study, we report experiments designed to identify the region(s) of the RyR molecule, participating in this reciprocal interaction. Using epitope-specific antibodies, we identified a RyR tryptic fragment that specifically bound the intact immobilized RyR. Three overlapping RyR fragments encompassing this epitope, expressed using an in vitro mammalian expression system, were immunoprecipitated by RyR. To refine the binding regions, smaller RyR fragments were expressed as glutathione S-transferase (GST) fusion proteins, and their binding to RyR was monitored using a "sandwich" enzyme-linked immunosorbent assay. Three GST-RyR fusion proteins demonstrated specific binding, dependent upon ionic strength. Binding was greatest at 50-150 mm NaCl for two GST-RyR constructs, and a third GST-RyR construct demonstrated maximum binding between 150 and 450 mm NaCl. The binding at high NaCl concentration suggested involvement of a hydrophobic interaction. In silico analysis of secondary structure showed evidence of coil regions in two of these RyR fragment sequences, which might explain these data. In GST pull-down assays, these same three fragments captured RyR2, and two of them retained RyR1. These results identify a region at the center of the linear RyR (residues 2540-3207 of human RyR2) which is able to bind to the RyR oligomer. This region may constitute a specific subdomain participating in RyR-RyR interaction.

Amino Acid Sequence↗

PubMed2004

Role of FKBP12.6 in hypoxia- and norepinephrine-induced Ca2+ release and contraction in pulmonary artery myocytes.

The cellular and molecular processes underlying the regulation of ryanodine receptor (RyR) Ca(2+) release in smooth muscle cells (SMCs) are incompletely understood. Here we show that FKBP12.6 proteins are expressed in pulmonary artery (PA) smooth muscle and associated with type-2 RyRs (RyR2), but not RyR1, RyR3, or IP(3) receptors (IP(3)Rs) in PA sarcoplasmic reticulum. Application of FK506, which binds to FKBPs and dissociates these proteins from RyRs, induced an increase in [Ca(2+)](i) and Ca(2+)-activated Cl(-) and K(+) currents in freshly isolated PASMCs, whereas cyclosporin, an agent known to inhibit calcineurin but not to interact with FKBPs, failed to induce an increase in [Ca(2+)](i). FK506-induced [Ca(2+)](i) increase was completely blocked by the RyR antagonist ruthenium red and ryanodine, but not the IP(3)R antagonist heparin. Hypoxic Ca(2+) response and hypoxic vasoconstriction were significantly enhanced in FKBP12.6 knockout mouse PASMCs. FK506 or rapamycin pretreatment also enhanced hypoxic increase [Ca(2+)](i), but did not alter caffeine-induced Ca(2+) release (SR Ca(2+) content) in PASMCs. Norepinephrine-induced Ca(2+) release and force generation were also markedly enhanced in PASMCs from FKBP12.6 null mice. These findings suggest that FKBP12.6 plays an important role in hypoxia- and neurotransmitter-induced Ca(2+) and contractile responses by regulating the activity of RyRs in PASMCs.

Animals↗

PubMed2003

Oligomerization of the cardiac ryanodine receptor C-terminal tail.

The C-terminal 100 amino acids of the RyR (ryanodine receptor), referred to as the C-terminal tail, is a highly conserved sequence that is present in all known RyR isoforms and which has been implicated in channel function. Deleting the final 15 amino acids from the full-length skeletal muscle RyR resulted in an inactive channel, attributed to impaired assembly of a tetrameric RyR complex [Gao, Tripathy, Lu and Meissner (1997) FEBS Lett. 412, 223-226]. To account for these observations, the C-terminal tail itself may be an important molecular determinant of oligomerization. Alternatively, the large N-terminal cytoplasmic domain may fold back upon itself to interact with the C-terminal tail to provide a correctly folded tetrameric structure. We explored these possibilities for RyR2 (cardiac RyR) using the yeast two-hybrid interaction assay and in vitro translation followed by immunoprecipitation and chemical cross-linking. The data indicate that the C-terminal tail of RyR2 is capable of self-tetramerization. Moreover, a truncated C-terminal tail, lacking the final 15 amino acids, failed to self-associate. These observations suggest that the intrinsic ability of the RyR C-terminal tail to self-tetramerize may be vitally important for the oligomeric assembly of the native RyR channel.

Amino Acid Sequence↗

PubMed2003

Ryanodine receptor mutations associated with stress-induced ventricular tachycardia mediate increased calcium release in stimulated cardiomyocytes.

Ca2+ release from the sarcoplasmic reticulum mediated by the cardiac ryanodine receptor (RyR2) is a fundamental event in cardiac muscle contraction. RyR2 mutations suggested to cause defective Ca2+ channel function have recently been identified in catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD) affected individuals. We report expression of three CPVT-linked human RyR2 (hRyR2) mutations (S2246L, N4104K, and R4497C) in HL-1 cardiomyocytes displaying correct targeting to the endoplasmic reticulum. N4104K also localized to the Golgi apparatus. Phenotypic characteristics including intracellular Ca2+ handling, proliferation, viability, RyR2:FKBP12.6 interaction, and beat rate in resting HL-1 cells expressing mutant hRyR2 were indistinguishable from wild-type (WT) hRyR2. However, Ca2+ release was augmented in cells expressing mutant hRyR2 after RyR activation (caffeine and 4-chloro-m-cresol) or beta-adrenergic stimulation (isoproterenol). RyR2:FKBP12.6 interaction remained intact after caffeine or 4-CMC activation, but was dramatically disrupted by isoproterenol or forskolin, an activator of adenylate cyclase. Isoproterenol and forskolin elevated cyclic-AMP to similar magnitudes in all cells and were associated with equivalent hyperphosphorylation of mutant and WT hRyR2. CPVT-linked mutations in hRyR2 did not alter resting cardiomyocyte phenotype but mediated augmented Ca2+ release on RyR-agonist or beta-AR stimulation. Furthermore, equivalent interaction between mutant and WT hRyR2 and FKBP12.6 was demonstrated.

Adenylyl Cyclases↗

PubMed2003

Dysregulated ryanodine receptors mediate cellular toxicity: restoration of normal phenotype by FKBP12.6.

Ca2+ homeostasis is a vital cellular control mechanism in which Ca2+ release from intracellular stores plays a central role. Ryanodine receptor (RyR)-mediated Ca2+ release is a key modulator of Ca2+ homeostasis, and the defective regulation of RyR is pathogenic. However, the molecular events underlying RyR-mediated pathology remain undefined. Cells stably expressing recombinant human RyR2 (Chinese hamster ovary cells, CHOhRyR2) had similar resting cytoplasmic Ca2+ levels ([Ca2+]c) to wild-type CHO cells (CHOWT) but exhibited increased cytoplasmic Ca2+ flux associated with decreased cell viability and proliferation. Intracellular Ca2+ flux increased with human RyR2 (hRyR2) expression levels and determined the extent of phenotypic modulation. Co-expression of FKBP12.6, but not FKBP12, or incubation of cells with ryanodine suppressed intracellular Ca2+ flux and restored normal cell viability and proliferation. Restoration of normal phenotype was independent of the status of resting [Ca2+]c or ER Ca2+ load. Heparin inhibition of endogenous inositol trisphosphate receptors (IP3R) had little effect on intracellular Ca2+ handling or viability. However, purinergic stimulation of endogenous IP3R resulted in apoptotic cell death mediated by hRyR2 suggesting functional interaction occurred between IP3R and hRyR2 Ca2+ release channels. These data demonstrate that defective regulation of RyR causes altered cellular phenotype via profound perturbations in intracellular Ca2+ signaling and highlight a key modulatory role of FKBP12.6 in hRyR2 Ca2+ channel function.

Adenosine Triphosphate↗

PubMed2003

In situ modulation of the human cardiac ryanodine receptor (hRyR2) by FKBP12.6.

The ryanodine receptor complex (RyR), a large oligomeric assembly that functions as a Ca(2+)-release channel in the sarcoplasmic reticulum (SR)/endoplasmic reticulum (ER), comprises four RyR subunits and four FK506-binding proteins (FKBP). The precise mode of interaction and modulation of the cardiac RyR (RyR2) channel by FKBP12/FKBP12.6 remains to be fully defined. We have generated a series of Chinese-hamster ovary (CHO) cell lines stably expressing discrete levels of recombinant human RyR2 (hRyR2) (CHO(hRyR2)). Confocal microscopy of CHO(hRyR2) cells co-expressing either FKBP12 or FKBP12.6 demonstrated that FKBP12.6 was sequestered from the cytoplasm to ER membranes as the cellular levels of hRyR2 increased. There was negligible hRyR2-induced subcellular redistribution of FKBP12. The magnitude of Ca(2+) release in CHO(hRyR2) cells in response to stimulation by 4-chloro- m -cresol was in direct proportion to the expression levels of hRyR2. However, in CHO(hRyR2) cells co-expressing FKBP12.6, Ca(2+) release triggered by the addition of 4-chloro- m -cresol was markedly decreased. In contrast, co-expression of FKBP12 did not affect agonist-induced Ca(2+) release in CHO(hRyR2) cells. Resting cytoplasmic [Ca(2+)] in CHO(hRyR2) remained unaltered after co-expression of FKBP12 or FKBP12.6, but estimation of the ER Ca(2+) load status showed that co-expression of FKBP12.6, but not FKBP12, promoted superfilling of the ER Ca(2+) store which could not be released by RyR2 after agonist activation. The effects of FKBP12.6 on hRyR2-mediated intracellular Ca(2+) handling could be antagonized using rapamycin (5 microM). These results suggest that FKBP12.6 associates with hRyR2 in situ to modulate precisely the functionality of hRyR2 Ca(2+)-release channel.

Animals↗

PubMed2003

Ryanodine stores and calcium regulation in the inner segments of salamander rods and cones.

Despite the prominent role played by intracellular Ca2+ stores in the regulation of neuronal Ca2+ homeostasis and in invertebrate photoreception, little is known about their contribution to the control of free Ca2+ concentration ([Ca2+]i) in the inner segments of vertebrate photoreceptors. Previously, caffeine-sensitive intracellular Ca2+ stores were shown to play a role in regulating glutamate release from photoreceptors. To understand the properties of these intracellular stores better we used pharmacological approaches that alter the dynamics of storage and release of Ca2+ from intracellular compartments. Caffeine evoked readily discernible changes in [Ca2+]i in the inner segments of rods, but not cones. Caffeine-evoked Ca2+ responses in cone inner segments were unmasked in the presence of inhibitors of the plasma membrane Ca2+ ATPases (PMCAs) and mitochondrial Ca2+ sequestration. Caffeine-evoked responses were blocked by ryanodine, a selective blocker of Ca2+ release and by cyclopiazonic acid, a blocker of Ca2+ sequestration into the endoplasmic reticulum. These two inhibitors also substantially reduced the amplitude of depolarization-evoked [Ca2+]i increases, providing evidence for Ca2+-induced Ca2+ release (CICR) in rods and cones. The magnitude and kinetics of caffeine-evoked Ca2+ elevation depended on the basal [Ca2+]i, PMCA activity and on mitochondrial function. These results reveal an intimate interaction between the endoplasmic reticulum, voltage-gated Ca2+ channels, PMCAs and mitochondrial Ca2+ stores in photoreceptor inner segments, and suggest a role for CICR in the regulation of synaptic transmission.

Ambystoma↗

PubMed2003

Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta-cells (MIN6).

We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001) J. Cell Biol. 155, 41-51) that ryanodine receptors (RyR) are present on insulin-containing secretory vesicles. Here we show that pancreatic islets and derived beta-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core secretory vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca(2+) concentrations in the secretory vesicle compartment from 40.1 +/- 6.7 to 90.4 +/- 14.8 microm (n = 4, p < 0.01), while having no effect on ER Ca(2+) concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca(2+)-mobilizing agent, decreased dense core secretory vesicle but not ER free Ca(2+) concentrations in permeabilized MIN6 beta-cells, and flash photolysis of caged NAADP released Ca(2+) from a thapsigargin-insensitive Ca(2+) store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 +/- 0.51-fold stimulation to no significant glucose effect; n = 3, p < 0.01), we conclude that RyR I-mediated Ca(2+)-induced Ca(2+) release from secretory vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion.

Animals↗

PubMed2002

Isoform-dependent formation of heteromeric Ca2+ release channels (ryanodine receptors).

Three ryanodine receptor (RyR) isoforms, RyR1, RyR2, and RyR3, are expressed in mammalian tissues. It is unclear whether RyR isoforms are capable of forming heteromeric channels. To investigate their ability to form heteromeric channels, we co-expressed different RyR isoforms in HEK293 cells and examined their interactions biochemically and functionally. Immunoprecipitation studies revealed that RyR2 is able to interact physically with RyR3 and RyR1 in HEK293 cells and that RyR1 does not interact with RyR3. Co-expression of a ryanodine binding deficient mutant of RyR2, RyR2 (I4827T), with RyR3 (wt) restored [(3)H]ryanodine binding to the mutant. Interactions between RyR isoforms were further assessed by complementation analysis using mutants RyR2 (I4827T), RyR2 (E3987A), RyR3 (I4732T), RyR3 (E3885A), and RyR1 (E4032A), all of which are deficient in caffeine response. Caffeine-induced Ca(2+) release was restored in HEK293 cells co-transfected with mutants RyR2 (I4827T) and RyR3 (E3885A), RyR2 (E3987A) and RyR3 (I4732T), or RyR2 (I4827T) and RyR1 (E4032A), but not with RyR1 (E4032A) and RyR3 (I4732T), indicating that mutants of RyR2 and RyR3, or RyR2 and RyR1, but not RyR1 and RyR3, are able to complement each other. Co-expression of RyR3 (wt) and a pore mutant of RyR2, RyR2 (G4824A), produced regulatable single channels with intermediate unitary conductances. These observations demonstrate that RyR2 is capable of forming functional heteromeric channels with RyR3 and RyR1, whereas RyR1 is incapable of forming heteromeric channels with RyR3.

Caffeine↗

PubMed2002

PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development.

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca(2+) oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca(2+) release. The 'sperm factor' hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca(2+) transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLC zeta, that triggers Ca(2+) oscillations in mouse eggs indistinguishable from those at fertilisation. PLC zeta removal from sperm extracts abolishes Ca(2+) release in eggs. Moreover, the PLC zeta content of a single sperm was sufficient to produce Ca(2+) oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLC zeta as the molecular trigger for development of a fertilised egg into an embryo.

Amino Acid Sequence↗