Lumenal Ca2 Research Articles

Multiscale Modeling of Dyadic Structure-Function Relation in Ventricular Cardiac Myocytes

Cardiovascular disease is often related to defects of subcellular components in cardiac myocytes, specifically in the dyadic cleft, which include changes in cleft geometry and channel placement. Modeling of these pathological changes requires both spatially resolved cleft as well as whole cell level descriptions. We use a multiscale model to create dyadic structure-function relationships to explore the impact of molecular changes on whole cell electrophysiology and calcium cycling. This multiscale model incorporates stochastic simulation of individual L-type calcium channels and ryanodine receptor channels, spatially detailed concentration dynamics in dyadic clefts, rabbit membrane potential dynamics, and a system of partial differential equations for myoplasmic and lumenal free Ca2+ and Ca2+-binding molecules in the bulk of the cell.

Endoplasmic Reticulum Lumenal Indicators in Drosophila Reveal Effects of HSP-Related Mutations on Endoplasmic Reticulum Calcium Dynamics.

Genes for endoplasmic reticulum (ER)-shaping proteins are among the most commonly mutated in hereditary spastic paraplegia (HSP). Mutation of these genes in model organisms can lead to disruption of the ER network. To investigate how the physiological roles of the ER might be affected by such disruption, we developed tools to interrogate its Ca2+ signaling function. We generated GAL4-driven Ca2+ sensors targeted to the ER lumen, to record ER Ca2+ fluxes in identified Drosophila neurons. Using GAL4 lines specific for Type Ib or Type Is larval motor neurons, we compared the responses of different lumenal indicators to electrical stimulation, in axons and presynaptic terminals. The most effective sensor, ER-GCaMP6-210, had a Ca2+ affinity close to the expected ER lumenal concentration. Repetitive nerve stimulation generally showed a transient increase of lumenal Ca2+ in both the axon and presynaptic terminals. Mutants lacking neuronal reticulon and REEP proteins, homologs of human HSP proteins, showed a larger ER lumenal evoked response compared to wild type; we propose mechanisms by which this phenotype could lead to neuronal dysfunction or degeneration. Our lines are useful additions to a Drosophila Ca2+ imaging toolkit, to explore the physiological roles of ER, and its pathophysiological roles in HSP and in axon degeneration more broadly.

Multiscale Modeling of Dyadic Structure-Function Relation in Ventricular Cardiac Myocytes

Cardiovascular disease is often related to defects of subcellular components in cardiac myocytes, specifically in the dyadic cleft, which include changes in cleft geometry and channel placement. Modeling of these pathological changes requires both spatially resolved cleft as well as whole cell level descriptions. We use a multiscale model to create dyadic structure-function relationships to explore the impact of molecular changes on whole cell electrophysiology and calcium cycling. This multiscale model incorporates stochastic simulation of individual L-type calcium channels and ryanodine receptor channels, spatially detailed concentration dynamics in dyadic clefts, rabbit membrane potential dynamics, and a system of partial differential equations for myoplasmic and lumenal free Ca2+ and Ca2+-binding molecules in the bulk of the cell. We found action potential duration, systolic, and diastolic [Ca2+] to respond most sensitively to changes in L-type calcium channel current. The ryanodine receptor channel cluster structure inside dyadic clefts was found to affect all biomarkers investigated. The shape of clusters observed in experiments by Jayasinghe et al. and channel density within the cluster (characterized by mean occupancy) showed the strongest correlation to the effects on biomarkers.

A pH-correctable, DNA-based fluorescent reporter for organellar calcium.

It is extremely challenging to quantitate lumenal Ca2+ in acidic Ca2+ stores of the cell because all Ca2+ indicators are pH sensitive, and Ca2+ transport is coupled to pH in acidic organelles. We have developed a fluorescent DNA-based reporter, CalipHluor, that is targetable to specific organelles. By ratiometrically reporting lumenal pH and Ca2+ simultaneously, CalipHluor functions as a pH-correctable Ca2+ reporter. By targeting CalipHluor to the endolysosomal pathway, we mapped lumenal Ca2+ changes during endosomal maturation and found a surge in lumenal Ca2+ specifically in lysosomes. Using lysosomal proteomics and genetic analysis, we found that catp-6, a Caenorhabditis elegans homolog of ATP13A2, was responsible for lysosomal Ca2+ accumulation-an example of a lysosome-specific Ca2+ importer in animals. By enabling the facile quantification of compartmentalized Ca2+, CalipHluor can expand the understanding of subcellular Ca2+ importers.

How Does Calcium Overload Generate Calcium Waves in Heart?

Calcium-induced calcium (Ca2+) release (CICR) is the fundamental signaling mechanism that enables the voltage-sensitive L-type Ca2+ channels (LCCs) in the sarcolemma to activate the cellular [Ca2+]i transient and thus contraction in the heart. In CICR the LCC Ca2+ influx activates nearby Ca2+ release channels (ryanodine receptors, RyR2s) across a 15 nm “subspace” gap via CICR, just as the Ca2+ released from any open RyR2 can activate other RyR2s located within the same RyR2 cluster. The junctional ensemble of LCCs and the opposing RyR2s constitute the cardiac “Ca2+ release unit” or CRU. When a CRU is activated by any means it produces a Ca2+ spark. Excitation-contraction (EC) coupling is the process by which CRUs are activated quasi-synchronously by the voltage-activated LCCs in a cell during the action potential (AP). Importantly, CICR also occurs independently when the probabilistic opening of RyR2s produce a Ca2+ spark in the absence of an LCC trigger. Ca2+ sparks and individual RyR2 openings constitute the endogenous Ca2+ leak pathway from the sarcoplasmic reticulum (SR), the “cellular Ca2+ store”. However, in pathological conditions the Ca2+ leak can become unstable and produce cell-wide Ca2+ waves and life-threatening Ca2+-dependent arrhythmias. During SR Ca2+ overload, it has been suggested that a putatively novel phenomena dubbed “Store Overload Induced Ca2+ Release” or “SOICR”, that is distinct from CICR, may develop. SOICR's distinctiveness hinges on the molecular ability of SR lumenal Ca2+ to directly activate RyR2 openings without the need for elevated Ca2+ in the subspace to produce CICR. To investigate this hypothesis, we extend our existing model of RyR2 gating to include a formulation for SOICR whereby SR Ca2+ can directly activate a RyR2 channel to open. Using a multi-scale modeling approach that spans from single channel to whole-cell and spatial simulations, we show that both CICR and SOICR gating modes can indeed activate RyR2 channels and modify Ca2+ spark dynamics in a manner consistent with experimental observations. However, detailed comparison of Ca2+ wave generation and CRU-to-CRU Ca2+ wave propagation shows that CICR alone is a sufficient, and necessary, mechanism to explain Ca2+ release in heart under both physiological and pathological conditions.

A multiscale computational model of spatially resolved calcium cycling in cardiac myocytes: from detailed cleft dynamics to the whole cell concentration profiles.

Mathematical modeling of excitation-contraction coupling (ECC) in ventricular cardiac myocytes is a multiscale problem, and it is therefore difficult to develop spatially detailed simulation tools. ECC involves gradients on the length scale of 100 nm in dyadic spaces and concentration profiles along the 100 μm of the whole cell, as well as the sub-millisecond time scale of local concentration changes and the change of lumenal Ca2+ content within tens of seconds. Our concept for a multiscale mathematical model of Ca2+ -induced Ca2+ release (CICR) and whole cardiomyocyte electrophysiology incorporates stochastic simulation of individual LC- and RyR-channels, spatially detailed concentration dynamics in dyadic clefts, rabbit membrane potential dynamics, and a system of partial differential equations for myoplasmic and lumenal free Ca2+ and Ca2+-binding molecules in the bulk of the cell. We developed a novel computational approach to resolve the concentration gradients from dyadic space to cell level by using a quasistatic approximation within the dyad and finite element methods for integrating the partial differential equations. We show whole cell Ca2+-concentration profiles using three previously published RyR-channel Markov schemes.

Ca2+/H+ exchange, lumenal Ca2+ release and Ca2+/ATP coupling ratios in the sarcoplasmic reticulum ATPase

The Ca2+ transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca2+ in intracellular membrane bound compartments, thereby lowering cytosolic Ca2+ to induce relaxation. The stored Ca2+ is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca2+, whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca2+. We review here biochemical and biophysical evidence demonstrating that release of bound Ca2+ into the lumen of SR requires Ca2+/H+ exchange at the low affinity Ca2+ sites. Rise of lumenal Ca2+ above its dissociation constant from low affinity sites, or reduction of the H+ concentration by high pH, prevent Ca2+/H+ exchange. Under these conditions Ca2+ release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca2+pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.

Altered expression and splicing of Ca2+ metabolism genes in myotonic dystrophies DM1 and DM2

Myotonic dystrophy types 1 and 2 (DM1 and DM2) are multisystem disorders caused by similar repeat expansion mutations, with similar yet distinct clinical features. Aberrant splicing of multiple effector genes, as well as dysregulation of transcription and translation, has been suggested to underlie different aspects of the complex phenotypes in DM1 and DM2. Ca(2+) plays a central role in both muscle contraction and control of gene expression, and recent expression profiling studies have indicated major perturbations of the Ca(2+) signalling pathways in DM. Here we have further investigated the expression of genes and proteins involved in Ca(2+) metabolism in DM patients, including Ca(2+) channels and Ca(2+) binding proteins. We used patient muscle biopsies to analyse mRNA expression and splicing of genes by microarray expression profiling and RT-PCR. We studied protein expression by immunohistochemistry and immunoblotting. Most of the genes studied showed mRNA up-regulation in expression profiling. When analysed by immunohistochemistry the Ca(2+) release channel ryanodine receptor was reduced in DM1 and DM2, as was calsequestrin 2, a sarcoplasmic reticulum lumen Ca(2+) storage protein. Abnormal splicing of ATP2A1 was more pronounced in DM2 than DM1. We observed abnormal mRNA and protein expression in DM affecting several proteins involved in Ca(2+) metabolism, with some differences between DM1 and DM2. Our protein expression studies are suggestive of a post-transcriptional defect(s) in the myotonic dystrophies.

Ca2+ Release to Lumen from ADP-sensitive Phosphoenzyme E1PCa2 without Bound K+ of Sarcoplasmic Reticulum Ca2+-ATPase

During Ca(2+) transport by sarcoplasmic reticulum Ca(2+)-ATPase, the conformation change of ADP-sensitive phosphoenzyme (E1PCa(2)) to ADP-insensitive phosphoenzyme (E2PCa(2)) is followed by rapid Ca(2+) release into the lumen. Here, we find that in the absence of K(+), Ca(2+) release occurs considerably faster than E1PCa(2) to E2PCa(2) conformation change. Therefore, the lumenal Ca(2+) release pathway is open to some extent in the K(+)-free E1PCa(2) structure. The Ca(2+) affinity of this E1P is as high as that of the unphosphorylated ATPase (E1), indicating the Ca(2+) binding sites are not disrupted. Thus, bound K(+) stabilizes the E1PCa(2) structure with occluded Ca(2+), keeping the Ca(2+) pathway to the lumen closed. We found previously (Yamasaki, K., Wang, G., Daiho, T., Danko, S., and Suzuki, H. (2008) J. Biol. Chem. 283, 29144-29155) that the K(+) bound in E2P reduces the Ca(2+) affinity essential for achieving the high physiological Ca(2+) gradient and to fully open the lumenal Ca(2+) gate for rapid Ca(2+) release (E2PCa(2) → E2P + 2Ca(2+)). These findings show that bound K(+) is critical for stabilizing both E1PCa(2) and E2P structures, thereby contributing to the structural changes that efficiently couple phosphoenzyme processing and Ca(2+) handling.

Stable Structural Analog of Ca2+-ATPase ADP-insensitive Phosphoenzyme with Occluded Ca2+ Formed by Elongation of A-domain/M1′-linker and Beryllium Fluoride Binding

We have developed a stable analog for the ADP-insensitive phosphoenzyme intermediate with two occluded Ca(2+) at the transport sites (E2PCa(2)) of sarcoplasmic reticulum Ca(2+)-ATPase. This is normally a transient intermediate state during phosphoenzyme isomerization from the ADP-sensitive to ADP-insensitive form and Ca(2+) deocclusion/release to the lumen; E1PCa(2) --> E2PCa(2) --> E2P + 2Ca(2+). Stabilization was achieved by elongation of the Glu(40)-Ser(48) loop linking the Actuator domain and M1 (1st transmembrane helix) with four glycine insertions at Gly(46)/Lys(47) and by binding of beryllium fluoride (BeF(x)) to the phosphorylation site of the Ca(2+)-bound ATPase (E1Ca(2)). The complex E2Ca(2)xBeF(3)(-) was also produced by lumenal Ca(2+) binding to E2xBeF(3)(-) (E2P ground state analog) of the elongated linker mutant. The complex was stable for at least 1 week at 25 degrees C. Only BeF(x), but not AlF(x) or MgF(x), produced the E2PCa(2) structural analog. Complex formation required binding of Mg(2+), Mn(2+), or Ca(2+) at the catalytic Mg(2+) site. Results reveal that the phosphorylation product E1PCa(2) and the E2P ground state (but not the transition states) become competent to produce the E2PCa(2) transient state during forward and reverse phosphoenzyme isomerization. Thus, isomerization and lumenal Ca(2+) release processes are strictly coupled with the formation of the acylphosphate covalent bond at the catalytic site. Results also demonstrate the critical structural roles of the Glu(40)-Ser(48) linker and of Mg(2+) at the catalytic site in these processes.

Effects of Divalent Current Carriers on Voltage-Dependence of RyR2 Channels

Voltage-dependent modulation of cardiac ryanodine receptors (RyR2) was studied in planar lipid bilayers. Different earth alkaline cations (M2+: Mg2+, Ca2+, Sr2+, Ba2+) were utilized as current carriers. When added to the cytosolic surface of the channels, Ca2+ (EC50 ∼2μM) or Sr2+ (EC50 ∼20μM) interacted with cytosolic high affinity (HA) sites and activated the channels. Neither Mg2+ nor Ba2+ activated RyR2 but only Mg2+ interfered with Ca2+/Sr2+ action. Fully activated RyR2 (100-200μM cytosolic Ca2+) were inhibited with low affinity (LA) by all M2+ (similar IC50, ranging 3.6-5.7mM) suggesting that LA inhibitory sites do not distinguish M2+ identity. We found that RyR2 were much more active with lumenal Ca2+/Sr2+ versus lumenal Ba2+/Mg2+. However increasing lumenal→cytosolic M2+ flux by increasing lumenal holding voltage (Vm) never increased open probability (Po). In contrast, increasing Vm decreased Po in half of the RyR2 exposed to lumenal Ca2+/Sr2+ and in all RyR2 exposed to lumenal Ba2+/Mg2. This suggests that lumenal flux does not reach HA but LA sites. An effect of Vm is evident in all channels displaying modal gating (low and high Po mode) but not in RyR2 where high Po dominates. Indeed, high Po mode is much more abundant with lumenal Ca2+/Sr2+. Moreover, increasing cytosolic Ca2+ activated all channels and removed the effect of Vm. In summary, modulation of RyR2 gating by M2+ flux is complex and seems to reflect lumenal M2+-dependent stabilization of high Po and low Po mode (the latter being voltage-dependent). Supported by NIH R01 GM078665 to JAC.

Formation of the Stable Structural Analog of ADP-sensitive Phosphoenzyme of Ca2+-ATPase with Occluded Ca2+ by Beryllium Fluoride: STRUCTURAL CHANGES DURING PHOSPHORYLATION AND ISOMERIZATION

As a stable analog for ADP-sensitive phosphorylated intermediate of sarcoplasmic reticulum Ca(2+)-ATPase E1PCa(2).Mg, a complex of E1Ca(2).BeF(x), was successfully developed by addition of beryllium fluoride and Mg(2+) to the Ca(2+)-bound state, E1Ca(2). In E1Ca(2).BeF(x), most probably E1Ca(2).BeF(3)(-), two Ca(2+) are occluded at high affinity transport sites, its formation required Mg(2+) binding at the catalytic site, and ADP decomposed it to E1Ca(2), as in E1PCa(2).Mg. Organization of cytoplasmic domains in E1Ca(2).BeF(x) was revealed to be intermediate between those in E1Ca(2).AlF(4)(-) ADP (transition state of E1PCa(2) formation) and E2.BeF(3)(-).(ADP-insensitive phosphorylated intermediate E2P.Mg). Trinitrophenyl-AMP (TNP-AMP) formed a very fluorescent (superfluorescent) complex with E1Ca(2).BeF(x) in contrast to no superfluorescence of TNP-AMP bound to E1Ca(2).AlF(x). E1Ca(2).BeF(x) with bound TNP-AMP slowly decayed to E1Ca(2), being distinct from the superfluorescent complex of TNP-AMP with E2.BeF(3)(-), which was stable. Tryptophan fluorescence revealed that the transmembrane structure of E1Ca(2).BeF(x) mimics E1PCa(2).Mg, and between those of E1Ca(2).AlF(4)(-).ADP and E2.BeF(3)(-). E1Ca(2).BeF(x) at low 50-100 microm Ca(2+) was converted slowly to E2.BeF(3)(-) releasing Ca(2+), mimicking E1PCa(2).Mg --> E2P.Mg + 2Ca(2+). Ca(2+) replacement of Mg(2+) at the catalytic site at approximately millimolar high Ca(2+) decomposed E1Ca(2).BeF(x) to E1Ca(2). Notably, E1Ca(2).BeF(x) was perfectly stabilized for at least 12 days by 0.7 mm lumenal Ca(2+) with 15 mm Mg(2+). Also, stable E1Ca(2).BeF(x) was produced from E2.BeF(3)(-) at 0.7 mm lumenal Ca(2+) by binding two Ca(2+) to lumenally oriented low affinity transport sites, as mimicking the reverse conversion E2P. Mg + 2Ca(2+) --> E1PCa(2).Mg.

Silencing genes of sarcoplasmic reticulum proteins clarifies their roles in excitation–contraction coupling

Silencing genes of sarcoplasmic reticulum proteins clarifies their roles in excitation–contraction coupling

Knockdown of stromal interaction molecule 1 attenuates store-operated Ca2+ entry and Ca2+ responses to acute hypoxia in pulmonary arterial smooth muscle

Stromal interaction molecule 1 (STIM1) is a recently discovered membrane-spanning protein thought to sense lumenal Ca(2+) in sarco(endo)plasmic reticulum (SR/ER) and transduce activation of Ca(2+)-permeable store-operated channels (SOC) in plasmalemma in response to SR/ER Ca(2+) depletion. To evaluate the role of STIM1 and a closely related protein, STIM2, in Ca(2+) signaling of rat distal pulmonary arterial smooth muscle cells (PASMC) during hypoxia, we used fluorescent microscopy and the Ca(2+)-sensitive dye, fura 2, to measure basal intracellular Ca(2+) concentration ([Ca(2+)](i)), store-operated Ca(2+) entry (SOCE), and increases in [Ca(2+)](i) caused by acute hypoxia (4% O(2)) or depolarization (60 mmol/l KCl) in cells treated with small interfering RNA targeted to STIM1 (siSTIM1) or STIM2 (siSTIM2). As determined by real-time quantitative PCR analysis (qPCR), STIM1 mRNA was 200-fold more abundant than STIM2 in untreated control PASMC. siSTIM1 and siSTIM2 caused specific and significant knockdown of both mRNA measured by qPCR and protein measured by Western blotting. siSTIM1 markedly inhibited SOCE and abolished the sustained [Ca(2+)](i) response to hypoxia but did not alter the initial transient [Ca(2+)](i) response to hypoxia, the [Ca(2+)](i) response to depolarization, or basal [Ca(2+)](i). The only effect of siSTIM2 was a smaller inhibition of SOCE. We conclude that STIM1 was quantitatively more important than STIM2 in activation of SOC in rat distal PASMC and that the increase in [Ca(2+)](i) induced by acute hypoxia in these cells required SR Ca(2+) release and STIM1-dependent activation of SOC.

Expression of the P2X7 Receptor Increases the Ca2+ Content of the Endoplasmic Reticulum, Activates NFATc1, and Protects from Apoptosis

The P2X(7) receptor is known for the cytotoxic activity because of its ability to cause opening of non-selective pores in the plasma membrane and activate apoptotic caspases. A key factor of P2X(7)-dependent cytotoxicity is the massive intracellular Ca(2+) increase triggered by its activation. Here we show that P2X(7) transfection increased the ability of the endoplasmic reticulum to accumulate, store, and release Ca(2+). This caused a larger agonist-stimulated increase in cytosol and mitochondrial Ca(2+) in P2X(7) transfectants than in mock transfected cells. P2X(7) transfectants survived and even proliferated in serum-free conditions and were resistant to apoptosis triggered by ceramide, staurosporin, or intracellular Zn(2+) chelation. Finally, the nuclear factor of activated T cells complex 1 (NFATc1) was strongly activated in the P2X(7) transfectants. These observations support our previous finding that the P2X(7) receptor under tonic conditions of stimulation, i.e. those observed in response to basal ATP release, has an anti-apoptotic or even growth promoting rather than cytotoxic activity.

Ryanodine Receptor Structure: Progress and Challenges

Ryanodine Receptor Structure: Progress and Challenges

Roles of Tyr122-hydrophobic Cluster and K+ Binding in Ca2+-releasing Process of ADP-insensitive Phosphoenzyme of Sarcoplasmic Reticulum Ca2+-ATPase

Tyr(122)-hydrophobic cluster (Y122-HC) is an interaction network formed by the top part of the second transmembrane helix and the cytoplasmic actuator and phosphorylation domains of sarcoplasmic reticulum Ca(2+)-ATPase. We have previously found that Y122-HC plays critical roles in the processing of ADP-insensitive phosphoenzyme (E2P) after its formation by the isomerization from ADP-sensitive phosphoenzyme (E1PCa(2)) (Wang, G., Yamasaki, K., Daiho, T., and Suzuki, H. (2005) J. Biol. Chem. 280, 26508-26516). Here, we further explored kinetic properties of the alanine-substitution mutants of Y122-HC to examine roles of Y122-HC for Ca(2+) release process in E2P. In the steady state, the amount of E2P decreased so that of E1PCa(2) increased with increasing lumenal Ca(2+) concentration in the mutants with K(0.5) 110-320 microm at pH 7.3. These lumenal Ca(2+) affinities in E2P agreed with those estimated from the forward and lumenal Ca(2+)-induced reverse kinetics of the E1PCa(2)-E2P isomerization. K(0.5) of the wild type in the kinetics was estimated to be 1.5 mM. Thus, E2P of the mutants possesses significantly higher affinities for lumenal Ca(2+) than that of the wild type. The kinetics further indicated that the rates of lumenal Ca(2+) access and binding to the transport sites of E2P were substantially slowed by the mutations. Therefore, the proper formation of Y122-HC and resulting compactly organized structure are critical for both decreasing Ca(2+) affinity and opening the lumenal gate, thus for Ca(2+) release from E2PCa(2). Interestingly, when K(+) was omitted from the medium of the wild type, the properties of the wild type became similar to those of Y122-HC mutants. K(+) binding likely functions via producing the compactly organized structure, in this sense, similarly to Y122-HC.

Calcium, calcium-sensing receptor and colon cancer

There is much evidence that dietary Ca 2+ loading reduces colon cell proliferation and carcinogenesis in humans and rodents, but during carcinogenesis it becomes ineffective or even tumor-promoting. We are beginning to see how Ca 2+ balances the continuous massive cell production in colon crypts by driving the terminal differentiation and eventually the apoptosis of the cells mainly on the mucosal surface, and how this Ca 2+ control is lost during colon carcinogenesis. The rapid proliferation of the transit-amplifying (TA) progeny of the colon stem cells is driven by the so-called “Wnt” signaling mechanism, which involves the stimulation of proliferogenic genes such as those for c-Myc and cyclin D1 and the silencing of the gene for the cell cycle-stopping p21 Cip1/WAF1 protein by nuclear β-catenin • Tcf-4 complexes. TA cells avoid mitotic damage and premature apoptosis by expressing the protein survivin. It appears that TA cell cycling stops and terminal differentiation starts when the cells reach a higher level in the crypt where there is enough lumenal Ca 2+ to stimulate the expression and activation of CaSRs (Ca 2+-sensing receptors), the signals from which stimulate the expression of E-cadherin. Along with this, the APC (adenomatous polyposis coli) protein appears and some of it enters the nucleus. There it makes the TA cells susceptible to the eventual apoptotic balancing by stopping survivin expression and the β-catenin • Tcf-4 complex from driving further cell cycling by releasing β-catenin from the nucleus, and delivering it to cytoplasmic APC • axin • GSK-3β complexes for ultimate proteasomal destruction. Cytoplasmic β-catenin is then prevented from returning to the nucleus by either being intercepted and destroyed by APC • axin • GSK-3β complexes or locked by the emerging E-cadherin into membrane adherens junctions which tie the cell into the sheet of proliferatively shut-down cells with APC-dependent cytoskeletons moving to the mouth of the crypt and onto the flat mucosal surface. A common first step in sporadic colon carcinogenesis is the loss of functional APC which disorients upwardly directed migration and causes the retention of nuclear β-catenin and proliferogenic β-catenin • Tcf-4 complexes as well as genomic instability. Eventually the balance between cell proliferation and terminal differentiation and death is radically tipped in favour of proliferation by the appearance of apoptosis-resistant, survivin-expressing clones of Ca 2+-insensitve cells which are locked into the proliferative, mutation-prone mode because of CaSR-disabling gene mutations which prevent the stimulation of E-cadherin expression and terminal differentiation.

A calcium-induced calcium release mechanism mediated by calsequestrin

Calcium ( Ca 2 + )-induced Ca 2 + release (CICR) is widely accepted as the principal mechanism linking electrical excitation and mechanical contraction in cardiac cells. The CICR mechanism has been understood mainly based on binding of cytosolic Ca 2 + with ryanodine receptors (RyRs) and inducing Ca 2 + release from the sarcoplasmic reticulum (SR). However, recent experiments suggest that SR lumenal Ca 2 + may also participate in regulating RyR gating through calsequestrin (CSQ), the SR lumenal Ca 2 + buffer. We investigate how SR Ca 2 + release via RyR is regulated by Ca 2 + and calsequestrin (CSQ). First, a mathematical model of RyR kinetics is derived based on experimental evidence. We assume that the RyR has three binding sites, two cytosolic sites for Ca 2 + activation and inactivation, and one SR lumenal site for CSQ binding. The open probability ( P o ) of the RyR is found by simulation under controlled cytosolic and SR lumenal Ca 2 + . Both peak and steady-state P o effectively increase as SR lumenal Ca 2 + increases. Second, we incorporate the RyR model into a CICR model that has both a diadic space and the junctional SR (jSR). At low jSR Ca 2 + loads, CSQs are more likely to bind with the RyR and act to inhibit jSR Ca 2 + release, while at high SR loads CSQs are more likely to detach from the RyR, thereby increasing jSR Ca 2 + release. Furthermore, this CICR model produces a nonlinear relationship between fractional jSR Ca 2 + release and jSR load. These findings agree with experimental observations in lipid bilayers and cardiac myocytes.

Halothane modulation of skeletal muscle ryanodine receptors: dependence on Ca2+, Mg2+, and ATP

Malignant hyperthermia (MH) susceptibility is a genetic disorder of skeletal muscle associated with mutations in the ryanodine receptor isoform 1 (RyR1) of sarcoplasmic reticulum (SR). In MH-susceptible skeletal fibers, RyR1-mediated Ca(2+) release is highly sensitive to activation by the volatile anesthetic halothane. Indeed, studies with isolated RyR1 channels (using simple Cs(+) solutions) found that halothane selectively affects mutated but not wild-type RyR1 function. However, studies in skeletal fibers indicate that halothane can also activate wild-type RyR1-mediated Ca(2+) release. We hypothesized that endogenous RyR1 agonists (ATP, lumenal Ca(2+)) may increase RyR1 sensitivity to halothane. Consequently, we studied how these agonists affect halothane action on rabbit skeletal RyR1 reconstituted into planar lipid bilayers. We found that cytosolic ATP is required for halothane-induced activation of the skeletal RyR1. Unlike RyR1, cardiac RyR2 (much less sensitive to ATP) responded to halothane even in the absence of this agonist. ATP-dependent halothane activation of RyR1 was enhanced by cytosolic Ca(2+) (channel agonist) and counteracted by Mg(2+) (channel inhibitor). Dantrolene, a muscle relaxant used to treat MH episodes, did not affect RyR1 or RyR2 basal activity and did not interfere with halothane-induced activation. Studies with skeletal SR microsomes confirmed that halothane-induced RyR1-mediated SR Ca(2+) release is enhanced by high ATP-low Mg(2+) in the cytosol and by increased SR Ca(2+) load. Thus, physiological or pathological processes that induce changes in cellular levels of these modulators could affect RyR1 sensitivity to halothane in skeletal fibers, including the outcome of halothane-induced contracture tests used to diagnose MH susceptibility.