Calcium-sensitive Photoprotein Aequorin Research Articles

Identification of Chemical and Pharmacological Chaperones for Correction of Trafficking-Deficient Mutant Cyclic Nucleotide-Gated A3 Channels.

Trafficking deficiency caused by missense mutations is a well known phenomenon that occurs for mutant, misfolded proteins. Typically, the misfolded protein is retained by the protein quality-control system and degraded by the endoplasmic reticulum-associated protein degradation pathway and thus does not reach its destination, although residual function of the protein may be preserved. Chemical and pharmacological chaperones can improve the targeting of trafficking-deficient proteins and thus may be promising candidates for therapeutic applications. Here, we report the application of a cellular bioassay based on the bioluminescent calcium reporter aequorin to quantify surface expression of mutant CNGA3 channels associated with the autosomal recessively inherited retinal disease achromatopsia. A screening of 77 compounds enabled the identification of effective chemical and pharmacological chaperones that result in a 1.5- to 4.8-fold increase of surface expression of mutant CNGA3. Using selected compounds, we confirmed that the rescue of the defective trafficking is not limited to a single mutation in CNGA3. Active compounds and our structure-activity correlated data for the dihydropyridine compound class may provide valuable information for developing a treatment of the trafficking defect in achromatopsia. SIGNIFICANCE STATEMENT: This study describes a novel luminescence-based assay to detect the surface expression of mutant trafficking-deficient CNGA3 channels based on the calcium-sensitive photoprotein aequorin. Using this assay for a compound screening, this study identifies novel chemical and pharmacological chaperones that restore the surface localization of mutant trafficking-deficient CNGA3 channels. The results from this work may serve as starting point for the development of potent compounds that rescue trafficking deficiencies in the autosomal recessively inherited retinal disease achromatopsia.

The Claudin Family Protein FigA Mediates Ca2+ Homeostasis in Response to Extracellular Stimuli in Aspergillus nidulans and Aspergillus fumigatus.

The claudin family protein Fig1 is a unique fungal protein that is involved in pheromone-induced calcium influx and membrane fusion during the mating of Saccharomyces cerevisiae and Candida albicans. Whether and how Fig1 regulates Ca2+ homeostasis in response to extracellular stimuli is poorly understood. Previously, we found Aspergillus nidulans FigA, a homolog of Fig1 in S. cerevisiae, similar to the high-affinity calcium uptake system, is required for normal growth under low-Ca2+ minimal medium. In this study, using the calcium-sensitive photoprotein aequorin to monitor cytosolic free calcium concentration ([Ca2+]c) in living cells, we found that the FigA dysfunction decreases the transient [Ca2+]c induced by a high extracellular calcium stress. Furthermore, FigA acts synergistically with CchA (a high-affinity Ca2+ channel) to coordinate cytoplasmic Ca2+ influx in response to an extracellular Ca2+ stimulus. Moreover, FigA mediates ER stress-induced transient [Ca2+]c in the presence or absence of extracellular calcium. Most importantly, these [Ca2+]c responses mediated by FigA are closely related to its conserved claudin superfamily motif, which is also required for hyphal growth and asexual development in A. nidulans. Finally, the function of FigA in Aspergillus fumigatus, the most common airborne human fungal pathogen was studied. The result showed that the two FigA homologous in A. nidulans and A. fumigatus have a large degree of functional homology not only in asexual development but also in regulating transient [Ca2+]c. Our study expands the knowledge of claudin family protein FigA in Ca2+ homeostasis in response to extracellular stimuli.

The Lectin Chaperone Calnexin Is Involved in the Endoplasmic Reticulum Stress Response by Regulating Ca2+ Homeostasis in Aspergillus nidulans.

The Ca2+-mediated signaling pathway is crucial for environmental adaptation in fungi. Here we show that calnexin, a molecular chaperone located in the endoplasmic reticulum (ER), plays an important role in regulating the cytosolic free calcium concentration ([Ca2+]c) in Aspergillus nidulans Inactivation of calnexin (ClxA) in A. nidulans caused severe defects in hyphal growth and conidiation under ER stress caused by the ER stress-inducing agent dithiothreitol (DTT) or high temperature. Importantly, defects in the ΔclxA mutant were restored by the addition of extracellular calcium. Furthermore, the CchA/MidA complex (the high-affinity Ca2+ channels), calcineurin (calcium/calmodulin-dependent protein phosphatase), and PmrA (secretory pathway Ca2+ ATPase) were required for extracellular calcium-based restoration of the DTT/thermal stress sensitivity in the ΔclxA mutant. Interestingly, the ΔclxA mutant exhibited markedly reduced conidium formation and hyphal growth defects under the low-calcium condition, which is similar to defects caused by mutations in MidA/CchA. Moreover, the phenotypic defects were further exacerbated in the ΔclxA ΔmidA ΔcchA mutant, which suggested that ClxA and MidA/CchA are both required under the calcium-limiting condition. Using the calcium-sensitive photoprotein aequorin to monitor [Ca2+]c in living cells, we found that ClxA and MidA/CchA complex synergistically coordinate transient increase in [Ca2+]c in response to extracellular calcium. Moreover, ClxA, in particular its luminal domain, plays a role in mediating the transient [Ca2+]c in response to DTT-induced ER stress in the absence of extracellular calcium, indicating ClxA may mediate calcium release from internal calcium stores. Our findings provide new insights into the role of calnexin in the regulation of calcium-mediated response in fungal ER stress adaptation.IMPORTANCE Calnexin is a well-known molecular chaperone conserved from yeast to humans. Although it contains calcium binding domains, little is known about the role of calnexin in Ca2+ regulation. In this study, we demonstrate that calnexin (ClxA) in the filamentous fungus Aspergillus nidulans, similar to the high-affinity calcium uptake system (HACS), is required for normal growth and conidiation under the calcium-limiting condition. The ClxA dysfunction decreases the transient cytosolic free calcium concentration ([Ca2+]c) induced by a high extracellular calcium or DTT-induced ER stress. Our findings provide the direct evidence that calnexin plays important roles in regulating Ca2+ homeostasis in addition to its role as a molecular chaperone in fungi. These results provide new insights into the roles of calnexin and expand knowledge of fungal stress adaptation.

A putative mitochondrial calcium uniporter in A. fumigatus contributes to mitochondrial Ca2+ homeostasis and stress responses

Ca2+ uptake into mitochondria plays a central role in cell physiology by stimulating ATP production, shaping cytosolic Ca2+ transients and regulating cell survival or death. Although this system has been studied extensively in mammalian cells, the physiological implications of Ca2+ uptake into mitochondria in fungal cells are still unknown. In this study, a bi-directional best-hit BLASTP search revealed that the genome of Aspergillus fumigatus encodes a homolog of a putative mitochondrial Ca2+ uniporter (MCU) and a mitochondrial carrier protein AGC1/MICU1 homolog. Both putative homologs are mitochondrially localized and required for the response to azole and oxidative stress such that the loss of either McuA or AgcA results in reduced susceptibility to azole and oxidative stress, suggesting a role in environmental stress adaptation. Overexpressing mcuA restores the azole-resistance phenotype of the ΔagcA strain to wild-type levels, but not vice versa, indicating McuA plays a dominant role during these stress responses. Using a mitochondrially targeted version of the calcium-sensitive photoprotein aequorin, we found that only mcuA deletion leads to dysfunctional [Ca2+]mt and [Ca2+]c homeostasis, suggesting that McuA, but not AgcA, contributes to Ca2+ uptake into mitochondria. Further point-mutation experiments combined with extracellular Ca2+ chelator treatment verified that two predicted Ca2+-binding sites in McuA are required for Ca2+ uptake into mitochondria and stress responses through the regulation of [Ca2+]c homeostasis.

Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca2+ Homeostasis in Aspergillus nidulans.

The eukaryotic calcium/calmodulin-dependent protein phosphatase calcineurin is crucial for the environmental adaption of fungi. However, the mechanism of coordinate regulation of the response to salt stress by calcineurin and the high-affinity calcium channel CchA in fungi is not well understood. Here we show that the deletion of cchA suppresses the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans Additionally, the hypersensitivity of the ΔcnaA strain to extracellular calcium and cell-wall-damaging agents can be suppressed by cchA deletion. Using the calcium-sensitive photoprotein aequorin to monitor the cytoplasmic Ca(2+) concentration ([Ca(2+)]c) in living cells, we found that calcineurin negatively regulates CchA on calcium uptake in response to external calcium in normally cultured cells. However, in salt-stress-pretreated cells, loss of either cnaA or cchA significantly decreased the [Ca(2+)]c, but a deficiency in both cnaA and cchA switches the [Ca(2+)]c to the reference strain level, indicating that calcineurin and CchA synergistically coordinate calcium influx under salt stress. Moreover, real-time PCR results showed that the dysfunction of cchA in the ΔcnaA strain dramatically restored the expression of enaA (a major determinant for sodium detoxification), which was abolished in the ΔcnaA strain under salt stress. These results suggest that double deficiencies of cnaA and cchA could bypass the requirement of calcineurin to induce enaA expression under salt stress. Finally, YvcA, a member of the transient receptor potential channel (TRPC) protein family of vacuolar Ca(2+) channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption.IMPORTANCE The feedback inhibition relationship between calcineurin and the calcium channel Cch1/Mid1 has been well recognized from yeast. Interestingly, our previous study (S. Wang et al., PLoS One 7:e46564, 2012, http://dx.doi.org/10.1371/journal.pone.0046564) showed that the deletion of cchA could suppress the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans In this study, our findings suggest that fungi are able to develop a unique mechanism for adapting to environmental salt stress. Compared to cells cultured normally, the NaCl-pretreated cells had a remarkable increase in transient [Ca(2+)]c Furthermore, we show that calcineurin and CchA are required to modulate cellular calcium levels and synergistically coordinate calcium influx under salt stress. Finally, YvcA, a member of of the TRPC family of vacuolar Ca(2+) channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption. The findings in this study provide insights into the complex regulatory links between calcineurin and CchA to maintain cytoplasmic Ca(2+) homeostasis in response to different environments.

Cold-induced cytosolic free calcium ion concentration changes in wheat

Relatively little is known about changes in the cytosolic free calcium ion concentration ([Ca(2+)](c)) in monocotyledonous plants. Therefore, we produced transgenic winter wheat lines stably expressing the calcium-sensitive photoprotein aequorin constitutively in the cytosol. [Ca(2+)](c) was detected in vivo by luminometry, and [Ca(2+)](c) elevations were imaged at video rate. Experiments with the transgenic seedlings focused on potential changes in [Ca(2+)](c) during cold exposure. Temperature-induced changes in [Ca(2+)](c) were found to be more dependent on the change in temperature (dT dt(-1)) than on the absolute value of temperature. [Ca(2+)](c) increased only at cooling rates higher than 8 degrees Cmin(-1), indicating that an overall cellular [Ca(2+)](c) increase is of minor relevance as a signal for cold acclimation in wheat under ecological conditions. The results are discussed with regard to the so-called 'calcium signature hypothesis'.

Characterization of the 5-HT2b Receptor in Evaluation of Aequorin Detection of Calcium Mobilization for Miniaturized GPCR High-Throughput Screening

Fluorescent detection of calcium mobilization has been used successfully to identify modulators of G-protein-coupled receptors (GPCRs); however, inherent issues with fluorescence may limit its potential for high-throughput screening miniaturization. The data presented here demonstrate that the calcium-sensitive photoprotein aequorin (AequoScreen), when compared with FLUO-4 in the same cellular background, allows for miniaturization of functional kinetic calcium flux assays, in which the rank order of potency and efficacy was maintained for a series of diverse small-molecule modulators. Small-volume (<10 microL) 384- and 1536-well aequorin assays were implemented by integration of acoustic dispensing (Echo 550) and kinetic flash luminometry (CyBi Lumax). The enhanced high signal-to-background ratios observed relative to fluorescence were readily manipulated by altering per-well cell densities and yielded acceptable screening statistics in miniaturized format for both agonist and antagonist screening scenarios. In addition, the authors demonstrate the feasibility of using agonist concentrations less than EC(50) in a miniaturized antagonist assay. These features, coupled with improved sample handling, should enhance sensitivity and provide the benefits of miniaturization including cost reduction and throughput gains.

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin: Catalytic properties for the oxidation of coelenterazine as an oxygenase

Blue fluorescent protein from the calcium-binding photoprotein aequorin (BFP-aq) is a complex of Ca 2+-bound apoaequorin and coelenteramide, and shows luminescence activity like a luciferase, catalyzing the oxidation of coelenterazine with molecular oxygen. To understand the catalytic properties of BFP-aq, various fluorescent proteins (FP-aq) have been prepared from semi-synthetic aequorin and characterized in comparison with BFP-aq. FP-aq has luciferase activity and could be regenerated into native aequorin by incubation with coelenterazine. The results from substrate specificity studies of FP-aq using various coelenterazine analogues have suggested that the oxidation of coelenterazine by BFP-aq in the luciferase reaction and the regeneration process to aequorin might involve the same catalytic site of BFP-aq.

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin is a heat resistant enzyme, catalyzing the oxidation of coelenterazine

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin (BFP-aq) was prepared and determined to be a heat resistant enzyme, catalyzing the luminescent oxidation of coelenterazine (luciferin) with molecular oxygen as a general luciferase. After treatment with excess ethylenediaminetetraacetic acid to remove Ca 2+ from BFP-aq, the blue fluorescence shifted to a greenish fluorescence. This greenish fluorescent protein (gFP-aq) was identified as a non-covalent complex of apoaequorin with coelenteramide (oxyluciferin) in a molar ratio of 1:1. By incubation with coelenterazine in the absence of reducing reagents, gFP-aq was converted to aequorin at 25 °C. BFP-aq and gFP-aq possessing both fluorescence and luminescence activities may work as novel reporter proteins.

Perception of environmental signals by a marine diatom.

Diatoms are a key component of marine ecosystems and are extremely important for the biogeochemical cycling of silica and as contributors to global fixed carbon. However, the answers to fundamental questions such as what diatoms can sense in their environment, how they respond to external signals, and what factors control their life strategies are largely unknown. We generated transgenic diatom cells containing the calcium-sensitive photoprotein aequorin to determine whether changes in calcium homeostasis are used to respond to relevant environmental stimuli. Our results reveal sensing systems for detecting and responding to fluid motion (shear stress), osmotic stress, and iron, a key nutrient that controls diatom abundance in the ocean.

Power-frequency electromagnetic fields and the capacitative calcium entry system in SV40-transformed Swiss 3T3 cells.

The present study was designed to test the hypothesis that a 60 Hz electromagnetic field could affect the influx of calcium ions across the plasma membrane through the so-called capacitative calcium entry system. Recordings of cytosolic calcium-ion concentrations in SV40-transformed Swiss 3T3 cells were obtained in real time during exposure to magnetic fields ranging from 0.3-50 mT or to sham conditions using the calcium-sensitive photoprotein aequorin. This was done for cell populations whose capacitative entry system was activated by either bradykinin or thapsigargin under a variety of experimental conditions. No effects of the magnetic field were observed on bradykinin-induced calcium transients and, with the exception of a small but statistically significant increase observed in experiments performed at 50 mT, no effects of the fields were observed on baseline calcium levels prior to or after such transients. The magnetic fields also had no effects on the size or kinetics of any of the thapsigargin-induced calcium transients. Overall, the data fail to support the hypothesis tested in this work.

Connexin-Aequorin Chimerae Report Cytoplasmic Calcium Environments along Trafficking Pathways Leading to Gap Junction Biogenesis in Living COS-7 Cells

The cytoplasmic calcium environments along membrane trafficking pathways leading to gap junction intercellular communication channels at the plasma membrane were studied. Connexins, the constitutive proteins of gap junctions, were fused at their carboxyl terminus to the calcium-sensitive photoprotein aequorin. The cellular location of the chimeric proteins was determined by immunolocalization and subcellular fractionation. The generation of functional gap junctions by the connexin chimerae was monitored by the ability of the cells to exchange small dyes. Although aequorin fused to connexin-26 was nonfunctional, its ability to report Ca2+ and to form functional gap junctions was rescued by replacement of its cytoplasmic carboxyl tail with that of connexin-43. In COS-7 cells expressing these connexin-aequorin chimerae, calcium levels below the plasma membrane were higher (approximately 5 microM) than those in the cytoplasm (approximately 100 nM); gap junctions were able to transfer dyes under these conditions. Cytoplasmic levels of free calcium surrounding the ERGIC/Golgi reported by connexin-43 chimera (approximately 420 nM) were twice those measured by connexin-32 chimera (approximately 200 nM); both chimerae measured calcium levels substantially higher than those reported by a connexin-26 chimera (approximately 130 nM). Dispersion of the ERGIC and Golgi complex by brefeldin A led to a marked reduction in calcium levels. The results show that the various connexin chimerae were located in spatially different subcellular stores and that the ERGIC/Golgi regions of the cell maintain heterogeneous cytoplasmic domains of calcium. The implications of the subplasma-membrane Ca2+ levels on the gating of gap junctions are discussed.

Sources and sites of action of calcium in the regulation of aldosterone biosynthesis.

The role of free calcium as a crucial intracellular messenger in the stimulation of aldosterone biosynthesis by various agonists is well established. Using electropermeabilized or Ca(2+)-clamped adrenal zona glomerulosa (ZG) cells, we have previously shown that Ca2+ entry into the mitochondrial matrix is required for the activation of steroidogenesis. We now describe the use of various strategies to answer the following questions: 1. Which pathway does Ca2+ follow before triggering steroidogenesis? 2. Which step of steroidogenesis is under the control of Ca2+? The first approach combined the patch-clamp method, in the perforated patch configuration, with microfluorimetry of Ca2+; in the second approach, ZG cells were transiently transfected with a chimeric cDNA encoding for the calcium-sensitive photoprotein aequorin linked to a mitochondrial targeting presequence; in a third approach, ZG mitochondria were isolated and fractionated into outer membranes, contact sites and inner membranes and the effect of prior exposure of the ZG cells to a physiologically elevated intracellular calcium concentration or to angiotensin II (Ang II) on cholesterol content was then examined in those three mitochondrial fractions. The results of these combined approaches allow us to propose the following scheme: The source of calcium which is predominantly responsible for mediating the steroidogenic effect of potassium appears to be funneled through the T-type calcium channels to close proximity of the mitochondria. This signal, as well as that triggered by Ang II, appears to be relayed within the mitochondrial matrix. This rise of mitochondrial calcium is associated with a transfer of free cholesterol from the outer to the inner mitochondrial membrane, via the contact sites. Thus the main role of the calcium messenger is to promote intramitochondrial cholesterol transfer and supply to the P450scc enzyme.

Fertilization and early embryology: Oscillations in intracellular free calcium induced by spermatozoa in human oocytes at fertilization

Calcium has an important role in the events of egg activation and early preimplantation development. We investigated changes in intracellular calcium concentration in human oocytes at fertilization using the calcium-sensitive photoprotein aequorin. Oocytes were donated for research by patients undergoing in-vitro fertilization treatment in the Department of Obstetrics and Gynaecology. Cumulus cells, and in some cases zonae pellucidae, were removed by appropriate enzyme treatment. Single oocytes were micro-injected with aequorin and incubated in a chamber perfused with pre-equilibrated culture medium in a photomultiplier system. Eleven zona-intact and 15 zona-free oocytes were incubated with sperm, and oocytes from each group were incubated without sperm as controls. Dramatic transient increases in intracellular free calcium concentration were recorded in three zona-intact and seven zona-free oocytes, thought to be the first direct measurements of intracellular changes in human oocytes at fertilization. The amplitude (up to 2.5 microM), duration (120 s) and frequency (every 10-35 min) of these transients were similar in zona-intact and zona-free oocytes. They resemble those recorded in mouse oocytes, which may therefore be a suitable model for biochemical events at human fertilization.

Modulation of free Ca oscillations in single hepatocytes by changes in extracellular K +, Na + and Ca 2+

Single rat hepatocytes, microinjected with the calcium-sensitive photoprotein aequorin, when stimulated with either phenylephrine or arg 8-vasopressin exhibit agonist-specific oscillations in cytosolic free calcium levels (free Ca). In the majority of the cells examined adding excess potassium chloride, sodium chloride or choline chloride abolished transient behaviour. However, in cells that continued to oscillate the transient parameters were subtly modified by these treatments. In experiments using phenylephrine as the agonist, adding excess potassium chloride to the superfusate significantly reduced transient length, increased the rate of transient rise and reduced the smoothed peak free Ca level without significantly altering the intertransient resting free Ca level or the falling time constant. The possible mechanisms by which these alterations may occur are discussed.

Electric field-directed cell shape changes, displacement, and cytoskeletal reorganization are calcium dependent.

C3H/10T1/2 mouse embryo fibroblasts were stimulated by a steady electric field ranging up to 10 V/cm. Some cells elongated and aligned perpendicular to the field direction. A preferential positional shift toward the cathode was observed which was inhibited by the calcium channel blocker D-600 and the calmodulin antagonist trifluoperazine. Rhodaminephalloidin labeling of actin filaments revealed a field-induced disorganization of the stress fiber pattern, which was reduced when stimulation was conducted in calcium-depleted buffer or in buffer containing calcium antagonist CoCl2, calcium channel blocker D-600, or calmodulin antagonist trifluoperazine. Treatment with calcium ionophore A23187 had similar effects, except that the presence of D-600 did not reduce the stress fiber disruption. The calcium-sensitive photoprotein aequorin was used to monitor changes in intracellular-free calcium. Electric stimulation caused an increase of calcium to the micromolar range. This increase was inhibited by calcium-depleted buffer or by CoCl2, and was reduced by D-600. A calcium-dependent mechanism is proposed to explain the observed field-directed cell shape changes, preferential orientation, and displacement.

The effects of changes of pH on intracellular calcium transients in mammalian cardiac muscle.

The calcium-sensitive photoprotein aequorin was micro-injected into cells of rat, ferret, rabbit and cat papillary muscles. Aequorin light emission is a function of free intracellular calcium concentration. The changes in intracellular calcium concentration [( Ca2+]i) and tension accompanying changes of pH have been studied. When the solution perfusing the papillary muscle was changed from Tyrode solution equilibrated with 5% CO2 to Tyrode solution equilibrated with 15% CO2, developed tension showed a rapid fall followed by a slower rise to a steady state which was less than the control. However the calcium transient associated with each contraction increased monophasically to a new steady state. When the external pH was held constant during exposure to 15% CO2 (by increasing the [HCO3-]), the initial fall of tension was reduced and the slow recovery of tension was greater than when CO2 alone was changed. The amplitude of the calcium transient increased monophasically to a new steady state which was greater than control, but less than when [CO2] alone was increased. If [HCO3-] was decreased while maintaining [CO2] at 5%, there was a slow monophasic decline in developed tension, and a small increase in peak light. Alkaloses produced by changing the [HCO3-]/[CO2] ratio produced similar results but the changes observed were in the opposite direction to those described above. The effects of changes of pHo can be explained if pHi affects tension by two mechanisms. The first mechanism, which is responsible for the rapid change in tension, is not associated with a change in [Ca2+]i. The second mechanism leads to a slower and smaller change in tension, in the opposite direction to the first, and is due to a change in the intracellular calcium transient.

External calcium and contractility in single giant muscle fibres

Isometric tension and intracellular calcium movements detected using the calcium sensitive photoprotein aequorin have been studied on single striated fibres. The decrease of tension and light response upon deprivation of external calcium would support the notion that calcium ions are involved in the transmission of the electrical potential change from the tubular system to the sarcoplasmic reticulum, by occupying sites on the external (extracellular) surface of tubular system in direct correspondence with the terminal sacs of the sarcoplasmic reticulum.

Measurement of Intracellular Free Calcium in Monkey Kidney Cells with Aequorin

A method has been developed for the measurement of intracellular free calcium in mammalian cells. The calcium-sensitive photoprotein aequorin can be incorporated into isolated cells by hypo-osmotic treatment without altering the cell viability, permeability, or metabolism. Intracellular calcium activity (Cai2+) was monitored in a perfusion system. In monkey kidney cells (LLC-MK2), Cai2+ is approximately 57 nanomoles per liter. Changes in Cai2+ with time can also be followed: exposure of the cells to anaerobiosis or the calcium ionophore A23187 reversibly increases Cai2+. The method has also been successfully tested in rat hepatocytes.

The effects of muscle length on intracellular calcium transients in mammalian cardiac muscle.

1. The calcium-sensitive photoprotein aequorin was micro-injected into cells of rat and cat ventricular muscles. The resulting light emission is a function of intracellular free calcium concentration ([Ca2+]i). The transient increases in [Ca2+]i that accompany contraction were monitored. 2. After an increase in muscle length, the developed tension increased immediately and then showed a slow increase over a period of minutes. The peak [Ca2+]i in each contraction was initially unchanged after an increase in muscle length but then showed a slow increase with a time course similar to that of the slow tension change. 3. As a consequence of these slow changes, the shape of the tension-length relation depends on the procedure used to determine it and this change in shape can be attributed to changes in activation. 4. Immediately after an increase in muscle length the calcium transient was abbreviated. 5. When a quick release was performed during a contraction, a short-lived increase in the [Ca2+]i was observed following the release. 6. The two previous observations can both be explained if the binding constant of troponin for calcium is a function of developed tension.