scholarly journals Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Nordine Helassa ◽  
Borbala Podor ◽  
Alan Fine ◽  
Katalin Török
Keyword(s):  
Intracellular Calcium ◽  
Action Potentials ◽  
Rational Design ◽  
Calcium Transients ◽  
Calcium Dynamics ◽  
Binding Interface ◽  
Calcium Indicators ◽  
Slow Kinetics ◽  
Decay Times

Abstract Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6f u displaying fluorescence rise and decay times (t 1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6f u revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t 1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6f u is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators.

Periodic synchronized bursting and intracellular calcium transients elicited by low magnesium in cultured cortical neurons

1993 ◽  
Vol 70 (4) ◽  
pp. 1606-1616 ◽  
Author(s):  
H. P. Robinson ◽  
M. Kawahara ◽  
Y. Jimbo ◽  
K. Torimitsu ◽  
Y. Kuroda ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Voltage Clamp ◽  
Cortical Neurons ◽  
Action Potentials ◽  
Calcium Transients ◽  
Resting Potential ◽  
Current Clamp ◽  
Postsynaptic Currents ◽  
Decay Times ◽  
Spike Bursts

1. In Mg(2+)-free external solution, rat cortical neurons in cultured networks entered a stable firing mode, consisting of regular bursts of action potentials superimposed on long-lasting depolarizations. The average separation between bursts varied from culture to culture, but was usually between 5 and 20 s. The distribution of burst intervals followed a Gaussian or normal distribution, with a standard deviation of typically 10% of the average burst period. 2. A gradually depolarizing pacemaker potential was never observed between bursts, but the threshold for action potentials during the quiescent phase was > or = 10 mV above the resting potential. No progressive change in conductance or excitability was observed during the quiescent period. Intracellular stimulation of action potentials did not reproduce the long-lasting depolarization. 3. Switching from current clamp to voltage clamp at the resting potential revealed large postsynaptic currents, mainly excitatory but with a small inhibitory component, at the same phase and frequency as the spike bursts, showing that periodic synaptic input is responsible for the burst-depolarizations. The current could be eliminated by local application of 2-amino-5-phosphonovaleric acid (APV) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the postsynaptic cell. In the presence of tetrodotoxin, irregular miniature excitatory postsynaptic currents were observed. 4. A fluorescent calcium indicator (fluo-3, 100 microM) was included in the whole-cell pipette solution, to allow simultaneous electrical and calcium measurements in the same cell. In current clamp, transient intracellular calcium increases were found, which were synchronized to the spike bursts. The Ca2+ rise lasted as long as the action potential burst, and was followed by an exponential decay considerably slower than that of the membrane potential. Calcium transients disappeared during voltage clamp at the resting potential, suggesting that calcium influx through voltage-dependent calcium channels greatly exceeds that through synaptic channels. 5. Multisite Ca2+ recording, after loading with fluo-3 acetoxymethyl (AM) ester, revealed that the onsets of burst-related calcium transients were synchronized in all active cells of each view-field, to within approximately 20 ms. Occasionally, secondary rhythms were observed in which only a subset of cells participated. The times to peak and the decay times of calcium transients varied among synchronized cells. 6. The pharmacology of the burst-related calcium transients was investigated by bath application of a variety of compounds.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular calcium levels in the Plasmodium berghei ookinete

Parasitology ◽  
2008 ◽  
Vol 135 (12) ◽  
pp. 1355-1362 ◽  
Author(s):  
I. SIDÉN-KIAMOS ◽  
C. LOUIS
Keyword(s):  
Intracellular Calcium ◽  
Plasmodium Berghei ◽  
Calcium Concentration ◽  
Insect Cells ◽  
Ionophore A23187 ◽  
Rodent Malaria Parasite ◽  
Calcium Indicators ◽  
Intracellular Ca

SUMMARYOokinetes are the motile and invasive stages of Plasmodium parasites in the mosquito host. Here we explore the role of intracellular Ca2+ in ookinete survival and motility as well as in the formation of oocysts in vitro in the rodent malaria parasite Plasmodium berghei. Treatment with the Ca2+ ionophore A23187 induced death of the parasite, an effect that could be prevented if the ookinetes were co-incubated with insect cells before incubation with the ionophore. Treatment with the intracellular calcium chelator BAPTA/AM resulted in increased formation of oocysts in vitro. Calcium imaging in the ookinete using fluorescent calcium indicators revealed that the purified ookinetes have an intracellular calcium concentration in the range of 100 nm. Intracellular calcium levels decreased substantially when the ookinetes were incubated with insect cells and their motility was concomitantly increased. Our results suggest a pleiotropic role for intracellular calcium in the ookinete.

Intracellular calcium dynamics and cerebral injury: modeling various insults in vitro

1993 ◽  
Vol 613 (1) ◽  
pp. 156-159 ◽  
Author(s):  
Myung H. Kim-Lee ◽  
Bradford T. Stokes ◽  
Douglas K. Anderson
Keyword(s):  
Intracellular Calcium ◽  
Cerebral Injury ◽  
Calcium Dynamics ◽  
Intracellular Calcium Dynamics

Activity-Related Calcium Dynamics in Motoneurons of the Nucleus Hypoglossus From Mouse

1999 ◽  
Vol 82 (6) ◽  
pp. 2936-2946 ◽  
Author(s):  
Mario B. Lips ◽  
Bernhard U. Keller
Keyword(s):  
Quantitative Analysis ◽  
Action Potential ◽  
Calcium Binding ◽  
Calcium Influx ◽  
Binding Capacity ◽  
Calcium Transients ◽  
Calcium Dynamics ◽  
The Brain

A quantitative analysis of activity-related calcium dynamics was performed in motoneurons of the nucleus hypoglossus in the brain stem slice preparation from mouse by simultaneous patch-clamp and microfluorometric calcium measurements. Motoneurons were analyzed under in vitro conditions that kept them in a functionally intact state represented by rhythmic, inspiratory-related bursts of excitatory postsynaptic currents and associated action potential discharges. Bursts of electrical activity were paralleled by somatic calcium transients resulting from calcium influx through voltage-activated calcium channels, where each action potential accounted for a calcium-mediated charge influx around 2 pC into the somatic compartment. Under in vivo conditions, rhythmic-respiratory activity in young mice occurred at frequencies up to 5 Hz, demonstrating the necessity for rapid calcium elevation and recovery in respiratory-related neurons. The quantitative analysis of hypoglossal calcium homeostasis identified an average extrusion rate, but an exceptionally low endogenous calcium binding capacity as cellular parameters accounting for rapid calcium signaling. Our results suggest that dynamics of somatic calcium transients 1) define an upper limit for the maximum frequency of respiratory-related burst discharges and 2) represent a potentially dangerous determinant of intracellular calcium profiles during pathophysiological and/or excitotoxic conditions.

scholarly journals Intracellular calcium dynamics at the core of endocardial stationary spiral waves in Langendorff-perfused rabbit hearts

2008 ◽  
Vol 295 (1) ◽  
pp. H297-H304 ◽  
Author(s):  
Liang Tang ◽  
Gyo-Seung Hwang ◽  
Hideki Hayashi ◽  
Juan Song ◽  
Masahiro Ogawa ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Transmembrane Potential ◽  
Spiral Wave ◽  
In Vitro Models ◽  
Spiral Waves ◽  
Calcium Dynamics ◽  
The Core ◽  
Intracellular Calcium Dynamics ◽  
The Right

In vitro models of sustained monomorphic ventricular tachycardia (MVT) are rare and do not usually show spiral reentry on the epicardium. We hypothesized that MVT is associated with the spiral wave in the endocardium and that this stable reentrant propagation is supported by a persistently elevated intracellular calcium (Cai) transient at the core of the spiral wave. We performed dual optical mapping of transmembrane potential ( Vm) and Cai dynamics of the right ventricular (RV) endocardium in Langendorff-perfused rabbit hearts ( n = 12). Among 64 induced arrhythmias, 55% were sustained MVT (>10 min). Eighty percent of MVT showed stationary spiral waves (>10 cycles, cycle length: 128 ± 14.6 ms) in the endocardial mapped region, anchoring to the anatomic discontinuities. No reentry activity was observed in the epicardium. During reentry, the amplitudes of Vm and Cai signals were higher in the periphery and gradually decreased toward the core. At the core, maximal Vm and Cai amplitudes were 42.95 ± 5.89% and 43.95 ± 9.46%, respectively, of the control ( P < 0.001). However, the trough of the Vm and Cai signals at the core were higher than those in the periphery, indicating persistent Vm and Cai elevations during reentry. BAPTA-AM, a calcium chelator, significantly reduced the maximal Cai transient amplitude and prevented sustained MVT and spiral wave formation in the mapped region. These findings indicate that endocardial spiral waves often anchor to anatomic discontinuities causing stable MVT in normal rabbit ventricles. The spiral core is characterized by diminished Vm and Cai amplitudes and persistent Vm and Cai elevations during reentry.

scholarly journals Calcium confusion--is the variability in calcium response by Sertoli cells to specific hormones meaningful or simply redundant?

2000 ◽  
Vol 167 (1) ◽  
pp. 1-5 ◽  
Author(s):  
FF Rommerts ◽  
FM Lyng ◽  
E von Ledebur ◽  
L Quinlan ◽  
GR Jones ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Sertoli Cells ◽  
Cellular Response ◽  
Culture Conditions ◽  
Calcium Transients ◽  
Tight Coupling ◽  
Calcium Signals ◽  
Experimental Conditions ◽  
Negative Findings

When results of more than ten different studies on hormone-induced calcium signals in Sertoli cells are taken together, a wide variety of responses emerges. The reported changes range from increased concentrations, via no response at all, to decreased calcium concentrations. Minor variations in cell isolation techniques, culture conditions, or techniques for measuring the intracellular calcium could explain some of these differences. However, erratic variations in response are also observed within research groups under very similar experimental conditions. Such 'negative' findings are mainly reported orally and do not further penetrate the scientific community. As hormone-dependent calcium responses evidently may depend very much on the context of the cells, calcium transients would appear to be unreliable bioassay principles with which to detect the primary actions of FSH and effectors such as androgens on Sertoli cells. A more important biological question is whether these sometimes opposed calcium transients are connected with a particular cellular response. To date there is no evidence for such a tight coupling in Sertoli cells, implying that, at least under in vitro conditions, calcium signals might even be redundant altogether. Such calcium variability is probably not unique to Sertoli cells, and the aim of this commentary is to promote an open debate that may help to transform the current state of 'calcium confusion' into a better understanding of the intracellular calcium language.

Development of instrumentation to measure rapid calcium transits in neonatal rat ventricular myocytes

1988 ◽  
Vol 46 ◽  
pp. 44-45
Author(s):  
H. K. Hagler ◽  
A. C. Morris ◽  
L. M. Buja
Keyword(s):  
Intracellular Calcium ◽  
Cell Injury ◽  
Ventricular Myocytes ◽  
Calcium Transients ◽  
Neonatal Rat ◽  
Free Calcium ◽  
Total Calcium ◽  
X Ray ◽  
Calcium Indicators ◽  
Electron Microscopes

Changes in the intracellular calcium levels in cardiac myocytes are important in the regulation of normal cardiac function and have been implicated in contributing to irreversible cell injury with ischemia or hypoxia. Intracellular measurement of total calcium changes with subcellular resolution have become routine using rapid cryofixation, cryosectioning, cryotransfer and energy dispersive x-ray microanalysis in analytical electron microscopes. The x-ray microanalysis technique measures total calcium changes within subcellular compartments, but does not distinguish between the bound and free calcium. With the successful development of fluorescent calcium indicators which may be introduced into cells without significantly buffering the intracellular calcium levels, it has become possible to measure rapid calcium transients during contraction. The primary requirements in the development of a system to utilize the fluorescent calcium indicators were to resolve calcium transients in individual cells (since the response to perturbations such as hypoxia is heterogeneous) and develop a system which would be flexible enough to accommodate new indicators as they become available.

scholarly journals 234.Calcium involvement in glucose induced GLUT3 expression in preimplantation mouse embryos

2004 ◽  
Vol 16 (9) ◽  
pp. 234
Author(s):  
K. Gardner ◽  
M. Pantaleon ◽  
P. L. Kaye
Keyword(s):  
Intracellular Calcium ◽  
Calcium Release ◽  
Glucose Sensing ◽  
Mouse Embryos ◽  
Calcium Transients ◽  
Cell Stage ◽  
Nutrient Environment ◽  
Glucose Exposure ◽  
Voltage Gated Ion Channels

Despite their inability to utilise glucose for energy prior to compaction (E3), mouse embryos have a requirement for at least a brief glucose exposure to permit normal development. In the absence of this glucose pulse in vitro, we and others have found that embryos cleave to form morulae but fail to form blastocysts and subsequently degenerate. These embryos do not develop the capacity to utilise glucose preferentially and are unable to adapt to their nutrient environment and utilise alternate substrates (1). This inability to utilise glucose is due to failure to express GLUT3 at compaction (2). Brief glucose exposure prior to the 8-cell stage is sufficient to permit the embryo to undergo compaction, express GLUT3 and ultimately form a blastocyst, suggesting that glucose induces metabolic differentiation of the developing embryo. In this study we have explored the role of intracellular calcium in response to glucose given its central role in pancreatic glucose induced signalling events. Zygotes were cultured in the presence and absence of glucose and treated with either calcium mobilising agents, ethanol or ionomycin at 54�h post hCG or with the intracellular calcium chelator BAPTA-AM. Embryos were fixed and assayed for GLUT3 expression individually at 96�h post hCG using confocal immunofluorescence. Release of intracellular calcium by either ethanol or ionomycin, activated GLUT3 expression in a glucose like manner (P�<�0.01) suggesting that calcium transients may be involved in glucose sensing. Moreover, buffering of calcium with the calcium chelator BAPTA-AM interfered with the ability of glucose to activate GLUT3 expression (P�<�0.05), suggesting that glucose exposure does result in calcium transients that affect GLUT3 expression. It is unclear whether these calcium transients occur as a result of influx of extracellular calcium via voltage-gated ion channels or the release of calcium from intracellular stores via inositol triphosphate-gated calcium release channels in the endoplasmic reticulum. (1) Martin and Leese (1995) Mol. Reprod. Dev. 40, 436–443. (2) Pantaleon et al. (2001) Proc 32nd Annual SRB Conference, Gold Coast, Qld. A42.

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