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Eduardo Ríos

Publications and source records attributed to Eduardo Ríos.

16 recordsLinked to original sources
PubMed2006

The changes in Ca2+ sparks associated with measured modifications of intra-store Ca2+ concentration in skeletal muscle.

In cardiac muscle and amphibian skeletal muscle, the intracellular Ca2+ release that signals contractile activation proceeds by discrete local packets, which result in Ca2+ sparks. The remarkably stereotyped duration of these release events requires a robustly timed termination mechanism. In cardiac muscle the mechanism of spark termination appears to crucially involve depletion of Ca2+ in the lumen of the sarcoplasmic reticulum (SR), but in skeletal muscle, the mechanism is unknown. We used SEER (shifted excitation and emission ratioing of fluorescence) of SR-trapped mag-indo-1 and confocal imaging of fluorescence of cytosolic rhod-2 to image Ca2+ sparks while reversibly changing and measuring [Ca2+] in the SR ([Ca2+]SR) of membrane-permeabilized frog skeletal muscle cells. Sparks were collected in cells immersed in a solution promoting production of events at moderate frequency. Just after permeabilization, event frequency was zero, and in 10 minutes it reached close to a steady value. Controlled interventions modified [Ca2+]SR reversibly between a low value (299 microM on average in 10 experiments) and a high value (433 microM, a 45% average increase). This change increased sparks frequency by 93%, spatial width by 7%, rise time by 10%, and peak amplitude by 38% (provided that it was calculated in absolute terms, rather than normalized by resting fluorescence). The changes in event frequency and amplitude were statistically significant. The "strength" of the effect of [Ca2+]SR on frequency, quantified by decomposition of variance, was <6%. While the average change in [Ca2+]SR was limited, it reached up to 200% in individual fibers, without causing massive Ca2+ release or an increase of >3.5-fold in event frequency. Taken together with existing evidence that depletion is modest during Ca2+ sparks or release elicited by an action potential, the mild effects of [Ca2+]SR reported here do not support a major role of depletion in either the termination of sparks or the strong inactivation that terminates Ca2+ release at the global level in frog skeletal muscle.

Animals↗
PubMed2006

Depletion "skraps" and dynamic buffering inside the cellular calcium store.

Ca2+ signals, produced by Ca2+ release from cellular stores, switch metabolic responses inside cells. In muscle, Ca2+ sparks locally exhibit the rapid start and termination of the cell-wide signal. By imaging Ca2+ inside the store using shifted excitation and emission ratioing of fluorescence, a surprising observation was made: Depletion during sparks or voltage-induced cell-wide release occurs too late, continuing to progress even after the Ca2+ release channels have closed. This finding indicates that Ca2+ is released from a "proximate" compartment functionally in between store lumen and cytosol. The presence of a proximate compartment also explains a paradoxical surge in intrastore Ca2+, which was recorded upon stimulation of prolonged, cell-wide Ca2+ release. An intrastore surge upon induction of Ca2+ release was first reported in subcellular store fractions, where its source was traced to the store buffer, calsequestrin. The present results update the evolving concept, largely due to N. Ikemoto and C. Kang, of calsequestrin as a dynamic store. Given the strategic location and reduction of dimensionality of Ca2+-adsorbing linear polymers of calsequestrin, they could deliver Ca2+ to the open release channels more efficiently than the luminal store solution, thus constituting the proximate compartment. When store depletion becomes widespread, the polymers would collapse to increase store [Ca2+] and sustain the concentration gradient that drives release flux.

Animals↗
PubMed2005

A probable role of dihydropyridine receptors in repression of Ca2+ sparks demonstrated in cultured mammalian muscle.

To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca(2+) release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca(2+) release. Murine primary cultures were confocally imaged for Ca(2+) detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca(2+) sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic mice (mdg) that lack DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency, a high-Ca(2+) saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 microM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca(2+) saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca(2+)] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca(2+)] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca(2+) release channels and/or their susceptibility to Ca(2+)-induced activation, thereby suppressing the production of Ca(2+) sparks.

Animals↗
PubMed2005

Confocal imaging of [Ca2+] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence.

Intracellular calcium signals regulate multiple cellular functions. They depend on release of Ca2+ from cellular stores into the cytosol, a process that appears to be tightly controlled by changes in [Ca2+] within the store. A method to image free [Ca2+] within cellular organelles was devised, which provided the first quantitative confocal images of [Ca2+] inside the sarcoplasmic reticulum (SR) of skeletal muscle. The method exploits, for greater sensitivity, the dual spectral shifts that some fluorescent dyes undergo upon binding Ca2+. It was implemented with mag-indo-1 trapped in the intracellular organelles of frog skeletal muscle and validated showing that it largely monitors [Ca2+] in a caffeine-sensitive compartment with the structure of the SR cisternae. A tentative calibration in situ demonstrated an increase in the dye's dissociation constant, not unlike that observed for other dyes in cellular environments. This increase, together with other characteristics of the ratioing method, placed the half-signal [Ca2+] near 1 mM, a value suitable for cellular stores. Demonstrated advantages of the technique include accuracy (that of a calibrated ratiometric method), dynamic range and sensitivity (from the combination of two spectral shifts), spatial and temporal resolution, and compatibility with a vast array of visible dyes to monitor diverse aspects of cellular function. SEER (shifted excitation and emission ratioing) also provides a [Ca2+]-independent measure of dye concentration in the cell. Store and mitochondrial [Ca2+] ([Ca2+]SR and [Ca2+]mito could be measured separately using the high spatial resolution of SEER. Evolution of [Ca2+]SR was followed upon changes in cytosolic [Ca2+] ([Ca2+]cyto). At [Ca2+]cyto = 100 nM, [Ca2+]mito remained near the lower limit of detection and [Ca2+]SR stabilized at values that were submillimolar according to our tentative calibration. Steady [Ca2+]SR was only slightly higher in 800 nM [Ca2+]cyto, and essentially did not decrease unless [Ca2+]cyto was reduced below 10 nM. While the increase of [Ca2+]SR was limited by loss through Ca2+ release channels, its decrease in low [Ca2+]cyto was largely dependent on leaks through the SR Ca2+ pump.

Animals↗
PubMed2005

Concerted vs. sequential. Two activation patterns of vast arrays of intracellular Ca2+ channels in muscle.

To signal cell responses, Ca(2+) is released from storage through intracellular Ca(2+) channels. Unlike most plasmalemmal channels, these are clustered in quasi-crystalline arrays, which should endow them with unique properties. Two distinct patterns of local activation of Ca(2+) release were revealed in images of Ca(2+) sparks in permeabilized cells of amphibian muscle. In the presence of sulfate, an anion that enters the SR and precipitates Ca(2+), sparks became wider than in the conventional, glutamate-based solution. Some of these were "protoplatykurtic" (had a flat top from early on), suggesting an extensive array of channels that activate simultaneously. Under these conditions the rate of production of signal mass was roughly constant during the rise time of the spark and could be as high as 5 microm(3) ms(-1), consistent with a release current >50 pA since the beginning of the event. This pattern, called "concerted activation," was observed also in rat muscle fibers. When sulfate was combined with a reduced cytosolic [Ca(2+)] (50 nM) these sparks coexisted (and interfered) with a sequential progression of channel opening, probably mediated by Ca(2+)-induced Ca(2+) release (CICR). Sequential propagation, observed only in frogs, may require parajunctional channels, of RyR isoform beta, which are absent in the rat. Concerted opening instead appears to be a property of RyR alpha in the amphibian and the homologous isoform 1 in the mammal.

Animals↗
PubMed2004

The quantal nature of Ca2+ sparks and in situ operation of the ryanodine receptor array in cardiac cells.

Intracellular Ca(2+) release in many types of cells is mediated by ryanodine receptor Ca(2+) release channels (RyRCs) that are assembled into two-dimensional paracrystalline arrays in the endoplasmic/sarcoplasmic reticulum. However, the in situ operating mechanism of the RyRC array is unknown. Here, we found that the elementary Ca(2+) release events, Ca(2+) sparks from individual RyRC arrays in rat ventricular myocytes, exhibit quantized Ca(2+) release flux. Analysis of the quantal property of Ca(2+) sparks provided a view of unitary Ca(2+) current and gating kinetics of the RyRC in intact cells and revealed that spark activation involves dynamic recruitment of small, variable cohorts of RyRCs. Intriguingly, interplay of RyRCs in multichannel sparks renders an unusual, thermodynamically irreversible mode of channel gating that is unshared by an RyRC acting solo, nor by RyRCs in vitro. Furthermore, an array-based inhibitory feedback, overriding the regenerative Ca(2+)-induced Ca(2+) release of RyRCs, provides a supramolecular mechanism for the microscopic stability of intracellular Ca(2+) signaling.

Animals↗
PubMed2004

How source content determines intracellular Ca2+ release kinetics. Simultaneous measurement of [Ca2+] transients and [H+] displacement in skeletal muscle.

In skeletal muscle, the waveform of Ca(2+) release under clamp depolarization exhibits an early peak. Its decay reflects an inactivation, which locally corresponds to the termination of Ca(2+) sparks, and is crucial for rapid control. In cardiac muscle, both the frequency of spontaneous sparks (i.e., their activation) and their termination appear to be strongly dependent on the Ca(2+) content in the sarcoplasmic reticulum (SR). In skeletal muscle, no such role is established. Seeking a robust measurement of Ca(2+) release and a way to reliably modify the SR content, we combined in the same cells the "EGTA/phenol red" method (Pape et al., 1995) to evaluate Ca(2+) release, with the "removal" method (Melzer et al., 1987) to evaluate release flux. The cytosol of voltage-clamped frog fibers was equilibrated with EGTA (36 mM), antipyrylazo III, and phenol red, and absorbance changes were monitored simultaneously at three wavelengths, affording largely independent evaluations of Delta[H(+)] and Delta[Ca(2+)] from which the amount of released Ca(2+) and the release flux were independently derived. Both methods yielded mutually consistent evaluations of flux. While the removal method gave a better kinetic picture of the release waveform, EGTA/phenol red provided continuous reproducible measures of calcium in the SR (Ca(SR)). Steady release permeability (P), reached at the end of a 120-ms pulse, increased as Ca(SR) was progressively reduced by a prior conditioning pulse, reaching 2.34-fold at 25% of resting Ca(SR) (four cells). Peak P, reached early during a pulse, increased proportionally much less with SR depletion, decreasing at very low Ca(SR). The increase in steady P upon depletion was associated with a slowing of the rate of decay of P after the peak (i.e., a slower inactivation of Ca(2+) release). These results are consistent with a major inhibitory effect of cytosolic (rather than intra-SR) Ca(2+) on the activity of Ca(2+) release channels.

Action Potentials↗
PubMed2004

Regulation of Ca2+ sparks by Ca2+ and Mg2+ in mammalian and amphibian muscle. An RyR isoform-specific role in excitation-contraction coupling?

Ca2+ and Mg2+ are important mediators and regulators of intracellular Ca2+ signaling in muscle. The effects of changes of cytosolic [Ca2+] or [Mg2+] on elementary Ca2+ release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca2+ sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca2+] or [Mg2+] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca2+] (reaching peaks >100 microM) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca2+] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca2+] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca2+] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca2+] inside the SR. In the frog only, increases in cytosolic [Ca2+] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg2+] caused a fall of spark frequency, but with striking species differences. In the rat, but not the frog, sparks were observed at 4-40 mM [Mg2+]. Reducing [Mg2+] below 2 mM, which should enable the RyR channel's activation (CICR) site to bind Ca2+, caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg2+ are hallmarks of CICR. Their absence in the rat suggests that CICR requires RyR3 para-junctional clusters, present only in the frog. The observed frequency of sparks corresponds to a channel open probability of 10(-7) in the frog or 10(-8) in the rat. Together with the failure of photorelease to induce activation directly, this indicates a basal inhibition of channels in situ. It is proposed that relief of this inhibition could be the mechanism by which increased SR load increases spark frequency.

Action Potentials↗
PubMed2004

Control of dual isoforms of Ca2+ release channels in muscle.

Here we compare excitation-contraction coupling in single muscle cells of frogs and rats. Because amphibians have isoform 3 (or 3) of the ryanodine receptor/Ca2+ release channel, in addition to 1 (alpha), which is also present in the mammal, any extra feature present in the frog may in principle be attributed to isoform 3. Ca2+ release under voltage clamp depolarization has a peak and a steady phase in both taxonomic classes, but the peak is more marked in the frog, where the ratio of amplitudes of the two phases is voltage-dependent. This dependence is a hallmark of CICR. Confocal imaging identified Ca2+ sparks in the frog, but not in the voltage-clamped rat cells. Because Ca2+ sparks involve CICR both observations indicate that the contribution of CICR is minor or null in the mammal. The "couplon" model well accounts for observations in the frog, but assumes a structure that we now know to be valid only for the rat. A revised model is proposed, whereby the isoform 3 channels, located parajunctionally, are activated by CICR and contribute its characteristic global and local features. Several issues regarding the roles of different channels remain open to further study.

Animals↗
PubMed2003

Unitary Ca2+ current through mammalian cardiac and amphibian skeletal muscle ryanodine receptor Channels under near-physiological ionic conditions.

Ryanodine receptor (RyR) channels from mammalian cardiac and amphibian skeletal muscle were incorporated into planar lipid bilayers. Unitary Ca2+ currents in the SR lumen-to-cytosol direction were recorded at 0 mV in the presence of caffeine (to minimize gating fluctuations). Currents measured with 20 mM lumenal Ca2+ as exclusive charge carrier were 4.00 and 4.07 pA, respectively, and not significantly different. Currents recorded at 1-30 mM lumenal Ca2+ concentrations were attenuated by physiological [K+] (150 mM) and [Mg2+] (1 mM), in the same proportion (approximately 55%) in mammalian and amphibian channels. Two amplitudes, differing by approximately 35%, were found in amphibian channel studies, probably corresponding to alpha and beta RyR isoforms. In physiological [Mg2+], [K+], and lumenal [Ca2+] (1 mM), the Ca2+ current was just less than 0.5 pA. Comparison of this value with the Ca2+ flux underlying Ca2+ sparks suggests that sparks in mammalian cardiac and amphibian skeletal muscles are generated by opening of multiple RyR channels. Further, symmetric high concentrations of Mg2+ substantially reduced the current carried by 10 mM Ca2+ (approximately 40% at 10 mM Mg2+), suggesting that high Mg2+ may make sparks smaller by both inhibiting RyR gating and reducing unitary current.

Algorithms↗
PubMed2003

Two components of voltage-dependent inactivation in Ca(v)1.2 channels revealed by its gating currents.

Voltage-dependent inactivation (VDI) was studied through its effects on the voltage sensor in Ca(v)1.2 channels expressed in tsA 201 cells. Two kinetically distinct phases of VDI in onset and recovery suggest the presence of dual VDI processes. Upon increasing duration of conditioning depolarizations, the half-distribution potential (V(1/2)) of intramembranous mobile charge was negatively shifted as a sum of two exponential terms, with time constants 0.5 s and 4 s, and relative amplitudes near 50% each. This kinetics behavior was consistent with that of increment of maximal charge related to inactivation (Qn). Recovery from inactivation was also accompanied by a reduction of Qn that varied with recovery time as a sum of two exponentials. The amplitudes of corresponding exponential terms were strongly correlated in onset and recovery, indicating that channels recover rapidly from fast VDI and slowly from slow VDI. Similar to charge "immobilization," the charge moved in the repolarization (OFF) transient became slower during onset of fast VDI. Slow VDI had, instead, hallmarks of interconversion of charge. Confirming the mechanistic duality, fast VDI virtually disappeared when Li(+) carried the current. A nine-state model with parallel fast and slow inactivation pathways from the open state reproduces most of the observations.

Adaptation, Physiological↗
PubMed2002

Initiation and termination of calcium sparks in skeletal muscle

Three main paths to derive the Ca2+ release flux underlying Ca2+ sparks are reviewed here: Some properties of release flux can be inferred from an examination of spark morphology. Others from model simulations, which generate sparks assuming an ion source within a cytoplasm-like medium. Finally, the release flux can be derived from the fluorescence transient by generalizing an algorithm developed earlier for global or whole cell signals. The transient and spatially limited nature of sparks adds many uncertainties to the process. These methods yield estimates between 1.4 and 30 pA, not clearly greater in skeletal than in cardiac muscle. At their low end, the estimates are consistent with generation of sparks by one or two ryanodine receptor channels, but the results are easier to explain if several channels, from as little as four to as many as 60, cooperate in their generation. How release flux determines spark shape and time course has been understood largely through simulations. The rise time of sparks corresponds to active release time. Both release flux and release time may vary among individual sparks, leading to their varied size and shape. Release flux turns off abruptly, therefore the decay of sparks is determined by Ca2+ removal and diffusion. Spatial width increases with release time (rise time). That its experimentally determined value is too large compared with simulations, remains the single most important question in the interpretation of shape. Sparks are not the sole form of local fluorescence transients. When channel opening drugs are present, or sometimes spontaneously, sparks may be prolonged by embers. If the release flux calculated during an ember corresponds to a single open channel, then the release underlying a spark must require many open channels. The continued examination of Ca2+ release flux appears to be an essential requisite for the interpretation of sparks and their place in calcium signaling.

Animals↗
PubMed2002

Thermodynamically irreversible gating of ryanodine receptors in situ revealed by stereotyped duration of release in Ca(2+) sparks.

For a single or a group of Markov channels gating reversibly, distributions of open and closed times should be the sum of positively weighted decaying exponentials. Violation of this microscopic reversibility has been demonstrated previously on a number of occasions at the single channel level, and has been attributed to possible channel coupling to external sources of free energy. Here we show that distribution of durations of Ca(2+) release underlying Ca(2+) sparks in intact cardiac myocytes exhibits a prominent mode at approximately 8 ms. Analysis of the cycle time for repetitive sparks at hyperactive sites revealed no intervals briefer than approximately 35 ms and a mode at approximately 90 ms. These results indicate that, regardless of whether Ca(2+) sparks are single-channel or multi-channel in origin, they are generated by thermodynamically irreversible stochastic processes. In contrast, data from planar lipid bilayer experiments were consistent with reversible gating of RyR under asymmetric cis (4 microM) and trans Ca(2+) (10 mM), suggesting that the irreversibility for Ca(2+) spark genesis may reside at a supramolecular level. Modeling suggests that Ca(2+)-induced Ca(2+) release among adjacent RyRs may couple the external energy derived from Ca(2+) gradients across the SR to RyR gating in situ, and drive the irreversible generation of Ca(2+) sparks.

Animals↗
PubMed2002

Intracellular Ca(2+) release as irreversible Markov process.

In striated muscles, intracellular Ca(2+) release is tightly controlled by the membrane voltage sensor. Ca(2+) ions are necessary mediators of this control in cardiac but not in skeletal muscle, where their role is ill-understood. An intrinsic gating oscillation of Ca(2+) release-not involving the voltage sensor-is demonstrated in frog skeletal muscle fibers under voltage clamp. A Markov model of the Ca(2+) release units is shown to reproduce the oscillations, and it is demonstrated that for Markov processes to have oscillatory transients, its transition rates must violate thermodynamic reversibility. Such irreversibility results in permanent cycling of the units through a ring of states, which requires a source of free energy. Inhibition of the oscillation by 20 to 40 mM EGTA or partial depletion of Ca(2+) in the sarcoplasmic reticulum (SR) identifies the SR [Ca(2+)] gradient as the energy source, and indicates a location of the critical Ca(2+)-sensing site at distances greater than 35 nm from the open channel. These results, which are consistent with a recent demonstration of irreversibility in gating of cardiac Ca(2+) sparks, (Wang, S.-Q., L.-S. Song, L. Xu, G. Meissner, E. G. Lakatta, E. Ríos, M. D. Stern, and H. Cheng. 2002. Biophys. J. 83:242-251) exemplify a cell-wide oscillation caused by coupling between ion permeation and channel gating.

Algorithms↗