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.

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.

Entrada capacitativa de calcio: un mecanismo nuevo de entrada de calcio en células cardiacas / Capacitative calcium entry: a new mechanism for calcium entry in cardiac cells

Las células cardiacas requieren de una apropiada regulación del calcio intracelular para cumplir su función contráctil. Los mecanismos que regulan la entrada de calcio al interior celular, descritos hasta ahora, incluyen canales voltaje dependientes, bombas e intercambiadores. Este trabajo presenta evidencias de la presencia de un mecanismo adicional de entrada de calcio que es activado por el vaciamiento de los depósitos intracelulares de calcio. Se utilizaron células ventriculares aisladas enzimaticamente, del corazón de ratas, y cargadas con el indicador de calcio Fura-2. El vaciamiento del retículo sarcoplasmático (RS) se obtuvo activando los canales de liberación de calcio con Clorocresol (CmC), un agonista de los receptores de ryanodina. O inhibiendo la Ca2+ -ATPasa del RS con Tapsigagina. El vaciamiento del RS indujo un aumento del calcio intracelular al colocar las células en un medio externo con calcio. Este mecanismo de entrada de calcio es permeable al manganeso y muestra una inhibición > 80 por ciento en presencia de SKF-96365. Estas evidencias indican que en miocitos ventriculares de rata existe una mecanismo de entrada de calcio similar a la denominada corriente capacitativa. La velocidad de activación de este mecanismo depende del tiempo transcurrido desde que se vació el RS, sugiriendo un acoplamiento indirecto entre los canales capacitativos y el RS.

Cardiac cells need to regulate the intracellular calcium level to function as a pump. Calcium entry at the plasmalemma is regulated by proteins that functions as channels, pumps or exchangers. This work has evidence that in cardiac cells there is a new mechanism for calcium entry. The mechanism is activated after the intracellular stores are depleted. We used rat enzimatically isolated ventricular cells loaded with Fura-2. The sarcoplasmic reticulum (SR) calcium stores were depleted by activating the calcium release channels with Chlorocresol, a ryanodine receptor agonist. Or by decreasing the calcium uptake with Thapsigargin. After store depletion was completed, expousing the cells to extracellular calcium induced a raise in [Ca2+]i. This calcium entry mechanism is permeable to Manganese and is inhibited by a capacitative calcium entry blocker (SKF-96365). These evidences support the presence of a capacitative calcium entry mechanism.Our results also show that gating of this mechanism depends on the time elapsed after store depletion, being faster at longer time after depletion. This result may suggest an indirect coupling between the calcium entry mechanism and RS.

Inactivation of Ca2+ transients in amphibian and mammalian muscle fibres.

MagFluo-4 fluorescence (Ca2+) transients associated with action potentials were measured in intact muscle fibres, manually dissected from toads ( Leptodactylus insularis ) or enzymatically dissociated from mice. In toads, the decay phase of the Ca2+ transients is described by a single exponential with a time constant ( tau ) of about 7 ms. In mice, a double exponential function with tau 's of 1.5 and 15.5 ms, respectively gives a better fit. In both species the amplitude of Ca2+ transients diminished during repetitive stimulation: in amphibian muscle fibres, the decrease was about 20% with 1 Hz stimulation and 55% at 10 Hz. In mammalian fibres, repetitive stimulation causes a less conspicuous decrease of the transient amplitude: 10% at 1 Hz and 15% at 10 Hz. During tetanic stimulation at 100 Hz the transient amplitude decays to 20% in toad fibres and 40% in mouse fibres. This decrease could be associated with the phenomenon of inactivation of Ca2+ release, described by other authors. Recovery from inactivation, studied by a double stimuli protocol also indicates that in toad fibres the ability to release Ca2+ is abolished to a greater extent than in mouse fibres. In fact the ratio between the amplitudes of the second and first transient, when they are separated by a 10 ms interval, is 0.29 for toad and 0.58 for mouse fibres. In toad fibres, recovery from inactivation, to about 80 % of the initial value, occurs with a tau of 32 ms at 22 degrees C; while in mouse fibres recovery from inactivation is almost complete and occurs with a tau of 36 ms under the same conditions. The results indicate that Ca2+ release in enzymatically dissociated mammalian muscle fibres inactivates to a smaller extent than in intact amphibian muscle fibres.

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.

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.