scholarly journals Dihydropyridine Receptors as Voltage Sensors for a Depolarization-evoked, IP3R-mediated, Slow Calcium Signal in Skeletal Muscle Cells

2002 ◽  
Vol 121 (1) ◽  
pp. 3-16 ◽  
Author(s):  
Roberto Araya ◽  
José L. Liberona ◽  
J. César Cárdenas ◽  
Nora Riveros ◽  
Manuel Estrada ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Calcium Release ◽  
Calcium Transient ◽  
Voltage Sensor ◽  
Calcium Transients ◽  
Early Gene ◽  
Calcium Signal ◽  
Calcium Signals ◽  
Transfected Cells ◽  
Voltage Dependent

The dihydropyridine receptor (DHPR), normally a voltage-dependent calcium channel, functions in skeletal muscle essentially as a voltage sensor, triggering intracellular calcium release for excitation-contraction coupling. In addition to this fast calcium release, via ryanodine receptor (RYR) channels, depolarization of skeletal myotubes evokes slow calcium waves, unrelated to contraction, that involve the cell nucleus (Jaimovich, E., R. Reyes, J.L. Liberona, and J.A. Powell. 2000. Am. J. Physiol. Cell Physiol. 278:C998–C1010). We tested the hypothesis that DHPR may also be the voltage sensor for these slow calcium signals. In cultures of primary rat myotubes, 10 μM nifedipine (a DHPR inhibitor) completely blocked the slow calcium (fluo-3-fluorescence) transient after 47 mM K+ depolarization and only partially reduced the fast Ca2+ signal. Dysgenic myotubes from the GLT cell line, which do not express the α1 subunit of the DHPR, did not show either type of calcium transient following depolarization. After transfection of the α1 DNA into the GLT cells, K+ depolarization induced slow calcium transients that were similar to those present in normal C2C12 and normal NLT cell lines. Slow calcium transients in transfected cells were blocked by nifedipine as well as by the G protein inhibitor, pertussis toxin, but not by ryanodine, the RYR inhibitor. Since slow Ca2+ transients appear to be mediated by IP3, we measured the increase of IP3 mass after K+ depolarization. The IP3 transient seen in control cells was inhibited by nifedipine and was absent in nontransfected dysgenic cells, but α1-transfected cells recovered the depolarization-induced IP3 transient. In normal myotubes, 10 μM nifedipine, but not ryanodine, inhibited c-jun and c-fos mRNA increase after K+ depolarization. These results suggest a role for DHPR-mediated calcium signals in regulation of early gene expression. A model of excitation-transcription coupling is presented in which both G proteins and IP3 appear as important downstream mediators after sensing of depolarization by DHPR.

Caffeine attenuates contraction-induced diminutions of the intracellular calcium transient in mouse lumbrical muscle ex vivo

2019 ◽  
Vol 97 (5) ◽  
pp. 429-435 ◽  
Author(s):  
Ian C. Smith ◽  
Rene Vandenboom ◽  
A. Russell Tupling
Keyword(s):  
Skeletal Muscle ◽  
Intracellular Calcium ◽  
Calcium Release ◽  
Ex Vivo ◽  
Calcium Transient ◽  
Calcium Transients ◽  
Inotropic Effects ◽  
Intracellular Calcium Transient ◽  
Lumbrical Muscle ◽  
Lumbrical Muscles

The amount of calcium released from the sarcoplasmic reticulum in skeletal muscle rapidly declines during repeated twitch contractions. In this study, we test the hypothesis that caffeine can mitigate these contraction-induced declines in calcium release. Lumbrical muscles were isolated from male C57BL/6 mice and loaded with the calcium-sensitive indicator, AM-furaptra. Muscles were then stimulated at 8 Hz for 2.0 s in the presence or absence of 0.5 mM caffeine, at either 30 °C or 37 °C. The amplitude and area of the furaptra-based intracellular calcium transients and force produced during twitch contractions were calculated. For each of these measures, the values for twitch 16 relative to twitch 1 were higher in the presence of caffeine than in the absence of caffeine at both temperatures. We conclude that caffeine can attenuate contraction-induced diminutions of calcium release during repeated twitch contractions, thereby contributing to the inotropic effects of caffeine.

scholarly journals Relationship of calcium transients to calcium currents and charge movements in myotubes expressing skeletal and cardiac dihydropyridine receptors.

1994 ◽  
Vol 103 (1) ◽  
pp. 125-147 ◽  
Author(s):  
J García ◽  
T Tanabe ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Calcium Channel ◽  
Calcium Current ◽  
Calcium Release ◽  
Voltage Dependence ◽  
Calcium Transient ◽  
Calcium Currents ◽  
Calcium Transients ◽  
Charge Movement ◽  
The Relationship

In both skeletal and cardiac muscle, the dihydropyridine (DHP) receptor is a critical element in excitation-contraction (e-c) coupling. However, the mechanism for calcium release is completely different in these muscles. In cardiac muscle the DHP receptor functions as a rapidly-activated calcium channel and the influx of calcium through this channel induces calcium release from the sarcoplasmic reticulum (SR). In contrast, in skeletal muscle the DHP receptor functions as a voltage sensor and as a slowly-activating calcium channel; in this case, the voltage sensor controls SR calcium release. It has been previously demonstrated that injection of dysgenic myotubes with cDNA (pCAC6) encoding the skeletal muscle DHP receptor restores the slow calcium current and skeletal type e-c coupling that does not require entry of external calcium (Tanabe, Beam, Powell, and Numa. 1988. Nature. 336:134-139). Furthermore, injection of cDNA (pCARD1) encoding the cardiac DHP receptor produces rapidly activating calcium current and cardiac type e-c coupling that does require calcium entry (Tanabe, Mikami, Numa, and Beam. 1990. Nature. 344:451-453). In this paper, we have studied the voltage dependence of, and the relationship between, charge movement, calcium transients, and calcium current in normal skeletal muscle cells in culture. In addition, we injected pCAC6 or pCARD1 into the nuclei of dysgenic myotubes and studied the relationship between the restored events and compared them with those of the normal cells. Charge movement and calcium currents were recorded with the whole cell patch-clamp technique. Calcium transients were measured with Fluo-3 introduced through the patch pipette. The kinetics and voltage dependence of the charge movement, calcium transients, and calcium current in dysgenic myotubes expressing pCAC6 were qualitatively similar to the ones elicited in normal myotubes: the calcium transient displayed a sigmoidal dependence on voltage and was still present after the addition of 0.5 mM Cd2+ + 0.1 mM La3+. In contrast, the calcium transient in dysgenic myotubes expressing pCARD1 followed the amplitude of the calcium current and thus showed a bell shaped dependence on voltage. In addition, the transient had a slower rate of rise than in pCAC6-injected myotubes and was abolished completely by the addition of Cd2+ + La3+.

Depolarization-induced slow calcium transients activate early genes in skeletal muscle cells

2003 ◽  
Vol 284 (6) ◽  
pp. C1438-C1447 ◽  
Author(s):  
Maria Angélica Carrasco ◽  
Nora Riveros ◽  
Juan Rı́os ◽  
Marioly Müller ◽  
Francisco Torres ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gene Activation ◽  
Muscle Cells ◽  
Camp Response Element Binding ◽  
Early Gene ◽  
Calcium Signals ◽  
Creb Phosphorylation ◽  
Early Genes ◽  
Erk Phosphorylation ◽  
Element Binding Protein

The signaling mechanisms by which skeletal muscle electrical activity leads to changes in gene expression remain largely undefined. We have reported that myotube depolarization induces calcium signals in the cytosol and nucleus via inositol 1,4,5-trisphosphate (IP3) and phosphorylation of both ERK1/2 and cAMP-response element-binding protein (CREB). We now describe the calcium dependence of P-CREB and P-ERK induction and of the increases in mRNA of the early genes c- fos, c- jun, and egr-1. Increased phosphorylation and early gene activation were maintained in the absence of extracellular calcium, while the increase in intracellular calcium induced by caffeine could mimic the depolarization stimulus. Depolarization performed either in the presence of the IP3 inhibitors 2-aminoethoxydiphenyl borate or xestospongin C or on cells loaded with BAPTA-AM, in which slow calcium signals were abolished, resulted in decreased activation of the early genes examined. Both early gene activation and CREB phosphorylation were inhibited by ERK phosphorylation blockade. These data suggest a role for calcium in the transcription-related events that follow membrane depolarization in muscle cells.

scholarly journals Dissociation of charge movement from calcium release and calcium current in skeletal myotubes by gabapentin

2002 ◽  
Vol 283 (3) ◽  
pp. C941-C949 ◽  
Author(s):  
Kris J. Alden ◽  
Jesús Garcı́a
Keyword(s):  
Skeletal Muscle ◽  
Calcium Current ◽  
Calcium Release ◽  
Maximum Amplitude ◽  
Charge Movement ◽  
Initial Voltage ◽  
Extra Charge ◽  
Voltage Dependent ◽  
Integral Role ◽  
Skeletal Myotubes

The skeletal muscle L-type calcium channel or dihydropyridine receptor (DHPR) plays an integral role in excitation-contraction (E-C) coupling. Its activation initiates three sequential events: charge movement (Qr), calcium release, and calcium current ( I Ca,L). This relationship suggests that changes in Qr might affect release and I Ca,L. Here we studied the effect of gabapentin (GBP) on the three events generated by DHPRs in skeletal myotubes in culture. GBP specifically binds to the α2/δ1 subunit of the brain and skeletal muscle DHPR. Myotubes were stimulated with a protocol that included a depolarizing prepulse to inactivate voltage-dependent proteins other than DHPRs. Gabapentin (50 μM) significantly increased Qr while decreasing the rate of rise of calcium transients. Gabapentin also reduced the maximum amplitude of the I Ca,L (as we previously reported) without modifying the kinetics of activation. Exposure of GBP-treated myotubes to 10 μM nifedipine prevented the increase of Qr promoted by this drug, indicating that the extra charge recorded originated from DHPRs. Our data suggest that GBP dissociates the functions of the DHPR from the initial voltage-sensing step and implicates a role for the α2/δ1 subunit in E-C coupling.

scholarly journals Contractions of dysgenic skeletal muscle triggered by a potentiated, endogenous calcium current.

1991 ◽  
Vol 97 (4) ◽  
pp. 687-696 ◽  
Author(s):  
B A Adams ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Sarcoplasmic Reticulum ◽  
Calcium Current ◽  
Calcium Release ◽  
Voltage Sensor ◽  
Bay K 8644 ◽  
Direct Electrical Stimulation ◽  
External Calcium ◽  
Muscular Dysgenesis

The dihydropyridine (DHP) receptor of normal skeletal muscle is hypothesized to function as the voltage sensor for excitation-contraction (E-C) coupling, and also as the calcium channel underlying a slowly activating, DHP-sensitive current (termed ICa-s). Skeletal muscle from mice with muscular dysgenesis lacks both E-C coupling and ICa-s. However, dysgenic skeletal muscle does express a small DHP-sensitive calcium current (termed ICa-dvs) which is kinetically and pharmacologically distinct from ICa-s. We have examined the ability of ICa-dys, or the DHP receptor underlying it, to couple depolarization and contraction. Under most conditions ICa-dys is small (approximately 1 pA/pF) and dysgenic myotubes do not contract in response to sarcolemmal depolarization. However, in the combined presence of the DHP agonist Bay K 8644 (1 microM) and elevated external calcium (10 mM), ICa-dys is strongly potentiated and some dysgenic myotubes contract in response to direct electrical stimulation. These contractions are blocked by removing external calcium, by adding 0.5 mM cadmium to the bath, or by replacing Bay K 8644 with the DHP antagonist (+)-PN 200-110. Only myotubes having a density of ICa-dys greater than approximately 4 pA/pF produce detectible contractions, and the strength of contraction is positively correlated with the density of ICa-dys. Thus, unlike the contractions of normal myotubes, the contractions of dysgenic myotubes require calcium entry. These results demonstrate that the DHP receptor underlying ICa-dys is unable to function as a "voltage sensor" that directly couples membrane depolarization to calcium release from the sarcoplasmic reticulum.

scholarly journals An allosteric model of the molecular interactions of excitation-contraction coupling in skeletal muscle.

1993 ◽  
Vol 102 (3) ◽  
pp. 449-481 ◽  
Author(s):  
E Ríos ◽  
M Karhanek ◽  
J Ma ◽  
A González
Keyword(s):  
Skeletal Muscle ◽  
Calcium Release ◽  
Kinetic Equations ◽  
Voltage Sensor ◽  
Charge Movement ◽  
Calcium Release Channel ◽  
Open Probability ◽  
Release Channel ◽  
Voltage Sensors ◽  
Function Relationship

A contact interaction is proposed to exist between the voltage sensor of the transverse tubular membrane of skeletal muscle and the calcium release channel of the sarcoplasmic reticulum. This interaction is given a quantitative formulation inspired in the Monod, Wyman, and Changeux model of allosteric transitions in hemoglobin (Monod, J., J. Wyman, and J.-P. Changeux. 1965. Journal of Molecular Biology. 12:88-118), and analogous to one proposed by Marks and Jones for voltage-dependent Ca channels (Marks, T. N., and S. W. Jones. 1992. Journal of General Physiology. 99:367-390). The allosteric protein is the calcium release channel, a homotetramer, with two accessible states, closed and open. The kinetics and equilibrium of this transition are modulated by voltage sensors (dihydropyridine receptors) pictured as four units per release channel, each undergoing independent voltage-driven transitions between two states (resting and activating). For each voltage sensor that moves to the activating state, the tendency of the channel to open increases by an equal (large) factor. The equilibrium and kinetic equations of the model are solved and shown to reproduce well a number of experimentally measured relationships including: charge movement (Q) vs. voltage, open probability of the release channel (Po) vs. voltage, the transfer function relationship Po vs. Q, and the kinetics of charge movement, release activation, and deactivation. The main consequence of the assumption of allosteric coupling is that primary effects on the release channel are transmitted backward to the voltage sensor and give secondary effects. Thus, the model reproduces well the effects of perchlorate, described in the two previous articles, under the assumption that the primary effect is to increase the intrinsic tendency of the release channel to open, with no direct effects on the voltage sensor. This modification of the open-closed equilibrium of the release channel causes a shift in the equilibrium dependency of charge movement with voltage. The paradoxical slowing of charge movement by perchlorate also results from reciprocal effects of the channel on the allosterically coupled voltage sensors. The observations of the previous articles plus the simulations in this article constitute functional evidence of allosteric transmission.

189 CHARACTERIZATION OF THE FIRST SPERM-INDUCED CALCIUM TRANSIENT IN PIG OOCYTES

2016 ◽  
Vol 28 (2) ◽  
pp. 225
Author(s):  
C. Wang ◽  
Z. Machaty
Keyword(s):  
Calcium Concentration ◽  
Calcium Transient ◽  
Calcium Oscillations ◽  
Calcium Transients ◽  
Intracellular Free Calcium ◽  
Calcium Signal ◽  
Free Calcium ◽  
Essential Information ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration

Fertilization in mammals is associated with repetitive elevations in the oocytes’ intracellular free calcium concentration. The elevations are triggered by the fertilizing sperm and are responsible for stimulating embryo development. In mouse oocytes, the sperm-induced calcium signal starts with a calcium rise that is larger and longer in duration than any succeeding transients. It also has unique characteristics: it begins with a rapid increase for 2–3 s followed by a shoulder, which is an inflection point that represents a brief decline in the rise of calcium levels. Once calcium level reaches its maximum, it decreases but remains elevated for several minutes while it is superimposed by several smaller calcium spikes. In bovine oocytes the situation is somewhat different. In this species, the first sperm-induced calcium transient is larger than the additional spikes but it lacks the sustained elevation phase and is not superimposed by small calcium rises. In the present study our purpose was to characterise the first sperm-induced calcium transient in pig oocytes. Oocytes were obtained from ovaries of prepubertal gilts collected at an abattoir and matured in vitro for 44 h. Mature oocytes were loaded with the calcium indicator dye fura-2; subsequently, they were either IVF or used for intracytoplasmic sperm injection (ICSI). Changes in their intracellular free calcium concentration were then immediately monitored using InCyt Im2, a dual-wavelength fluorescence imaging system. Characteristics of the first transients (including amplitude and duration) were compared to those of the additional ones using Student’s t-test. We found that in oocytes that underwent IVF (n = 11), the oscillations started 83.4 ± 23.2 min after adding the sperm to the oocytes. In the ICSI group (n = 10 oocytes) the calcium oscillations started sooner, 27.1 ± 17.7 min after injection. The average peak amplitude and the mean interval between the calcium transients varied among individual oocytes, but no significant differences were found between the IVF and ICSI groups (which on average were fluorescence ratio of 2.6 ± 1.1 and 23.5 ± 11.4 min, respectively; P > 0.1). The oscillation patterns showed slight differences between individual oocytes in terms of spike frequency, which has been described before and may be due to variations in the amount of sperm-derived activating factor present in the ooplasm. Most importantly, in all oocytes measured, the initial calcium spike showed no differences when compared to subsequent calcium transients: its amplitude and duration was similar to the additional transients. This points at potential species-specific differences in the regulation of calcium signalling in oocytes and provides essential information for the better understanding of the fertilization process. This work was supported by Agriculture and Food Research Initiative Competitive Grant 2011–67015–30006 from the USDA National Institute of Food and Agriculture.

scholarly journals Measurement of calcium transients and slow calcium current in myotubes.

1994 ◽  
Vol 103 (1) ◽  
pp. 107-123 ◽  
Author(s):  
J García ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Calcium Current ◽  
Calcium Transient ◽  
Calcium Currents ◽  
Calcium Transients ◽  
Patch Clamp Technique ◽  
Slow Increase ◽  
Adult Skeletal Muscle ◽  
Calcium Indicator ◽  
Cultured Myotubes

The purpose of this study was to characterize excitation-contraction (e-c) coupling in myotubes for comparison with e-c coupling of adult skeletal muscle. The whole cell configuration of the patch clamp technique was used in conjunction with the calcium indicator dye Fluo-3 to study the calcium transients and slow calcium currents elicited by voltage clamp pulses in cultured myotubes obtained from neonatal mice. Cells were held at -80 mV and stimulated with 15-20 ms test depolarizations preceded and followed by voltage steps designed to isolate the slow calcium current. The slow calcium current had a threshold for activation of about 0 mV; the peak amplitude of the current reached a maximum at 30 to 40 mV a and then declined for still stronger depolarizations. The calcium transient had a threshold of about -10 mV, and its amplitude increased as a sigmoidal function of test potential and did not decrease again even for test depolarizations sufficiently strong (> or = 50 mV) that the amplitude of the slow calcium current became very small. Thus, the slow calcium current in myotubes appears to have a negligible role in the process of depolarization-induced release of intracellular calcium and this process in myotubes is essentially like that in adult skeletal muscle. After repolarization, however, the decay of the calcium transient in myotubes was very slow (hundreds of ms) compared to adult muscle, particularly after strong depolarizations that triggered larger calcium transients. Moreover, when cells were repolarized after strong depolarizations, the transient typically continued to increase slowly for up to several tens of ms before the onset of decay. This continued increase after repolarization was abolished by the addition of 5 mM BAPTA to the patch pipette although the rapid depolarization-induced release was not, suggesting that the slow increase might be a regenerative response triggered by the depolarization-induced release of calcium. The addition of either 0.5 mM Cd2+ + 0.1 mM La3+ or the dihydropyridine (+)-PN 200-110 (1 microM) reduced the amplitude of the calcium transient by mechanisms that appeared to be unrelated to the block of current that these agents produce. In the majority of cells, the decay of the transient was accelerated by the addition of the heavy metals or the dihydropyridine, consistent with the idea that the removal system becomes saturated for large calcium releases and becomes more efficient when the size of the release is reduced.

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