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Extracellular adenosine 5'-triphosphate (ATP) has profound effects on membrane conductance and on the intracellular free [Ca(2+)] ([Ca(2+)](i)) in cultured skeletal muscle cells. The aim of the present study was to examine the occurrence and to characterize the properties of such responses during mammalian muscle development in vivo. The effect of ATP (0.2 mM) was tested on membrane current and [Ca(2+)](i) in freshly isolated pre- and post-natal mouse skeletal muscle cells. Pre-natal cells were from 14- to 19-day-old fetuses. In pre- and early post-natal cells, very small elevations of [Ca(2+)](i) (50 nM) following ATP application could be detected with the fluorescent indicator fura-2. A clear subsarcolemmal rise in [Ca(2+)] was however associated to the presence of ATP, as demonstrated by increased activity of plasma membrane Ca(2+)-activated K(+) channels in cells bathed in a depolarizing, high-calcium-containing solution. In cells voltage-clamped at -80 mV in external Tyrode, ATP induced an inward current associated with an increased membrane conductance. The mean maximal amplitude of the ATP-induced current was -0.84 +/- 0.07 A/F ( n=39). The response to ATP was still present after birth, although its amplitude tended to decrease with post-natal development and was completely absent in muscle cells from 3- to 6-month-old mice. The ATP-induced current could be abolished reversibly by suramin. Our results suggest that, over the range of developmental stages examined, skeletal muscle cells display an ionotropic purinergic signalling pathway with functional properties qualitatively consistent with what is observed in cultured myotubes.

The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were studied on fast twitch muscles of rodents. To identify the site of action of the drug, single fibers for voltage-clamp measurements, heavy SR vesicles for calcium efflux studies and solubilized SR calcium release channels/RYRs for lipid bilayer studies were isolated. Using the double Vaseline-gap or the silicone-clamp technique, dantrolene was found to suppress the depolarization-induced elevation in intracellular calcium concentration ([Ca2+]i) by inhibiting the release of calcium from the SR. The suppression of [Ca2+]i was dose-dependent, with no effect at or below 1 microM and a 53 +/- 8% (mean +/- SEM, n = 9, cut fibers) attenuation at 0 mV with 25 microM of extracellularly applied dantrolene. The drug was not found to be more effective if injected than if applied extracellularly. Calculating the SR calcium release revealed an equal suppression of the steady (53 +/- 8%) and of the early peak component (46 +/- 6%). The drug did not interfere with the activation of the voltage sensor in as much as the voltage dependence of both intramembrane charge movements and the L-type calcium currents (I(Ca)) were left, essentially, unaltered. However, the inactivation of I(Ca) was slowed fourfold, and the conductance was reduced from 200 +/- 16 to 143 +/- 8 SF(-1) (n = 10). Dantrolene was found to inhibit thymol-stimulated calcium efflux from heavy SR vesicles by 44 +/- 10% (n = 3) at 12 microM. On the other hand, dantrolene failed to affect the isolated RYR incorporated into lipid bilayers. The channel displayed a constant open probability for as long as 30-50 min after the application of the drug. These data locate the binding site for dantrolene to be on the SR membrane, but be distinct from the purified RYR itself.

When the endoplasmic reticulum (ER) calcium store is depleted, a Ca2+ influx is activated from the extracellular milieu to refill the intracellular stores. This well-regulated Ca2+ uptake mechanism, called store-operated Ca2+ entry (SOCE), depends on the cooperation of several proteins as STIM1, Orai1 and TRPC1. The role of STIM1 as the calcium sensor of the ER and Orai1 as the Ca2+ influx channel is well-known from the recent publications, but the function of TRPC1 as a store-operated channel remains elusive.Here TRPC1 was overexpressed by liposome-mediated transfection in C2C12 mouse skeletal muscle cell line. Overexpression was confirmed at mRNA level by RT-PCR and at protein level by immunostaining and Western-blot. The SOCE mechanism was studied by measuring the changes in [Ca2+]i evoked by the re-addition of 1.8 mM [Ca2+]e following the SERCA-inhibition by thapsigargin. As a result of TRPC1 overexpression, the amplitude and the maximum of the derivative of SOCE was significantly increased. When YM-58483, the antagonist of TRPC1 was used, these differences were eliminated, moreover in TRPC1-overexpressing myotubes the SOCE was slightly but not significantly lower, suggesting the downregulation of the STIM1-Orai1 system. This decrease in the expression level of STIM1 was confirmed by Western-blot together with the downregulation of SERCA. As a consequence a reduction in maximal Ca2+ uptake, and a higher resting [Ca2+]i following the transients evoked by 120 mM KCl were detected. Morphological changes caused by the overexpression of TRPC1 were also observed. The differentiation of the myoblasts started later, and the myotubes were thinner in TRPC1-overexpressing cultures.Our results suggest that enhancing the expression level of TRPC1 increases SOCE and has a negative feedback effect on the STIM1-Orai1 system, suggesting a cooperation between these proteins.

Capsaicin (100 nM to 1 microM) and anandamide (200 nM to 10 microM) caused a transient increase in fluorescence of fura-2 loaded cultured small trigeminal neurones of rats measured with a ratiometric technique. The percentage of cells responding to capsaicin at 100 nM, 330 nM and 1 microM was 47.4%, 45.3%, and 70.4%, respectively. Averaged peak value of fluorescense ratio (R) at 340 and 380 nm excitation was slightly dose dependent. Peaks of anandamide-induced transients were R = 0.2 at 200 nM and 0.16 at 10 microM. Near 40% of capsaicin-sensitive cells responded also to anandamide. Anandamide (200 nM) inhibited the capsaicin-induced calcium influx. The results suggest that anandamide increases intracellular calcium and inhibits capsaicin-evoked calcium transients.

1. Single muscle fibres were dissociated enzymatically from the extensor digitorum communis muscle of rats. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation-contraction coupling were studied under voltage clamp conditions in the presence and absence of the local anaesthetic tetracaine. 2. Changes in intracellular calcium concentration ([Ca2+]i) were monitored using the calcium sensitive dyes antipyrylazo III and fura-2 and the rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) was calculated. Tetracaine decreased the maximal attained [Ca2+]i and suppressed, in a dose-dependent manner, both the early peak and the steady level of Rrel in the voltage range examined. 3. The concentration dependence of the effects on the two kinetic components of Rrel were almost identical with a half-effective concentration (K50) of 70 and 71 microM and a Hill coefficient (nH) of 2.7 and 2.3 for the peak and the steady level, respectively. Furthermore, the drug did not alter the peak to steady level ratio up to a concentration (50 microM) that caused a 35 +/- 5 % reduction in calcium release. Higher concentrations did suppress the ratio but the degree of suppression was voltage independent. 4. Tetracaine (50 microM) neither influenced the total available intramembrane charge nor altered its membrane potential dependence. It shifted the transfer function, the normalized SR permeability versus normalized charge to the right, indicating that similar charge transfer caused a smaller increase in SR permeability. 5. To explore the site of action of tetracaine further the ryanodine receptor (RyR) calcium release channel of the SR was purified and reconstituted into planar lipid bilayers. The reconstituted channel had a conductance of 511 +/- 14 pS (n = 8) in symmetric 250 mM KCl that was not affected by tetracaine. Tetracaine decreased the open probability of the channel in a concentration-dependent manner with K50 = 68 microM and nH = 1.5. 6. These experiments show that tetracaine suppresses SR calcium release in enzymatic isolated mammalian skeletal muscle fibres. This effect is due, presumably, to the decreased open probability of the RyR in the presence of the drug. Since both the inactivating peak and the steady level of Rrel were equally affected by tetracaine, our observations suggest that there is a tight coupling between these kinetic components of SR calcium release in mammalian skeletal muscle.

1. Single muscle fibres were dissociated enzymatically from the extensor digitorum longus and communis muscles of rats and guinea-pigs. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation-contraction coupling were studied under voltage clamp conditions. 2. The voltage dependence of intramembrane charge movement followed a two-state Boltzmann distribution with maximal available charge of 26.1 +/- 1.5 and 26.1 +/- 1.3 nC microF-1, mid-point voltage of -35.1 +/- 5.0 and -42.2 +/- 1.2 mV and steepness of 16.7 +/- 2.2 and 17.0 +/- 1.9 mV (means +/- S.E.M., n = 7 and 4) in rats and guinea-pigs, respectively. 3. Intracellular calcium concentration ([Ca2+]i) was monitored using the calcium-sensitive dyes antipyrylazo III, fura-2 and mag-fura-5. Resting [Ca2+]i was similar in rats and guinea-pigs with 125 +/- 18 and 115 +/- 8 nM (n = 10 and 9), respectively, while the maximal increase for a 100 ms depolarization to 0 mV was larger in rats (6.3 +/- 1.0 microM; n = 7), than in guinea-pigs (2.8 +/- 0.3; n = 4). 4. The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) displayed an early peak followed by a fast and a slow decline to a quasi maintained steady level. After normalizing Rrel to the estimated SR calcium content (1.2 +/- 0.1 and 0.9 +/- 0.1 mM in rats and guinea-pigs, respectively) and correcting for depletion of calcium in the SR the peak and steady levels at 0 mV, respectively, were found to be 2.50 +/- 0.08 and 0.81 +/- 0.06% ms-1 in rats and 2.43 +/- 0.25 and 0.88 +/- 0.01% ms-1 in guinea-pigs. The voltage dependence was essentially the same in both species, but different from that in amphibians. 5. These experiments show that enzymatic isolation yields functionally intact mammalian skeletal muscle fibres for Vaseline gap experiments. The data also suggest a close connection in the regulation of the different kinetic components of SR calcium release in mammalian skeletal muscle.

Intracellular calcium release induced by transient applications of phosphoinositide agonists was measured using adherent single HaCaT keratino- cytes loaded with the acetoxymethyl derivative of fura- 2. Application of ATP, bradykinin and formyl-Met- Leu-Phe (fMLP) resulted in a transient increase in in- tracellular calcium concentration ((Ca 2+ )i) with an av- erage half-width of 40 ± 21 s and a decay time constant of 15 ± 10 s (mean ± SD, n = 108), irrespective of the agonist applied. The cells could be classified into two groups: in 53% of the cells repeated stimulation brought about a progressively smaller change in (Ca 2+ )i (type 1 cells), whereas in the remaining cells the amplitude of the calcium transients was essentially un- changed (type 2 cells). Furthermore, calcium tran- sients in type 1 cells had broader half-widths and slower decays. No difference was found between the agonists in respect of the characteristics of the evoked calcium transient within each subpopulation. How- ever, bradykinin and fMLP desensitized some cells. These results indicate that the activation of the inositol trisphospate transduction pathway by different ago- nists induces a characteristic elevation of (Ca 2+ )i within a given cell. Our results demonstrate that cul- tured HaCaT keratinocytes are heterogeneous in re- spect of the calcium transients evoked by the activa- tors of this second messenger system.

Cytoplasmic pH has major effects on most cellular processes in skeletal muscle, including its ability to develop force. Protons are continuously extruded from the cytoplasm against their electrochemical gradient as shown by the considerably more alkaline pH in the resting muscle than the predicted pH value if protons were distributed passively. We aimed to determine the contribution of the t-system proton extrusion mechanisms to this gradient and the diffusive proton flux of the t-system. To do this we trapped 10 mM of the pH-sensitive dye HPTS inside the tubular (t-) system of mechanically skinned fibres from the rat extensor digitorum longus muscles and continuously imaged dye fluorescence during changes in internal solution pH, [Na+], [K+] and [ATP] by confocal microscopy. Calibrations using monensin showed that in normally polarized fibres with 36 mM [Na+]cyto that pHt-sys was 7.50 ± 0.12 (n=3), 7.91 ± 0.20 (n=3) and 8.31 ± 0.29 (n=4) at pH-cyto of 6.8, 7.2 and 7.5, respectively. In the presence of 162 mM [Na+]cyto, with or without amiloride, the pHt-sys and [Na+]t-sys were similar to cytoplasmic values. The addition of 50 µM amiloride to normally polarized fibres with 36 mM [Na+]cyto increased pHt-sys further indicating that the Na+-H+ exchanger (NHE) was the major protein responsible for extruding protons from the cytoplasm. The pH difference across the t-system membrane at rest is reduced by NHE activity, which moves protons against the inward diffusive proton flux of the resting muscle fibre. We calculated the diffusive proton flux across the t-system to be 2.7 +/- 0.9 e-4 m/s (mean +/- SEM, n=3).

Kv1.3, a voltage-gated potassium channel found in human T cells, enters a non-conducting state during prolonged depolarization by the slow P/C type inactivation, whose exact mechanism is yet to be determined. We have previously shown that acidic extracellular pH (pHe) slows the inactivation process in the presence of low extracellular potassium concentration ([K+]e), whereas speeds it in the presence of high [K+]e. Our aim was to generate a gating scheme, based on a Markov model, which is able to explain the experimental results as a continuous function of pHe and [K+]e.We developed a new computer program based on the Gillespie algorithm that is able to generate simulated records from given gating schemes and compare the resulting curve to the original recording, i.e. it provides an indicator for the goodness of a given scheme. Optimizing of the fitted parameters was achieved using a genetic algorithm. The algorithm was implemented in parallel and run in multi-core and multi-processor environments.As the program enabled us to optimize the gating scheme for more than one measurement record at a time, we could conclude that our simple model having two open microstates and one inactivation pathway from both of them was unable to explain the effects of pHe and [K+]e. Introduction of a new binary parameter and therefore the duplication of inactivation pathways was needed to fully explain the experimental results. We therefore assume that not only the binding of a single potassium ion influences the speed of inactivation, but the conducting pore can have two conformations with different conduction and inactivation properties depending on the [K+]e.

1. Intramembrane charge movements and changes in intracellular calcium concentration were recorded simultaneously in voltage clamped cut skeletal muscle fibres of the frog in the presence and absence of tetracaine. 2. Extracellular application of 20 microM tetracaine reduced the increase in myoplasmic [Ca2+]. The effect on the underlying calcium release flux from the sarcoplasmic reticulum was to suppress the peak of the release while sparing the steady level attained at the end of 100 ms clamp depolarizations. 3. While the peak of the release flux at corresponding voltages was reduced by 62% after the addition of tetracaine, the rate of inactivation was the same when the pulses elicited release fluxes of similar amplitude. 4. Higher concentrations of tetracaine, 0.2 mM, abolished the calcium signal in stretched fibres whereas in slack fibres this concentration left a non-inactivating calcium release flux. 5. Lowering the extracellular pH antagonized the effect of the drug both on charge movements and on calcium signals. The permanently charged analogue tetracaine methobromide lacked effects on excitation-contraction coupling. 6. These results imply that the two kinetic components of calcium release flux have very different tetracaine sensitivities. They are also consistent with an intracellular site of action of the drug at low concentration. Taken together they strongly suggest that the inactivating and non-inactivating components of calcium release correspond to different pathways: one that inactivates, is sensitive to tetracaine and is controlled by calcium, and another that does not inactivate, is much less sensitive to tetracaine and is directly controlled by voltage.

Skeletal muscle contraction is initiated by the increase in the intracellular calcium concentration ([Ca2+]) following the depolarization of the surface- and transverse-(T-) tubular membranes (see the review by Ebashi, 1991). Calcium ions are released into the myoplasm from an intracellular store, the sarcoplasmic reticulum (SR), through the ryanodine receptor calcium release channel (Imagawa et al., 1987; Inui et al., 1987). The voltage sensitive step that links channel opening to depolarization is the displacement of permanent charges (charge movement, Schneider and Chandler, 1973) found in the T-tubular membrane and localized in the DHP receptor (Rios and Pizarro, 1991).

Four manifestations of excitation-contraction (E-C) coupling were derived from measurements in cut skeletal muscle fibers of the frog, voltage clamped in a Vaseline-gap chamber: intramembranous charge movement currents, myoplasmic [Ca2+] transients, flux of calcium release from the sarcoplasmic reticulum (SR), and the intrinsic optical transparency change that accompanies calcium release. In attempts to suppress Ca release by direct effects on the SR, three interventions were applied: (a) a conditioning pulse that causes calcium release and inhibits release in subsequent pulses by Ca-dependent inactivation; (b) a series of brief, large pulses, separated by long intervals (greater than 700 ms), which deplete Ca2+ in the SR; and (c) intracellular application of the release channel blocker ruthenium red. All these reduced calcium release flux. None was expected to affect directly the voltage sensor of the T-tubule; however, all of them reduced or eliminated a component of charge movement current with the following characteristics: (a) delayed onset, peaking 10-20 ms into the pulse; (b) current reversal during the pulse, with an inward phase after the outward peak; and (c) OFF transient of smaller magnitude than the ON, of variable polarity, and sometimes biphasic. When the total charge movement current had a visible hump, the positive phase of the current eliminated by the interventions agreed with the hump in timing and size. The component of charge movement current blocked by the interventions was greater and had a greater inward phase in slack fibers with high [EGTA] inside than in stretched fibers with no EGTA. Its amplitude at -40 mV was on average 0.26 A/F (SEM 0.03) in slack fibers. The waveform of release flux determined from the Ca transients measured simultaneously with the membrane currents had, as described previously (Melzer, W., E. Ríos, and M. F. Schneider. 1984. Biophysical Journal. 45:637-641), an early peak followed by a descent to a steady level during the pulse. The time at which this peak occurred was highly correlated with the time to peak of the current suppressed, occurring on average 6.9 ms later (SEM 0.73 ms). The current suppressed by the above interventions in all cases had a time course similar to the time derivative of the release flux; specifically, the peak of the time derivative of release flux preceded the peak of the current suppressed by 0.7 ms (SEM 0.6 ms). The magnitude of the current blocked was highly correlated with the inhibitory effect of the interventions on Ca2+ release flux.(ABSTRACT TRUNCATED AT 400 WORDS)

Thymol is widely used as a general antiseptic and antioxidant compound in the medical practice and industry, and also as a stabilizer to several therapeutic agents, including halothane. Thus intoxication with thymol may occur in case of ingestion or improper anesthesia. In the present study, therefore, concentration-dependent effects of thymol (30–600 micro-grams) were studied on calcium and potassium currents in enzymatically isolated rat skeletal muscle fibers using the double vaseline gap voltage clamp technique. Thymol suppressed both Ca and K currents in a concentration-dependent manner, the EC50 values were 193 ± 26 and 93 ± 11 μM, with Hill coefficients of 2.52 ± 0.29 and 1.51 ± 0.18, respectively. Thymol had a biphasic effect on Ca current kinetics: time to peak current and the time constant for inactivation increased at lower (100–200 μM) but decreased below their control values at higher (600 μM) concentrations. Inactivation of K current was also significantly accelerated by thymol (200–300 μM). These effects of thymol developed rapidly and were partially reversible. In spite of the marked effects on the time-dependent properties, thymol caused no change in the current-voltage relationship of Ca and K peak currents. Present results revealed marked suppression of Ca and K currents in skeletal muscle, similar to results obtained previously in cardiac cells. Furthermore, it is possible that part of the suppressive effects of halothane on Ca and K currents, observed experimentally, may be attributed to the concomitant presence of thymol in the superfusate.

1. The effects of perchlorate ions (1-8 mM) on intramembrane charge movement, myoplasmic Antipyrylazo III Ca2+ transients and contractile activation were examined in voltage-clamped cut skeletal muscle fibres of the frog. 2. Perchlorate shifted both the voltage dependence of charge movement and the rheobase of the strength-duration relation for contraction threshold towards more negative membrane potentials. 3. Both charge movements and myoplasmic Ca2+ transients were much slower at the new rheobase in the presence of perchlorate than in the control solution but there was no change in the threshold amount of charge or in the calculated peak binding of Ca2+ to troponin C. 4. The peak release rate had a steeper voltage dependence than the non-linear charge, but a lower concentration (2 mM) of perchlorate shifted both voltage dependences equally without altering the maxima in the amount of charge and in the rate of Ca2+ release. 5. The voltage dependence of the difference between total charge and charge at the threshold of Ca2+ transients agreed well with the voltage dependence of the rate of Ca2+ release in both the presence and absence of perchlorate. 6. It is concluded that the effect of perchlorate on contractile activation can be accounted for by its action on the intramembrane charge movement responsible for contraction, without significant effects on subsequent Ca2+ release from the sarcoplasmic reticulum or on Ca2+ binding to regulatory sites of troponin C.

Antipyrylazo III calcium transients from voltage-clamped, cut skeletal muscle fibers of the frog were recorded, and the calcium binding to the regulatory sites of troponin C was calculated. The strength-duration curve for the contraction threshold was determined. It was found that the increase in myoplasmic calcium concentration necessary to produce the same level of contractile activation, i.e., the just visible movement, was approximately 60% higher at more positive membrane potentials resulting from short depolarizing pulses than at rheobase. However, using biochemical data for the kON and kOFF rate coefficients of the binding sites, the calculated maximums of the calcium binding curves were about the same at different voltages, and the time to maximum saturation was roughly equal to the latency of the contractions. To characterize the calcium binding in intact fibers more accurately, those values of the kON and kOFF rate coefficients that gave equal peak saturations during threshold movement at different membrane potentials were determined.

The role of elevated intracellular calcium concentration ([Ca2+]) in activating calcium release from the sarcoplasmic reticulum (SR) was studied in skeletal muscle fibers microinjected with strong calcium buffers. After the injection of 3.8 +/- 0.5 mM (mean +/- S.E. of mean, n = 16) BAPTA (1,2-bis[o-aminophenoxy]ethane- N,N,N',N'-tetraacetic acid) or 2.2-2.8 mM fura-2 the normal increase in [Ca2+] during a depolarizing pulse was virtually eliminated. Even though calcium was released from the SR the kinetics of this release were markedly altered: the extensive buffering selectively eliminated the early peak component of SR calcium release with no effect on the maintained steady level. Microinjections of similar volumes but with low concentrations of fura-2 had no significant effect on the release waveform. The calcium released by voltage-dependent activation during depolarization may thus be involved in activating further calcium release, that is, in a calcium-induced calcium release mechanism.

Measurements of intracellular free magnesium concentration ([Mg2+]i) were performed on enzymatically isolated skeletal muscle fibers from mice, using the fluorescent ratiometric indicator mag-indo-1. An original procedure was developed to calibrate the dye response within the fibers: fibers were first permeabilized with saponin in the presence of a given extracellular magnesium concentration and were then embedded in silicone grease. The dye was then pressure microinjected into the saponin-permeabilized silicone-embedded fibers, and fluorescence was measured. The results show that for all tested [Mg2+], the value of the measured fluorescence ratio was higher than that found in aqueous solutions. Furthermore, the apparent binding curve that could be fit to the in vivo ratio data was shifted toward higher [Mg2+] by a factor of approximately 2. Using the in vivo calibration parameters, the mean resting [Mg2+]i was found to be 1.53 +/- 0.16 mM (n = 7). In an attempt to gain insight into the myoplasmic magnesium buffering capacity, we measured, together with mag-indo-1 fluorescence, the current elicited by the application of carbamylcholine (CCh) to the endplate of isolated fibers, in the presence of a high extracellular magnesium concentration. The results show that, under these conditions, a change in [Mg2+]i displaying a time course and amplitude qualitatively consistent with the CCh-induced inward current can be measured.

The effects of tetracaine on charge movements and on antipyrylazo III signals monitoring intracellular delta [Ca2+] were compared in cut frog semitendinosus muscle fibers in a single vaseline gap-voltage clamp. Low tetracaine concentrations (25-40 microM) markedly reduced delta [Ca2+] signals and shifted the rheobase. However, they neither influenced charge movement nor that peak delta [Ca2+] value associated with the contractile threshold. Higher tetracaine concentrations (100-200 microM) partly inhibited charge movements in cut fibers. They separated a steeply voltage-sensitive charge, some of whose features resembled 'q gamma' reported in intact fibers, and whose movement preceded delta [Ca2+] signals at threshold. These findings: (a) directly confirm an earlier suggestion that tetracaine acts on steps in excitation-contraction coupling rather than myofilament activation; (b) show that tetracaine at low concentrations can directly interfere with sarcoplasmic reticular calcium release without modifying charge movement; (c) show that the tetracaine-sensitive charge, first found in intact fibers, also exists in cut fibers; and (d) make it unlikely that tetracaine-sensitive charge transfer is a consequence of Ca2+ release as suggested on earlier occasions.

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Background As an essential trace element selenium plays a significant role in many physiological functions of the organs. It is found within muscles as selenocystein in selenoprotein N, which is involved in redox-modulated calcium homeostasis and in protection against oxidative stress. Methods The effects of two different selenium compounds (selenate and NanoSe in 0.5 and 5 ppm concentration for two weeks) on muscle properties of mice were examined by measuring in vivo muscle performance, in vitro force in soleus (SOL) and extensor digitorum longus (EDL) muscles and changes in intracellular Ca2+ concentration in single fibers from flexor digitorum brevis (FDB) muscle.. Western-blot analysis on muscle lysates of EDL and SOL were used to measure the selenoprotein N expression. Control mice received 0.3 ppm Se. Results While the grip force did not change, 5 ppm selenium diets significantly increased the speed of voluntary running and the daily distance covered. Both forms of selenium increased significantly the amplitude of single twitches in EDL and SOL muscle in a concentration dependent manner. Selenate increased fatigue resistance in SOL. The amplitude of the calcium transients evoked by KCl depolarization increased significantly from the control of 343 ± 44 nM to 671 ± 51 nM in the presence of 0.5 ppm selenate in FDB fibers. In parallel, the rate of calcium release during short depolarizations increased significantly from 28.4 ± 2.2 to 45.5 ± 3.8 and 52.1 ± 1.9 μM/ms in the presence of 0.5 ppm NanoSe and selenate, respectively. In 0.5 ppm concentration both selenium compounds increased significantly the selenoprotein N expression only in EDL muscle. Conclusions Selenium supplementation augments calcium release from the sarcoplasmic reticulum thus improves skeletal muscle performance. These effects are accompanied by the increased selenoprotein N expression in the muscles which could result in increased oxidative stress tolerance in case of long lasting contraction. Electronic supplementary material The online version of this article (doi:10.1186/s12986-016-0134-6) contains supplementary material, which is available to authorized users.

It was shown previously that the Ca analogue Gd inhibits RyR1 gating symmetrically with a Kd about 5.5 microM and Hill coefficient (nH) of 4 both on cis and trans side using single channel electrophysiology. We further tested the RyR1-lanthanide interaction using two lanthanides - having an ionic radii between Ca2+ and Gd3+ - by bilayer measurements and ryanodine binding experiments. Cis inhibition of RyR1 by Eu was characterized by a binding constant of Kd=167±5 nM and an nH of 2±0.1 while trans inhibition exhibits Kd=4.8±0.2 microM and nN of 5.2±1.2. The inhibition constants for Sm on the cis side are Kd=64.3±2.5 nM and nH=2.2±0.2 while on the trans side Kd=6.15±0.13 microM and nH=4.68±0.45. Inhibition by Eu and Sm are potential and polarity dependent in contrast to Gd due to the differences in ionic radii of these lanthanides. Increasing the ionic radius from 0.938 (Gd) to 0.964 (Sm) increased the binding affinity from 5.6 microM to 64.3 nM revealing that the size of Ca binding pocket is only slightly higher than the ionic radius of Sm. Ryanodine (Ry) binding experiments revealed that lanthanides bind - at least partially - to the regulatory Ca binding site because the dose response curve of 3H Ry binding starts with an increase of Ry binding, which amounts to about 40% for Eu and 70% for Sm of basic Ry binding. A model has been proposed for one possible spatial arrangement of lanthanide and calcium binding sites of the RyR1 pore based on the ionic radii of Ca and the tested lanthanides. Supported by OTKA 81923.

CaV1.1e is the calcium channel splice variant of embryonic skeletal muscle. It functions as voltage sensor in EC coupling, but in contrast to the adult CaV1.1a variant it also supports sizeable calcium currents activating at the same membrane potential as SR calcium release. Mis-splicing of CaV1.1 results in elevated levels of the CaV1.1e variant in mature muscle that correlate with the severity of muscle weakness in patients suffering from dystrophic myotonia. Therefore we hypothesize that exclusive expression of CaV1.1e in exon 29 knockout mice will cause muscle weakness. In muscles of wildtype mice expression of CaV1.1e declines after birth. However, two weeks after sciatic nerve resection CaV1.1e transcript levels are upregulated in paralyzed muscles, indicative of an activity-dependent regulation of CaV1.1e splicing. Quantitative RT-PCR analysis demonstrates that exon 29 knockouts express exclusively the CaV1.1e splice variant at levels comparable to total CaV1.1 in wildtype muscle. Combined voltage clamp and fluorescence calcium recordings of isolated muscle fibers from exon 29 knockout mice revealed a substantial influx-dependent component of the calcium transient. This not only alters myoplasmic calcium handling, but is further expected to prolong the muscle action potential and consequently reduce the tetanic fusion frequency. Surprisingly, these severe alterations of the muscle fiber properties were not accompanied by a dystrophic myotonia phenotype in knockout mice. A battery of behavioral tests (home-cage activity, voluntary and forced running, grip strength and rotarod tests) revealed no signs of muscle weakness or altered motor activity compared to wildtype siblings. Thus, development of dystrophic myotonia may require the combinatorial action of several mis-spliced genes. Alternatively, strong compensatory mechanisms may cause the absence of the disease phenotype in exon 29 knockout mice. Support: FWF P23479, W1101, OTKA-NN107765, TAMOP-4.2.2./B.-10/1-2010-0024.