Frog Skeletal Muscle Fibers Research Articles

Quantal Properties of Voltage-Dependent Ca2+ Release in Frog Skeletal Muscle Persist After Reduction of [Ca2+] in the Sarcoplasmic Reticulum.

In skeletal muscle, the Ca2+ release flux elicited by a voltage clamp pulse rises to an early peak that inactivates rapidly to a much lower steady level. Using a double pulse protocol the fast inactivation follows an arithmetic rule: if the conditioning depolarization is less than or equal to the test depolarization, then decay (peak minus steady level) in the conditioning release is approximately equal to suppression (unconditioned minus conditioned peak) of the test release. This is due to quantal activation by voltage, analogous to the quantal activation of IP3 receptor channels. Two mechanisms are possible. One is the existence of subsets of channels with different sensitivities to voltage. The other is that the clusters of Ca2+-gated Ryanodine Receptor (RyR) β in the parajunctional terminal cisternae might constitute the quantal units. These Ca2+-gated channels are activated by the release of Ca2+ through the voltage-gated RyR α channels. If the RyR β were at the basis of quantal release, it should be modified by strong inhibition of the primary voltage-gated release. This was attained in two ways, by sarcoplasmic reticulum (SR) Ca2+ depletion and by voltage-dependent inactivation. Both procedures reduced global Ca2+ release flux, but SR Ca2+ depletion reduced the single RyR current as well. The effect of both interventions on the quantal properties of Ca2+ release in frog skeletal muscle fibers were studied under voltage clamp. The quantal properties of release were preserved regardless of the inhibitory maneuver applied. These findings put a limit on the role of the Ca2+-activated component of release in generating quantal activation.

High Time Resolution Analysis of Voltage-Dependent and Voltage-Independent Calcium Sparks in Frog Skeletal Muscle Fibers.

In amphibian skeletal muscle calcium (Ca2+) sparks occur both as voltage-dependent and voltage-independent ligand-activated release events. However, whether their properties and their origin show similarities are still in debate. Elevated K+, constant Cl– content solutions were used to initiate small depolarizations of the resting membrane potential to activate dihydropyridine receptors (DHPR) and caffeine to open ryanodine receptors (RyR) on intact fibers. The properties of Ca2+ sparks observed under control conditions were compared to those measured on depolarized cells and those after caffeine treatment. Calcium sparks were recorded on intact frog skeletal muscle fibers using high time resolution confocal microscopy (x-y scan: 30 Hz). Sparks were elicited by 1 mmol/l caffeine or subthreshold depolarization to different membrane potentials. Both treatments increased the frequency of sparks and altered their morphology. Images were analyzed by custom-made computer programs. Both the amplitude (in ΔF/F0; 0.259 ± 0.001 vs. 0.164 ± 0.001; n = 24942 and 43326, respectively; mean ± SE, p < 0.001) and the full width at half maximum (FWHM, in μm; parallel with fiber axis: 2.34 ± 0.01 vs. 1.92 ± 0.01, p < 0.001; perpendicular to fiber axis: 2.08 ± 0.01 vs. 1.68 ± 0.01, p < 0.001) of sparks was significantly greater after caffeine treatment than on depolarized cells. 9.8% of the sparks detected on depolarized fibers and about one third of the caffeine activated sparks (29.7%) overlapped with another one on the previous frame on x-y scans. Centre of overlapping sparks travelled significantly longer distances between consecutive frames after caffeine treatment then after depolarization (in μm; 1.66 ± 0.01 vs. 0.95 ± 0.01, p < 0.001). Our results suggest that the two types of ryanodine receptors, the junctional RyRs controlled by DHPRs and the parajunctional RyRs are activated independently, using alternate ways, with the possibility of cooperation between neighboring release channels.

The dynamics of Ca2+ within the sarcoplasmic reticulum of frog skeletal muscle. A simulation study

In skeletal muscle, Ca2+ release from the sarcoplasmic reticulum (SR) triggers contraction. In this study we develop a two compartment model to account for the Ca2+ dynamics in frog skeletal muscle fibers. The two compartments in the model correspond to the SR and the cytoplasm, where the myofibrils are placed. We use a detailed model for the several Ca2+ binding proteins in the cytoplasm in line with previous models. As a new feature, Ca2+ binding sites within the SR, attributed to calsequestrin, are modeled based on experimentally obtained properties. The intra SR Ca2+ buffer shows cooperativity, well represented by a Hill equation with parameters that depend on the initial [Ca2+] in the SR ([Ca2+]SR). The number of total sites as well as the [Ca2+]SR of half saturation are reduced as the resting [Ca2+]SR is reduced, on the other hand the Hill number is not changed. The buffer power remained roughly constant.The release process is activated by a voltage dependent mechanism that increases the Ca2+ permeability of the SR. We use the permeability time course and amplitude experimentally obtained during a voltage clamp pulse to drive the simulations. This model successfully reproduces the SR and cytoplasmic transients observed.Additionally, we simulate [Ca2+] SR transients in the case of high concentration of extrinsic Ca2+ buffers added to the cytoplasm to explore what properties of the permeability are necessary to account for the experimentally observed [Ca2+]SR transients. The main novelty of the model, the intra SR Ca2+ buffer, is crucial for reproducing the experimental observations and it would be of use in future theoretical studies of excitation contraction coupling in skeletal muscle.

Increased sodium channel use-dependent inhibition by a new potent analogue of tocainide greatly enhances in vivo antimyotonic activity

Although the sodium channel blocker, mexiletine, is the first choice drug in myotonia, some myotonic patients remain unsatisfied due to contraindications, lack of tolerability, or incomplete response. More therapeutic options are thus needed for myotonic patients, which require clinical trials based on solid preclinical data. In previous structure-activity relationship studies, we identified two newly-synthesized derivatives of tocainide, To040 and To042, with greatly enhanced potency and use-dependent behavior in inhibiting sodium currents in frog skeletal muscle fibers. The current study was performed to verify their potential as antimyotonic agents. Patch-clamp experiments show that both compounds, especially To042, are greatly more potent and use-dependent blockers of human skeletal muscle hNav1.4 channels compared to tocainide and mexiletine. Reduced effects on F1586C hNav1.4 mutant suggest that the compounds bind to the local anesthetic receptor, but that the increased hindrance and lipophilia of the N-substituent may further strengthen drug-receptor interaction and use-dependence. Compared to mexiletine, To042 was 120 times more potent to block hNav1.4 channels in a myotonia-like cellular condition and 100 times more potent to improve muscle stiffness in vivo in a previously-validated rat model of myotonia. To explore toxicological profile, To042 was tested on hERG potassium currents, motor coordination using rotarod, and C2C12 cell line for cytotoxicity. All these experiments suggest a satisfactory therapeutic index for To042. This study shows that, owing to a huge use-dependent block of sodium channels, To042 is a promising candidate drug for myotonia and possibly other membrane excitability disorders, warranting further preclinical and human studies.

Differential effects of contractile potentiators on action potential-induced Ca2+ transients of frog and mouse skeletal muscle fibres.

Muscle fibres, isolated from frog tibialis anterior and mouse flexor digitorum brevis (FDB) were loaded with the fast dye MagFluo-4 to study the effects of potentiators caffeine, nitrate, Zn2+ and perchlorate on Ca2+ transients elicited by single action potentials. Overall, the potentiators doubled the transients amplitude and prolonged by about 1.5-fold their decay time. In contrast, as shown here for the first time, nitrate and Zn2+, but not caffeine, activated a late, secondary component of the transient rising phase of frog but not mouse, fibres. The rise time was increased from 1.9ms in normal solution (NR) to 3.3ms (nitrate) and 4.4ms (Zn2+). In NR, a single exponential, fitted the rising phase of calcium transients of frog (τ1=0.47ms) and mouse (τ1=0.28ms). In nitrate and Zn2+ only frog transients showed a secondary exponential component, τ2=0.72ms (nitrate) and 0.94ms, (Zn2+). We suggest that nitrate and Zn2+ activate a late slower component of the ΔF/F signals of frog but not of mouse fibres, possibly promoting Ca2+ induced Ca2+ release at level of the RyR3, that in frog muscle fibres are localized in the para-junctional region of the triads and are absent in mouse FDB muscle fibres.

Excitation contraction uncoupling by high intracellular [Ca2+] in frog skeletal muscle: a voltage clamp study.

Raising the intracellular [Ca2+] ([Ca2+]i) was previously found to produce uncoupling between the electrical depolarization of the transverse tubules and contraction in skinned muscle fibers. Here we study the effect of elevated [Ca2+]i in voltage clamped cut fibers of frog skeletal muscle to establish how the charge movement, a measure of the activation of the dihydropyridine receptors (DHPR)-voltage sensors, and Ca2+ release, a consequence of the opening of the ryanodine receptor (RyR)-release channels, were affected. [Ca2+]i was raised by various procedures (pharmacological release from the sarcoplasmic reticulum, application of high [Ca2+]i intracellular solution, permeabilization of the plasma membrane by a Ca2+ ionophore) all of which produced impairment of excitation-contraction coupling. The charge movement was reduced from 20.2±1.24 to 9.9±0.94 nC/μF meanwhile the Ca2+ release flux was reduced from 13.5+0.7 to 2.2±0.3μM/ms (n=33). This suggests that a significant fraction of the DHPRs that remained functional, could not activate RyRs, and were therefore presumably disconnected. These results are broadly consistent with the original reports in skinned fibers. Uncoupling was prevented by the addition to the intracellular solution of the protease inhibitor leupeptin. In approximately 40% of the uncoupled cells we observed that the [Ca2+]i transient continued to rise after the voltage clamp pulse was turned off. This loss of control by membrane voltage suggests that the uncoupled release channels might have another mechanism of activation, likely by Ca2+.

Compartment calcium model of frog skeletal muscle during activation

Skeletal muscle contraction is triggered by a rise in calcium (Ca2+) concentration in the myofibrillar space. The objective of this study was to develop a voltage dependent compartment model of Ca2+ dynamics in frog skeletal muscle fibers. The compartment model corresponds to the myofibrillar space (MS) and a calcium store, the sarcoplasmic reticulum (SR). Ca2+ is released from the SR to the MS based on the voltage and is able to bind to several proteins in the MS. We use a detailed model to account for voltage dependent Ca2+ release and inactivation. With this model, we are able to match previous experimental data for Ca2+ release and binding to proteins for an applied (fixed) voltage. We explore the sensitivity of parameters in the model and illustrate the importance of inactivation of the SR; during a long depolarization, the SR must be inactivated in order to achieve realistic Ca2+ concentrations in the MS. A Hodgkin Huxley type model was also developed to describe voltage at the surface membrane using electrophysiological data from previous experiments. This voltage model was then used as the time dependent voltage to determine Ca2+ release from the SR. With this fully coupled model, we were able to match previous experimental results for Ca2+ concentrations for a given applied current. Additionally, we examined simulated Ca2+ concentrations in the case of twitch and tetanus, corresponding to different applied currents. The developed model is robust and reproduces many aspects of voltage dependent calcium signaling in frog skeletal muscle fibers. This modeling framework provides a platform for future studies of excitation contraction coupling in skeletal muscle fibers.

Efficient Automatic Analysis of High-Speed Confocal Images Containing Localized Calcium Release Events

High-speed confocal microscopy is a relatively new method used to investigate localized calcium release events, most notably calcium sparks. Line scan images can reliably be recorded at speeds up to 60000 lines/s while for X-Y images 120 frame/s rate is achievable. The increased amount of recorded data raises the necessity of efficient automatic analysis methods. A set of images recorded on frog skeletal muscle cells after caffeine treatment was used to test the described methods.We present a collection of methods useful for the automatic analysis of high speed line scan and X-Y recordings, mostly based on stationary wavelet transform (SWT). Our one-dimensional SWT based ember-detection method was adapted to detect sparks on high-speed line scan recordings. The centerline of detected sparks is automatically marked on each scan line to assess the spatial and temporal characteristics of the spark. 3.66% of all sparks (n=23704) in the test image set had complex spatial properties revealed by high-speed imaging. Amplitude and full width at half maximum (FWHM) is determined for each scan line to calculate the amount of calcium released (signal mass).On X-Y image series two-dimensional SWT was used to denoise the images and to detect sparks on each frame. The orientation of the skeletal muscle cell and the position of sarcomeres are determined using fast Fourier-transform on the stationary components of the image series. The position of the center for each spark is determined relatively to the sarcomeres. FWHM is calculated in the axes perpendicular (FWHMX) and parallel (FWHMY) to the sarcomeres. For the test dataset (n=22426), FWHMX/FWHMY is 1.22.The above-described methods enable us to automatically analyze high-speed confocal images and to reveal some of their properties which were hidden on conventional confocal images.

A study of store dependent Ca2+ influx in frog skeletal muscle

Ca²⁺ influx across the plasma membrane upon drastic reduction of the sarcoplasmic reticulum Ca²⁺ content was studied in voltage clamped frog skeletal muscle fibers. Depletion was produced by the application of 30 μM cyclopiazonic acid (CPA) in Ca²⁺-free, [Mg²⁺] = 8 mM external salines and produced an increase in resting free myoplasmic [Ca²⁺]. Once depletion was attained the external solution was changed to one containing the same concentration of the drug but with Ca²⁺ instead of Mg²⁺. Of 27 fibers studied only nine showed a secondary increase in free myoplasmic [Ca²⁺] upon readmitting Ca²⁺ in the external perfusate. In the presence of CPA the resting myoplasmic [Ca²⁺] in Ca²⁺-free external saline was 0.08 ± 0.01 μM (Mean ± SEM), and in Ca²⁺-containing external saline 0.10 ± 0.02 μM when the intracellular solution contained [EGTA] = 5 mM (n = 18). In cells with lower (0.5 mM) intracellular [EGTA] resting [Ca²⁺] went from 0.35 +/- 0.08 μM in Ca²⁺-free external solution to 0.42 +/- 0.12 μM upon reapplication of Ca²⁺(n = 9). In both cases the differences between means were not statistically significant (paired t test, p = 0.13 in high EGTA and p = 0.25 in low EGTA). In the nine fibers that showed a secondary increase of resting [Ca²⁺] the holding current measured at -90 mV did not significantly change. These results suggest the Ca²⁺ entry secondary to store depletion is a labile mechanism in frog skeletal muscle and when present does not have an obvious electrical manifestation.

Role of Ca2+ in the rapid cooling-induced Ca2+ release from sarcoplasmic reticulum in ferret cardiac muscles

Rapid lowering of the solution temperature (rapid cooling, RC) from 24 to 3°C within 3 s releases considerable amounts of Ca2+ from the sarcoplasmic reticulum (SR) in mammalian cardiac muscles. In this study, we investigated the intracellular mechanism of RC-induced Ca2+ release, especially the role of Ca2+, in ferret ventricular muscle. Saponin-treated skinned trabeculae were placed in a glass capillary, and the amount of Ca2+ released from the SR by RC and caffeine (50 mM) was measured with fluo-3. It was estimated that in the presence of ATP about 45% of the Ca2+ content in the SR was released by RC. The amount of SR Ca2+ released by RC was unchanged by the replacement of ATP by AMP-PCP (a non-hydrolysable ATP analogue and agonist for the ryanodine receptor but not for the Ca2+ pump of SR), suggesting that the suppression of the Ca2+ pump of SR at low temperature might not be a major mechanism in RC-induced Ca2+ release. The free Ca2+ concentration of the solution used for triggering RC-induced Ca2+ release was estimated to be only about 20 nM with fluo-3 or aequorin. When this solution was applied to the preparation at 3°C, only a small amount of Ca2+ was released from SR presumably by the Ca2+-induced Ca2+ release (CICR) mechanism. Thus, in mammalian cardiac muscles, RC releases a part of the (<50%) stored Ca2+ contained in the SR, and the mechanism of RC-induced Ca2+ release may differ from that of CICR, which is thought to play a role in frog skeletal muscle fibres that express ryanodine receptors of different types.

Non-crossbridge calcium-dependent stiffness in slow and fast skeletal fibres from mouse muscle

We showed previously that force development in frog and FDB mouse skeletal muscle fibres is preceded by an increase of fibre stiffness occurring well before crossbridge attachment and force generation. This stiffness increase, referred to as static stiffness, is due to a Ca(2+)-dependent stiffening of a non-crossbridge sarcomere structure which we suggested could be attributed to the titin filaments. To investigate further the role of titin in static stiffness, we measured static stiffness properties at 24 and 35°C in soleus and EDL mouse muscle fibres which are known to express different titin isoforms. We found that static stiffness was present in both soleus and EDL fibres, however, its value was about five times greater in EDL than in soleus fibres. The rate of development of static stiffness on stimulation increased with temperature and was slightly faster in EDL than in soleus in agreement with previously published data on the time course of the intracellular Ca(2+) transients in these muscles. The present results show that the presence of a non-crossbridge Ca(2+)-dependent stiffening of the muscle fibre is a physiological general characteristic of skeletal muscle. Static stiffness depends on fibre type, being greater and developing faster in fast than in slow fibres. Our observations are consistent with the idea that titin stiffening on contraction improves the sarcomere structure stability. Such an action in fact seems to be more important in EDL fast fibre than in soleus slow fibres.

Different Capabilities for CICR of Skeletal Muscle of Amphibians and Mammals, Demonstrated through the Response to Artificial Ca Sparks

To probe CICR (Endo, Phys. Rev. 2009), single skeletal muscle cells were stimulated with artificial Ca2+ sparks generated by spot 2-photon breakdown of NDBF-EGTA, while their Ca2+ response was monitored by confocal imaging of fluorescence of fluo 4FF. The cells had their plasmalemma permeabilized. Sparks were applied either inside cells or slightly outside, a method that avoids photodamage and allows measurement of the triggering [Ca2+] at precisely the time when the cellular response is elicited. Frog skeletal muscle fibers usually responded to stimuli > 1 μM with a propagating, all-or-none Ca2+ wave. The moving wavefront peaked at 3-5 μM; the calculated Ca2+ release flux peaked at 50-100 mM/s and the wave velocity was ∼100 μm/s (and slightly faster in the axial direction). In other cases a “frustrated” response was generated, which stopped after propagating for 20-30 μm, usually following progressive reduction in Ca2+ release flux. In striking contrast, the same stimuli failed to elicit a response when applied to cells of the mouse FDB muscle. Variants of the stimulus included “whorls” (2-P radiation in scribbles that covered regions of ∼3 μm diameter), involving much greater amounts of Ca2+ than an artificial spark. Whether placed outside or inside the cells, even at intensities that caused persistent cell damage, whorls elicited neither the fully propagated nor the frustrated response. These results directly demonstrate that frog muscle is capable of CICR but do not prove a physiologic role, as they were obtained on permeabilized cells. In conclusion, mammalian muscle does not have the CICR capability, which other works have associated with the presence of RyR isoform 3 (Pouvreau et al., PNAS 2007; Legrand et al., J. Physiol. 2008). Supported by NIAMS and NHLBI (NIH).

Two inhibitors of store operated Ca2+ entry suppress excitation contraction coupling in frog skeletal muscle

Two drugs, 2-APB and SKF-96365, commonly used to block Store Operated Ca(2+) Entry (SOCE) were found to have inhibitory effects at different levels of the Excitation Contraction Coupling (ECC) process in frog skeletal muscle fibers. Treatment with either drug suppressed Ca(2+) release from the Sarcoplasmic Reticulum, but this effect was not due to inhibition of SOCE as it occurred in Ca(2+)-free conditions. 2-APB applied extracellularly at 100 microM, the usual concentration to suppress SOCE, reversibly reduced the charge movement elicited by pulses in the range between -45 and -35 mV from 7.99 +/- 0.73 nC/microF (N = 17) before drug application to 6.27 +/- 0.68 nC/microF in the presence of 2-APB. This effect was mostly on the delayed Q(gamma) component. In fibers treated with the SERCA ATPase inhibitor CPA the Q(gamma) component disappeared, under this condition the application of 2-APB did not suppress the remaining charge movement. Thus the effect of 2-APB on charge movement currents seemed to be secondary to the suppression of Ca(2+) release, likely occurring directly on the release channels. No significant suppression of ECC was observed for concentration below 20 muM. 2-APB also inhibited the L-type Ca(2+) current (20 +/- 4%, N = 8). On the other hand SKF-96365 had a direct effect on the voltage sensor promoting its voltage dependent inactivation. Applied at 20 muM, a typical concentration used for inhibiting SOCE, to fibers held at -80 mV inhibited the charge moved in response to pulses ranging -45 to -30 mV from 7.95 +/- 2.59 nC/microF to 3.48 +/- 0.9 nC/microF (N = 12). A parallel reduction of Ca(2+) release was observed. Wash out was drastically increased by hyperpolarization of the holding potential to -100 mV. SKF-96365 also inhibited the L-type Ca(2+) current (41 +/- 8%, N = 4) and increased its rate of inactivation.

Open channel block and beyond

After settling down from the excitement of seeing single channels open and close in the membrane of a living cell for the first time (Neher & Sakmann, 1976), the question that arose was what could these extraordinary recordings tell us? At the time we had no idea how many different applications we would soon find for the patch clamp technique. Most of these applications followed from the subsequent improvement of the gigaseal, which dramatically increased the power of the patch clamp. But as a ‘first application’ for single channel recording, in an article in The Journal of Physiology, Neher & Steinbach (1978) turned to a question about the mechanism of action of local anaesthetics. It was known that local anaesthetics reduce the current through the membranes of excitable cells. The endplate current at the neuromuscular synapse becomes smaller in the presence of a local anaesthetic, and its decay follows a double-exponential time course. These results could be accounted for by a model in which a channel first opens, and a local anaesthetic molecule then binds to a site within the open channel to block it (Ruff, 1977). These drugs could thus be pictured as acting like a plug in the drain of a bathtub. This model fitted the data well, and most researchers regarded it as very appealing. Neher and Steinbach noted a number of studies that had invoked a sequential mechanism of channel blockade, but this mechanism was not generally accepted as rigorously proven. The key result of two exponentials in the decay of the synaptic current indicated that the drug must induce the appearance of a new kinetic state of the channel. From an analysis of macroscopic currents alone, it was very difficult to decide whether the new state was an open or closed channel, or whether entry into the new state occurs from an open or closed channel. Single channel recordings from a frog skeletal muscle fibre Patch electrode solution containing 0.1 μm suberyldicholine alone in A, together with 5 μm (B), 10 μm (C), and 50 μm (D) QX-222. (Reproduced from Fig. 2 of Neher & Steinbach, 1978.) QX-222 and QX314 are both charged, and if their binding site resides within the channel, then the transmembrane potential should influence binding. The single channel data permitted a direct determination of the blocking and unblocking rates (F and G), and these rates did indeed depend on voltage. The magnitude of this voltage dependence suggested that the drug traverses 78% of the membrane potential upon binding. This study established open channel block as an important mechanism of drug action. But the study had much greater significance in providing the first illustration of the remarkable power of single channel recording in the elucidation of kinetic mechanism. By observing the closed state, the open state and the blocked state directly, some very strong statements could be made about how these states interconvert. For any ion channel mechanism, the single-molecule transitions contain information not readily extracted from the macroscopic currents that report the activity of populations of channels. This work thus initiated a new era of mechanistic studies of ion channels. A rigorous mathematical formulation of single-channel kinetics provided the essential theoretical tools for interpreting complex lifetime distributions (Colquhoun & Hawkes, 1982). Single channel recordings revealed correlations in the lifetimes of adjacent gating intervals that added to the information content uniquely available in this form of data (Jackson et al. 1983), and again a theoretical framework served to guide these studies (Fredkin et al. 1985). The enduring contribution of this paper was its illustration of the extraordinary power of single-channel recording in model discrimination and the elucidation of kinetic mechanism. The many applications of single-channel kinetics that followed this work brought our understanding of ion channels to a new level of detail. These studies offer valuable lessons that should prove useful in the study of other forms of single-molecule kinetics, most notably single-molecule fluorescence.

CICR and Calcium-Dependent Inactivation, Quantified Through the Response to Artificial Ca Sparks in Single Muscle Cells

Local calcium stimuli (artificial sparks) generated by 2-photon breakdown of the cage NDBF-EGTA were applied to evoke Ca release from the SR in single skeletal or cardiac muscle cells undergoing fast Ca imaging with the low affinity dye fluo 4FF. The figure shows selected sequential images of the Ca transient generated by a frog skeletal muscle fiber with permeabilized plasmalemma, in response to a spark (elicited outside the fiber to avoid photodamage). Two types of responses were observed: (i) an all-or-none wave -shown- that propagates over the entire cell and (ii) graded responses, which fail to propagate. Release analysis (Rios, JGP 1999; Figueroa, this meeting) separates SR release from simple diffusion of photo-released Ca into cells. The technique yields a sensitive measure of threshold [Ca2+] for release activation, which in the example (0.3 mM [Mg2+]cyto) was 1 μM, and can monitor inactivation by combining multiple stimuli. Modeling of these responses aims at describing quantitatively the properties of activation, as well as the roles of inactivation and depletion in the control of Ca release. Other details and acknowledgments are presented elsewhere (Figueroa, this meeting.)View Large Image | View Hi-Res Image | Download PowerPoint Slide

Three Types of Single Voltage-Dependent Potassium Channels in the Sarcolemma of Frog Skeletal Muscle

Patch-clamp experiments in the sarcolemma of frog skeletal muscle evidenced the presence of three types of voltage-dependent single-channel K(+) currents. According to their unitary conductance at a membrane voltage of +40 mV, we classified them as 16-, 13-, and 7-pS K(+) channels. The 16-pS K(+) channels are active close to a membrane voltage of -80 mV and they do not become inactivated during voltage pulses of 100 ms. Within 10 min after beginning the recording, these channels developed rundown with an exponential time course. The 13-pS K(+) channels are active near -60 mV; upon a 100-ms depolarization, they exhibited inactivation with an approximate exponential time course. The 7-pS K(+) channels were recorded at voltages positive to 0 mV. In patches containing all three types of K(+) channels, the ensemble average currents resemble the kinetic properties of the macroscopic delayed rectifier K(+) currents recorded in skeletal muscle and other tissues. In conclusion, the biophysical properties of unitary K(+) currents suggest that these single-channel K(+) currents may underlie the macroscopic delayed K(+) currents in frog skeletal muscle fibers. In addition, since the 16- and 13-pS channels were more frequently recorded, both are the main contributors to the delayed K(+) currents.

Load Dependence of Structural Changes in the Myosin Filament during Muscle Activation

The M3 and M6 X-ray reflections from the myosin filaments of skeletal muscle correspond to axial periodicities SM3 and SM6 which are 14.34 and 7.19 nm at rest and 14.58 and 7.31 nm at the tetanus plateau (force T0). This ∼1.5% periodicity increase is much larger than the instantaneous filament compliance, and is probably due to an activation-dependent change in filament structure. SM6 increases faster than SM3 during force development (Brunello et al., J. Physiol. 577:971, 2006) and the M6 intensity at T0 is much higher than expected from the myosin motor conformation (Huxley et al. J. Mol. Biol., 363:743, 2006), suggesting that the M6 mainly originates from other filament components. To better understand these structural changes, we recorded X-ray patterns from intact fibers of frog skeletal muscle (∼2.15 μm sarcomere length, 4°C) during tetanic contraction under full force control. Force was held near zero for 50 ms after the start of stimulation, increased within 5 ms to T0 and held there for 230 ms, then returned to zero. During the initial fiber shortening at zero force SM3 was almost constant but SM6 increased by 0.6%. SM3 and SM6 increased to the tetanus plateau values listed above within 10 ms of the force step to T0, and decreased to steady values of 14.38 nm and 7.24 nm within 20 ms of the force step to zero. These results show that i, the full increase in SM3 and SM6 on activation is triggered by the force increase; ii, activation at zero force produces a partial (0.6%) increase in the periodicity of the structure responsible for the M6 reflection. Supported by NIH (5R01AR49033), MiUR and CNISM (Italy) and MRC (UK).

Structural Changes in the Myosin Motors During Activation of Skeletal Muscle

Structural changes in the myosin motors during the transition from the resting state to the plateau of isometric contraction were investigated by X-ray interference from single fibers of frog skeletal muscle. Isolated intact fibers (2.1μm sarcomere length, 4°C) were mounted vertically at beamline ID2 of the ESRF synchrotron (Grenoble, France) between a loudspeaker motor and a capacitance force transducer. 2D diffraction patterns were collected on a CCD detector 10 m from the preparation with 5 ms time resolution. During the development of the isometric tetanus, the intensity of the M3 reflection, originating from the axial repeat of the myosin motors, first decreases to 30% (at 50 ms) of its resting value, then increases to a steady value 70% of that at rest. The M3 reflection has a major peak with spacing 14.34 nm at rest and two peaks with mean spacing 14.57 nm at the tetanus plateau (Linari et al., Proc. Natl. Acad. Sci. USA 97:7226, 2000). The changes in the fine structure of the M3 reflection during activation were best fit by a structural model in which (1) all thick filaments have the same mean spacing at a given time during activation (2) the number of active motors increases in proportion to the isometric force (Brunello et al., J. Physiol. 577:971 2006), (3) the conformation of the active motors is independent of the level of force and strain in the thick filament.Supported by MiUR Italy, MRC UK, CNISM, EMBO, ESRF, EMBL.

Charged Surface Area of Maurocalcine Determines Its Interaction with the Skeletal Ryanodine Receptor

The 33 amino acid scorpion toxin maurocalcine (MCa) has been shown to modify the gating of the skeletal-type ryanodine receptor (RyR1). Here we explored the effects of MCa and its mutants ([Ala 8]MCa, [Ala 19]MCa, [Ala 20]MCa, [Ala 22]MCa, [Ala 23]MCa, and [Ala 24]MCa) on RyR1 incorporated into artificial lipid bilayers and on elementary calcium release events (ECRE) in rat and frog skeletal muscle fibers. The peptides induced long-lasting subconductance states (LLSS) on RyR1 that lasted for several seconds. However, their average length and frequency were decreased if the mutation was placed farther away in the 3D structure from the critical 24Arg residue. The effect was strongly dependent on the direction of the current through the channel. If the direction was similar to that followed by calcium during release, the peptides were 8- to 10-fold less effective. In fibers long-lasting calcium release events were observed after the addition of the peptides. The average length of these events correlated well with the duration of LLSS. These data suggest that the effect of the peptide is governed by the large charged surface formed by residues Lys 20, Lys 22, Arg 23, Arg 24, and Lys 8. Our observations also indicate that the results from bilayer experiments mimic the in situ effects of MCa on RyR1.

Synchronization of Na/K pump molecules by an oscillating electric field

Synchronization of the Na/K pump molecules in a cell membrane was studied in frog skeletal muscle fibers using double Vaseline-gap voltage-clamp techniques. We found that the pumping rate of naturally random-paced pump molecules can be artificially synchronized by a pulsed, symmetric, oscillating membrane potential with a frequency comparable to the physiological turnover rate. The synchronized pump currents show separated outward and inward components, where the magnitude of the outward component is about three times the randomly-paced pump currents, and the magnitude-ratio of the outward to inward pump currents is close to 3:2, which reflects the stoichiometric ratio of the pump molecules. Once synchronized, the pumping rate is restricted to the field frequency, and the pump currents are mainly dependent on the field frequency, but not the field strength. In contrast to previous work, which by restraining the pumps at a presteady state succeeded in triggering the steps of the pump cycle only individually and between interruptions, here we synchronize the pumps running continuously and in a normal running mode.