Type I males of the Pacific midshipman fish ( Porichthys notatus ) vibrate their swimbladder to generate mating calls, or “hums,” that attract females to their nests. In contrast to the intermittent calls produced by male Atlantic toadfish ( Opsanus tau ), which occur with a duty cycle (calling time divided by total time) of only 3–8%, midshipman can call continuously for up to an hour. With 100% duty cycles and frequencies of 50–100 Hz (15°C), the superfast muscle fibers that surround the midshipman swimbladder may contract and relax as many as 360,000 times in 1 h. The energy for this activity is supported by a large volume of densely packed mitochondria that are found in the peripheral and central regions of the fiber. The remaining fiber cross section contains contractile filaments and a well-developed network of sarcoplasmic reticulum (SR) and triadic junctions. Here, to understand quantitatively how Ca 2+ is managed by midshipman fibers during calling, we measure (a) the Ca 2+ pumping-versus-pCa and force-versus-pCa relations in skinned fiber bundles and (b) changes in myoplasmic free [Ca 2+ ] (Δ[Ca 2+ ]) during stimulated activity of individual fibers microinjected with the Ca 2+ indicators Mag-fluo-4 and Fluo-4. As in toadfish, the force–pCa relation in midshipman is strongly right-shifted relative to the Ca 2+ pumping–pCa relation, and contractile activity is controlled in a synchronous, not asynchronous, fashion during electrical stimulation. SR Ca 2+ release per action potential is, however, approximately eightfold smaller in midshipman than in toadfish. Midshipman fibers have a larger time-averaged free [Ca 2+ ] during activity than toadfish fibers, which permits faster Ca 2+ pumping because the Ca 2+ pumps work closer to their maximum rate. Even with midshipman’s sustained release and pumping of Ca 2+ , however, the Ca 2+ energy cost of calling (per kilogram wet weight) is less than twofold more in midshipman than in toadfish.

We are wired with conducting cables called axons that rapidly transmit electrical signals (e.g., “Ouch!”) from, for example, the toe to the spinal cord. Because of the high internal resistance of axons (salt water rather than copper), a signal must be reinforced after traveling a short distance. Reinforcement is accomplished by ion channels, Na channels for detecting the signal and reinforcing it by driving it further positive (to near 50 mV) and K channels for then restoring it to the resting level (near −70 mV). The signal is called an action potential and has a duration of roughly a millisecond. The return of membrane voltage (V m ) to the resting level after an action potential is facilitated by “inactivation” of the Na channels: i.e., an internal particle diffuses into the mouth of any open Na channel and temporarily blocks it. Some types of K channels also show inactivation after being open for a time. N-type inactivation of K channels has a relatively fast time course and involves diffusion of the N-terminal of one of the channel’s four identical subunits into the channel’s inner mouth, if it is open. This mechanism is similar to Na channel inactivation. Both Na and K channels also display slower inactivation processes. C inactivation in K channels involves changes in the channel’s outer mouth, the “selectivity filter,” whose normal function is to prevent Na + ions from entering the K channel. C inactivation deforms the filter so that neither K + nor Na + can pass.

The ryanodine receptor (RyR)1 isoform of the sarcoplasmic reticulum (SR) Ca 2+ release channel is an essential component of all skeletal muscle fibers. RyR1s are detectable as “junctional feet” (JF) in the gap between the SR and the plasmalemma or T-tubules, and they are required for excitation–contraction (EC) coupling and differentiation. A second isoform, RyR3, does not sustain EC coupling and differentiation in the absence of RyR1 and is expressed at highly variable levels. Anatomically, RyR3 expression correlates with the presence of parajunctional feet (PJF), which are located on the sides of the SR junctional cisternae in an arrangement found only in fibers expressing RyR3. In frog muscle fibers, the presence of RyR3 and PJF correlates with the occurrence of Ca 2+ sparks, which are elementary SR Ca 2+ release events of the EC coupling machinery. Here, we explored the structural and functional roles of RyR3 by injecting zebrafish ( Danio rerio ) one-cell stage embryos with a morpholino designed to specifically silence RyR3 expression. In zebrafish larvae at 72 h postfertilization, fast-twitch fibers from wild-type (WT) tail muscles had abundant PJF. Silencing resulted in a drop of the PJF/JF ratio, from 0.79 in WT fibers to 0.03 in the morphants. The frequency with which Ca 2+ sparks were detected dropped correspondingly, from 0.083 to 0.001 sarcomere −1 s −1 . The few Ca 2+ sparks detected in morphant fibers were smaller in amplitude, duration, and spatial extent compared with those in WT fibers. Despite the almost complete disappearance of PJF and Ca 2+ sparks in morphant fibers, these fibers looked structurally normal and the swimming behavior of the larvae was not affected. This paper provides important evidence that RyR3 is the main constituent of the PJF and is the main contributor to the SR Ca 2+ flux underlying Ca 2+ sparks detected in fully differentiated frog and fish fibers.

The mating call of the Atlantic toadfish is generated by bursts of high-frequency twitches of the superfast twitch fibers that surround the swimbladder. At 16°C, a calling period can last several hours, with individual 80–100-Hz calls lasting ∼500 ms interleaved with silent periods (intercall intervals) lasting ∼10 s. To understand the intracellular movements of Ca 2+ during the intercall intervals, superfast fibers were microinjected with fluo-4, a high-affinity fluorescent Ca 2+ indicator, and stimulated by trains of 40 action potentials at 83 Hz, which mimics fiber activity during calling. The fluo-4 fluorescence signal was measured during and after the stimulus trains; the signal was also simulated with a kinetic model of the underlying myoplasmic Ca 2+ movements, including the binding and transport of Ca 2+ by the sarcoplasmic reticulum (SR) Ca 2+ pumps. The estimated total amount of Ca 2+ released from the SR during a first stimulus train is ∼6.5 mM (concentration referred to the myoplasmic water volume). At 40 ms after cessation of stimulation, the myoplasmic free Ca 2+ concentration ([Ca 2+ ]) is below the threshold for force generation (∼3 µM), yet the estimated concentration of released Ca 2+ remaining in the myoplasm (Δ[Ca M ]) is large, ∼5 mM, with ∼80% bound to parvalbumin. At 10 s after stimulation, [Ca 2+ ] is ∼90 nM (three times the assumed resting level) and Δ[Ca M ] is ∼1.3 mM, with 97% bound to parvalbumin. Ca 2+ movements during the intercall interval thus appear to be strongly influenced by (a) the accumulation of Ca 2+ on parvalbumin and (b) the slow rate of Ca 2+ pumping that ensues when parvalbumin lowers [Ca 2+ ] near the resting level. With repetitive stimulus trains initiated at 10-s intervals, Ca 2+ release and pumping come quickly into balance as a result of the stability (negative feedback) supplied by the increased rate of Ca 2+ pumping at higher [Ca 2+ ].

Single twitch fibers from frog leg muscles were isolated by dissection and micro-injected with furaptra, a rapidly responding fluorescent Ca 2+ indicator. Indicator resting fluorescence (F R ) and the change evoked by an action potential (ΔF) were measured at long sarcomere length (16°C); ΔF/F R was scaled to units of Δf CaD , the change in fraction of the indicator in the Ca 2+ -bound form. Δf CaD was simulated with a multicompartment model of the underlying myoplasmic Ca 2+ movements, and the results were compared with previous measurements and analyses in mouse fast-twitch fibers. In frog fibers, sarcoplasmic reticulum (SR) Ca 2+ release evoked by an action potential appears to be the sum of two components. The time course of the first component is similar to that of the entire Ca 2+ release waveform in mouse fibers, whereas that of the second component is severalfold slower; the fractional release amounts are ∼0.8 (first component) and ∼0.2 (second component). Similar results were obtained in frog simulations with a modified model that permitted competition between Mg 2+ and Ca 2+ for occupancy of the regulatory sites on troponin. An anatomical basis for two release components in frog fibers is the presence of both junctional and parajunctional SR Ca 2+ release channels (ryanodine receptors [RyRs]), whereas mouse fibers (usually) have only junctional RyRs. Also, frog fibers have two RyR isoforms, RyRα and RyRβ, whereas the mouse fibers (usually) have only one, RyR1. Our simulations suggest that the second release component in frog fibers functions to supply extra Ca 2+ to activate troponin, which, in mouse fibers, is not needed because of the more favorable location of their triadic junctions (near the middle of the thin filament). We speculate that, in general, parajunctional RyRs permit increased myofilament activation in fibers whose triadic junctions are located at the z-line.

In skeletal muscle fibers, action potentials elicit contractions by releasing calcium ions (Ca 2+ ) from the sarcoplasmic reticulum. Experiments on individual mouse muscle fibers micro-injected with a rapidly responding fluorescent Ca 2+ indicator dye reveal that the amount of Ca 2+ released is three- to fourfold larger in fast-twitch fibers than in slow-twitch fibers, and the proportion of the released Ca 2+ that binds to troponin to activate contraction is substantially smaller.

Ca 2+ release from the sarcoplasmic reticulum (SR) of skeletal muscle takes place at the triadic junctions; following release, Ca 2+ spreads within the sarcomere by diffusion. Here, we report multicompartment simulations of changes in sarcomeric Ca 2+ evoked by action potentials (APs) in fast-twitch fibers of adult mice. The simulations include Ca 2+ complexation reactions with ATP, troponin, parvalbumin, and the SR Ca 2+ pump, as well as Ca 2+ transport by the pump. Results are compared with spatially averaged Ca 2+ transients measured in mouse fibers with furaptra, a low-affinity, rapidly responding Ca 2+ indicator. The furaptra Δf CaD signal (change in the fraction of the indicator in the Ca 2+ -bound form) evoked by one AP is well simulated under the assumption that SR Ca 2+ release has a peak of 200–225 μM/ms and a FDHM of ∼1.6 ms (16°C). Δf CaD elicited by a five-shock, 67-Hz train of APs is well simulated under the assumption that in response to APs 2–5, Ca 2+ release decreases progressively from 0.25 to 0.15 times that elicited by the first AP, a reduction likely due to Ca 2+ inactivation of Ca 2+ release. Recovery from inactivation was studied with a two-AP protocol; the amplitude of the second release recovered to >0.9 times that of the first with a rate constant of 7 s −1 . An obvious feature of Δf CaD during a five-shock train is a progressive decline in the rate of decay from the individual peaks of Δf CaD . According to the simulations, this decline is due to a reduction in available Ca 2+ binding sites on troponin and parvalbumin. The effects of sarcomere length, the location of the triadic junctions, resting [Ca 2+ ], the parvalbumin concentration, and possible uptake of Ca 2+ by mitochondria were also investigated. Overall, the simulations indicate that this reaction-diffusion model, which was originally developed for Ca 2+ sparks in frog fibers, works well when adapted to mouse fast-twitch fibers stimulated by APs.

The properties of Ca 2+ sparks in frog intact skeletal muscle fibers depolarized with 13 mM [K + ] Ringer's are well described by a computational model with a Ca 2+ source flux of amplitude 2.5 pA (units of current) and duration 4.6 ms (18 °C; Model 2 of Baylor et al., 2002 ). This result, in combination with the values of single-channel Ca 2+ current reported for ryanodine receptors (RyRs) in bilayers under physiological ion conditions, 0.5 pA ( Kettlun et al., 2003 ) to 2 pA ( Tinker et al., 1993 ), suggests that 1–5 RyR Ca 2+ release channels open during a voltage-activated Ca 2+ spark in an intact fiber. To distinguish between one and greater than one channel per spark, sparks were measured in 8 mM [K + ] Ringer's in the absence and presence of tetracaine, an inhibitor of RyR channel openings in bilayers. The most prominent effect of 75–100 μM tetracaine was an approximately sixfold reduction in spark frequency. The remaining sparks showed significant reductions in the mean values of peak amplitude, decay time constant, full duration at half maximum (FDHM), full width at half maximum (FWHM), and mass, but not in the mean value of rise time. Spark properties in tetracaine were simulated with an updated spark model that differed in minor ways from our previous model. The simulations show that (a) the properties of sparks in tetracaine are those expected if tetracaine reduces the number of active RyR Ca 2+ channels per spark, and (b) the single-channel Ca 2+ current of an RyR channel is ≤1.2 pA under physiological conditions. The results support the conclusion that some normal voltage-activated sparks (i.e., in the absence of tetracaine) are produced by two or more active RyR Ca 2+ channels. The question of how the activation of multiple RyRs is coordinated is discussed.