Na+ and K+ channels: history and structure.

In this perspective, we discuss the physiological roles of Na and K channels, emphasizing the importance of the K channel for cellular homeostasis in animal cells and of Na and K channels for cellular signaling. We consider the structural basis of Na and K channel gating in light of recent structural and electrophysiological findings.

Small Ca2+ releases enable hour-long high-frequency contractions in midshipman swimbladder muscle.

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 Ca2+ is managed by midshipman fibers during calling, we measure (a) the Ca2+ pumping-versus-pCa and force-versus-pCa relations in skinned fiber bundles and (b) changes in myoplasmic free [Ca2+] (Δ[Ca2+]) during stimulated activity of individual fibers microinjected with the Ca2+ indicators Mag-fluo-4 and Fluo-4. As in toadfish, the force-pCa relation in midshipman is strongly right-shifted relative to the Ca2+ pumping-pCa relation, and contractile activity is controlled in a synchronous, not asynchronous, fashion during electrical stimulation. SR Ca2+ release per action potential is, however, approximately eightfold smaller in midshipman than in toadfish. Midshipman fibers have a larger time-averaged free [Ca2+] during activity than toadfish fibers, which permits faster Ca2+ pumping because the Ca2+ pumps work closer to their maximum rate. Even with midshipman's sustained release and pumping of Ca2+, however, the Ca2+ energy cost of calling (per kilogram wet weight) is less than twofold more in midshipman than in toadfish.

A perspective on Na and K channel inactivation.

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 (Vm) 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.

Structural and functional properties of ryanodine receptor type 3 in zebrafish tail muscle.

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.

Intracellular calcium movements during relaxation and recovery of superfast muscle fibers of the toadfish swimbladder.

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[CaM]) 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[CaM] 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+)].

Comparison of myoplasmic calcium movements during excitation-contraction coupling in frog twitch and mouse fast-twitch muscle fibers.

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 (FR) and the change evoked by an action potential (ΔF) were measured at long sarcomere length (16°C); ΔF/FR was scaled to units of ΔfCaD, the change in fraction of the indicator in the Ca(2+)-bound form. ΔfCaD 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 RyRß, 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.

Intracellular calcium movements during excitation-contraction coupling in mammalian slow-twitch and fast-twitch muscle fibers.

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.

Measurement and simulation of myoplasmic calcium transients in mouse slow-twitch muscle fibres.

Bundles of intact fibres from soleus muscles of adult mice were isolated by dissection and one fibre within a bundle was micro-injected with either furaptra or mag-fluo-4, two low-affinity rapidly responding Ca(2+) indicators. Fibres were activated by action potentials to elicit changes in indicator fluorescence (ΔF), a monitor of the myoplasmic free Ca(2+) transient ([Ca(2+)]), and changes in fibre tension. All injected fibres appeared to be slow-twitch (type I) fibres as inferred from the time course of their tension responses. The full-duration at half-maximum (FDHM) of ΔF was found to be essentially identical with the two indicators; the mean value was 8.4 ± 0.3 ms (±SEM) at 16°C and 5.1 ± 0.3 ms at 22°C. The value at 22°C is about one-third that reported previously in enzyme-dissociated slow-twitch fibres that had been AM-loaded with mag-fluo-4: 12.4 ± 0.8 ms and 17.2 ± 1.7 ms. We attribute the larger FDHM in enzyme-dissociated fibres either to an alteration of fibre properties due to the enzyme treatment or to some error in the measurement of ΔF associated with AM loading. ΔF in intact fibres was simulated with a multi-compartment reaction-diffusion model that permitted estimation of the amount and time course of Ca(2+) release from the sarcoplasmic reticulum (SR), the binding and diffusion of Ca(2+) in the myoplasm, the re-uptake of Ca(2+) by the SR Ca(2+) pump, and Δ[Ca(2+)] itself. In response to one action potential at 16°C, the following estimates were obtained: 107 µm for the amount of Ca(2+) release; 1.7 ms for the FDHM of the release flux; 7.6 µm and 4.9 ms for the peak and FDHM of spatially averaged Δ[Ca(2+)]. With five action potentials at 67 Hz, the estimated amount of Ca(2+) release is 186 µm. Two important unknown model parameters are the on- and off-rate constants of the reaction between Ca(2+) and the regulatory sites on troponin; values of 0.4 × 10(8) m(-1) s(-1) and 26 s(-1), respectively, were found to be consistent with the ΔF measurements.

Paying the piper: the cost of Ca2+ pumping during the mating call of toadfish.

Superfast fibres of toadfish swimbladder muscle generate a series of superfast Ca(2+) transients, a necessity for high-frequency calling. How is this accomplished with a relatively low rate of Ca(2+) pumping by the sarcoplasmic reticulum (SR)? We hypothesized that there may not be complete Ca(2+) saturation and desaturation of the troponin Ca(2+) regulatory sites with each twitch during calling. To test this, we determined the number of regulatory sites by measuring the concentration of troponin C (TNC) molecules, 33.8 µmol per kg wet weight. We then estimated how much SR Ca(2+) is released per twitch by measuring the recovery oxygen consumption in the presence of a crossbridge blocker, N-benzyl-p-toluene sulphonamide (BTS). The results agreed closely with SR release estimates obtained with a kinetic model used to analyse Ca(2+) transient measurements. We found that 235 µmol of Ca(2+) per kg muscle is released with the first twitch of an 80 Hz stimulus (15(o)C). Release per twitch declines dramatically thereafter such that by the 10th twitch release is only 48 µmol kg(-1) (well below the concentration of TNC Ca(2+) regulatory sites, 67.6 µmol kg(-1)). The ATP usage per twitch by the myosin crossbridges remains essentially constant at ∼25 µmol kg(-1) throughout the stimulus period. Hence, for the first twitch, ∼80% of the energy goes into pumping Ca(2+) (which uses 1 ATP per 2 Ca(2+) ions pumped), but by the 10th and subsequent twitches the proportion is ∼50%. Even though by the 10th stimulus the Ca(2+) release per twitch has dropped 5-fold, the Ca(2+) remaining in the SR has declined by only ∼18%; hence dwindling SR Ca(2+) content is not responsible for the drop. Rather, inactivation of the Ca(2+) release channel by myoplasmic Ca(2+) likely explains this reduction. If inactivation did not occur, the SR would run out of Ca(2+) well before the end of even a 40-twitch call. Hence, inactivation of the Ca(2+) release channel plays a critical role in swimbladder muscle during normal in vivo function.

Calcium indicators and calcium signalling in skeletal muscle fibres during excitation-contraction coupling.

During excitation-contraction coupling in skeletal muscle, calcium ions are released into the myoplasm by the sarcoplasmic reticulum (SR) in response to depolarization of the fibre's exterior membranes. Ca(2+) then diffuses to the thin filaments, where Ca(2+) binds to the Ca(2+) regulatory sites on troponin to activate muscle contraction. Quantitative studies of these events in intact muscle preparations have relied heavily on Ca(2+)-indicator dyes to measure the change in the spatially-averaged myoplasmic free Ca(2+) concentration (Δ[Ca(2+)]) that results from the release of SR Ca(2+). In normal fibres stimulated by an action potential, Δ[Ca(2+)] is large and brief, requiring that an accurate measurement of Δ[Ca(2+)] be made with a low-affinity rapidly-responding indicator. Some low-affinity Ca(2+) indicators monitor Δ[Ca(2+)] much more accurately than others, however, as reviewed here in measurements in frog twitch fibres with sixteen low-affinity indicators. This article also examines measurements and simulations of Δ[Ca(2+)] in mouse fast-twitch fibres. The simulations use a multi-compartment model of the sarcomere that takes into account Ca(2+)'s release from the SR, its diffusion and binding within the myoplasm, and its re-sequestration by the SR Ca(2+) pump. The simulations are quantitatively consistent with the measurements and appear to provide a satisfactory picture of the underlying Ca(2+) movements.

Low-affinity Ca2+ indicators compared in measurements of skeletal muscle Ca2+ transients.

The low-affinity fluorescent Ca(2+) indicators OGB-5N, Fluo-5N, fura-5N, Rhod-5N, and Mag-fluo-4 were evaluated for their ability to accurately track the kinetics of the spatially averaged free Ca(2+) transient (Delta[Ca(2+)]) in skeletal muscle. Frog single fibers were injected with one of the above indicators and, usually, furaptra (previously shown to rapidly track Delta[Ca(2+)]). In response to an action potential, the full duration at half-maximum of the indicator's fluorescence change (DeltaF) was found to be larger with OGB-5N, Fluo-5N, fura-5N, and Rhod-5N than with furaptra; thus, these indicators do not track Delta[Ca(2+)] with kinetic fidelity. In contrast, the DeltaF time course of Mag-fluo-4 was identical to furaptra's; thus, Mag-fluo-4 also yields reliable kinetic information about Delta[Ca(2+)]. Mag-fluo-4's DeltaF has a larger signal/noise ratio than furaptra's (for similar indicator concentrations), and should thus be more useful for tracking Delta[Ca(2+)] in small cell volumes. However, because the resting fluorescence of Mag-fluo-4 probably arises largely from indicator that is bound with Mg(2+), the amplitude of the Mag-fluo-4 signal, and its calibration in Delta[Ca(2+)] units, is likely to be more sensitive to variations in [Mg(2+)] than furaptra's.

Comparison of the myoplasmic calcium transient elicited by an action potential in intact fibres of mdx and normal mice.

The myoplasmic free [Ca2+] transient elicited by an action potential (Delta[Ca2+]) was compared in fast-twitch fibres of mdx (dystrophin null) and normal mice. Methods were used that maximized the likelihood that any detected differences apply in vivo. Small bundles of fibres were manually dissected from extensor digitorum longus muscles of 7- to 14-week-old mice. One fibre within a bundle was microinjected with furaptra, a low-affinity rapidly responding fluorescent calcium indicator. A fibre was accepted for study if it gave a stable, all-or-nothing fluorescence response to an external shock. In 18 normal fibres, the peak amplitude and the full-duration at half-maximum (FDHM) of Delta[Ca2+] were 18.4 +/- 0.5 microm and 4.9 +/- 0.2 ms, respectively (mean +/- s.e.m.; 16 degrees C). In 13 mdx fibres, the corresponding values were 14.5 +/- 0.6 microm and 4.7 +/- 0.2 ms. The difference in amplitude is statistically highly significant (P = 0.0001; two-tailed t test), whereas the difference in FDHM is not (P = 0.3). A multi-compartment computer model was used to estimate the amplitude and time course of the sarcoplasmic reticulum (SR) calcium release flux underlying Delta[Ca2+]. Estimates were made based on several differing assumptions: (i) that the resting myoplasmic free Ca2+ concentration ([Ca2+]R) and the total concentration of parvalbumin ([Parv(T)]) are the same in mdx and normal fibres, (ii) that [Ca2+](R) is larger in mdx fibres, (iii) that [Parv(T)] is smaller in mdx fibres, and (iv) that [Ca2+]R is larger and [Parv(T)] is smaller in mdx fibres. According to the simulations, the 21% smaller amplitude of Delta[Ca2+] in mdx fibres in combination with the unchanged FDHM of Delta[Ca2+] is consistent with mdx fibres having a approximately 25% smaller flux amplitude, a 6-23% larger FDHM of the flux, and a 9-20% smaller total amount of released Ca2+ than normal fibres. The changes in flux are probably due to a change in the gating of the SR Ca2+-release channels and/or in their single channel flux. The link between these changes and the absence of dystrophin remains to be elucidated.

Simulation of Ca2+ movements within the sarcomere of fast-twitch mouse fibers stimulated by action potentials.

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 Deltaf(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 microM/ms and a FDHM of approximately 1.6 ms (16 degrees C). Deltaf(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 Deltaf(CaD) during a five-shock train is a progressive decline in the rate of decay from the individual peaks of Deltaf(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.

Effects of tetracaine on voltage-activated calcium sparks in frog intact skeletal muscle fibers.

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 degrees 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 microM 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

Malaria parasites are rapidly killed by dantrolene derivatives specific for the plasmodial surface anion channel.

Dantrolene was recently identified as a novel inhibitor of the plasmodial surface anion channel (PSAC), an unusual ion channel on Plasmodium falciparum-infected human red blood cells. Because dantrolene is used clinically, has a high therapeutic index, and has desirable chemical synthetic properties, it may be a lead compound for antimalarial development. However, dantrolene derivatives would need to preferentially interact with PSAC over the sarcoplasmic reticulum (SR) Ca2+ release channel to avoid unwanted side effects from antimalarial therapy. Furthermore, dantrolene's modest affinity for PSAC (K(m) of 1.2 microM) requires improvement. In this study, we tested 164 derivatives of dantrolene to examine whether these hurdles can be surmounted. A simple screen for PSAC block defined the minimal scaffold needed and identified compounds with > or =5-fold higher affinity. Single-channel patch-clamp recordings on infected human red blood cells with two derivatives also revealed increased blocking affinity that resulted from slower unbinding from a site on the extracellular face of PSAC. We tested these derivatives in a frog skeletal muscle contractility assay and found that, in contrast to dantrolene, they had little or no effect on SR Ca2+ release. Finally, these blockers kill in vitro parasite cultures at lower concentrations than dantrolene, consistent with an essential role for PSAC. Because, as a class, these derivatives fulfil the requirements for drug leads and can be studied with simple screening technology, more extensive medicinal chemistry is warranted to explore antimalarial development.

Measurement and Interpretation of Cytoplasmic

Ca(2+)-indicator dyes are widely used in biology yet difficult to characterize inside cells. Studies in skeletal muscle fibers provide important information about indicator behavior and about Ca(2+) signaling within the cytoplasm.