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In JoVE (1)
Other Publications (16)
- Pflügers Archiv : European Journal of Physiology
- Biophysical Journal
- The Journal of Physiology
- The Journal of Physiology
- Protein Expression and Purification
- The Journal of General Physiology
- Biological Research
- The Journal of General Physiology
- The Journal of Physiology
- Proceedings of the National Academy of Sciences of the United States of America
- Journal of Muscle Research and Cell Motility
- American Journal of Physiology. Cell Physiology
- The Journal of General Physiology
- The Journal of Physiology
- Human Molecular Genetics
- The Journal of General Physiology
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Articles by Marino DiFranco in JoVE
JoVE General
DNA Transfectie van zoogdieren skeletspieren met behulp van In Vivo Elektroporatie
Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles
We beschrijven gedetailleerde procedures voor de efficiënte transfectie van plasmide DNA in de vezels van mond spieren van levende muizen met behulp van elektroporatie en de daaropvolgende visualisatie van eiwitexpressie met behulp van fluorescentie microscopie.
Other articles by Marino DiFranco on PubMed
Characterization of the Calcium Release Domains During Excitation-contraction Coupling in Skeletal Muscle Fibres
The spatiotemporal properties of the Ca2+ release process in skeletal muscle fibres were determined using a confocal spot detection system. The low-affinity, fluorescent Ca2+ indicator Oregon Green 488 BAPTA-5N (OGB-5N) was used to record localized, action potential-induced fluorescence signals from consecutive locations separated by 200 nm within a single sarcomere. Three-dimensional reconstructions of the Ca2+ transients illustrated the existence of fluorescence domains around Ca2+ release sites, which are centred at the T-tubules. By constructing isochronal plots, it was estimated that the earliest detectable full width at half-maximum (FWHM) of the Ca2+ domains was 0.77+/-0.08 microm and increased rapidly with time to 1.4+/-0.04 microm at peak (17-18 degrees C). A delay of 0.64+/-0.1 ms was observed between the onset of the fluorescence transients at the Z- and M-lines. Deconvolution of fluorescence transients gave estimates of approximately 9 and 2 microM for the peak [Ca2+] changes at the Z and M-lines, respectively. Our results are compatible with the possibility that action potential stimulation elicits Ca2+ release from a region of the sarcoplasmic reticulum (SR) broader than the T-SR junction.
Comparison Between the Predictions of Diffusion-reaction Models and Localized Ca2+ Transients in Amphibian Skeletal Muscle Fibers
We developed a three-dimensional cylindrical diffusion-reaction model of a single amphibian myofibril in which Ca(2+) release occurred only at the Z-line. The model incorporated diffusion of Ca(2+), Mg(2+), and all relevant buffer species, as well as the kinetic binding reactions between the buffers and appropriate ions. Model data was blurred according to a Gaussian approximation of the point spread function of the microscope and directly compared with experimental data obtained using the confocal spot methodology. The flux parameters were adjusted until the simulated Z-line transient matched the experimental one. This model could not simultaneously predict key parameters of the experimental M- and Z-line transients, even when model parameters were adjusted to unreasonably extreme values. Even though the model was accurate in predicting the Z-line transient under conditions of high [EGTA], it predicted a significantly narrower Ca(2+) domain than observed experimentally. We modified the model to incorporate a broader band of release centered at the Z-line. This extended release model was superior both in simultaneously predicting critical features of the Z- and M-line transients as well as the domain profile under conditions of high [EGTA]. We conclude that a model of release occurring exclusively at the Z-line cannot explain our experimental data and suggest that Ca(2+) may be released from a broader region of the sarcoplasmic reticulum than just the T-tubule-sarcoplasmic reticulum junction.
The Action Potential-evoked Sarcoplasmic Reticulum Calcium Release is Impaired in Mdx Mouse Muscle Fibres
The mdx mouse, a model of the human disease Duchenne muscular dystrophy, has skeletal muscle fibres which display incompletely understood impaired contractile function. We explored the possibility that action potential-evoked Ca(2+) release is altered in mdx fibres. Action potential-evoked Ca(2+)-dependent fluorescence transients were recorded, using both low and high affinity Ca(2+) indicators, from enzymatically isolated fibres obtained from extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of normal and mdx mice. Fibres were immobilized using either intracellular EGTA or N-benzyl-p-toluene sulphonamide, an inhibitor of the myosin II ATPase. We found that the amplitude of the action potential-evoked Ca(2+) transients was significantly decreased in mdx mice with no measured difference in that of the surface action potential. In addition, Ca(2+) transients recorded from mdx fibres in the absence of EGTA also displayed a marked prolongation of the slow decay phase. Model simulations of the action potential-evoked transients in the presence of high EGTA concentrations suggest that the reduction in the evoked sarcoplasmic reticulum Ca(2+) release flux is responsible for the decrease in the peak of the Ca(2+) transient in mdx fibres. Since the myoplasmic Ca(2+) concentration is a critical regulator of muscle contraction, these results may help to explain the weakness observed in skeletal muscle fibres from mdx mice and, possibly, Duchenne muscular dystrophy patients.
Propagation in the Transverse Tubular System and Voltage Dependence of Calcium Release in Normal and Mdx Mouse Muscle Fibres
Using a two-microelectrode voltage clamp technique, we investigated possible mechanisms underlying the impaired excitation-contraction coupling in skeletal muscle fibres of the mdx mouse, a model of the human disease Duchenne muscular dystrophy. We evaluated the role of the transverse tubular system (T-system) by using the potentiometric indicator di-8 ANEPPS, and that of the sarcoplasmic reticulum (SR) Ca2+ release by measuring Ca2+ transients with a low affinity indicator in the presence of high EGTA concentrations under voltage clamp conditions. We observed minimal differences in the T-system structure and the T-system electrical propagation was not different between normal and mdx mice. Whereas the maximum Ca2+ release elicited by voltage pulses was reduced by approximately 67% in mdx fibres, in agreement with previous results obtained using AP stimulation, the voltage dependence of SR Ca2+ release was identical to that seen in normal fibres. Taken together, our data suggest that the intrinsic ability of the sarcoplasmic reticulum to release Ca2+ may be altered in the mdx mouse.
Quantitative Evaluation of Mammalian Skeletal Muscle As a Heterologous Protein Expression System
The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of approximately 1mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.
Calcium Release Domains in Mammalian Skeletal Muscle Studied with Two-photon Imaging and Spot Detection Techniques
The spatiotemporal characteristics of the Ca(2+) release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 muM of the low-affinity Ca(2+) indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca(2+) chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca(2+)] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks ( approximately 23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca(2+) transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest DeltaF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca(2+) release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in approximately 1.4 ms and dissipate within approximately 4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca(2+) transients peaked at T-tubule locations, was 0.62 mum, similar to the 0.61 mum measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca(2+) domains in adult mammalian muscle fibers are confined to Ca(2+) release sites located at the junctional sarcoplasmic reticulum (SR).
Modulation by Caffeine of Calcium-release Microdomains in Frog Skeletal Muscle Fibers
The effects of caffeine on the process of excitation-contraction coupling in amphibian skeletal muscle fibers were investigated using the confocal spot detection technique. This method permits to carefully discriminate between caffeine effects on the primary sources of Ca2+ release at the Z-lines where the triads are located and secondary actions on other potential Ca Release sources. Our results demonstrate that 0.5 mM caffeine potentiates and prolongs localized action-potential evoked Ca2+ transients recorded at the level of the Z-lines, but that 1mM only prolongs them. The effects at both doses are reversible. At the level of the M-line, localized Ca2+ transients displayed more variability in the presence of 1 mM caffeine than in control conditions. At this dose of caffeine, extra-junctional sources of Ca2+ release also were observed occasionally.
Voltage-dependent Dynamic FRET Signals from the Transverse Tubules in Mammalian Skeletal Muscle Fibers
Two hybrid voltage-sensing systems based on fluorescence resonance energy transfer (FRET) were used to record membrane potential changes in the transverse tubular system (TTS) and surface membranes of adult mice skeletal muscle fibers. Farnesylated EGFP or ECFP (EGFP-F and ECFP-F) were used as immobile FRET donors, and either non-fluorescent (dipicrylamine [DPA]) or fluorescent (oxonol dye DiBAC(4)(5)) lipophilic anions were used as mobile energy acceptors. Flexor digitorum brevis (FDB) muscles were transfected by in vivo electroporation with pEGFP-F and pECFP-F. Farnesylated fluorescent proteins were efficiently expressed in the TTS and surface membranes. Voltage-dependent optical signals resulting from resonance energy transfer from fluorescent proteins to DPA were named QRET transients, to distinguish them from FRET transients recorded using DiBAC(4)(5). The peak DeltaF/F of QRET transients elicited by action potential stimulation is twice larger in fibers expressing ECFP-F as those with EGFP-F (7.1% vs. 3.6%). These data provide a unique experimental demonstration of the importance of the spectral overlap in FRET. The voltage sensitivity of QRET and FRET signals was demonstrated to correspond to the voltage-dependent translocation of the charged acceptors, which manifest as nonlinear components in current records. For DPA, both electrical and QRET data were predicted by radial cable model simulations in which the maximal time constant of charge translocation was 0.6 ms. FRET signals recorded in response to action potentials in fibers stained with DiBAC(4)(5) exhibit DeltaF/F amplitudes as large as 28%, but their rising phase was slower than those of QRET signals. Model simulations require a time constant for charge translocation of 1.6 ms in order to predict current and FRET data. Our results provide the basis for the potential use of lipophilic ions as tools to test for fast voltage-dependent conformational changes of membrane proteins in the TTS.
Somatic Ca2+ Transients Do Not Contribute to Inspiratory Drive in PreBötzinger Complex Neurons
PreBötzinger Complex (preBötC) neurons are postulated to underlie respiratory rhythm generation. The inspiratory phase of the respiratory cycle in vitro results from preBötC neurons firing synchronous bursts of action potentials (APs) on top of 10-20 mV, 0.3-0.8 s inspiratory drive potentials. Is the inspiratory drive in individual neurons simply the result of the passive integration of inspiratory-modulated synaptic currents or do active processes modulate these currents? As somatic Ca(2+) is known to increase during inspiration, we hypothesized that it affects inspiratory drive. We combined whole cell recording in an in vitro slice preparation with Ca(2+) microfluorometry to detect single inspiratory neuron somatic Ca(2+) transients with high temporal resolution ( approximately mus). In neurons loaded with either Fluo-4 or Oregon Green BAPTA 5 N, we observed Ca(2+) transients associated with each AP. During inspiration, significant somatic Ca(2+) influx was a direct consequence of activation of voltage-gated Ca(2+) channels by APs. However, when we isolated the inspiratory drive potential in active preBötC neurons (by blocking APs with intracellular QX-314 or by hyperpolarization), we did not detect somatic Ca(2+) transients; yet, the parameters of inspiratory drive were the same with or without APs. We conclude that, in the absence of APs, somatic Ca(2+) transients do not shape the somatic inspiratory drive potential. This suggests that in preBötC neurons, substantial and widespread somatic Ca(2+) influx is a consequence of APs during the inspiratory phase and does not contribute substantively to the inspiratory drive potential. Given evidence that the Ca(2+) buffer BAPTA can significantly reduce inspiratory drive, we hypothesize that dendritic Ca(2+) transients amplify inspiratory-modulated synaptic currents.
Dystrophic Skeletal Muscle Fibers Display Alterations at the Level of Calcium Microdomains
The spatiotemporal properties of the Ca(2+)-release process in skeletal muscle fibers from normal and mdx fibers were determined using the confocal-spot detection technique. The Ca(2+) indicator OGB-5N was used to record action potential-evoked fluorescence signals at consecutive locations separated by 200 nm along multiple sarcomeres of FDB fibers loaded with 10- and 30-mM EGTA. Three-dimensional reconstructions of fluorescence transients demonstrated the existence of microdomains of increased fluorescence around the Ca(2+)-release sites in both mouse strains. The Ca(2+) microdomains in mdx fibers were regularly spaced along the fiber axis, displaying a distribution similar to that seen in normal fibers. Nevertheless, both preparations differed in that in 10-mM EGTA Ca(2+) microdomains had smaller amplitudes and were wider in mdx fibers than in controls. In addition, Ca(2+)-dependent fluorescence transients recorded at selected locations within the sarcomere of mdx muscle fibers were not only smaller, but also slower than their counterparts in normal fibers. Notably, differences in the spatial features of the Ca(2+) microdomains recorded in mdx and normal fibers, but not in the amplitude and kinetics of the Ca(2+) transients, were eliminated in 30-mM EGTA. Our results consistently demonstrate that Ca(2+)-release flux calculated from release sites in mdx fibers is uniformly impaired with respect to those normal fibers. The Ca(2+)-release reduction is consistent with that previously measured using global detection techniques.
Effects of Membrane Depolarization and Changes in Extracellular [K(+)] on the Ca (2+) Transients of Fast Skeletal Muscle Fibers. Implications for Muscle Fatigue
Repetitive activation of skeletal muscle fibers leads to a reduced transmembrane K(+) gradient. The resulting membrane depolarization has been proposed to play a major role in the onset of muscle fatigue. Nevertheless, raising the extracellular K(+) K(+)(O) concentration ([K(+)](O)) to 10 mM potentiates twitch force of rested amphibian and mammalian fibers. We used a double Vaseline gap method to simultaneously record action potentials (AP) and Ca(2+) transients from rested frog fibers activated by single and tetanic stimulation (10 pulses, 100 Hz) at various [K(+)](O) and membrane potentials. Depolarization resulting from current injection or raised [K(+](O) produced an increase in the resting [Ca(2+)]. Ca(2+) transients elicited by single stimulation were potentiated by depolarization from -80 to -60 mV but markedly depressed by further depolarization. Potentiation was inversely correlated with a reduction in the amplitude, overshoot and duration of APs. Similar effects were found for the Ca(2+) transients elicited by the first pulse of 100 Hz trains. Depression or block of Ca(2+) transient in response to the 2nd to 10th pulses of 100 Hz trains was observed at smaller depolarizations as compared to that seen when using single stimulation. Changes in Ca(2+) transients along the trains were associated with impaired or abortive APs. Raising [K(+)](O) to 10 mM potentiated Ca(2+) transients elicited by single and tetanic stimulation, while raising [K(+)](O) to 15 mM markedly depressed both responses. The effects of 10 mM K(+)(O) on Ca(2+) transients, but not those of 15 mM K(+)(O), could be fully reversed by hyperpolarization. The results suggests that the force potentiating effects of 10 mM K(+)(O) might be mediated by depolarization dependent changes in resting [Ca(2+)] and Ca(2+) release, and that additional mechanisms might be involved in the effects of 15 mM K(+)(O) on force generation.
Excitation-contraction Coupling Alterations in Mdx and Utrophin/dystrophin Double Knockout Mice: a Comparative Study
The double knockout mouse for utrophin and dystrophin (utr(-/-)/mdx) has been proposed to be a better model of Duchenne Muscular Dystrophy (DMD) than the mdx mouse because the former displays more similar muscle pathology to that of the DMD patients. In this paper the properties of action potentials (APs) and Ca(2+) transients elicited by single and repetitive stimulation were studied to understand the excitation-contraction (EC) coupling alterations observed in muscle fibers from mdx and utr(-/-)/mdx mice. Based on the comparison of the AP durations with those of fibers from wild-type (WT) mice, fibers from both mdx and utr(-/-)/mdx mice could be divided in two groups: fibers with WT-like APs (group 1) and fibers with significantly longer APs (group 2). Although the proportion of fibers in group 2 was larger in utr(-/-)/mdx (36%) than in mdx mice (27%), the Ca(2+) release elicited by single stimulation was found to be similarly depressed (32-38%) in utr(-/-)/mdx and mdx fibers compared with WT counterparts regardless of the fiber's group. Stimulation at 100 Hz revealed that, with the exception of those from utr(-/-)/mdx mice, group 1 fibers were able to sustain Ca(2+) release for longer than group 2 fibers, which displayed an abrupt limitation even at the onset of the train. The differences in behavior between fibers in groups 1 and 2 became almost unnoticeable at 50 Hz stimulation. In general, fibers from utr(-/-)/mdx mice seem to display more persistent alterations in the EC coupling than those observed in the mdx model.
Chloride Currents from the Transverse Tubular System in Adult Mammalian Skeletal Muscle Fibers
Chloride fluxes are the main contributors to the resting conductance of mammalian skeletal muscle fibers. ClC-1, the most abundant chloride channel isoform in this preparation, is believed to be responsible for this conductance. However, the actual distribution of ClC-1 channels between the surface and transverse tubular system (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltageclamped enzymatically dissociated short fibers using a two-microelectrode configuration and simultaneously recorded chloride currents (I(Cl)) and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride to enhance the magnitude of inward I(Cl), and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and I(Cl) acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be I(Cl) dependent since its magnitude varied in close correlation with the amplitude and time course of I(Cl). While the properties of I(Cl), and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (P(Cl)) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if P(Cl) was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded I(Cl) arises from TTS contributions.
Functional Expression of Transgenic 1sDHPR Channels in Adult Mammalian Skeletal Muscle Fibres
We investigated the effects of the overexpression of two enhanced green fluorescent protein (EGFP)-tagged α1sDHPR variants on Ca2+ currents (ICa), charge movements (Q) and SR Ca2+ release of muscle fibres isolated from adult mice. Flexor digitorum brevis (FDB)muscles were transfected by in vivo electroporation with plasmids encoding for EGFP-α1sDHPR-wt and EGFP-α1sDHPR-T935Y (an isradipine-insensitive mutant). Two-photon laser scanning microscopy (TPLSM) was used to study the subcellular localization of transgenic proteins, while ICa, Q and Ca2+ release were studied electrophysiologically and optically under voltage-clamp conditions. TPLSM images demonstrated that most of the transgenic α1sDHPR was correctly targeted to the transverse tubular system (TTS). Immunoblotting analysis of crude extracts of transfected fibres demonstrated the synthesis of bona fide transgenic EGFP-α1sDHPR-wt in quantities comparable to that of native α1sDHPR. Though expression of both transgenic variants of the alpha subunit of the dihydropyridine receptor (α1sDHPR) resulted in ∼50% increase in Q, they surprisingly had no effect on the maximal Ca2+ conductance (gCa) nor the SR Ca2+ release. Nonetheless, fibres expressing EGFP-α1sDHPR-T935Y exhibited up to 70% isradipine-insensitive ICa (ICa-ins) with a right-shifted voltage dependence compared to that in control fibres. Interestingly, Qand SRCa2+ release also displayed right-shifted voltage dependence in fibres expressing EGFP-α1sDHPR-T935Y. In contrast, the midpoints of the voltage dependence of gCa, Q and Ca2+ release were not different from those in control fibres and in fibres expressing EGFP-α1sDHPR-wt. Overall, our results suggest that transgenic α1sDHPRs are correctly trafficked and inserted in the TTS membrane, and that a substantial fraction of the mworks as conductive Ca2+ channels capable of physiologically controlling the release of Ca2+ from the SR. A plausible corollary of this work is that the expression of transgenic variants of the α1sDHPR leads to the replacement of native channels interacting with the ryanodine receptor 1 (RyR1), thus demonstrating the feasibility of molecular remodelling of the triads in adult skeletal muscle fibres.
Pathogenity of Some Limb Girdle Muscular Dystrophy Mutations Can Result from Reduced Anchorage to Myofibrils and Altered Stability of Calpain 3
Calpain 3 (CAPN3) is a muscle-specific, calcium-dependent proteinase that is mutated in Limb Girdle Muscle Dystrophy type 2A. Most pathogenic missense mutations in LGMD2A affect CAPN3's proteolytic activity; however, two mutations, D705G and R448H, retain activity but nevertheless cause muscular dystrophy. Previously, we showed that D705G and R448H mutations reduce CAPN3s ability to bind to titin in vitro. In this investigation, we tested the consequence of loss of titin binding in vivo and examined whether this loss can be an underlying pathogenic mechanism in LGMD2A. To address this question, we created transgenic mice that express R448H or D705G in muscles, on wild-type (WT) CAPN3 or knock-out background. Both mutants were readily expressed in insect cells, but when D705G was expressed in skeletal muscle, it was not stable enough to study. Moreover, the D705G mutation had a dominant negative effect on endogenous CAPN3 when expressed on a WT background. The R448H protein was stably expressed in muscles; however, it was more rapidly degraded in muscle extracts compared with WT CAPN3. Increased degradation of R448H was due to non-cysteine, cellular proteases acting on the autolytic sites of CAPN3, rather than autolysis. Fractionation experiments revealed a significant decrease of R448H from the myofibrillar fraction, likely due to the mutant's inability to bind titin. Our data suggest that R448H and D705G mutations affect both CAPN3s anchorage to titin and its stability. These studies reveal a novel mechanism by which mutations that spare enzymatic activity can still lead to calpainopathy.
The Na Conductance in the Sarcolemma and the Transverse Tubular System Membranes of Mammalian Skeletal Muscle Fibers
Na (and Li) currents and fluorescence transients were recorded simultaneously under voltage-clamp conditions from mouse flexor digitorum brevis fibers stained with the potentiometric dye di-8-ANEPPS to investigate the distribution of Na channels between the surface and transverse tubular system (TTS) membranes. In fibers rendered electrically passive, voltage pulses resulted in step-like fluorescence changes that were used to calibrate the dye response. The effects of Na channel activation on the TTS voltage were investigated using Li, instead of Na, because di-8-ANEPPS transients show anomalies in the presence of the latter. Na and Li inward currents (I(Na), I(Li); using half of the physiological ion concentration) showed very steep voltage dependences, with no reversal for depolarizations beyond the calculated equilibrium potential, suggesting that most of the current originates from a noncontrolled membrane compartment. Maximum peak I(Li) was ∼ 30% smaller than for I(Na), suggesting a Li-blocking effect. I(Li) activation resulted in the appearance of overshoots in otherwise step-like di-8-ANEPPS transients. Overshoots had comparable durations and voltage dependence as those of I(Li). Simultaneously measured maximal overshoot and peak I(Li) were 54 ± 5% and 773 ± 53 µA/cm(2), respectively. Radial cable model simulations predicted the properties of I(Li) and di-8-ANEPPS transients when TTS access resistances of 10-20 Ω cm(2), and TTS-to-surface Na permeability density ratios in the range of 40:60 to 70:30, were used. Formamide-based osmotic shock resulted in incomplete detubulation. However, results from a subpopulation of treated fibers (low capacitance) provide confirmatory evidence that a significant proportion of I(Li), and the overshoot in the optical signals, arises from the TTS in normal fibers. The quantitative evaluation of the distribution of Na channels between the sarcolemma and the TTS membranes, as provided here, is crucial for the understanding of the radial and longitudinal propagation of the action potential, which ultimately govern the mechanical activation of muscle in normal and diseased conditions.
