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Loss-of-function mutations in the ClC-1 Cl(-) channel trigger skeletal muscle hyperexcitability in myotonia congenita. For reasons that remain unclear, the severity of the myotonic symptoms can vary markedly even among patients with identical ClC-1 mutations, and may become exacerbated during pregnancy and with diuretic treatment. Since both these conditions are associated with hypomagnesemia and hypocalcemia, we explored whether extracellular Mg(2+) and Ca(2+) ([Mg(2+)]o and [Ca(2+)]o) can affect myotonia. Experimental myotonia was induced in isolated rat muscles by ClC-1 inhibition and effects of [Mg(2+)]o or [Ca(2+)]o on myotonic contractions were determined. Both cations dampened myotonia within their physiological concentration ranges. Thus, myotonic contractile activity was 6-fold larger at 0.3 than at 1.2 mM [Mg(2+)]o and 82-fold larger at 0.3 than at 1.27 mM [Ca(2+)]o. In intracellular recordings of action potentials, the threshold for action potential excitation was raised by 4-6 mV when [Mg(2+)]o was elevated from 0.6 to 3 mM, compatible with an increase in the depolarization of the membrane potential necessary to activate the Na(+) channels. Supporting this notion, mathematical simulations showed that myotonia went from appearing with normal Cl(-) channel function to disappearing in the absence of Cl(-) channel function when Na(+) channel activation was depolarized by 6 mV. In conclusion, variation in serum Mg(2+) and Ca(2+) may contribute to phenotypic variation in myotonia congenita patients., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Dihydropyridine (DHPR) and ryanodine receptors (RyRs) are central to transduction of transverse (T) tubular membrane depolarisation initiated by surface action potentials into release of sarcoplasmic reticular (SR) Ca2+ in skeletal muscle excitation-contraction coupling. Electronmicroscopic methods demonstrate an orderly positioning of such tubular DHPRs relative to RyRs in the SR at triad junctions where their membranes come into close proximity. Biochemical and genetic studies associated expression of specific, DHPR and RyR, isoforms with the particular excitation-contraction coupling processes and related elementary Ca2+ release events found respectively in skeletal and cardiac muscle. Physiological studies of intramembrane charge movements potentially related to voltage triggering of Ca2+ release demonstrated a particular qγ charging species identifiable with DHPRs through its T-tubular localization, pharmacological properties, and steep voltage-dependence paralleling Ca2+ release. Its nonlinear kinetics implicated highly co-operative conformational events in its transitions in response to voltage change. The effects of DHPR and RyR agonists and antagonists upon this intramembrane charge in turn implicated reciprocal rather than merely unidirectional DHPR-RyR interactions in these complex reactions. Thus, following membrane potential depolarization, an orthograde qγ-DHPR-RyR signaling likely initiates conformational alterations in the RyR with which it makes contact. The latter changes could then retrogradely promote further qγ-DHPR transitions through reciprocal co-operative allosteric interactions between receptors. These would relieve the resting constraints on both further, delayed, nonlinear qγ-DHPR charge transfers and on RyR-mediated Ca2+ release. They would also explain the more rapid charging and recovery qγ transients following larger depolarizations and membrane potential repolarization to the resting level.

Activation of skeletal muscle fibers requires rapid sarcolemmal action potential (AP) conduction to ensure uniform excitation along the fiber length, as well as successful tubular excitation to initiate excitation-contraction coupling. In our companion paper in this issue, Pedersen et al. (2011. J. Gen. Physiol. doi:10.1085/jgp.201010510) quantify, for subthreshold stimuli, the influence upon both surface conduction velocity and tubular (t)-system excitation of the large changes in resting membrane conductance (G(M)) that occur during repetitive AP firing. The present work extends the analysis by developing a multi-compartment modification of the charge-difference model of Fraser and Huang to provide a quantitative description of the conduction velocity of actively propagated APs; the influence of voltage-gated ion channels within the t-system; the influence of t-system APs on ionic homeostasis within the t-system; the influence of t-system ion concentration changes on membrane potentials; and the influence of Phase I and Phase II G(M) changes on these relationships. Passive conduction properties of the novel model agreed with established linear circuit analysis and previous experimental results, while key simulations of AP firing were tested against focused experimental microelectrode measurements of membrane potential. This study thereby first quantified the effects of the t-system luminal resistance and voltage-gated Na(+) channel density on surface AP propagation and the resultant electrical response of the t-system. Second, it demonstrated the influence of G(M) changes during repetitive AP firing upon surface and t-system excitability. Third, it showed that significant K(+) accumulation occurs within the t-system during repetitive AP firing and produces a baseline depolarization of the surface membrane potential. Finally, it indicated that G(M) changes during repetitive AP firing significantly influence both t-system K(+) accumulation and its influence on the resting membrane potential. Thus, the present study emerges with a quantitative description of the changes in membrane potential, excitability, and t-system ionic homeostasis that occur during repetitive AP firing in skeletal muscle.

Skeletal muscle activation requires action potential (AP) initiation followed by its sarcolemmal propagation and tubular excitation to trigger Ca(2+) release and contraction. Recent studies demonstrate that ion channels underlying the resting membrane conductance (G(M)) of fast-twitch mammalian muscle fibers are highly regulated during muscle activity. Thus, onset of activity reduces G(M), whereas prolonged activity can markedly elevate G(M). Although these observations implicate G(M) regulation in control of muscle excitability, classical theoretical studies in un-myelinated axons predict little influence of G(M) on membrane excitability. However, surface membrane morphologies differ markedly between un-myelinated axons and muscle fibers, predominantly because of the tubular (t)-system of muscle fibers. This study develops a linear circuit model of mammalian muscle fiber and uses this to assess the role of subthreshold electrical properties, including G(M) changes during muscle activity, for AP initiation, AP propagation, and t-system excitation. Experimental observations of frequency-dependent length constant and membrane-phase properties in fast-twitch rat fibers could only be replicated by models that included t-system luminal resistances. Having quantified these resistances, the resulting models showed enhanced conduction velocity of passive current flow also implicating elevated AP propagation velocity. Furthermore, the resistances filter passive currents such that higher frequency current components would determine sarcolemma AP conduction velocity, whereas lower frequency components excite t-system APs. Because G(M) modulation affects only the low-frequency membrane impedance, the G(M) changes in active muscle would predominantly affect neuromuscular transmission and low-frequency t-system excitation while exerting little influence on the high-frequency process of sarcolemmal AP propagation. This physiological role of G(M) regulation was increased by high Cl(-) permeability, as in muscle endplate regions, and by increased extracellular [K(+)], as observed in working muscle. Thus, reduced G(M) at the onset of exercise would enhance t-system excitation and neuromuscular transmission, whereas elevated G(M) after sustained activity would inhibit these processes and thereby accentuate muscle fatigue.

Regulation of cell volume is a fundamental property of all animal cells and is of particular importance in skeletal muscle where exercise is associated with a wide range of cellular changes that would be expected to influence cell volume. These complex electrical, metabolic and osmotic changes, however, make rigorous study of the consequences of individual factors on muscle volume difficult despite their likely importance during exercise. Recent charge-difference modelling of cell volume distinguishes three major aspects to processes underlying cell volume control: (i) determination by intracellular impermeant solute; (ii) maintenance by metabolically dependent processes directly balancing passive solute and water fluxes that would otherwise cause cell swelling under the influence of intracellular membrane-impermeant solutes; and (iii) volume regulation often involving reversible short-term transmembrane solute transport processes correcting cell volumes towards their normal baselines in response to imposed discrete perturbations. This review covers, in turn, the main predictions from such quantitative analysis and the experimental consequences of comparable alterations in extracellular pH, lactate concentration, membrane potential and extracellular tonicity. The effects of such alterations in the extracellular environment in resting amphibian muscles are then used to reproduce the intracellular changes that occur in each case in exercising muscle. The relative contributions of these various factors to the control of cell volume in resting and exercising skeletal muscle are thus described.

The membrane potential measured by intracellular electrodes, E(m), is the sum of the transmembrane potential difference (E(1)) between inner and outer cell membrane surfaces and a smaller potential difference (E(2)) between a volume containing fixed charges on or near the outer membrane surface and the bulk extracellular space. This study investigates the influence of E(2) upon transmembrane ion fluxes, and hence cellular electrochemical homeostasis, using an integrative approach that combines computational and experimental methods. First, analytic equations were developed to calculate the influence of charges constrained within a three-dimensional glycocalyceal matrix enveloping the cell membrane outer surface upon local electrical potentials and ion concentrations. Electron microscopy confirmed predictions of these equations that extracellular charge adsorption influences glycocalyceal volume. Second, the novel analytic glycocalyx formulation was incorporated into the charge-difference cellular model of Fraser and Huang to simulate the influence of extracellular fixed charges upon intracellular ionic homeostasis. Experimental measurements of E(m) supported the resulting predictions that an increased magnitude of extracellular fixed charge increases net transmembrane ionic leak currents, resulting in either a compensatory increase in Na(+)/K(+)-ATPase activity, or, in cells with reduced Na(+)/K(+)-ATPase activity, a partial dissipation of transmembrane ionic gradients and depolarization of E(m).

This study used Rana temporaria sartorius muscles to examine the effect of fatiguing electrical stimulation on the gap between the T-tubular and sarcoplasmic reticular membranes (T-SR distance) and the T-tubule diameter and compared this with corresponding effects on resting fibres exposed to a range of extracellular conditions that each replicate one of the major changes associated with muscular activity: membrane depolarisation, isotonic volume increase, acidification and intracellular lactate accumulation. Following each treatment, muscles were immersed in isotonic fixative solution and processed for transmission electron microscopy (TEM). Mean T-SR distances were estimated from orthogonal intercepts to provide estimates of diffusion distances between T and SR membranes and T-tubule diameter was estimated by measuring its shortest axis in the sampled J-SR complexes. Measurements from muscles fatigued by low frequency intermittent stimulation showed significant (P << 0.05) reversible increases in both T-SR distance and T-tubule diameter from 15.97 +/- 0.37 nm to 20.15 +/- 0.56 nm and from 15.44 +/- 0.60 nm to 22.26 +/- 0.84 nm (n = 40, 30) respectively. Exposure to increasing concentrations of extracellular [K(+)] in the absence of Cl(-) to produce membrane depolarisation without accompanying cell swelling reduced T-SR distance and increased T-tubule diameter, whilst comparable increases in [K(+)](e) in the presence of Cl(-) suggested that isotonic cell swelling has the opposite effect. Acidification alone, produced by NH(4)Cl addition and withdrawal, also decreased T-SR distance and T-tubule diameter. A similar reduction in T-SR distance occurred following exposure to extracellular Na-lactate where such acidification was accompanied by elevations of intracellular lactate, but these conditions produced a significant swelling of T-tubules attributable to movement of lactate from the cell into the T-tubules. This study thus confirms previous reports of significant increases in T-SR distance and T-tubule diameter following stimulation. However, of membrane depolarisation, isotonic cell swelling, intracellular acidification and lactate accumulation, only isotonic cell swelling increases T-SR distance whilst membrane depolarisation and intracellular lactate likely contribute to the observed increases in T-tubule diameter.

The effects of alterations in intracellular calcium homeostasis on surface membrane excitability were investigated in resting Rana temporaria sartorius muscle. This was prompted by initial results from a fatiguing stimulation protocol study that demonstrated a fibre subpopulation in which action potential generation in response to a standard 1.5 V electrical stimulus failed despite mean membrane potentials [E (m), -69+/-2.3 mV (n=14)] compatible with spike firing in a control set of quiescent muscle fibres. Intracellular micro-electrode recordings showed a similar reversible loss of excitability, attributable to an increased threshold, despite only small (7.1+/-1.8 mV) positive changes in E (m) after approximately 60-min exposures to nominally 0 Ca(2+) Ringer solutions in which Ca(2+) was replaced by Mg(2+). This effect was not reproduced by addition of Mg(2+) to the Ringer solution and persisted under conditions of Cl(-) deprivation. The effects of three pharmacological agents, cyclopiazonic acid (CPA), caffeine and 4-chloro-m-cresol (4-CmC), each known to deplete store Ca(2+) and increase cytosolic Ca(2+) through contrasting mechanisms without influencing E (m), were then investigated. All three agents produced a more rapid, but nevertheless still reversible, loss of membrane excitability than in 0 Ca(2+) Ringer solution alone. This reduction in membrane excitability persisted in fibres studied in solutions containing a normal [Ca(2+)] following prior depletion of store Ca(2+) using CPA- and 4-CmC-containing solutions. These novel findings suggest that sarcoplasmic reticulum Ca(2+) content profoundly influences surface membrane excitability, thereby providing a potential mechanism by which spike firing fails in well-polarised fibres during fatigue.

The combined effects of intracellular lactate and proton accumulation on cell volume, Vc, were investigated in resting Rana temporaria striated muscle fibres. Intracellular lactate and H+ concentrations were simultaneously increased by exposing resting muscle fibres to extracellular solutions that contained 20-80 mm sodium lactate. Cellular H+ and lactate entry was confirmed using pH-sensitive electrodes and 1H-NMR, respectively, and effects on Vc were measured using confocal microscope xz-scanning. Exposure to extracellular lactate up to 80 mm produced significant changes in pH and intracellular lactate (from a pH of 7.24 +/- 0.03, n = 8, and 4.65 +/- 1.07 mm, n = 6, respectively, in control fibres, to 6.59 +/- 0.03, n = 4, and 26.41 +/- 0.92 mm, n = 3, respectively) that were comparable to those observed following fatiguing stimulation (6.30-6.70 and 18.04 +/- 1.78 mm, n = 6, respectively). Yet, the increase in intracellular osmolarity expected from such an increase in intracellular lactate did not significantly alter Vc. Simulation of these experimental results, modified from the charge difference model of Fraser & Huang, demonstrated that such experimental manoeuvres produced changes in intracellular [H+] and [lactate] comparable to those observed during muscle fatigue, and accounted for this paradoxical conservation of Vc through balancing negative osmotic effects resulting from the net cation efflux that would follow a titration of intracellular membrane-impermeant anions by the intracellular accumulation of protons. It demonstrated that with established physiological values for intracellular buffering capacity and the permeability ratio of lactic acid and anionic lactate, P(LacH): P(Lac-), this would provide a mechanism that precisely balanced any effect on cell volume resulting from lactate accumulation during exercise.

The effect of electrical stimulation on cell volume, V (c), and its relationship to membrane potential, E (m), was investigated in Rana temporaria striated muscle. Confocal microscope xz-plane scanning and histology of plastic sections independently demonstrated significant and reversible increases in V (c) of 19.8+/-0.62% (n=3) and 27.1+/-8.62% (n=3), respectively, after a standard stimulation protocol. Microelectrode measurements demonstrated an accompanying membrane potential change, DeltaE (m), of +23.6+/-0.98 mV (n=3). The extent to which this DeltaE (m) might contribute to the observed changes in V (c) was explored in quiescent muscle exposed to variations in extracellular potassium concentration, [K(+)](e). E (m) and V (c) varied linearly with log [K(+)](e) and [K(+)](e), respectively, in the range 2.5-15 mM (R (2)=0.99 and 0.96), and these results were used to reconstruct an approximately linear relationship between V (c) and E (m) (DeltaV (c)=0.85E (m)+68.53; R (2)=0.99) and hence derive the DeltaV (c) expected from the DeltaE (m) during stimulation. This demonstrated that both the time course and magnitude of the increase and recovery of V (c) observed in active muscles could be reproduced by the corresponding [K(+)](e)-induced depolarisation in quiescent muscles, suggesting that the depolarisation associated with membrane activity makes a substantial contribution to the cell swelling during exercise. Furthermore, conditions of Cl(-) deprivation abolished the relationship between E (m) and V (c), supporting a mechanism in which the depolarisation of E (m) drives a passive redistribution of Cl(-) and hence cellular entry of Cl(-) and K(+) and an accompanying, osmotically driven, increase in V (c).

The influence of extracellular hypotonicity on the relationship between cell volume (V(c)) and resting membrane potential (E(m)) was investigated in Rana temporaria skeletal muscle. V(c) was measured by confocal microscope imaging of fibres through their transverse (xz) planes, and E(m) was determined using standard microelectrode techniques. Hypotonic solutions first elicited a rapid increase in fibre volume, DeltaV(R+) that fulfilled expectations of simple osmotic behaviour described in earlier reports. However, this was consistently followed by a slow increase in V(c) (DeltaV(S+)) to 10-15% above osmotic predictions. Longer (>1 h) exposures to hypotonic solutions permitted a subsequent slow decrease in V(c) (DeltaV(S-)), the eventual magnitude of which exceeded that of the preceding DeltaV(S+). Restoration of isotonic conditions elicited a prompt recovery in V(c) that matched simple osmotic predictions and thus left a net change in V(c). Such alterations in V(c) attributable to DeltaV(S+) then gradually reversed, while those due to DeltaV(S-) persisted. Both DeltaV(S+) and DeltaV(S-) persisted under conditions of Cl- deprivation. The depolarization of E(m) that accompanied DeltaV(R+) was consistent with dilution of intracellular [K(+)]. E(m) did not significantly alter during the subsequent DeltaV(S) transients. These empirical features of DeltaV(S+) and DeltaV(S-) were analysed using the quantitative charge-difference model of Fraser and Huang, published in 2004. This attributed the DeltaV(S+) to an electroneutral increase in the effective osmotic activity of normally membrane-impermeant intracellular anions. In contrast, the DeltaV(S-) could only be explained by an efflux of such anions and was accordingly comparable to organic anion-dependent regulatory volume decreases reported in other cell types.

The relationship between cell volume (V(c)) and membrane potential (E(m)) in Rana temporaria striated muscle fibres was investigated under different conditions of intracellular acidification. Confocal microscope xz-scanning monitored the changes in V(c), whilst conventional KCl and pH-sensitive microelectrodes measured E(m) and intracellular pH (pH(i)), respectively. Applications of Ringer solutions with added NH(4)Cl induced rapid reductions in V(c) that rapidly reversed upon their withdrawal. These could be directly attributed to the related alterations in extracellular tonicity. However: (1) a slower and persistent decrease in V(c) followed the NH(4)Cl withdrawal, leaving V(c) up to 10% below its resting value; (2) similar sustained decreases in resting V(c) were produced by the addition and subsequent withdrawal of extracellular solutions in which NaCl was isosmotically replaced with NH(4)Cl; (3) the same manoeuvres also produced a marked intracellular acidification, that depended upon the duration of the preceding exposure to NH(4)Cl, of up to 0.53 +/- 0.10 pH units; and (4) the corresponding reductions in V(c) similarly increased with this exposure time. These reductions in V(c) persisted and became more rapid with Cl(-) deprivation, thus excluding mechanisms involving either direct or indirect actions of pH(i) upon Cl(-)-dependent membrane transport. However they were abolished by the Na(+),K(+)-ATPase inhibitor ouabain. The E(m) changes that accompanied the addition and withdrawal of NH(4)(+) conformed to a Nernst equation modified to include realistic NH(4)(+) permeability terms, and thus the withdrawal of NH(4)(+) restored E(m) to close to control values despite a persistent change in V(c). Finally these E(m) changes persisted and assumed faster kinetics with Cl(-) deprivation. The relative changes in V(c), E(m) and pH(i) were compared to predictions from the recent model of Fraser and Huang published in 2004 that related steady-state values of V(c) and E(m) to the mean charge valency (z(x)) of intracellular membrane-impermeant anions, X(-)(i). By assuming accepted values of intracellular buffering capacity (beta(i)), intracellular acidification was shown to produce quantitatively predictable decreases in V(c). These findings thus provide experimental evidence that titration of the anionic z(x) by increased intracellular [H(+)] causes cellular volume decrease in the presence of normal Na(+),K(+)- ATPase activity, with Cl(-)-dependent membrane fluxes only influencing the kinetics of such changes.

A recently reported stabilization ('splinting') of the resting membrane potential ( Em) observed in amphibian skeletal muscle fibres despite extracellular hyperosmotic challenge has been attributed to high resting ratios of membrane Cl- to K+ permeability ( P Cl/ P K) combined with elevations of their intracellular Cl- concentrations, [Cl-]i, above electrochemical equilibrium by diuretic-sensitive cation-Cl-, Na-Cl (NCC) and/or Na-K-2Cl (NKCC), co-transporter activity. The present experiments localized this co-transporter activity by investigating the effects of established detubulation procedures on Em splinting. They exposed fibres to introduction and subsequent withdrawal of 400 mM extracellular glycerol, high divalent cation concentrations, and cooling. An abolition of tubular access of extracellularly added lissamine rhodamine fluorescence, visualized by confocal microscopy, and of the action potential afterdepolarization together confirmed successful transverse (T-) tubular detachment. Fibre volumes, V , of such detubulated fibres, determined using recently introduced confocal microscope-scanning methods, retained the simple dependence upon 1/[extracellular osmolarity], without significant evidence of the regulatory volume increases described in other cell types, previously established in intact fibres. However detubulation abolished the Em splinting shown by intact fibres. Em thus varied with extracellular osmolarity in detubulated fibres studied in standard, Cl(-)-containing, Ringer solutions and conformed to simple predictions from such changes in assuming that intracellular ion content was conserved and membrane potential change DeltaEm was principally determined by the K+ Nernst potential. Furthermore, cation--Cl- co-transport block brought about by [Cl-]o or [Na+]o deprivation, or inclusion of bumetanide (10 microM) and chlorothiazide (10 microM) in the extracellular fluid gave similar results. When taken together with previous reports of significant Cl- conductances in the surface membrane, these findings suggest a model that contrastingly suggests a T-tubular location for cation--Cl- co-transporter activity or its regulation.

Ultrastructural features of tubular-sarcoplasmic (T-SR) triad junctions and measures of cell volume following graded increases of extracellular tonicity were compared under physiological conditions recently shown to produce spontaneous release of intracellularly stored Ca2+ in fully polarized amphibian skeletal muscle fibres. The fibres were fixed using solutions of equivalent tonicities prior to processing for electron microscopy. The resulting anatomical sections demonstrated a partially reversible cell shrinkage corresponding to substantial increases in intracellular solute or ionic strength graded with extracellular tonicity. Serial thin sections through triad structures confirmed the presence of geometrically close but anatomically isolated transverse (T-) tubular and sarcoplasmic reticular (SR) membranes contrary to earlier suggestions for the development of luminal continuities between these structures in hypertonic solutions. They also quantitatively demonstrated accompanying decreases in T-SR distances, increased numbers of sections that showed closely apposed T and SR membranes, tubular luminal swelling and reductions in luminal volume of the junctional SR, all correlated with the imposed increases in extracellular osmolarity. Fully polarized fibres correspondingly showed elementary Ca(2+)-release events ('sparks', in 100 mM-sucrose-Ringer solution), sustained Ca2+ elevations and propagated Ca2+ waves (> or = 350-500 mM sucrose) following exposure to physiological Ringer solutions of successively greater tonicities. These were absent in hypotonic, isotonic or less strongly hypertonic (approximately 50 mM sucrose-Ringer) solutions. Yet exposure to hypotonic solutions also disrupted T-SR junctional anatomy. It increased the tubular diameters and T-SR distances and reduced their area of potential contact. The spontaneous release of intracellularly stored Ca2+ thus appears more closely to correlate with the expected changes in intracellular solute strength or a reduction in absolute T-SR distance rather than disruption of an optimal anatomical relationship between T and SR membranes taking place with either increases or decreases in extracellular tonicity.

Sartorius muscle fibres from cold-adapted Rana temporaria were exposed to variants of an established detubulation procedure (Koutsis et al. (1995) J Muscle Res Cell Motil 16, 519-528) to test the extent to which detubulation and tubular vacuolation phenomena could be separated using different conditions of osmotic shock. A control procedure was optimised to a 28-min exposure to 400 mM glycerol-Ringer. This was followed by a recovery step involving its replacement by a Ca2+/Mg(2+)-Ringer solution and steady cooling over 30 min from room temperature (approximately 18 degress C) to approximately 10 degress C, followed by the restoration of the normal Ringer solution. This procedure successfully abolished the action potential after-depolarisation component, reflecting a loss of tubular conduction ('detubulation') in 74.3 +/- 5.9% of the fibres studied. Omitting the cooling during the recovery step sharply reduced the incidence of detubulation. So did omitting either the high-[Ca2+] and/or [Mg2+] in the recovery solutions in test procedures, but to significantly different extents (P < 5%). Yet trapping of fluorescent Sulfhorhodamine B dye in 'closed' vacuoles persisted albeit with reduced proportions of fibre volume occupied by vacuoles. Furthermore, the variations in recovery conditions produced similar levels of vacuolation despite smaller vacuole sizes in the cooled fibres (P < 0.05). These findings demonstrate that fibre vacuolation and detubulation are phenomena that are potentially separable through varying the conditions of osmotic shock, with detubulation requiring significantly more stringent conditions than vacuolation.

The exposure of amphibian muscle to osmotic shock through the introduction and subsequent withdrawal of extracellular glycerol causes 'vacuolation' in the transverse tubules. Such manoeuvres can also electrically isolate the transverse tubules from the surface ('detubulation'), particular if followed by exposures to high extracellular [Ca2+] and/or gradual cooling. This study explored factors influencing vacuolation in Rana temporaria sartorius muscle. Vacuole formation was detected using phase contrast microscopy and through the trapping or otherwise of lissamine rhodamine dye fluorescence within such vacuoles. The preparations were also examined using electron microscopy, for penetration into the transverse tubules and tubular vacuoles of extracellular horseradish peroxidase introduced following the osmotic procedures. These comparisons distinguished for he first time two types of vacuole, 'open' and 'closed', whose lumina were respectively continuous with or detached from the remaining extracellular space. The vacuoles formed closed to and between the Z-lines, but subsequently elongated along the longitudinal axis of the muscle fibres. This suggested an involvement of tubular membrane material; the latter appeared particularly concentrated around such Z-lines in the electron-micrograph stereopairs of thick longitudinal sections. 'Open' vacuoles formed following osmotic shock produced by extracellular glycerol withdrawal from a glycerol-loaded fibre at a stage when one would expect a net water entry to the intracellular space. This suggests that vacuole formation requires active fluid transport into the tubular lumina in response to fibre swelling. 'Closed' vacuoles only formed when the muscle was subsequently exposed to high extracellular [Ca/+] and/or gradual cooling following the initial osmotic shock. Their densities were similar to those shown by 'open' vacuoles in preparations not so treated, suggesting that both vacuole types resulted from a single process initiated by glycerol withdrawal. However, vacuole 'closure' took place well after formation of 'open' vacuoles, over 25 min after glycerol withdrawal. Its time course closely paralleled the development of detubulation reported recently. It was irreversible, in contrast to the reversibility of 'open' vacuole formation. These findings identify electrophysiological 'detubulation' of striated muscle with 'closure' of initially 'open' vacuoles. The reversible formation of open vacuoles is compatible with some normal membrane responses to some physiological stresses such as fatigue, whereas irreversible formation of closed vacuoles might only be expected in pathological situations as in dystrophic muscle.