[Show abstract][Hide abstract] ABSTRACT: Transient increases in the intracellular calcium concentration, which are associated with electrical activation of neurones, control synapse-to-nucleus communication. Calcium signals differ in time and space but it is unclear exactly how this translates into stimulus-specific gene expression. Analysis of transcription induced by calcium transients with defined durations revealed that the evoked genomic responses, unlike those following neurotrophin exposure, are not all-or-none but graded events. The CRE-binding protein CREB, its coactivator CREB-binding protein (CBP), and SRE-interacting transcriptional regulators are fast on–off switches: their activities are induced by short-lasting calcium signals, remain active for the duration of the signal and are rapidly shut-off after calcium concentrations have returned to basal levels. CREB is switched on by a fast, nuclear calmodulin (CaM) kinase-dependent mechanism that mediates CREB phosphorylation on serine 133 within 30 s of calcium entry. The second calcium-activated route to CREB involves the MAP kinase/extracellular signal-regulated kinase (ERK1/2) cascade. This pathway can be triggered by brief, 30–60 s calcium transients. ERK1/2 activity peaks several minutes after calcium entry and can outlast the calcium transient. The shut-off of CREB and ERK1/2 involves rapid dephosphorylation of their activator sites. These properties of transcription factors and their regulating kinases and phosphatases provide a mechanism through which the duration of calcium signals specifies the magnitude of the transcriptional response. The decoding of temporal features of calcium transients is likely to contribute to impulse-specific gene expression.
Journal of Neurochemistry 01/2008; 79(4):849-858. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Heart failure leading to ventricular arrhythmogenesis is a major cause of clinical mortality and has been associated with a leak of sarcoplasmic reticular Ca(2+) into the cytosol due to increased open probabilities in cardiac ryanodine receptor Ca(2+)-release channels. Caffeine similarly increases such open probabilities, and so we explored its arrhythmogenic effects on intact murine hearts. A clinically established programmed electrical stimulation protocol adapted for studies of isolated intact mouse hearts demonstrated that caffeine (1 mM) increased the frequency of ventricular tachycardia from 0 to 100% yet left electrogram duration and latency unchanged during programmed electrical stimulation, thereby excluding slowed conduction as a cause of arrhythmogenesis. We then used fluorescence measurements of intracellular Ca(2+) concentration in isolated mouse ventricular cells to investigate parallel changes in Ca(2+) homeostasis associated with these arrhythmias. Both caffeine (1 mM) and FK506 (30 microM) reduced electrically evoked cytosolic Ca(2+) transients yet increased the frequency of spontaneous Ca(2+)-release events. Diltiazem (1 microM) but not nifedipine (1 microM) pretreatment suppressed these increases in frequency. Identical concentrations of diltiazem but not nifedipine correspondingly suppressed the arrhythmogenic effects of caffeine in whole hearts. These findings thus directly implicate spontaneous Ca(2+) waves in triggered arrhythmogenesis in intact hearts.
[Show abstract][Hide abstract] ABSTRACT: Many neuronal processes require gene activation by synaptically evoked Ca(2+) transients. Ca(2+)-dependent signal pathways activate some transcription factors outright, but here we report that such signals also potentiate the activation of nuclear receptors by their cognate hormone, and of CBF1 by Notch, transcription factors hitherto not thought to be Ca(2+)-responsive. This potentiation is occluded by histone deacetylase inhibition, indicating a mechanism involving inactivation of co-repressors associated with these transcription factors. Synaptic activity, acting via the nuclear Ca(2+)-dependent activation of CaM kinase IV, triggers the disruption of subnuclear domains containing class II histone deacetylases (HDACs) and silencing mediator of retinoic acid and thyroid hormone receptors (SMRT), a broad-specificity co-repressor which represses nuclear hormone receptors and CBF1. The sequential loss of class II HDACs and SMRT from the subnuclear domains, followed by nuclear export, is associated with disruption of SMRT interaction with its target transcription factors and sensitization of these factors to their activating signal. Counterbalancing these changes, protein phosphatase 1 promotes nuclear localization of SMRT and inactivation of nuclear receptors and CBF1. Thus, the synaptically controlled kinase-phosphatase balance of the neuron determines the efficacy of SMRT-mediated repression and the signal-responsiveness of a variety of transcription factors.
Journal of Neurochemistry 05/2005; 93(1):171-85. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ventricular arrhythmogenesis leading to sudden cardiac death remains responsible for significant mortality in conditions such as cardiac failure and the long-QT syndrome (LQTS). Arrhythmias may be accentuated by beta-adrenergic stimulation and, accordingly, the present study explored the possible effects of beta-adrenergic stimulation and L-type Ca(2+) channel blockade on ventricular arrhythmogenesis and Ca(2+) handling using the mouse heart as an experimental system. Studies in whole, Langendorff-perfused hearts using programmed electrical stimulation protocols adapted from clinical practice demonstrated sustained ventricular tachycardia following addition of 0.1 microM isoprenaline (n=15), whilst no arrhythmias were observed in the absence of the drug (n=15). Arrhythmias were suppressed by nifedipine or diltiazem pre-treatment (both 1 microM) (n=8 and 4 respectively) and were also induced by elevating external [Ca(2+)] (n=3). At the cellular level, 0.1 microM isoprenaline significantly increased normalized fluorescence (F/F(0)) in field-stimulated fluo-3-loaded mouse ventricular myocytes imaged using confocal microscopy, reflecting increases in sarcoplasmic reticulum Ca(2+) release (n=8). Elevated external [Ca(2+)] also increased F/F(0) (n=4) whilst 0.1 microM nifedipine or 0.1 microM diltiazem significantly decreased F/F(0) (n=13 and 6 respectively). Pre-treatment with 0.1 microM nifedipine or 0.1 microM diltiazem suppressed the increases in F/F(0) induced by 0.1 microM isoprenaline alone (n=14 and 6 respectively). The findings thus paralleled suppression of isoprenaline-induced arrhythmias seen with nifedipine or diltiazem at the whole-heart level. Taken together, the findings may have implications for the use of L-type Ca(2+) channel blockade in conditions associated with beta-adrenergically driven ventricular arrhythmias such as cardiac failure and LQTS.
Pflügers Archiv - European Journal of Physiology 12/2004; 449(2):150-8. · 4.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study investigated membrane transport mechanisms influencing relative changes in cell volume (V) and resting membrane potential (E(m)) following osmotic challenge in amphibian skeletal muscle fibres. It demonstrated a stabilization of E(m) despite cell shrinkage, which was attributable to elevation of intracellular [Cl(-)] above electrochemical equilibrium through Na(+)-Cl(-) and Na(+)-K(+)-2Cl(-) cotransporter action following exposures to extracellular hypertonicity. Fibre volumes (V) determined by confocal microscope x z - scanning of cutaneous pectoris muscle fibres varied linearly with [1/extracellular osmolarity], showing insignificant volume corrections, in fibres studied in Cl(-)-free, normal and Na(+)-free Ringer solutions and in the presence of bumetanide, chlorothiazide and ouabain. The observed volume changes following increases in extracellular tonicity were compared with microelectrode measurements of steady-state resting potentials (E(m)). Fibres in isotonic Cl(-)-free, normal and Na(+)-free Ringer solutions showed similar E(m) values consistent with previously reported permeability ratios P(Na)/P(K)(0.03-0.05) and P(Cl)/P(K) ( approximately 2.0) and intracellular [Na(+)], [K(+)] and [Cl(-)]. Increased extracellular osmolarities produced hyperpolarizing shifts in E(m) in fibres studied in Cl(-)-free Ringer solution consistent with the Goldman-Hodgkin-Katz (GHK) equation. In contrast, fibres exposed to hypertonic Ringer solutions of normal ionic composition showed no such E(m) shifts, suggesting a Cl(-)-dependent stabilization of membrane potential. This stabilization of E(m) was abolished by withdrawing extracellular Na(+) or by the combined presence of the Na(+)-Cl(-) cotransporter (NCC) inhibitor chlorothiazide (10 microM) and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC) inhibitor bumetanide (10 microM), or the Na(+)-K(+)-ATPase inhibitor ouabain (1 or 10 microM) during alterations in extracellular osmolarity. Application of such agents after such increases in tonicity only produced a hyperpolarization after a time delay, as expected for passive Cl(-) equilibration. These findings suggest a model that implicates the NCC and/or NKCC in fluxes that maintain [Cl(-)](i) above its electrochemical equilibrium. Such splinting of [Cl(-)](i) in combination with the high P(Cl)/P(K) of skeletal muscle stabilizes E(m) despite volume changes produced by extracellular hypertonicity, but at the expense of a cellular capacity for regulatory volume increases (RVIs). In situations where P(Cl)/P(K) is low, the same co-transporters would instead permit RVIs but at the expense of a capacity to stabilize E(m).
The Journal of Physiology 04/2004; 555(Pt 2):423-38. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A number of recent reports have suggested that ryanodine receptor (RyR)-Ca2+ release channels are gated by tubular depolarization in skeletal muscle through their direct coupling to intramembrane dihydropyridine receptor (DHPR)-voltage sensors. The qgama charge movement, which is inhibited by DHPR antagonists, is often regarded as the electrical signature for the voltage sensing process, yet pharmacological modifications of the RyR produce reciprocal upstream kinetic effects on an otherwise conserved qgamma charge. This study investigates the effect of DHPR-specific agonists upon intramembrane charge and the release of intracellularly stored Ca2+. We empirically demonstrate kinetic effects of FPL-64176 upon charge movements that closely resemble the consequences of previous interventions directed instead at the RyR. Increases in extracellular FPL-64176 concentration from 10 to 40 microM converted delayed qgamma transients to monotonic decays indistinguishable from the exponential qbeta current component. Yet total steady-state intramembrane charge and the steepness of its dependence upon test potential closely resembled previous reports from untreated fibres. These changes accompanied an appearance of transient cytosolic [Ca2+] elevations in confocal line-scans in fluo-3-loaded fibres studied in 10mM K+ and 40, but not 10 microM, FPL-64176 that resembled elementary Ca2+ release events ('sparks'). Pharmacological manipulations of the DHPR whose effects on intramembrane charge resembled those from manoeuvres directed at the RyR can thus produce downstream effects upon Ca2+ release.
Pflügers Archiv - European Journal of Physiology 04/2004; 447(6):922-7. · 4.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ionic currents in intact and detubulated frog sartorius muscle fibres were compared at room temperature using a loose-patch voltage clamp configuration in four experimental groups. The test fibres (i) were detubulated by a previously established osmotic shock protocol that involved the introduction and withdrawal of extracellular glycerol followed by exposure to Ca2+/Mg2+-Ringer solution and cooling. The control fibres were spared osmotic shock and (ii) simply studied in normal Ringer solution, (iii) exposed to 30 min of steady cooling to 9-10 degrees C before electrophysiological study or (iv) exposed to and studied in glycerol-Ringer solution. The presence or absence of detubulation was confirmed for all the experimental groups through assessing for the abolition or otherwise of the delayed after-depolarisation normally associated with action potential propagation into the transverse (T) tubules. All fibre groups showed similar resting potentials (-80 to -90 mV) thus ensuring consistent baseline voltages from which the voltage clamp steps were imposed. The intact muscle fibres in the three control groups (ii)-(iv) spared osmotic shock showed both inward Na+ and delayed rectifier outward (K+) currents. In contrast, patches from detubulated muscle fibres in the test group (i) showed only delayed outward currents, consistent with contrasting contributions to Na+ and K+ currents from regions of membrane affected or spared by the detubulation procedure. Nevertheless, the voltage dependence, maximum steady state amplitudes and timecourses of the delayed outward currents were conserved through all the experimental groups. These findings suggest that the surface as opposed to the tubular membrane contributes the greater part of the delayed rectifier current in amphibian skeletal muscle.
Journal of Muscle Research and Cell Motility 02/2004; 25(4-5):389-95. · 1.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The class II histone deacetylases, HDAC4 and HDAC5, directly bind to and repress myogenic transcription factors of the myocyte enhancer factor-2 (MEF-2) family thereby inhibiting skeletal myogenesis. During muscle differentiation, repression of gene transcription by MEF-2/HDAC complexes is relieved due to calcium/calmodulin-dependent (CaM) kinase-induced translocation of HDAC4 and HDAC5 to the cytoplasm. MEF-2 proteins and HDACs are also highly expressed in the nervous system and have been implicated in neuronal survival and differentiation. Here we investigated the possibility that the subcellular localization of HDACs, and thus their ability to repress target genes, is controlled by synaptic activity in neurones. We found that, in cultured hippocampal neurones, the localization of HDAC4 and HDAC5 is dynamic and signal-regulated. Spontaneous electrical activity was sufficient for nuclear export of HDAC4 but not of HDAC5. HDAC5 translocation to the cytoplasm was induced following stimulation of calcium flux through synaptic NMDA receptors or L-type calcium channels; glutamate bath application (stimulating synaptic and extrasynaptic NMDA receptors) antagonized nuclear export. Activity-induced nucleocytoplasmic shuttling of both HDACs was partially blocked by the CaM kinase inhibitor KN-62 with HDAC5 nuclear export being more sensitive to CaM kinase inhibition than that of HDAC4. Thus, the subcellular localization of HDACs in neurones is specified by neuronal activity; differences in the activation thresholds for HDAC4 and HDAC5 nuclear export provides a mechanism for input-specific gene expression.
Journal of Neurochemistry 05/2003; 85(1):151-9. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Muscle Research and Cell Motility 02/2003; 24(7):407-15. · 1.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calcium ions are ubiquitous second messengers that control diverse cellular functions. The versatility of Ca(2+) arises both from the ability of cells to employ a range of mechanisms to generate stimulus-induced Ca(2+) signals with defined characteristics and the existence of a large repertoire of Ca(2+) receptive proteins that mediate the effects of Ca(2+). In neurons, the regulation of gene expression by electrical activity-induced increases in Ca(2+) is critical for the long-term maintenance of neuronal adaptive responses. Different patterns of synaptic activity are able to generate Ca(2+) signals varying in their amplitude, temporal profile, spatial properties and source or site of entry. The information embedded in Ca(2+) signals is decoded by Ca(2+)-responsive transcriptional regulators, including protein kinases, phosphatases and transcription factors, with differing Ca(2+) sensitivities, kinetics of activation and deactivation, and subcellular localisation. The coordinated control of many transcriptional regulators by Ca(2+) signals determines the qualitative and quantitative nature of the genomic response.
European Journal of Pharmacology 08/2002; 447(2-3):131-40. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A hypothesis in which intramembrane charge reflects a voltage sensing process allosterically coupled to transitions in ryanodine receptor (RyR)-Ca(2+) release channels as opposed to one driven by release of intracellularly stored Ca(2+) would predict that such charging phenomena should persist in skeletal muscle fibres unable to release stored Ca(2+). Charge movement components were accordingly investigated in intact voltage-clamped amphibian fibres treated with known sarcoplasmic reticular (SR) Ca(2+)-ATPase inhibitors. Cyclopiazonic acid (CPA) pretreatment abolished Ca(2+) transients in fluo-3-loaded fibres following even prolonged applications of caffeine (10 mM) or K(+) (122 mM). Both CPA and thapsigargin (TG) transformed charge movements that included delayed (q(gamma)) "hump" components into simpler decays. However, steady-state charge-voltage characteristics were conserved to values (maximum charge, Q(max) approximately equal to 20-25 nC microF(-1); transition voltage, V* approximately equal to -40 to-50 mV; steepness factor, k approximately equal to 6-9 mV; holding voltage -90 mV) indicating persistent q(gamma) charge. The features of charge inactivation similarly suggested persistent q(beta) and q(gamma) charge contributions in CPA-treated fibres. Perchlorate (8.0 mM) restored the delayed kinetics shown by "on" q(gamma) charge movements, prolonged their "off" decays, conserved both Q(max) and k, yet failed to restore the capacity of such CPA-treated fibres for Ca(2+) release. Introduction of perchlorate (8.0 mM) or caffeine (0.2 mM) to tetracaine (2.0 mM)-treated fibres, also known to restore q(gamma) charge, similarly failed to restore Ca(2+) transients. Steady-state intramembrane q(gamma) charge thus persists with modified kinetics that can be restored to its normally complex waveform by perchlorate, even in intact muscle fibres unable to release Ca(2+). It is thus unlikely that q(gamma) charge movement is a consequence of SR Ca(2+) release rather than changes in tubular membrane potential.
The Journal of Physiology 04/2002; 539(Pt 3):869-82. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Muscle Research and Cell Motility 01/2002; 23(4):327-33. · 1.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A hypothesis in which intramembrane charge reflects a voltage sensing process allosterically coupled to transitions in ryanodine receptor (RyR)-Ca2+ release channels as opposed to one driven by release of intracellularly stored Ca2+ would predict that such charging phenomena should persist in skeletal muscle fibres unable to release stored Ca2+. Charge movement components were accordingly investigated in intact voltage-clamped amphibian fibres treated with known sarcoplasmic reticular (SR) Ca2+-ATPase inhibitors. Cyclopiazonic acid (CPA) pretreatment abolished Ca2+ transients in fluo-3-loaded fibres following even prolonged applications of caffeine (10 mM) or K+ (122 mM). Both CPA and thapsigargin (TG) transformed charge movements that included delayed (qγ) ‘hump’ components into simpler decays. However, steady-state charge-voltage characteristics were conserved to values (maximum charge, Qmax∼ 20–25 nC μF−1; transition voltage, V*∼−40 to −50 mV; steepness factor, k∼ 6–9 mV; holding voltage −90 mV) indicating persistent qγ charge. The features of charge inactivation similarly suggested persistent qβ and qγ charge contributions in CPA-treated fibres. Perchlorate (8.0 mm) restored the delayed kinetics shown by ‘on’qγ charge movements, prolonged their ‘off’ decays, conserved both Qmax and k, yet failed to restore the capacity of such CPA-treated fibres for Ca2+ release. Introduction of perchlorate (8.0 mm) or caffeine (0.2 mm) to tetracaine (2.0 mm)-treated fibres, also known to restore qγ charge, similarly failed to restore Ca2+ transients. Steady-state intramembrane qγ charge thus persists with modified kinetics that can be restored to its normally complex waveform by perchlorate, even in intact muscle fibres unable to release Ca2+. It is thus unlikely that qγ charge movement is a consequence of SR Ca2+ release rather than changes in tubular membrane potential.
The Journal of Physiology 12/2001; 539(3):869 - 882. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 1. Regenerative Ca2+ waves and oscillations indicative of calcium-induced calcium release (CICR) activity were induced in fully polarized, fluo-3-loaded, intact frog skeletal muscle fibres by exposure to hypertonic Ringer solutions. 2. The calcium waves persisted in fibres exposed to EGTA-containing solutions, during sustained depolarization of the membrane potential or following treatment with the dihydropyridine receptor (DHPR)-blocker nifedipine. 3. The waves were blocked by the ryanodine receptor (RyR)-specific agents ryanodine and tetracaine, and potentiated by caffeine. 4. In addition to these pharmacological properties, the amplitudes, frequency and velocity of such hypertonicity-induced waves closely resembled those of Ca2+ waves previously described in dyspedic skeletal myocytes expressing the cardiac RyR-2. 5. Quantitative transmission and freeze-fracture electronmicroscopy demonstrated a reversible cell shrinkage, transverse (T)-tubular luminal swelling and decreased T-sarcoplasmic reticular (SR) junctional gaps in fibres maintained in and then fixed using hypertonic solutions. 6. The findings are consistent with a hypothesis in which RyR-Ca2+ release channels can be partially liberated from their normal control by T-tubular DHPR-voltage sensors in hypertonic solutions, thereby permitting CICR to operate even in such fully polarized skeletal muscle fibres.
The Journal of Physiology 11/2001; 536(Pt 2):351-9. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 1. The influence of the transverse (T) tubules on surface action potential conduction was investigated by comparing electrophysiological and confocal microscopic assessments of tubular changes in osmotically shocked and control fibres from frog sartorius muscle. 2. The membrane-impermeant fluorescent dye, di-8-ANEPPs spread readily from the bathing extracellular solution into the tubular membranes in control, intact fibres. Prior exposure of muscles to a hypertonic glycerol-Ringer solution, its replacement by an isotonic Ca(2+)-Mg(2+) Ringer solution and cooling sharply reduced such access. In contrast, dye application in the course of this osmotic shock procedure stained the large tubular vacuoles hitherto associated with successful muscle detubulation. 3. Conduction velocities in intact, control fibres (1.91 +/- 0.048 m s(-1), mean +/- S.E.M., n = 32 fibres) agreed with earlier values reported at room temperature (18-21 degrees C) and were unaffected by prior episodes of steady cooling to 8-10 degrees C (1.91 +/- 0.043 m s(-1), n = 30). 4. Cooling to 11.5 degrees C reduced these velocities (1.47 +/- 0.081 m s(-1), n = 25) but action potential waveforms still included early overshoots and the delayed after-depolarizations associated with tubular electrical activity. 5. In contrast, action potentials from cooled, superficial fibres in osmotically shocked muscles lacked after-depolarization phases implying tubular detachment. Their mean conduction velocities (1.62 +/- 0.169 m s(-1), n = 25) were not significantly altered from values obtained in untreated controls or in intact fibres in muscle similarly treated with glycerol, in direct contrast to earlier results. 6. Cooling produced similar reductions in maximum rates of voltage change dV/dt in action potentials from all fibre groups with lower rates of change shown by detubulated fibres. 7. Use of an antibody to a conserved epitope of the alpha-subunit of voltage-gated sodium channels suggested a concentration of sodium channels close to the mouths of the T tubules. 8. These electrophysiological and anatomical findings are consistent with a partial independence of electrical events in the transverse tubules from those responsible for the rapid conduction of surface regenerative activity. 9. The findings are discussed in terms of a partial separation of the electrical activity propagated over the surface membrane, from the initiation of propagated activity within the T tubules, by the triggering of the sodium channels clustered selectively around the mouths of the T tubules.
The Journal of Physiology 10/2001; 535(Pt 2):579-90. · 4.38 Impact Factor