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ABSTRACT: Depolarization of skeletal muscle fibers induces sarcoplasmic reticulum (SR) Ca(2+) release and contraction that progressively decline while depolarization is maintained. Voltage-dependent inactivation of SR Ca(2+) release channels and SR Ca(2+) depletion are the two processes proposed to explain the decline of SR Ca(2+) release during long-lasting depolarizations. However, the relative contribution of these processes, especially under physiological conditions of activation, is not clearly established. Using Fura-2 and Fluo-5N to monitor cytosolic and SR Ca(2+) changes, respectively, in voltage-controlled mouse muscle fibers, we show that 2-min conditioning depolarizations reduce voltage-activated cytosolic Ca(2+) signals with a V1/2 of -53 mV but also induce SR Ca(2+) depletion that decreased the releasable pool of Ca(2+) with the same voltage sensitivity. In contrast, measurement of SR Ca(2+) changes indicated that SR Ca(2+) release channels were inactivated after SR had been depleted and in response to much higher depolarizations with a V1/2 of -13 mV. In response to trains of action potentials, cytosolic Ca(2+) signals decayed with time, whereas SR Ca(2+) changes remained stable over 1-min stimulation, demonstrating that SR Ca(2+) depletion is exclusively responsible for the decline of SR Ca(2+) release under physiological conditions of excitation. These results suggest that previous studies using steady-state inactivation protocols to investigate the voltage dependence of Ca(2+) release inactivation in fact probed the voltage dependence of SR Ca(2+) depletion, and that SR Ca(2+) depletion is the only process that leads to Ca(2+) release decline during continuous stimulation of skeletal muscle.
The Journal of General Physiology 05/2013; 141(5):557-65. · 3.84 Impact Factor
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ABSTRACT: Caveolins are plasma-membrane-associated proteins potentially involved in a variety of signalling pathways. Different mutations
in CAV3, the gene encoding for the muscle-specific isoform caveolin-3 (Cav-3), lead to muscle diseases, but the underlying molecular
mechanisms remain largely unknown. Here, we explored the functional consequences of a Cav-3 mutation (P104L) inducing the
1C type limb-girdle muscular dystrophy (LGMD 1C) in human on intracellular Ca2+ regulation of adult skeletal muscle fibres. A YFP-tagged human Cav-3P104L mutant was expressed in vivo in muscle fibres from mouse. Western blot analysis revealed that expression of this mutant led
to an ∼80% drop of the level of endogenous Cav-3. The L-type Ca2+ current density was found largely reduced in fibres expressing the Cav-3P104L mutant, with no change in the voltage dependence of activation and inactivation. Interestingly, the maximal density of intramembrane
charge movement was unaltered in the Cav-3P104L-expressing fibres, suggesting no change in the total amount of functional voltage-sensing dihydropyridine receptors (DHPRs).
Also, there was no obvious alteration in the properties of voltage-activated Ca2+ transients in the Cav-3P104L-expressing fibres. Although the actual role of the Ca2+ channel function of the DHPR is not clearly established in adult skeletal muscle, its specific alteration by the Cav-3P104L mutant suggests that it may be involved in the physiopathology of LGMD 1C.
Pflügers Archiv - European Journal of Physiology 04/2012; 457(2):361-375. · 4.46 Impact Factor
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ABSTRACT: Under resting conditions, external Ca(2+) is known to enter skeletal muscle cells, whereas Ca(2+) stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the sarcolemmal Ca(2+) entry and in the SR Ca(2+) leak is still a matter of debate, but several lines of evidence suggest that these Ca(2+) fluxes are up-regulated in Duchenne muscular dystrophy. We investigated here SR calcium permeation at resting potential and in response to depolarization in voltage-controlled skeletal muscle fibers from control and mdx mice, the mouse model of Duchenne muscular dystrophy. Using the cytosolic Ca(2+) dye Fura2, we first demonstrated that the rate of Ca(2+) increase in response to cyclopiazonic acid (CPA)-induced inhibition of SR Ca(2+)-ATPases at resting potential was significantly higher in mdx fibers, which suggests an elevated SR Ca(2+) leak. However, removal of external Ca(2+) reduced the rate of CPA-induced Ca(2+) increase in mdx and increased it in control fibers, which indicates an up-regulation of sarcolemmal Ca(2+) influx in mdx fibers. Fibers were then loaded with the low-affinity Ca(2+) dye Fluo5N-AM to measure intraluminal SR Ca(2+) changes. Trains of action potentials, chloro-m-cresol, and depolarization pulses evoked transient Fluo5N fluorescence decreases, and recovery of voltage-induced Fluo5N fluorescence changes were inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR Ca(2+) changes. Voltage dependence and magnitude of depolarization-induced SR Ca(2+) depletion were found to be unchanged in mdx fibers, but the rate of the recovery phase that followed depletion was found to be faster, indicating a higher SR Ca(2+) reuptake activity in mdx fibers. Overall, CPA-induced SR Ca(2+) leak at -80 mV was found to be significantly higher in mdx fibers and was potentiated by removal of external Ca(2+) in control fibers. The elevated passive SR Ca(2+) leak may contribute to alteration of Ca(2+) homeostasis in mdx muscle.
The Journal of General Physiology 03/2012; 139(3):209-18. · 3.84 Impact Factor
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ABSTRACT: When the intracellular calcium stores are depleted, a Ca(2+) influx is activated to refill these stores. This store-operated Ca(2+) entry (SOCE) depends on the cooperation of several proteins as STIM1, Orai1, and, possibly, TRPC1. To elucidate this role of TRPC1 in skeletal muscle, TRPC1 was overexpressed in C2C12 cells and SOCE was studied by measuring the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)). TRPC1 overexpression significantly increased both the amplitude and the maximal rate-of-rise of SOCE. When YM-58483, an inhibitor of TRPC1 was used, these differences were eliminated, moreover, SOCE was slightly suppressed. A decrease in the expression of STIM1 together with the downregulation of SERCA was confirmed by Western-blot. As a consequence, a reduction in maximal Ca(2+) uptake rate and a higher resting [Ca(2+)](i) following the Ca(2+) transients evoked by 120mM KCl were detected. Morphological changes also accompanied the overexpression of TRPC1. Differentiation of the myoblasts started later, and the myotubes were thinner in TRPC1-overexpressing cultures. For these changes the observed decrease in the nuclear expression of NFAT1 could be responsible. Our results suggest that enhanced expression of TRPC1 increases SOCE and has a negative effect on the STIM1-Orai1 system, indicating an interaction between these proteins.
Cell calcium 06/2011; 49(6):415-25. · 4.29 Impact Factor
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ABSTRACT: Caveolin-3 is the striated muscle specific isoform of the scaffolding protein family of caveolins and has been shown to interact with a variety of proteins, including ion channels. Mutations in the human CAV3 gene have been associated with several muscle disorders called caveolinopathies and among these, the P104L mutation (Cav-3(P104L)) leads to limb girdle muscular dystrophy of type 1C characterized by the loss of sarcolemmal caveolin. There is still no clear-cut explanation as to specifically how caveolin-3 mutations lead to skeletal muscle wasting. Previous results argued in favor of a role for caveolin-3 in dihydropyridine receptor (DHPR) functional regulation and/or T-tubular membrane localization. It appeared worth closely examining such a functional link and investigating if it could result from the direct physical interaction of the two proteins. Transient expression of Cav-3(P104L) or caveolin-3 specific siRNAs in C2C12 myotubes both led to a significant decrease of the L-type Ca(2+) channel maximal conductance. Immunolabeling analysis of adult skeletal muscle fibers revealed the colocalization of a pool of caveolin-3 with the DHPR within the T-tubular membrane. Caveolin-3 was also shown to be present in DHPR-containing triadic membrane preparations from which both proteins co-immunoprecipitated. Using GST-fusion proteins, the I-II loop of Ca(v)1.1 was identified as the domain interacting with caveolin-3, with an apparent affinity of 60nM. The present study thus revealed a direct molecular interaction between caveolin-3 and the DHPR which is likely to underlie their functional link and whose loss might therefore be involved in pathophysiological mechanisms associated to muscle caveolinopathies.
The international journal of biochemistry & cell biology 01/2011; 43(5):713-20. · 4.89 Impact Factor
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ABSTRACT: A number of G-protein-coupled receptors are expressed in skeletal muscle but their roles in muscle physiology and downstream effector systems remain poorly investigated. Here we explored the functional importance of the G-protein betagamma (Gbetagamma) signalling pathway on voltage-controlled Ca(2+) homeostasis in single isolated adult skeletal muscle fibres. A GFP-tagged Gbeta(1)gamma(2) dimer was expressed in vivo in mice muscle fibres. The GFP fluorescence pattern was consistent with a Gbeta(1)gamma(2) dimer localization in the transverse-tubule membrane. Membrane current and indo-1 fluorescence measurements performed under voltage-clamp conditions reveal a drastic reduction of both L-type Ca(2+) current density and of peak amplitude of the voltage-activated Ca(2+) transient in Gbeta(1)gamma(2)-expressing fibres. These effects were not observed upon expression of Gbeta(2)gamma(2), Gbeta(3)gamma(2) or Gbeta(4)gamma(2). Our data suggest that the G-protein beta(1)gamma(2) dimer may play an important regulatory role in skeletal muscle excitation-contraction coupling.
The Journal of Physiology 08/2010; 588(Pt 15):2945-60. · 4.72 Impact Factor
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Journal of Biological Chemistry 01/2010; 285(5):le2. · 4.77 Impact Factor
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Lama Al-Qusairi,
Norbert Weiss,
Anne Toussaint,
Céline Berbey,
Nadia Messaddeq,
Christine Kretz,
Despina Sanoudou,
Alan H Beggs, Bruno Allard,
Jean-Louis Mandel,
Jocelyn Laporte,
Vincent Jacquemond,
Anna Buj-Bello
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ABSTRACT: Skeletal muscle contraction is triggered by the excitation-contraction (E-C) coupling machinery residing at the triad, a membrane structure formed by the juxtaposition of T-tubules and sarcoplasmic reticulum (SR) cisternae. The formation and maintenance of this structure is key for muscle function but is not well characterized. We have investigated the mechanisms leading to X-linked myotubular myopathy (XLMTM), a severe congenital disorder due to loss of function mutations in the MTM1 gene, encoding myotubularin, a phosphoinositide phosphatase thought to have a role in plasma membrane homeostasis and endocytosis. Using a mouse model of the disease, we report that Mtm1-deficient muscle fibers have a decreased number of triads and abnormal longitudinally oriented T-tubules. In addition, SR Ca(2+) release elicited by voltage-clamp depolarizations is strongly depressed in myotubularin-deficient muscle fibers, with myoplasmic Ca(2+) removal and SR Ca(2+) content essentially unaffected. At the molecular level, Mtm1-deficient myofibers exhibit a 3-fold reduction in type 1 ryanodine receptor (RyR1) protein level. These data reveal a critical role of myotubularin in the proper organization and function of the E-C coupling machinery and strongly suggest that defective RyR1-mediated SR Ca(2+) release is responsible for the failure of muscle function in myotubular myopathy.
Proceedings of the National Academy of Sciences 11/2009; 106(44):18763-8. · 9.68 Impact Factor
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ABSTRACT: Extensive studies performed in nonexcitable cells and expression systems have shown that type 1 transient receptor potential canonical (TRPC1) channels operate mainly in plasma membranes and open through phospholipase C-dependent processes, membrane stretch, or depletion of Ca(2+) stores. In skeletal muscle, it is proposed that TRPC1 channels are involved in plasmalemmal Ca(2+) influx and stimulated by store depletion or membrane stretch, but direct evidence for TRPC1 sarcolemmal channel activity is not available. We investigated here the functional role of TRPC1 using an overexpressing strategy in adult mouse muscle fibers. Immunostaining for endogenous TRPC1 revealed a striated expression pattern that matched sarcoplasmic reticulum (SR) Ca(2+) pump immunolabeling. In cells expressing TRPC1-yellow fluorescent protein (YFP), the same pattern of expression was observed, compatible with a longitudinal SR localization. Resting electric properties, action potentials, and resting divalent cation influx were not altered in TRPC1-YFP-positive cells. Poisoning with the SR Ca(2+) pump blocker cyclopiazonic acid elicited a contracture of the fiber at the level of the overexpression site in presence and absence of external Ca(2+) which was not observed in control cells. Ca(2+) measurements indicated that resting Ca(2+) and the rate of Ca(2+) increase induced by cyclopiazonic acid were higher in the TRPC1-YFP-positive zone than in the TRPC1-YFP-negative zone and control cells. Ca(2+) transients evoked by 200-ms voltage clamp pulses decayed slower in TRPC1-YFP-positive cells. In contrast to previous hypotheses, these data demonstrate that TRPC1 operates as a SR Ca(2+) leak channel in skeletal muscle.
Journal of Biological Chemistry 10/2009; 284(52):36387-94. · 4.77 Impact Factor
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ABSTRACT: Ca2+ is known to enter skeletal muscle at rest and during activity. Except for the well-characterized Ca2+ entry through L-type channels, pathways involved in these Ca2+ entries remain elusive in adult muscle. This study investigates Ca2+ influx at rest and during activity using the method of Mn2+ quenching of fura-2 fluorescence on voltage-controlled adult skeletal muscle cells. Resting rate of Mn2+ influx depended on external [Mn2+] and membrane potential. At -80 mV, replacement of Mg2+ by Mn2+ gave rise to an outward current associated with an increase in cell input resistance. Calibration of fura-2 response indicated that Mn2+ influx was too small to be resolved as a macroscopic current. Partial depletion of the sarcoplasmic reticulum induced by a train of action potentials in the presence of cyclopiazonic acid led to a slight increase in resting Mn2+ influx but no change in cell input resistance and membrane potential. Trains of action potentials considerably increased Mn2+ entry through an electrically silent pathway independent of L-type channels, which provided 24% of the global Mn2+ influx at +30 mV under voltage-clamp conditions. Within this context, the nature and the physiological role of the Ca2+ pathways involved during muscle excitation still remain open questions.
Biophysical Journal 05/2009; 96(7):2648-57. · 3.65 Impact Factor
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ABSTRACT: Caveolins are plasma-membrane-associated proteins potentially involved in a variety of signalling pathways. Different mutations in CAV3, the gene encoding for the muscle-specific isoform caveolin-3 (Cav-3), lead to muscle diseases, but the underlying molecular mechanisms remain largely unknown. Here, we explored the functional consequences of a Cav-3 mutation (P104L) inducing the 1C type limb-girdle muscular dystrophy (LGMD 1C) in human on intracellular Ca(2+) regulation of adult skeletal muscle fibres. A YFP-tagged human Cav-3(P104L) mutant was expressed in vivo in muscle fibres from mouse. Western blot analysis revealed that expression of this mutant led to an approximately 80% drop of the level of endogenous Cav-3. The L-type Ca(2+) current density was found largely reduced in fibres expressing the Cav-3(P104L) mutant, with no change in the voltage dependence of activation and inactivation. Interestingly, the maximal density of intramembrane charge movement was unaltered in the Cav-3(P104L)-expressing fibres, suggesting no change in the total amount of functional voltage-sensing dihydropyridine receptors (DHPRs). Also, there was no obvious alteration in the properties of voltage-activated Ca(2+) transients in the Cav-3(P104L)-expressing fibres. Although the actual role of the Ca(2+) channel function of the DHPR is not clearly established in adult skeletal muscle, its specific alteration by the Cav-3(P104L) mutant suggests that it may be involved in the physiopathology of LGMD 1C.
Pflügers Archiv - European Journal of Physiology 06/2008; 457(2):361-75. · 4.46 Impact Factor
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ABSTRACT: The physiological properties and role of the type 3 ryanodine receptor (RyR3), a calcium release channel expressed in a wide variety of cell types, remain mysterious. We forced, in vivo, the expression of RyR3 in adult mouse skeletal muscle fibres using a GFP-RyR3 DNA construct. GFP fluorescence was found within spatially restricted regions of muscle fibres where it exhibited a sarcomere-related banded pattern consistent with a localization within or near the junctional sarcoplasmic reticulum membrane. Immunostaining confirmed the presence of RyR3 together with RyR1 within the GFP-positive areas. In approximately 90% of RyR3-positive fibres microinjected with the calcium indicator fluo-3, we detected repetitive spontaneous transient elevations of intracellular Ca2+ that persisted when fibres were voltage-clamped at -80 mV. These Ca2+ transients remained essentially confined to the RyR3 expression region. They ranged from wide local events to propagating Ca2+ waves and were in some cases associated with local contractile activity. When voltage-clamp depolarizations were applied while fluo-3 or rhod-2 fluorescence was measured within the RyR3-expressing region, no voltage-evoked 'spark-like' elementary Ca2+ release event could be detected. Still global voltage-activated Ca2+ release exhibited a prominent early peak within the RyR3-expressing regions. Measurements were also taken from muscles fibres expressing a GFP-RyR1 construct; positive fibres also yielded a local banded pattern of GFP fluorescence but exhibited no spontaneous Ca2+ release. Results demonstrate that RyR3 is a very potent source of voltage-independent Ca2+ release activity. Conversely we find no evidence that it could contribute to the production of discrete voltage-activated Ca2+ release events in differentiated mammalian skeletal muscle.
The Journal of Physiology 02/2008; 586(2):441-57. · 4.72 Impact Factor
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ABSTRACT: Odontoblasts are post-mitotic cells involved in the dentine formation throughout the life of the tooth and suspected to play
a role in tooth pain transmission. They are organized as a single layer of specialized cells along the interface between dental
pulp and calcified dentinal tubules into which run a cellular extension (odontoblast process) bathed in a liquid phase. Dense
sensory unmyelinated nerve fibres surrounded the odontoblast bodies, coiled around the cell processes and give to this complex
(nerve/odontoblast) a fundamental role as a barrier regulating molecules, fluid flow, ion transferts between dentine and pulp
following external stimuli (mechanical thermal, electrical, osmotic shock…). Thus, this unique spatial situation of odontoblasts
closely related with nerve endings and fluid movements suggest that odontoblasts could convert pain-evoking fluid displacement
within dentinal tubules into electrical signals via at least mechanosensitive ion channels. Along this line, two kinds of mechanosensitive K+ channels have been identified in human odontoblasts: I- TREK-1 channels belonging to the two-pore-domain potassium channel
family and expressed in the plasma membrane of coronal odontoblasts; II- high-conductance Ca2+ -activated potassium channels (KCa) activated by stretch of the membrane as well as osmotic shock. These findings strengthened by the recent evidence for excitable
properties of odontoblasts, concentration of mechanosensitive channels in the borderline between cell extension and bodies
and clustering of key molecules at the site of odontoblast-nerve contact strongly suggest that odontoblasts may operate as
sensor cells.
12/2007: pages 147-155;
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ABSTRACT: Caveolins are membrane scaffolding proteins that associate with and regulate a variety of signalling proteins, including ion channels. A deficiency in caveolin-3 (Cav-3), the major striated muscle isoform, is responsible for skeletal muscle disorders, such as limb-girdle muscular dystrophy 1C (LGMD 1C). The molecular mechanisms leading to the muscle wasting that characterizes this pathology are poorly understood. Here we show that a loss of Cav-3 induced by the expression of the LGMD 1C-associated mutant P104L (Cav-3(P104L)) provokes a reduction by half of the maximal conductance of the voltage-dependent L-type Ca(2+) channel in mouse primary cultured myotubes and fetal skeletal muscle fibres. Confocal immunomiscrocopy indicated a colocalization of Cav-3 and Ca(v)1.1, the pore-forming subunit of the L-type Ca(2+) channel, at the surface membrane and in the developing T-tubule network in control myotubes and fetal fibres. In myotubes expressing Cav-3(P104L), the loss of Cav-3 was accompanied by a 66% reduction in Ca(v)1.1 mean labelling intensity. Our results suggest that Cav-3 is involved in L-type Ca(2+) channel membrane function and localization in skeletal muscle cells and that an alteration of L-type Ca(2+) channels could be involved in the physiopathological mechanisms of caveolinopathies.
The Journal of Physiology 06/2007; 580(Pt.3):745-54. · 4.72 Impact Factor
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ABSTRACT: Control of membrane voltage and membrane current measurements are of strong interest for the study of numerous aspects of skeletal muscle physiology and pathophysiology. The silicone-clamp technique makes use of a conventional patch-clamp apparatus to achieve whole-cell voltage clamp of a restricted portion of a fully differentiated adult skeletal muscle fiber. The major part of an isolated muscle fiber is insulated from the extracellular medium with silicone grease, and the tip of a single microelectrode connected to the amplifier is then inserted within the fiber through the silicone layer. This method represents an alternative to the traditional vaseline-gap isolation and two or three microelectrode voltage-clamp techniques. This chapter reviews the main benefits of the silicone-clamp technique and provides detailed insights into its practical implementation.
Methods in molecular biology (Clifton, N.J.) 02/2007; 403:185-94.
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ABSTRACT: Odontoblasts are responsible for the dentin formation. They are suspected to play a role in tooth pain transmission as sensor cells because of their close relationship with nerve, but this role has never been evidenced. We demonstrate here that human odontoblasts in vitro produce voltage-gated tetrodotoxin-sensitive Na(+) currents in response to depolarization under voltage clamp conditions and are able to generate action potentials. Odontoblasts express neuronal isoforms of alpha2 and beta2 subunits of sodium channels. Co-cultures of odontoblasts with trigeminal neurons indicate a clustering of alpha2 and beta2 sodium channel subunits and, at the sites of cell-cell contact, a co-localization of odontoblasts beta2 subunits with peripherin. In vivo, sodium channels are expressed in odontoblasts. Ankyrin(G) and beta2 co-localize, suggesting a link for signal transduction between axons and odontoblasts. Evidence for excitable properties of odontoblasts and clustering of key molecules at the site of odontoblast-nerve contact strongly suggest that odontoblasts may operate as sensor cells that initiate tooth pain transmission.
Journal of Biological Chemistry 10/2006; 281(39):29002-10. · 4.77 Impact Factor
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ABSTRACT: In skeletal muscle, sarcoplasmic reticulum (SR) calcium release is controlled by the plasma membrane voltage through interactions between the voltage-sensing dihydropyridine receptor (DHPr) and the ryanodine receptor (RYr) calcium release channel. Maurocalcine (MCa), a scorpion toxin peptide presenting some homology with a segment of a cytoplasmic loop of the DHPr, has been previously shown to strongly affect the activity of the isolated RYr. We injected MCa into mouse skeletal muscle fibers and measured intracellular calcium under voltage-clamp conditions. Voltage-activated calcium transients exhibited similar properties in control and in MCa-injected fibers during the depolarizing pulses, and the voltage dependence of calcium release was similar under the two conditions. However, MCa was responsible for a pronounced sustained phase of Ca(2+) elevation that proceeded for seconds following membrane repolarization, with no concurrent alteration of the membrane current. The magnitude of the underlying uncontrolled extra phase of Ca(2+) release correlated well with the peak calcium release during the pulse. Results suggest that MCa binds to RYr that open on membrane depolarization and that this interaction specifically alters the process of repolarization-induced closure of the channels.
Biophysical Journal 10/2006; 91(6):2206-15. · 3.65 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: Odontoblasts are responsible for the dentin formation. They are suspected to play a role in tooth pain transmission as sensor
cells because of their close relationship with nerve, but this role has never been evidenced. We demonstrate here that human
odontoblasts in vitro produce voltage-gated tetrodotoxin-sensitive Na+ currents in response to depolarization under voltage clamp conditions and are able to generate action potentials. Odontoblasts
express neuronal isoforms of α2 and β2 subunits of sodium channels. Co-cultures of odontoblasts with trigeminal neurons indicate
a clustering of α2 and β2 sodium channel subunits and, at the sites of cell-cell contact, a co-localization of odontoblasts
β2 subunits with peripherin. In vivo, sodium channels are expressed in odontoblasts. AnkyrinG and β2 co-localize, suggesting a link for signal transduction between axons and odontoblasts. Evidence for excitable properties
of odontoblasts and clustering of key molecules at the site of odontoblast-nerve contact strongly suggest that odontoblasts
may operate as sensor cells that initiate tooth pain transmission.
Journal of Biological Chemistry 09/2006; 281(39):29002-29010. · 4.77 Impact Factor
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ABSTRACT: In skeletal muscle, sarcoplasmic reticulum (SR) Ca2+ depletion is suspected to trigger a calcium entry across the plasma membrane and recent studies also suggest that the opening of channels spontaneously active at rest and possibly involved in Duchenne dystrophy may be regulated by SR Ca2+ depletion. Here we simultaneously used the cell-attached and whole-cell voltage-clamp techniques as well as intracellular Ca2+ measurements on single isolated mouse skeletal muscle fibres to unravel any possible change in membrane conductance that would depend upon SR Ca2+ release and/or SR Ca2+ depletion. Delayed rectifier K+ single channel activity was routinely detected during whole-cell depolarizing pulses. In addition the activity of channels carrying unitary inward currents of approximately 1.5 pA at -80 mV was detected in 17 out of 127 and in 21 out of 59 patches in control and mdx dystrophic fibres, respectively. In both populations of fibres, large whole-cell depolarizing pulses did not reproducibly increase this channel activity. This was also true when, repeated application of the whole-cell pulses led to exhaustion of the Ca2+ transient. SR Ca2+ depletion produced by the SR Ca2+ pump inhibitor cyclopiazonic acid (CPA) also failed to induce any increase in the resting whole-cell conductance and in the inward single channel activity. Overall results indicate that voltage-activated SR Ca2+ release and/or SR Ca2+ depletion are not sufficient to activate the opening of channels carrying inward currents at negative voltages and challenge the physiological relevance of a store-operated membrane conductance in adult skeletal muscle.
The Journal of Physiology 09/2006; 575(Pt 1):69-81. · 4.72 Impact Factor
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ABSTRACT: The Caenorhabditis elegans SLO-1 channel belongs to the family of calcium-activated large conductance BK potassium channels. SLO-1 has been shown to be involved in neurotransmitter release and ethanol response. Here, we report that SLO-1 also has a critical role in muscles. Inactivation of the slo-1 gene in muscles leads to phenotypes similar to those caused by mutations of the dystrophin homologue dys-1. Notably, slo-1 mutations result in a progressive muscle degeneration when put into a sensitized genetic background. slo-1 localization was observed by gfp reporter gene in both the M-line and the dense bodies (Z line) of the C.elegans body-wall muscles. Using the inside-out configuration of the patch clamp technique on body-wall muscle cells of acutely dissected wild-type worms, we characterized a Ca2+-activated K+ channel that was identified unambiguously as SLO-1. Since neither the abundance nor the conductance of SLO-1 was changed significantly in dys-1 mutants compared to wild-type animals, it is likely that the inactivation of dys-1 causes a misregulation of SLO-1. All in all, these results indicate that SLO-1 function in C.elegans muscles is related to the dystrophin homologue DYS-1.
Journal of Molecular Biology 05/2006; 358(2):387-95. · 4.00 Impact Factor
