Marbella Quiñonez - Academia.edu (original) (raw)
Papers by Marbella Quiñonez
Acta científica venezolana
Biophysical Journal, 2014
Acta científica venezolana, 1995
Base de dados : LILACS. Pesquisa : 216748 [Identificador único]. Referências encontradas : 1 [ref... more Base de dados : LILACS. Pesquisa : 216748 [Identificador único]. Referências encontradas : 1 [refinar]. Mostrando: 1 .. 1 no formato [Detalhado]. página 1 de 1, 1 / 1, LILACS, seleciona. para imprimir. Fotocópia. experimental, Documentos relacionados. Id: 216748. ...
PLoS ONE, 2014
Background: Exercise intolerance in chronic heart failure (HF) has been attributed to abnormaliti... more Background: Exercise intolerance in chronic heart failure (HF) has been attributed to abnormalities of the skeletal muscles. Muscle function depends on intact excitation-contraction coupling (ECC), but ECC studies in HF models have been inconclusive, due to deficiencies in the animal models and tools used to measure calcium (Ca 2+ ) release, mandating investigations in skeletal muscle from HF patients. The purpose of this study was to test the hypothesis that Ca 2+ release is significantly impaired in the skeletal muscle of HF patients in whom exercise capacity is severely diminished compared to age-matched healthy volunteers.
The Journal of Physiology, 2014
r This paper provides a comprehensive electrophysiological characterization of the external [K + ... more r This paper provides a comprehensive electrophysiological characterization of the external [K + ] dependence and inward rectifying properties of Kir channels in fast skeletal muscle fibres of adult mice.
Journal of Visualized Experiments, 2009
A growing interest in cell biology is to express transgenically modified forms of essential prote... more A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver 1 , skeletal muscle 2,3 , and even the brain 4 . We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP 3 , calpain, FKBP12, β2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and α-actinin; and membrane proteins, i.e. α1s-DHPR, RyR1, cardiac Na/Ca 2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical 3 and functional evidence 5 . However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions 2,6-8 . Figure 1. Transfection efficiency of the in-vivo electroporation method. Brightfield (A) and fluorescence (B) images of an FDB muscle transfected with pmR-mCherry. Days after electroporation protocol: 12 days. Please click here for a larger version of figure 1.
The Journal of Physiology, 2013
• The role that the age-dependent expression of chloride channels (ClC-1) plays on the electrical... more • The role that the age-dependent expression of chloride channels (ClC-1) plays on the electrical properties of muscle fibres from normal and human skeletal actin (HSA) LR mice (a model of myotonic dystrophy) was studied using a combination of electrophysiological and optical techniques. • Chloride currents (I Cl ) are significantly smaller in fibres isolated from young (2 weeks old) HSA LR mice than from aged-matched control mice, but become statistically undistinguishable in adult (17 weeks old) mice. Thus, the severe ClC-1 channelopathy in young HSA LR animals slowly reverses with aging. • The maximal chloride conductance (g Cl,max ) is uniformly depressed in fibres of young HSA LR mice, but fibres from older animals show a wide range of g Cl,max values suggestive of a mosaic expression of ClC-1 channels in FDB muscles of these animals. • Regardless of the age of the animals, the chloride channelopathy does not affect the normal expression of ClC-1 channels at the sarcolemma and transverse tubular system membranes. • The membrane resistance (R m ) is lower than expected in young HSA LR animals due to an upregulation of an Rb-sensitive K conductance. In adult animals, differences in R m are negligible between fibres of both animal strains. • It is proposed that, while the hyperexcitability in young HSA LR mice can be accounted for by the reduction in g Cl,max , a mosaic expression of ClC-1 channels and/or alterations of other conductances may be the underlying causes in adult animals.
The Journal of Physiology, 2011
In mammalian skeletal muscle, the coupling between action potential activation and contraction is... more In mammalian skeletal muscle, the coupling between action potential activation and contraction is supposed to be ultimately mediated by the interaction of two ion channels, the L-type calcium channel (so-called dihydropyridine receptor channel) at the transverse tubular system, and the sarcoplasmic reticulum (SR) calcium release channel (so-called ryanodine receptor channel). This paper demonstrates that adult skeletal muscle fibres transfected in vivo with DNA plasmids are able to express functional transgenic dihydropyridine receptor channels. More importantly, the data suggest that transgenic dihydropyridine receptor channels replace native channels in their interaction with SR calcium release channels. Our findings open new avenues for structural and functional studies of the molecular interactions underlying excitation-contraction coupling within the physiologically relevant cellular context of adult mammalian skeletal muscle fibres.
![Research paper thumbnail of Effects of membrane depolarization and changes in extracellular [K+] on the Ca2+ transients of fast skeletal muscle fibers. Implications for muscle fatigue](https://attachments.academia-assets.com/40583592/thumbnails/1.jpg)
](Journal of Muscle Research and Cell Motility, 2010
Repetitive activation of skeletal muscle fibers leads to a reduced transmembrane K ? gradient. Th... more 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 þ o ) concentration (½K þ 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 ½K þ o and membrane potentials. Depolarization resulting from current injection or raised ½K þ 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 ½K þ o to 10 mM potentiated Ca 2? transients elicited by single and tetanic stimulation, while raising ½K þ o to 15 mM markedly depressed both responses. The effects of 10 mM K þ o on Ca 2? transients, but not those of 15 mM K þ o , could be fully reversed by hyperpolarization. The results suggests that the force potentiating effects of 10 mM K þ 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 þ o on force generation.
Journal of Membrane Biology, 1999
The role of methionine residues on the fast inactivation of the sodium channel from toad skeletal... more The role of methionine residues on the fast inactivation of the sodium channel from toad skeletal muscle fibers was studied with the mild oxidant chloramine-T (CT). Isolated segments of fibers were voltage clamped in a triple Vaseline gap chamber. Sodium current was isolated by replacing potassium ions by tetramethylammonium ions in the external and internal solutions. Externally applied chloramine-CT was found to render noninactivating a large fraction of sodium channels and to slow down the fast inactivation mechanism of the remainder fraction of inactivatable channels. The action of CT appeared to proceed first by slowing and then removing the fast inactivation mechanism. The voltage dependence of the steady-state inactivation of the inactivatable CT-treated currents was shifted +10 mV. CT also had a blocking effect on the sodium current, but was without effect on the activation mechanism. The effects of CT were time and concentration dependent and irreversible. The use of high CT concentrations and/or long exposure times was found to be deleterious to the fiber. This side effect precluded the complete removal of fast inactivation. The effects of CT on the fast inactivation of the sodium current can be explained assuming that at least two methionine residues are critically involved in the mechanism underlying this process.
The Journal of General Physiology, 2007
Two hybrid voltage-sensing systems based on fl uorescence resonance energy transfer (FRET) were u... more Two hybrid voltage-sensing systems based on fl uorescence 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 fi bers. Farnesylated EGFP or ECFP (EGFP-F and ECFP-F) were used as immobile FRET donors, and either non-fl uorescent (dipicrylamine [DPA]) or fl uorescent (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 fl uorescent proteins were effi ciently expressed in the TTS and surface membranes. Voltage-dependent optical signals resulting from resonance energy transfer from fl uorescent proteins to DPA were named QRET transients, to distinguish them from FRET transients recorded using DiBAC 4 (5). The peak ∆F/F of QRET transients elicited by action potential stimulation is twice larger in fi bers 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 fi bers stained with DiBAC 4 (5) exhibit ∆F/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.
The Journal of General Physiology, 2012
Cellular and Molecular Neurobiology, 1989
Page 1. Cellular and Molecular Neurobiology, Vol. 9, No. 2, 1989 Review Desensitization of the Ni... more Page 1. Cellular and Molecular Neurobiology, Vol. 9, No. 2, 1989 Review Desensitization of the Nicotinic Acetylcholine Receptor: Molecular Mechanisms and Effect of Modulators Enrique LM Ochoa, 1 Amitabha Chattopadhyay, ~ and Mark G. McNamee ~ ...
Biophysical Journal, 2012
Biophysical Journal, 2012
Biophysical Journal, 2010
Experiments were carried out to investigate the possibility that store operated Ca 2þ entry (SOCE... more Experiments were carried out to investigate the possibility that store operated Ca 2þ entry (SOCE) may be triggered by volatile anaesthetics in malignant hyperthermia susceptible (MHS) human skeletal muscle. Samples of vastus medialis muscle were obtained from patients undergoing assessment for malignant hyperthermia (MH) susceptibility using the standardised in vitro contracture test. All experiments were performed with institutional Research Ethics Committee approval and informed patient consent, according to the Declaration of Helsinki. Single fibres were mechanically skinned and confocal microscopy used to detect changes [Ca 2þ ] within the re-sealed t-tubules (with fluo-5N) or within the cytosol (with fluo-3). In normal fibres (MHN), exposure to 0.5 mM halothane failed to trigger SR Ca 2þ release, or to induce depletion of t-tubule Ca 2þ (n=8). However, in MHS fibres, 0.5 mM halothane induced both SR Ca 2þ release and a rapid depletion of t-tubule Ca 2þ , consistent with SOCE (n=8). In~20% of MHS fibres, SR Ca 2þ release took the form of a propagating Ca 2þ wave and this was associated with a corresponding SOCE wave of t-tubule Ca 2þ depletion. In MHN fibres, both SR Ca 2þ release and SOCE could be induced by 0.5 mM halothane when the cytosolic [Mg 2þ ] was decreased to 0.2 mM (n=6). In MHS fibres, SOCE was potently inhibited by inclusion of a STIM1 blocking antibody within the re-sealed t-tubules (n=6). These data suggest (i) that in MHS fibres the degree of SR Ca 2þ depletion induced by a clinically relevant level of volatile anaesthetic is sufficient to induce SOCE and (ii) that STIM1 located within the sarcolemma modulates SOCE.
Pfl�gers Archiv European Journal of Physiology, 1999
Here we describe an improved inverted double-grease-gap isolation chamber that allows the formati... more Here we describe an improved inverted double-grease-gap isolation chamber that allows the formation of grease seals of high mechanical stability and high electrical resistance. We also provide a detailed description of the procedure used to mount the muscle fibers onto the apparatus. The new chamber permits the electrophysiological study of muscle fibers using an inverted microscope and high-resolution objectives, thus complying with the requirements of modern fluorescence confocal detection methods. The simplicity and reliability of the mounting procedure make this chamber preferable over other gap isolation chambers currently used for simultaneous electrophysiological and optical studies of calcium release. Experimental results obtained from amphibian muscle fibers are presented to illustrate the performance of the chamber when using global fluorescence detection, confocal spot detection, and laser confocal scanning imaging.
Acta científica venezolana
Biophysical Journal, 2014
Acta científica venezolana, 1995
Base de dados : LILACS. Pesquisa : 216748 [Identificador único]. Referências encontradas : 1 [ref... more Base de dados : LILACS. Pesquisa : 216748 [Identificador único]. Referências encontradas : 1 [refinar]. Mostrando: 1 .. 1 no formato [Detalhado]. página 1 de 1, 1 / 1, LILACS, seleciona. para imprimir. Fotocópia. experimental, Documentos relacionados. Id: 216748. ...
PLoS ONE, 2014
Background: Exercise intolerance in chronic heart failure (HF) has been attributed to abnormaliti... more Background: Exercise intolerance in chronic heart failure (HF) has been attributed to abnormalities of the skeletal muscles. Muscle function depends on intact excitation-contraction coupling (ECC), but ECC studies in HF models have been inconclusive, due to deficiencies in the animal models and tools used to measure calcium (Ca 2+ ) release, mandating investigations in skeletal muscle from HF patients. The purpose of this study was to test the hypothesis that Ca 2+ release is significantly impaired in the skeletal muscle of HF patients in whom exercise capacity is severely diminished compared to age-matched healthy volunteers.
The Journal of Physiology, 2014
r This paper provides a comprehensive electrophysiological characterization of the external [K + ... more r This paper provides a comprehensive electrophysiological characterization of the external [K + ] dependence and inward rectifying properties of Kir channels in fast skeletal muscle fibres of adult mice.
Journal of Visualized Experiments, 2009
A growing interest in cell biology is to express transgenically modified forms of essential prote... more A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver 1 , skeletal muscle 2,3 , and even the brain 4 . We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP 3 , calpain, FKBP12, β2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and α-actinin; and membrane proteins, i.e. α1s-DHPR, RyR1, cardiac Na/Ca 2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical 3 and functional evidence 5 . However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions 2,6-8 . Figure 1. Transfection efficiency of the in-vivo electroporation method. Brightfield (A) and fluorescence (B) images of an FDB muscle transfected with pmR-mCherry. Days after electroporation protocol: 12 days. Please click here for a larger version of figure 1.
The Journal of Physiology, 2013
• The role that the age-dependent expression of chloride channels (ClC-1) plays on the electrical... more • The role that the age-dependent expression of chloride channels (ClC-1) plays on the electrical properties of muscle fibres from normal and human skeletal actin (HSA) LR mice (a model of myotonic dystrophy) was studied using a combination of electrophysiological and optical techniques. • Chloride currents (I Cl ) are significantly smaller in fibres isolated from young (2 weeks old) HSA LR mice than from aged-matched control mice, but become statistically undistinguishable in adult (17 weeks old) mice. Thus, the severe ClC-1 channelopathy in young HSA LR animals slowly reverses with aging. • The maximal chloride conductance (g Cl,max ) is uniformly depressed in fibres of young HSA LR mice, but fibres from older animals show a wide range of g Cl,max values suggestive of a mosaic expression of ClC-1 channels in FDB muscles of these animals. • Regardless of the age of the animals, the chloride channelopathy does not affect the normal expression of ClC-1 channels at the sarcolemma and transverse tubular system membranes. • The membrane resistance (R m ) is lower than expected in young HSA LR animals due to an upregulation of an Rb-sensitive K conductance. In adult animals, differences in R m are negligible between fibres of both animal strains. • It is proposed that, while the hyperexcitability in young HSA LR mice can be accounted for by the reduction in g Cl,max , a mosaic expression of ClC-1 channels and/or alterations of other conductances may be the underlying causes in adult animals.
The Journal of Physiology, 2011
In mammalian skeletal muscle, the coupling between action potential activation and contraction is... more In mammalian skeletal muscle, the coupling between action potential activation and contraction is supposed to be ultimately mediated by the interaction of two ion channels, the L-type calcium channel (so-called dihydropyridine receptor channel) at the transverse tubular system, and the sarcoplasmic reticulum (SR) calcium release channel (so-called ryanodine receptor channel). This paper demonstrates that adult skeletal muscle fibres transfected in vivo with DNA plasmids are able to express functional transgenic dihydropyridine receptor channels. More importantly, the data suggest that transgenic dihydropyridine receptor channels replace native channels in their interaction with SR calcium release channels. Our findings open new avenues for structural and functional studies of the molecular interactions underlying excitation-contraction coupling within the physiologically relevant cellular context of adult mammalian skeletal muscle fibres.
Journal of Muscle Research and Cell Motility, 2010
Repetitive activation of skeletal muscle fibers leads to a reduced transmembrane K ? gradient. Th... more 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 þ o ) concentration (½K þ 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 ½K þ o and membrane potentials. Depolarization resulting from current injection or raised ½K þ 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 ½K þ o to 10 mM potentiated Ca 2? transients elicited by single and tetanic stimulation, while raising ½K þ o to 15 mM markedly depressed both responses. The effects of 10 mM K þ o on Ca 2? transients, but not those of 15 mM K þ o , could be fully reversed by hyperpolarization. The results suggests that the force potentiating effects of 10 mM K þ 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 þ o on force generation.
Journal of Membrane Biology, 1999
The role of methionine residues on the fast inactivation of the sodium channel from toad skeletal... more The role of methionine residues on the fast inactivation of the sodium channel from toad skeletal muscle fibers was studied with the mild oxidant chloramine-T (CT). Isolated segments of fibers were voltage clamped in a triple Vaseline gap chamber. Sodium current was isolated by replacing potassium ions by tetramethylammonium ions in the external and internal solutions. Externally applied chloramine-CT was found to render noninactivating a large fraction of sodium channels and to slow down the fast inactivation mechanism of the remainder fraction of inactivatable channels. The action of CT appeared to proceed first by slowing and then removing the fast inactivation mechanism. The voltage dependence of the steady-state inactivation of the inactivatable CT-treated currents was shifted +10 mV. CT also had a blocking effect on the sodium current, but was without effect on the activation mechanism. The effects of CT were time and concentration dependent and irreversible. The use of high CT concentrations and/or long exposure times was found to be deleterious to the fiber. This side effect precluded the complete removal of fast inactivation. The effects of CT on the fast inactivation of the sodium current can be explained assuming that at least two methionine residues are critically involved in the mechanism underlying this process.
The Journal of General Physiology, 2007
Two hybrid voltage-sensing systems based on fl uorescence resonance energy transfer (FRET) were u... more Two hybrid voltage-sensing systems based on fl uorescence 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 fi bers. Farnesylated EGFP or ECFP (EGFP-F and ECFP-F) were used as immobile FRET donors, and either non-fl uorescent (dipicrylamine [DPA]) or fl uorescent (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 fl uorescent proteins were effi ciently expressed in the TTS and surface membranes. Voltage-dependent optical signals resulting from resonance energy transfer from fl uorescent proteins to DPA were named QRET transients, to distinguish them from FRET transients recorded using DiBAC 4 (5). The peak ∆F/F of QRET transients elicited by action potential stimulation is twice larger in fi bers 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 fi bers stained with DiBAC 4 (5) exhibit ∆F/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.
The Journal of General Physiology, 2012
Cellular and Molecular Neurobiology, 1989
Page 1. Cellular and Molecular Neurobiology, Vol. 9, No. 2, 1989 Review Desensitization of the Ni... more Page 1. Cellular and Molecular Neurobiology, Vol. 9, No. 2, 1989 Review Desensitization of the Nicotinic Acetylcholine Receptor: Molecular Mechanisms and Effect of Modulators Enrique LM Ochoa, 1 Amitabha Chattopadhyay, ~ and Mark G. McNamee ~ ...
Biophysical Journal, 2012
Biophysical Journal, 2012
Biophysical Journal, 2010
Experiments were carried out to investigate the possibility that store operated Ca 2þ entry (SOCE... more Experiments were carried out to investigate the possibility that store operated Ca 2þ entry (SOCE) may be triggered by volatile anaesthetics in malignant hyperthermia susceptible (MHS) human skeletal muscle. Samples of vastus medialis muscle were obtained from patients undergoing assessment for malignant hyperthermia (MH) susceptibility using the standardised in vitro contracture test. All experiments were performed with institutional Research Ethics Committee approval and informed patient consent, according to the Declaration of Helsinki. Single fibres were mechanically skinned and confocal microscopy used to detect changes [Ca 2þ ] within the re-sealed t-tubules (with fluo-5N) or within the cytosol (with fluo-3). In normal fibres (MHN), exposure to 0.5 mM halothane failed to trigger SR Ca 2þ release, or to induce depletion of t-tubule Ca 2þ (n=8). However, in MHS fibres, 0.5 mM halothane induced both SR Ca 2þ release and a rapid depletion of t-tubule Ca 2þ , consistent with SOCE (n=8). In~20% of MHS fibres, SR Ca 2þ release took the form of a propagating Ca 2þ wave and this was associated with a corresponding SOCE wave of t-tubule Ca 2þ depletion. In MHN fibres, both SR Ca 2þ release and SOCE could be induced by 0.5 mM halothane when the cytosolic [Mg 2þ ] was decreased to 0.2 mM (n=6). In MHS fibres, SOCE was potently inhibited by inclusion of a STIM1 blocking antibody within the re-sealed t-tubules (n=6). These data suggest (i) that in MHS fibres the degree of SR Ca 2þ depletion induced by a clinically relevant level of volatile anaesthetic is sufficient to induce SOCE and (ii) that STIM1 located within the sarcolemma modulates SOCE.
Pfl�gers Archiv European Journal of Physiology, 1999
Here we describe an improved inverted double-grease-gap isolation chamber that allows the formati... more Here we describe an improved inverted double-grease-gap isolation chamber that allows the formation of grease seals of high mechanical stability and high electrical resistance. We also provide a detailed description of the procedure used to mount the muscle fibers onto the apparatus. The new chamber permits the electrophysiological study of muscle fibers using an inverted microscope and high-resolution objectives, thus complying with the requirements of modern fluorescence confocal detection methods. The simplicity and reliability of the mounting procedure make this chamber preferable over other gap isolation chambers currently used for simultaneous electrophysiological and optical studies of calcium release. Experimental results obtained from amphibian muscle fibers are presented to illustrate the performance of the chamber when using global fluorescence detection, confocal spot detection, and laser confocal scanning imaging.