Calcineurin Controls Voltage-Dependent-Inactivation (VDI) of the Normal and Timothy Cardiac Channels

Ca2+-entry in the heart is tightly controlled by Cav1.2 inactivation, which involves Ca2+-dependent inactivation (CDI) and voltage-dependent inactivation (VDI) components. Timothy syndrome, a subtype-form of congenital long-QT syndrome, results from a nearly complete elimination of VDI by the G406R mutation in the α11.2 subunit of Cav1.2. Here, we show that a single (A1929P) or a double mutation (H1926A-H1927A) within the CaN-binding site at the human C-terminal tail of α11.2, accelerate the inactivation rate and enhances VDI of both wt and Timothy channels. These results identify the CaN-binding site as the long-sought VDI-regulatory motif of the cardiac channel. The substantial increase in VDI and the accelerated inactivation caused by the selective inhibitors of CaN, cyclosporine A and FK-506, which act at the same CaN-binding site, further support this conclusion. A reversal of enhanced-sympathetic tone by VDI-enhancing CaN inhibitors could be beneficial for improving Timothy syndrome complications such as long-QT and autism

TRPA1 Is a Polyunsaturated Fatty Acid Sensor in Mammals

Fatty acids can act as important signaling molecules regulating diverse physiological processes. Our understanding, however, of fatty acid signaling mechanisms and receptor targets remains incomplete. Here we show that Transient Receptor Potential Ankyrin 1 (TRPA1), a cation channel expressed in sensory neurons and gut tissues, functions as a sensor of polyunsaturated fatty acids (PUFAs) in vitro and in vivo. PUFAs, containing at least 18 carbon atoms and three unsaturated bonds, activate TRPA1 to excite primary sensory neurons and enteroendocrine cells. Moreover, behavioral aversion to PUFAs is absent in TRPA1-null mice. Further, sustained or repeated agonism with PUFAs leads to TRPA1 desensitization. PUFAs activate TRPA1 non-covalently and independently of known ligand binding domains located in the N-terminus and 5th transmembrane region. PUFA sensitivity is restricted to mammalian (rodent and human) TRPA1 channels, as the drosophila and zebrafish TRPA1 orthologs do not respond to DHA. We propose that PUFA-sensing by mammalian TRPA1 may regulate pain and gastrointestinal functions

Protein Phosphatase 2A Interacts with the Na+,K+-ATPase and Modulates Its Trafficking by Inhibition of Its Association with Arrestin

Background: The P-type ATPase family constitutes a collection of ion pumps that form phosphorylated intermediates during ion transport. One of the best known members of this family is the Na +,K +-ATPase. The catalytic subunit of the Na +,K +-ATPase includes several functional domains that determine its enzymatic and trafficking properties. Methodology/Principal Findings: Using the yeast two-hybrid system we found that protein phosphatase 2A (PP2A) catalytic C-subunit is a specific Na +,K +-ATPase interacting protein. PP-2A C-subunit interacted with the Na +,K +-ATPase, but not with the homologous sequences of the H +,K +-ATPase. We confirmed that the Na +,K +-ATPase interacts with a complex of A- and C-subunits in native rat kidney. Arrestins and G-protein coupled receptor kinases (GRKs) are important regulators of G-protein coupled receptor (GPCR) signaling, and they also regulate Na +,K +-ATPase trafficking through direct association. PP2A inhibits association between the Na +,K +-ATPase and arrestin, and diminishes the effect of arrestin on Na +,K +-ATPase trafficking. GRK phosphorylates the Na +,K +-ATPase and PP2A can at least partially reverse this phosphorylation. Conclusions/Significance: Taken together, these data demonstrate that the sodium pump belongs to a growing list of io

Biophysical Basis for Three Distinct Dynamical Mechanisms of Action Potential Initiation

Transduction of graded synaptic input into trains of all-or-none action potentials (spikes) is a crucial step in neural coding. Hodgkin identified three classes of neurons with qualitatively different analog-to-digital transduction properties. Despite widespread use of this classification scheme, a generalizable explanation of its biophysical basis has not been described. We recorded from spinal sensory neurons representing each class and reproduced their transduction properties in a minimal model. With phase plane and bifurcation analysis, each class of excitability was shown to derive from distinct spike initiating dynamics. Excitability could be converted between all three classes by varying single parameters; moreover, several parameters, when varied one at a time, had functionally equivalent effects on excitability. From this, we conclude that the spike-initiating dynamics associated with each of Hodgkin's classes represent different outcomes in a nonlinear competition between oppositely directed, kinetically mismatched currents. Class 1 excitability occurs through a saddle node on invariant circle bifurcation when net current at perithreshold potentials is inward (depolarizing) at steady state. Class 2 excitability occurs through a Hopf bifurcation when, despite net current being outward (hyperpolarizing) at steady state, spike initiation occurs because inward current activates faster than outward current. Class 3 excitability occurs through a quasi-separatrix crossing when fast-activating inward current overpowers slow-activating outward current during a stimulus transient, although slow-activating outward current dominates during constant stimulation. Experiments confirmed that different classes of spinal lamina I neurons express the subthreshold currents predicted by our simulations and, further, that those currents are necessary for the excitability in each cell class. Thus, our results demonstrate that all three classes of excitability arise from a continuum in the direction and magnitude of subthreshold currents. Through detailed analysis of the spike-initiating process, we have explained a fundamental link between biophysical properties and qualitative differences in how neurons encode sensory input

Modulation of calcium currents by pharmacological agents, naturally occuring toxins and phosphorylation in normal and abnormal cardiac and skeletal muscle

Résumé: Le but de ce travail est de comprendre la modulation des canaux calciques cardiaques par les antagonistes calciques, organique et inorganique ainsi que par certaines toxines et la phosphorylation. De plus, on a vérifié si les canaux calciques subissent des changements dans la cardiomypathie héréditaire du hamster et la dystrophyie musculaire de Duchenne (DMD). Dans cette étude, on a utilisé cinq différentes préparations cardiaques: cellules isolées du coeur 1) embryonnaire de poulet de trois jours, 2) embryonnaire de poulet de 10 jours, 3) foetal humain, 4) du hamster nouveau-né normal et 5) du hamster nouveau-né cardiomyopathique. De plus, on a aussi utilisé deux préparations des myotubes de muscle squelettique humain normal et dystrophique (DMD). Toutes les préparations cardiaques ont généralement exprimé deux types de canaux calciques: un courant calcique de type T Ica,T qui s'active et s'inactive rapidement et un courant ICa de type L Ica,L qui s'active et s'inactive lentement par rapport au type T. Le type T de ICa a un potentiel de demi-inactivation (Vo.5) situé 20 à 30 mV plus négativement que le type L. Pour cette raison, il est possible de séparer le type L du type T en maintenant le potentiel du clamp à -50 mV. A ce niveau de potentiel le ICa de type T est presque non mesurable (ou non disponible), par contre le type L est parfaitement disponible pour être activé par une dépolarisation de la membrane. De plus, le potentiel du pic de la courbe courant-voltage est plus négatif que celui du type L: entre -20 et -10 mV pour le type T et entre +10 et +20 mV pour le type L. Le type T et le type L des myocytes ventriculaires de l'embryon de poulet âgé de 10 jours et de fœtus humain sont sensibles aux bloqueurs inorganiques du canal calcique, le Mn2+ par contre, le Cd2+ bloque le type L et le Ni2+ semble être un bloqueur sélectif de ICa de type T. Les bloqueurs organiques des canaux calciques de la famille des dihydropiridines (DPH) comme la nifédipine et le PN 200-110 bloquent le ICa de type L de 50% à une concentration entre 5 et 10 µM et ce degré de bloquage dépend de la concentration extracellulaire en calcium. Le nouvel agent antiasmatique, l'azelastine, bloque le ICa,L a des concentrations de l'ordre du nanomolaire. Par contre avec cette concentration d'azelastine, cette drogue produit une importante augmentation du courant calcique de type T. L'apamine, une toxine du venin d'abeille bloque sélectivement et complément le ICa,L a une concentration de 10'°M. A notre connaissance, l'apamine est la première toxine naturelle qui semble être un bloqueur puissant et sélectif de courant Ca2+ de type L du muscle cardiaque. L'anesthésique local, bupivacaine, semble être un puissant bloqueur mais non sélectif du courant calcique de type L. Ces observations peuvent expliquer en partie la cardiotoxicité de cette rogue. Les activateurs de la protéine kinase dépendent de l'AMPc (PKA) stimule le ICa,L . Ces activateurs (AMPc, isoproterenol et forskoline) n'ont pas d'effet sur le ICa,L enregistré à 22°C. La dépendence de la température de la phosphorylation de ICa,L par le PKA peut expliquer en partie la valeur élevée du Q10 de ICa,L des cellules cardiaques. L'angiotensine II a des concentrations nanomolaires stimule d'une façon réversible le ICa,T . Une légère diminution de ICa,L par l'Ang II a été observée. Le mécanisme par lequel l'Ang II exerce son effet sur les courants Ca2+ peut être dû en partie à la stimulation de certains types de protein Kinase C. Il est à noter que des travaux supplémentaires sont nécessaires afin d'expliquer le mécanisme d'action de l'Ang II sur les courants calciques cardiaques. Dans la cardiomyopathie héréditaire du hamster, on a observé que le pourcentage de cellules qui montrent un courant de type T augmente. De plus, le courant de fenêtre de ICa,T des cellules cardiaques du hamster cardiomyopathique est 10 fois plus large que le courant de fenêtre de ICa,T des cellules cardiaques du hamster normal. Ces observations peuvent expliquer en partie l'augmentation de flux entrant calcique dans les cellules cardiaques du hamster cardiomyopathique. Il est intéressant de noter que le courant lent de type L subit des changements dans les myotubes de muscle squelettique dystrophique de Duchenne. La courbe de relation courant-voltage (I/V) ainsi que la courbe d'inactivation de IBa, dans les myotubes de DMD sont déplacés vers des potentiels plus positifs que ceux des myotubes normaux. En conclusion, cette étude nous a permis de confirmer que les propriétés électriques et pharmacologiques des canaux calciques de type T et de type L dans les cellules cardiaques de l'embryon de poulet âgé de 10 jours sont similaires à celles du hamster nouveau-né et à celles du fœtus humain. Ces propriétés électriques et pharmacologiques du canal T et du canal L cardiaques sont aussi similaires aux canaux calciques rapportés dans d'autres espèces animales. De plus, plusieurs nouvelles informations concernant la pharmacologie et la phathologie des canaux calciques ont été décrites dans ce travail. || Abstract: This study was aimed at understanding modulation of cardiac Ca2+ channels by inorganic and organic antagonists, phosphorylation and naturally occuring toxins. We also investigated possible abnormalities of Ca2+ currents in a model of congenital cardiomyopathy (Sirian hamster) and human Duchenne skeletal muscle distrophy. Three different cardiac preparations were used: 10-day-old chick embryo heart cells, fetal human ventricular myocytes and ventricular myocytes from normal and cardiomyopathic neonatal hamster. All three preparations expressed two types of Ca2+ channels: a rapidly activating and inactivating current T-type ICa, and a more slowly activating and inactivating Ca2+ current L-type. Typically, the T-type Ca2+ currents had V0.5 values 20 to 30 mV more negative than the L-type counterpart. Therefore, they could be effectively separated from L-type currents by holding the cells at more depolarizing holding potentials (i.e. -50 mV) at which T-type ICa, were almost completely inactivated and L-type ICa, were fully available for voltage activation. In addition, the T-type ICa, current-voltage relationship peaked at potential negative relative to that of ICa,L: -20 to -10 mV as opposed to +10 to +20 mV for the L-type ICa. The T- and L-type ICa, in 10-day-old chick embryo heart and fetal human ventricular myocytes were sensitive to mM ammounts of the inorganic Ca2+ blocker Mn2+. On the other hand, micromolar concentrations of Cd2+ blocked ICa,L in all preparations tested, while micromolar concentrations of Ni2+ appeared to selectively block ICa,T. Dihydropiridine (DHP) Ca2+ -antagonists, nifedipine and PN 200-110 selectively blocked ICa,L with K1/2 of 5 to 10 µM depending on extracellular [Ca2+]. Raising the extracellular Ca2+ concentration had a dramatic, allosteric-like effect on PN 200-110 action, reversing the DHP block and shifting the dose dependence of blockade to higher drug concentrations. The recently described antiasmatic agent, azelastin, was shown to block ICa,L at nanomolar concentrations. However, at these concentrations azelastin produced large activation of T-type ICa. The bee venom, apamin, was shown to selectively and completely block ICa,L at 1010M . The effect of apamin was similar in both 10-day-old chick embryo heart cells and fetal human ventricular myocytes. To our knowledge, apamin is the first naturally occurring toxin described that is both selective and potent blocker of ICa,L . The local anesthetic, bupivacaine, was shown to be a potent but non-selective blocker of ICa,L in 10-day-old chick embryo heart cells. This observation may explain the known cardiotoxic action of this drug. Potassium channel opener, cromakalim, was shown to induce up to 2 fold increases in Ica T at 10 8m without having a significant effect on ICa,L Therefore, this observation may explain the arrhythmic action of this drug. Activators of cAMP dependent protein kinase (PKA) have been shown to stimulate ICa,L in 10-day-old chick embryo heart single cells. Although, the stimulation is largely temperature sensitive. No effect of PKA activators: 8 Br-cAMP, isoproterenol or forskilin were noted at room temperature. This temperature sensitivity of ICa,L phosphorylation by PKA may explain in part the large Q10 (3.5) of ICa,L observed in this and other studies. Angiotensin II was shown to potently and reversibly stimulate ICa,T at nanomolar concentrations. An inhibition of ICa,L by ANG II was also noted. The mechanism by which ANG II exerts its action on Ca2+ currents presumably involves protein kinase C. However, more work is needed to completely elucidate the mechanism of ANG II action. The main difference between ICa of normal and cardiomyopathic hamsters is a larger proportion of ICa,T in cardiomyopathic hamsters' myocytes and 10 fold larger window currents of ICa,. in cardiomyopathic hamster ventricular myocytes. This observation may explain in part the additional Ca2+ influx observed in cardiomyopathic myocardium. ICa in Duchenne distrophic skeletal myoballs is also different from that in normal skeletal muscle in that the current-voltage relationship is shifted positively along the voltage axes. A similar shift was noted in the steady state inactivation curve. Also, an increase of the window L-type Ba2+ current was observed in DMD myoballs. Whether these changes reflect a developmental abnormality or genuine molecular changes in ICa,L remains to be established. In conclusion, this study touched on several aspects of L-type and T-type Ca2+-channels modulation by pharmacological agents and phosphorylation. Several novel findings have been described which reflect on the physiology of both normal and abnormal Ca2+- currents

Modulation of calcium currents by pharmacological agents, naturally occuring toxins and phosphorylation in normal and abnormal cardiac and skeletal muscle

Résumé: Le but de ce travail est de comprendre la modulation des canaux calciques cardiaques par les antagonistes calciques, organique et inorganique ainsi que par certaines toxines et la phosphorylation. De plus, on a vérifié si les canaux calciques subissent des changements dans la cardiomypathie héréditaire du hamster et la dystrophyie musculaire de Duchenne (DMD). Dans cette étude, on a utilisé cinq différentes préparations cardiaques: cellules isolées du coeur 1) embryonnaire de poulet de trois jours, 2) embryonnaire de poulet de 10 jours, 3) foetal humain, 4) du hamster nouveau-né normal et 5) du hamster nouveau-né cardiomyopathique. De plus, on a aussi utilisé deux préparations des myotubes de muscle squelettique humain normal et dystrophique (DMD). Toutes les préparations cardiaques ont généralement exprimé deux types de canaux calciques: un courant calcique de type T Ica,T qui s'active et s'inactive rapidement et un courant ICa de type L Ica,L qui s'active et s'inactive lentement par rapport au type T. Le type T de ICa a un potentiel de demi-inactivation (Vo.5) situé 20 à 30 mV plus négativement que le type L. Pour cette raison, il est possible de séparer le type L du type T en maintenant le potentiel du clamp à -50 mV. A ce niveau de potentiel le ICa de type T est presque non mesurable (ou non disponible), par contre le type L est parfaitement disponible pour être activé par une dépolarisation de la membrane. De plus, le potentiel du pic de la courbe courant-voltage est plus négatif que celui du type L: entre -20 et -10 mV pour le type T et entre +10 et +20 mV pour le type L. Le type T et le type L des myocytes ventriculaires de l'embryon de poulet âgé de 10 jours et de fœtus humain sont sensibles aux bloqueurs inorganiques du canal calcique, le Mn2+ par contre, le Cd2+ bloque le type L et le Ni2+ semble être un bloqueur sélectif de ICa de type T. Les bloqueurs organiques des canaux calciques de la famille des dihydropiridines (DPH) comme la nifédipine et le PN 200-110 bloquent le ICa de type L de 50% à une concentration entre 5 et 10 µM et ce degré de bloquage dépend de la concentration extracellulaire en calcium. Le nouvel agent antiasmatique, l'azelastine, bloque le ICa,L a des concentrations de l'ordre du nanomolaire. Par contre avec cette concentration d'azelastine, cette drogue produit une importante augmentation du courant calcique de type T. L'apamine, une toxine du venin d'abeille bloque sélectivement et complément le ICa,L a une concentration de 10'°M. A notre connaissance, l'apamine est la première toxine naturelle qui semble être un bloqueur puissant et sélectif de courant Ca2+ de type L du muscle cardiaque. L'anesthésique local, bupivacaine, semble être un puissant bloqueur mais non sélectif du courant calcique de type L. Ces observations peuvent expliquer en partie la cardiotoxicité de cette rogue. Les activateurs de la protéine kinase dépendent de l'AMPc (PKA) stimule le ICa,L . Ces activateurs (AMPc, isoproterenol et forskoline) n'ont pas d'effet sur le ICa,L enregistré à 22°C. La dépendence de la température de la phosphorylation de ICa,L par le PKA peut expliquer en partie la valeur élevée du Q10 de ICa,L des cellules cardiaques. L'angiotensine II a des concentrations nanomolaires stimule d'une façon réversible le ICa,T . Une légère diminution de ICa,L par l'Ang II a été observée. Le mécanisme par lequel l'Ang II exerce son effet sur les courants Ca2+ peut être dû en partie à la stimulation de certains types de protein Kinase C. Il est à noter que des travaux supplémentaires sont nécessaires afin d'expliquer le mécanisme d'action de l'Ang II sur les courants calciques cardiaques. Dans la cardiomyopathie héréditaire du hamster, on a observé que le pourcentage de cellules qui montrent un courant de type T augmente. De plus, le courant de fenêtre de ICa,T des cellules cardiaques du hamster cardiomyopathique est 10 fois plus large que le courant de fenêtre de ICa,T des cellules cardiaques du hamster normal. Ces observations peuvent expliquer en partie l'augmentation de flux entrant calcique dans les cellules cardiaques du hamster cardiomyopathique. Il est intéressant de noter que le courant lent de type L subit des changements dans les myotubes de muscle squelettique dystrophique de Duchenne. La courbe de relation courant-voltage (I/V) ainsi que la courbe d'inactivation de IBa, dans les myotubes de DMD sont déplacés vers des potentiels plus positifs que ceux des myotubes normaux. En conclusion, cette étude nous a permis de confirmer que les propriétés électriques et pharmacologiques des canaux calciques de type T et de type L dans les cellules cardiaques de l'embryon de poulet âgé de 10 jours sont similaires à celles du hamster nouveau-né et à celles du fœtus humain. Ces propriétés électriques et pharmacologiques du canal T et du canal L cardiaques sont aussi similaires aux canaux calciques rapportés dans d'autres espèces animales. De plus, plusieurs nouvelles informations concernant la pharmacologie et la phathologie des canaux calciques ont été décrites dans ce travail. || Abstract: This study was aimed at understanding modulation of cardiac Ca2+ channels by inorganic and organic antagonists, phosphorylation and naturally occuring toxins. We also investigated possible abnormalities of Ca2+ currents in a model of congenital cardiomyopathy (Sirian hamster) and human Duchenne skeletal muscle distrophy. Three different cardiac preparations were used: 10-day-old chick embryo heart cells, fetal human ventricular myocytes and ventricular myocytes from normal and cardiomyopathic neonatal hamster. All three preparations expressed two types of Ca2+ channels: a rapidly activating and inactivating current T-type ICa, and a more slowly activating and inactivating Ca2+ current L-type. Typically, the T-type Ca2+ currents had V0.5 values 20 to 30 mV more negative than the L-type counterpart. Therefore, they could be effectively separated from L-type currents by holding the cells at more depolarizing holding potentials (i.e. -50 mV) at which T-type ICa, were almost completely inactivated and L-type ICa, were fully available for voltage activation. In addition, the T-type ICa, current-voltage relationship peaked at potential negative relative to that of ICa,L: -20 to -10 mV as opposed to +10 to +20 mV for the L-type ICa. The T- and L-type ICa, in 10-day-old chick embryo heart and fetal human ventricular myocytes were sensitive to mM ammounts of the inorganic Ca2+ blocker Mn2+. On the other hand, micromolar concentrations of Cd2+ blocked ICa,L in all preparations tested, while micromolar concentrations of Ni2+ appeared to selectively block ICa,T. Dihydropiridine (DHP) Ca2+ -antagonists, nifedipine and PN 200-110 selectively blocked ICa,L with K1/2 of 5 to 10 µM depending on extracellular [Ca2+]. Raising the extracellular Ca2+ concentration had a dramatic, allosteric-like effect on PN 200-110 action, reversing the DHP block and shifting the dose dependence of blockade to higher drug concentrations. The recently described antiasmatic agent, azelastin, was shown to block ICa,L at nanomolar concentrations. However, at these concentrations azelastin produced large activation of T-type ICa. The bee venom, apamin, was shown to selectively and completely block ICa,L at 1010M . The effect of apamin was similar in both 10-day-old chick embryo heart cells and fetal human ventricular myocytes. To our knowledge, apamin is the first naturally occurring toxin described that is both selective and potent blocker of ICa,L . The local anesthetic, bupivacaine, was shown to be a potent but non-selective blocker of ICa,L in 10-day-old chick embryo heart cells. This observation may explain the known cardiotoxic action of this drug. Potassium channel opener, cromakalim, was shown to induce up to 2 fold increases in Ica T at 10 8m without having a significant effect on ICa,L Therefore, this observation may explain the arrhythmic action of this drug. Activators of cAMP dependent protein kinase (PKA) have been shown to stimulate ICa,L in 10-day-old chick embryo heart single cells. Although, the stimulation is largely temperature sensitive. No effect of PKA activators: 8 Br-cAMP, isoproterenol or forskilin were noted at room temperature. This temperature sensitivity of ICa,L phosphorylation by PKA may explain in part the large Q10 (3.5) of ICa,L observed in this and other studies. Angiotensin II was shown to potently and reversibly stimulate ICa,T at nanomolar concentrations. An inhibition of ICa,L by ANG II was also noted. The mechanism by which ANG II exerts its action on Ca2+ currents presumably involves protein kinase C. However, more work is needed to completely elucidate the mechanism of ANG II action. The main difference between ICa of normal and cardiomyopathic hamsters is a larger proportion of ICa,T in cardiomyopathic hamsters' myocytes and 10 fold larger window currents of ICa,. in cardiomyopathic hamster ventricular myocytes. This observation may explain in part the additional Ca2+ influx observed in cardiomyopathic myocardium. ICa in Duchenne distrophic skeletal myoballs is also different from that in normal skeletal muscle in that the current-voltage relationship is shifted positively along the voltage axes. A similar shift was noted in the steady state inactivation curve. Also, an increase of the window L-type Ba2+ current was observed in DMD myoballs. Whether these changes reflect a developmental abnormality or genuine molecular changes in ICa,L remains to be established. In conclusion, this study touched on several aspects of L-type and T-type Ca2+-channels modulation by pharmacological agents and phosphorylation. Several novel findings have been described which reflect on the physiology of both normal and abnormal Ca2+- currents