Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall ... more Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall upon which wall ingrowth papillae develop at right angles into the cytoplasm. The hypothesis that positional information directing construction of wall ingrowth papillae is mediated by Ca(2+) signals generated by spatiotemporal alterations in cytosolic Ca(2+) ([Ca(2+)]cyt) of cells trans-differentiating to a transfer cell morphology was tested. This hypothesis was examined using Vicia faba cotyledons. On transferring cotyledons to culture, their adaxial epidermal cells synchronously trans-differentiate to epidermal transfer cells. A polarized and persistent Ca(2+) signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca(2+) signal intensity, by withdrawing extracellular Ca(2+) or blocking Ca(2+) channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca(2+) signal polarity and pe...
Background-Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by mutations in... more Background-Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin (Casq2) and can be difficult to treat. The class Ic antiarrhythmic drug flecainide blocks RyR2 channels and prevents CPVT in mice and humans. It is not known whether other class I antiarrhythmic drugs also block RyR2 channels and to what extent RyR2 channel inhibition contributes to antiarrhythmic efficacy in CPVT. Methods and Results-We first measured the effect of all class I antiarrhythmic drugs marketed in the United States (quinidine, procainamide, disopyramide, lidocaine, mexiletine, flecainide, and propafenone) on single RyR2 channels incorporated into lipid bilayers. Only flecainide and propafenone inhibited RyR2 channels, with the S-enantiomer of propafenone having a significantly lower potency than R-propafenone or flecainide. In Casq2 Ϫ/Ϫ myocytes, the propafenone enantiomers and flecainide significantly reduced arrhythmogenic Ca 2ϩ waves at clinically relevant concentrations, whereas Na ϩ channel inhibitors without RyR2 blocking properties did not. In Casq2 Ϫ/Ϫ mice, 5 mg/kg R-propafenone or 20 mg/kg S-propafenone prevented exercise-induced CPVT, whereas procainamide (20 mg/kg) or lidocaine (20 mg/kg) were ineffective (nϭ5 to 9 mice, PϽ0.05). QRS duration was not significantly different, indicating a similar degree of Na ϩ channel inhibition. Clinically, propafenone (900 mg/d) prevented ICD shocks in a 22-year-old CPVT patient who had been refractory to maximal standard drug therapy and bilateral stellate ganglionectomy. Conclusions-RyR2 cardiac Ca 2ϩ release channel inhibition appears to determine efficacy of class I drugs for the prevention of CPVT in Casq2 Ϫ/Ϫ mice. Propafenone may be an alternative to flecainide for CPVT patients symptomatic on -blockers. (Circ Arrhythm Electrophysiol. 2011;4:128-135.) contributed equally to this work. The online-only Data Supplement is available at http://circep.ahajournals.org/cgi/content/full/CIRCEP.110.959916/DC1.
Journal of Molecular and Cellular Cardiology, 2010
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is linked to mutations in the cardia... more Catecholaminergic polymorphic ventricular tachycardia (CPVT) is linked to mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin. We recently found that the drug flecainide inhibits RyR2 channels and prevents CPVT in mice and humans. Here we compared the effects of flecainide and tetracaine, a known RyR2 inhibitor ineffective in CPVT myocytes, on arrhythmogenic Ca 2+ waves and elementary sarcoplasmic reticulum (SR) Ca 2+ release events, Ca 2+ sparks. In ventricular myocytes isolated from a CPVT mouse model, flecainide significantly reduced spark amplitude and spark width, resulting in a 40% reduction in spark mass. Surprisingly, flecainide significantly increased spark frequency. As a result, flecainide had no significant effect on spark-mediated SR Ca 2+ leak or SR Ca 2+ content. In contrast, tetracaine decreased spark frequency and spark-mediated SR Ca 2+ leak, resulting in a significantly increased SR Ca 2+ content. Measurements in permeabilized rat ventricular myocytes confirmed the different effects of flecainide and tetracaine on spark frequency and Ca 2+ waves. In lipid bilayers, flecainide inhibited RyR2 channels by open state block, whereas tetracaine primarily prolonged RyR2 closed times. The differential effects of flecainide and tetracaine on sparks and RyR2 gating can explain why flecainide, unlike tetracaine, does not change the balance of SR Ca 2+ fluxes. We suggest that the smaller spark mass contributes to flecainide's antiarrhythmic action by reducing the probability of saltatory wave propagation between adjacent Ca 2+ release units. Our results indicate that inhibition of the RyR2 open state provides a new therapeutic strategy to prevent diastolic Ca 2+ waves resulting in triggered arrhythmias, such as CPVT.
Calmodulin (CaM) mutations are associated with an autosomal dominant syndrome of ventricular arrh... more Calmodulin (CaM) mutations are associated with an autosomal dominant syndrome of ventricular arrhythmia and sudden death that can present with divergent clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CaM binds to and inhibits ryanodine receptor (RyR2) Ca release channels in the heart, but whether arrhythmogenic CaM mutants alter RyR2 function is not known. To gain mechanistic insight into how human CaM mutations affect RyR2 Ca channels. We studied recombinant CaM mutants associated with CPVT (N54I and N98S) or LQTS (D96V, D130G, and F142L). As a group, all LQTS-associated CaM mutants (LQTS-CaMs) exhibited reduced Ca affinity, whereas CPVT-associated CaM mutants (CPVT-CaMs) had either normal or modestly lower Ca affinity. In permeabilized ventricular myocytes, CPVT-CaMs at a physiological intracellular concentration (100 nmol/L) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Compared with wild-type CaM, CPVT-CaMs caused greater RyR2 single-channel open probability and showed enhanced binding affinity to RyR2. Even a 1:8 mixture of CPVT-CaM:wild-type-CaM activated Ca waves, demonstrating functional dominance. In contrast, LQTS-CaMs did not promote Ca waves and exhibited either normal regulation of RyR2 single channels (D96V) or lower RyR2-binding affinity (D130G and F142L). None of the CaM mutants altered Ca/CaM binding to CaM-kinase II. A small proportion of CPVT-CaM is sufficient to evoke arrhythmogenic Ca disturbances, whereas LQTS-CaMs do not. Our findings explain the clinical presentation and autosomal dominant inheritance of CPVT-CaM mutations and suggest that RyR2 interactions are unlikely to explain arrhythmogenicity of LQTS-CaM mutations.
We describe ATP-dependent inhibition of the 75-105-pS (in 250 mM Cl Ϫ ) anion channel (SCl) from ... more We describe ATP-dependent inhibition of the 75-105-pS (in 250 mM Cl Ϫ ) anion channel (SCl) from the sarcoplasmic reticulum (SR) of rabbit skeletal muscle. In addition to activation by Ca 2ϩ and voltage, inhibition by ATP provides a further mechanism for regulating SCl channel activity in vivo. Inhibition by the nonhydrolyzable ATP analog 5Ј-adenylylimidodiphosphate (AMP-PNP) ruled out a phosphorylation mechanism. Cytoplasmic ATP (ϳ1 mM) inhibited only when Cl Ϫ flowed from cytoplasm to lumen, regardless of membrane voltage. Flux in the opposite direction was not inhibited by 9 mM ATP. Thus ATP causes true, current rectification in SCl channels. Inhibition by cytoplasmic ATP was also voltage dependent, having a K I of 0.4 -1 mM at Ϫ40 mV (Hill coefficient ϳ2), which increased at more negative potentials. Luminal ATP inhibited with a K I of ϳ2 mM at ϩ40 mV, and showed no block at negative voltages. Hidden Markov model analysis revealed that ATP inhibition 1) reduced mean open times without altering the maximum channel amplitude, 2) was mediated by a novel, single, voltage-independent closed state (ϳ1 ms), and 3) was much less potent on lower conductance substates than the higher conductance states. Therefore, the SCl channel is unlikely to pass Cl Ϫ from cytoplasm to SR lumen in vivo, and balance electrogenic Ca 2ϩ uptake as previously suggested. Possible roles for the SCl channel in the transport of other anions are discussed.
The mammalian brain is a mechanosensitive organ that responds to different mechanical forces rang... more The mammalian brain is a mechanosensitive organ that responds to different mechanical forces ranging from intrinsic forces implicated in brain morphogenesis to extrinsic forces that can cause concussion and traumatic brain injury. However, little is known of the mechanosensors that transduce these forces. In this study we use cell-attached patch recording to measure single mechanically-gated (MG) channel currents and their affects on spike activity in identified neurons in neonatal mouse brain slices. We demonstrate that both neocortical and hippocampal pyramidal neurons express stretch-activated MG cation channels that are activated by suctions of $ 25 mm Hg, have a single channel conductance for inward current of 50-70 pS and show weak selectivity for alkali metal cations (i.e., Na þ oK þ oCs þ ). Significantly, single MG channel currents activated on the soma trigger spiking/action potentials in both neocortical and hippocampal pyramidal neurons. Not all neuron types studied here expressed MG channel currents. In particular, locus coeruleus and cerebellar Purkinje neurons showed no detectable MG channel activity. Moreover their robust rhythmic spike activity was resistant to mechanical modulation. Our observation that a single MG channel current can trigger spiking predicates the need for reassessment of the long held view that the impulse output of central neurons depends only upon their intrinsic voltage-gated channels and/or their integrated synaptic input. (O.P. Hamill). b r a i n r e s e a r c h 1 6 0 8 ( 2 0 1 5 ) 1 -1 3
This study examines the use of topical pharmacological agents as a snakebite first aid where slow... more This study examines the use of topical pharmacological agents as a snakebite first aid where slowing venom reaching the circulation prevents systemic toxicity. It is based on the fact that toxin molecules in most snake venoms are large molecules and generally first enter and traverse the lymphatic system before accessing the circulation. It follows on from a previous study where it was shown that topical application of a nitric oxide donor slowed lymph flow to a similar extent in humans and rats as well as increased the time to respiratory arrest for subcutaneous injection of an elapid venom (Pseudonaja textilis, Ptx; Eastern brown snake) into the hind feet of anaesthetized rats. The effects of topical application of the L-type Ca(2+) channel antagonist nifedipine and the local anesthetic lignocaine in inhibiting lymph flow and protecting against envenomation was examined in an anaesthetized rat model. The agents significantly increased dye-measured lymph transit times by 500% and 3...
Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall ... more Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall upon which wall ingrowth papillae develop at right angles into the cytoplasm. The hypothesis that positional information directing construction of wall ingrowth papillae is mediated by Ca(2+) signals generated by spatiotemporal alterations in cytosolic Ca(2+) ([Ca(2+)]cyt) of cells trans-differentiating to a transfer cell morphology was tested. This hypothesis was examined using Vicia faba cotyledons. On transferring cotyledons to culture, their adaxial epidermal cells synchronously trans-differentiate to epidermal transfer cells. A polarized and persistent Ca(2+) signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca(2+) signal intensity, by withdrawing extracellular Ca(2+) or blocking Ca(2+) channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca(2+) signal polarity and pe...
Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall ... more Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall upon which wall ingrowth papillae develop at right angles into the cytoplasm. The hypothesis that positional information directing construction of wall ingrowth papillae is mediated by Ca(2+) signals generated by spatiotemporal alterations in cytosolic Ca(2+) ([Ca(2+)]cyt) of cells trans-differentiating to a transfer cell morphology was tested. This hypothesis was examined using Vicia faba cotyledons. On transferring cotyledons to culture, their adaxial epidermal cells synchronously trans-differentiate to epidermal transfer cells. A polarized and persistent Ca(2+) signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca(2+) signal intensity, by withdrawing extracellular Ca(2+) or blocking Ca(2+) channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca(2+) signal polarity and pe...
Background-Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by mutations in... more Background-Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin (Casq2) and can be difficult to treat. The class Ic antiarrhythmic drug flecainide blocks RyR2 channels and prevents CPVT in mice and humans. It is not known whether other class I antiarrhythmic drugs also block RyR2 channels and to what extent RyR2 channel inhibition contributes to antiarrhythmic efficacy in CPVT. Methods and Results-We first measured the effect of all class I antiarrhythmic drugs marketed in the United States (quinidine, procainamide, disopyramide, lidocaine, mexiletine, flecainide, and propafenone) on single RyR2 channels incorporated into lipid bilayers. Only flecainide and propafenone inhibited RyR2 channels, with the S-enantiomer of propafenone having a significantly lower potency than R-propafenone or flecainide. In Casq2 Ϫ/Ϫ myocytes, the propafenone enantiomers and flecainide significantly reduced arrhythmogenic Ca 2ϩ waves at clinically relevant concentrations, whereas Na ϩ channel inhibitors without RyR2 blocking properties did not. In Casq2 Ϫ/Ϫ mice, 5 mg/kg R-propafenone or 20 mg/kg S-propafenone prevented exercise-induced CPVT, whereas procainamide (20 mg/kg) or lidocaine (20 mg/kg) were ineffective (nϭ5 to 9 mice, PϽ0.05). QRS duration was not significantly different, indicating a similar degree of Na ϩ channel inhibition. Clinically, propafenone (900 mg/d) prevented ICD shocks in a 22-year-old CPVT patient who had been refractory to maximal standard drug therapy and bilateral stellate ganglionectomy. Conclusions-RyR2 cardiac Ca 2ϩ release channel inhibition appears to determine efficacy of class I drugs for the prevention of CPVT in Casq2 Ϫ/Ϫ mice. Propafenone may be an alternative to flecainide for CPVT patients symptomatic on -blockers. (Circ Arrhythm Electrophysiol. 2011;4:128-135.) contributed equally to this work. The online-only Data Supplement is available at http://circep.ahajournals.org/cgi/content/full/CIRCEP.110.959916/DC1.
Journal of Molecular and Cellular Cardiology, 2010
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is linked to mutations in the cardia... more Catecholaminergic polymorphic ventricular tachycardia (CPVT) is linked to mutations in the cardiac ryanodine receptor (RyR2) or calsequestrin. We recently found that the drug flecainide inhibits RyR2 channels and prevents CPVT in mice and humans. Here we compared the effects of flecainide and tetracaine, a known RyR2 inhibitor ineffective in CPVT myocytes, on arrhythmogenic Ca 2+ waves and elementary sarcoplasmic reticulum (SR) Ca 2+ release events, Ca 2+ sparks. In ventricular myocytes isolated from a CPVT mouse model, flecainide significantly reduced spark amplitude and spark width, resulting in a 40% reduction in spark mass. Surprisingly, flecainide significantly increased spark frequency. As a result, flecainide had no significant effect on spark-mediated SR Ca 2+ leak or SR Ca 2+ content. In contrast, tetracaine decreased spark frequency and spark-mediated SR Ca 2+ leak, resulting in a significantly increased SR Ca 2+ content. Measurements in permeabilized rat ventricular myocytes confirmed the different effects of flecainide and tetracaine on spark frequency and Ca 2+ waves. In lipid bilayers, flecainide inhibited RyR2 channels by open state block, whereas tetracaine primarily prolonged RyR2 closed times. The differential effects of flecainide and tetracaine on sparks and RyR2 gating can explain why flecainide, unlike tetracaine, does not change the balance of SR Ca 2+ fluxes. We suggest that the smaller spark mass contributes to flecainide's antiarrhythmic action by reducing the probability of saltatory wave propagation between adjacent Ca 2+ release units. Our results indicate that inhibition of the RyR2 open state provides a new therapeutic strategy to prevent diastolic Ca 2+ waves resulting in triggered arrhythmias, such as CPVT.
Calmodulin (CaM) mutations are associated with an autosomal dominant syndrome of ventricular arrh... more Calmodulin (CaM) mutations are associated with an autosomal dominant syndrome of ventricular arrhythmia and sudden death that can present with divergent clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CaM binds to and inhibits ryanodine receptor (RyR2) Ca release channels in the heart, but whether arrhythmogenic CaM mutants alter RyR2 function is not known. To gain mechanistic insight into how human CaM mutations affect RyR2 Ca channels. We studied recombinant CaM mutants associated with CPVT (N54I and N98S) or LQTS (D96V, D130G, and F142L). As a group, all LQTS-associated CaM mutants (LQTS-CaMs) exhibited reduced Ca affinity, whereas CPVT-associated CaM mutants (CPVT-CaMs) had either normal or modestly lower Ca affinity. In permeabilized ventricular myocytes, CPVT-CaMs at a physiological intracellular concentration (100 nmol/L) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Compared with wild-type CaM, CPVT-CaMs caused greater RyR2 single-channel open probability and showed enhanced binding affinity to RyR2. Even a 1:8 mixture of CPVT-CaM:wild-type-CaM activated Ca waves, demonstrating functional dominance. In contrast, LQTS-CaMs did not promote Ca waves and exhibited either normal regulation of RyR2 single channels (D96V) or lower RyR2-binding affinity (D130G and F142L). None of the CaM mutants altered Ca/CaM binding to CaM-kinase II. A small proportion of CPVT-CaM is sufficient to evoke arrhythmogenic Ca disturbances, whereas LQTS-CaMs do not. Our findings explain the clinical presentation and autosomal dominant inheritance of CPVT-CaM mutations and suggest that RyR2 interactions are unlikely to explain arrhythmogenicity of LQTS-CaM mutations.
We describe ATP-dependent inhibition of the 75-105-pS (in 250 mM Cl Ϫ ) anion channel (SCl) from ... more We describe ATP-dependent inhibition of the 75-105-pS (in 250 mM Cl Ϫ ) anion channel (SCl) from the sarcoplasmic reticulum (SR) of rabbit skeletal muscle. In addition to activation by Ca 2ϩ and voltage, inhibition by ATP provides a further mechanism for regulating SCl channel activity in vivo. Inhibition by the nonhydrolyzable ATP analog 5Ј-adenylylimidodiphosphate (AMP-PNP) ruled out a phosphorylation mechanism. Cytoplasmic ATP (ϳ1 mM) inhibited only when Cl Ϫ flowed from cytoplasm to lumen, regardless of membrane voltage. Flux in the opposite direction was not inhibited by 9 mM ATP. Thus ATP causes true, current rectification in SCl channels. Inhibition by cytoplasmic ATP was also voltage dependent, having a K I of 0.4 -1 mM at Ϫ40 mV (Hill coefficient ϳ2), which increased at more negative potentials. Luminal ATP inhibited with a K I of ϳ2 mM at ϩ40 mV, and showed no block at negative voltages. Hidden Markov model analysis revealed that ATP inhibition 1) reduced mean open times without altering the maximum channel amplitude, 2) was mediated by a novel, single, voltage-independent closed state (ϳ1 ms), and 3) was much less potent on lower conductance substates than the higher conductance states. Therefore, the SCl channel is unlikely to pass Cl Ϫ from cytoplasm to SR lumen in vivo, and balance electrogenic Ca 2ϩ uptake as previously suggested. Possible roles for the SCl channel in the transport of other anions are discussed.
The mammalian brain is a mechanosensitive organ that responds to different mechanical forces rang... more The mammalian brain is a mechanosensitive organ that responds to different mechanical forces ranging from intrinsic forces implicated in brain morphogenesis to extrinsic forces that can cause concussion and traumatic brain injury. However, little is known of the mechanosensors that transduce these forces. In this study we use cell-attached patch recording to measure single mechanically-gated (MG) channel currents and their affects on spike activity in identified neurons in neonatal mouse brain slices. We demonstrate that both neocortical and hippocampal pyramidal neurons express stretch-activated MG cation channels that are activated by suctions of $ 25 mm Hg, have a single channel conductance for inward current of 50-70 pS and show weak selectivity for alkali metal cations (i.e., Na þ oK þ oCs þ ). Significantly, single MG channel currents activated on the soma trigger spiking/action potentials in both neocortical and hippocampal pyramidal neurons. Not all neuron types studied here expressed MG channel currents. In particular, locus coeruleus and cerebellar Purkinje neurons showed no detectable MG channel activity. Moreover their robust rhythmic spike activity was resistant to mechanical modulation. Our observation that a single MG channel current can trigger spiking predicates the need for reassessment of the long held view that the impulse output of central neurons depends only upon their intrinsic voltage-gated channels and/or their integrated synaptic input. (O.P. Hamill). b r a i n r e s e a r c h 1 6 0 8 ( 2 0 1 5 ) 1 -1 3
This study examines the use of topical pharmacological agents as a snakebite first aid where slow... more This study examines the use of topical pharmacological agents as a snakebite first aid where slowing venom reaching the circulation prevents systemic toxicity. It is based on the fact that toxin molecules in most snake venoms are large molecules and generally first enter and traverse the lymphatic system before accessing the circulation. It follows on from a previous study where it was shown that topical application of a nitric oxide donor slowed lymph flow to a similar extent in humans and rats as well as increased the time to respiratory arrest for subcutaneous injection of an elapid venom (Pseudonaja textilis, Ptx; Eastern brown snake) into the hind feet of anaesthetized rats. The effects of topical application of the L-type Ca(2+) channel antagonist nifedipine and the local anesthetic lignocaine in inhibiting lymph flow and protecting against envenomation was examined in an anaesthetized rat model. The agents significantly increased dye-measured lymph transit times by 500% and 3...
Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall ... more Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall upon which wall ingrowth papillae develop at right angles into the cytoplasm. The hypothesis that positional information directing construction of wall ingrowth papillae is mediated by Ca(2+) signals generated by spatiotemporal alterations in cytosolic Ca(2+) ([Ca(2+)]cyt) of cells trans-differentiating to a transfer cell morphology was tested. This hypothesis was examined using Vicia faba cotyledons. On transferring cotyledons to culture, their adaxial epidermal cells synchronously trans-differentiate to epidermal transfer cells. A polarized and persistent Ca(2+) signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca(2+) signal intensity, by withdrawing extracellular Ca(2+) or blocking Ca(2+) channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca(2+) signal polarity and pe...
Uploads
Papers by Derek Laver