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William A. Catterall - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Cardiac Calcium Channels in the Fight-or-Flight Response
Current molecular pharmacology, 2015Co-Authors: William A. CatterallAbstract:Intracellular calcium transients generated by activation of voltage-gated calcium (CaV) channels generate local signals, which initiate physiological processes such as secretion, synaptic transmission, and excitation-contraction coupling. Regulation of calcium entry through CaV channels is crucial for control of these physiological processes. In this article, I review experimental results that have emerged over several years showing that cardiac CaV1.2 channels form a local signaling complex, in which their proteolytically processed distal C-terminal domain, an A-Kinase Anchoring Protein, and cyclic AMP-dependent protein kinase (PKA) interact directly with the transmembrane core of the ion channel through the proximal C-terminal domain. This signaling complex is the substrate for β-adrenergic up-regulation of the CaV1.2 channel in the heart during the Fight-or-Flight Response. Protein phosphorylation of two sites at the interface between the distal and proximal C-terminal domains contributes importantly to control of basal CaV1.2 channel activity, and phosphorylation of Ser1700 by PKA at that interface up-regulates CaV1.2 activity in Response to β-adrenergic signaling. Thus, the intracellular C-terminal domain of CaV1.2 channels serves as a signaling platform, mediating beat-to-beat physiological regulation of channel activity and up-regulation by β-adrenergic signaling in the Fight-or-Flight Response.
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Basal and β-adrenergic regulation of the cardiac calcium channel CaV1.2 requires phosphorylation of serine 1700
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Ruth E Westenbroek, Todd Scheuer, William A. CatterallAbstract:L-type calcium (Ca2+) currents conducted by voltage-gated Ca2+ channel CaV1.2 initiate excitation–contraction coupling in cardiomyocytes. Upon activation of β-adrenergic receptors, phosphorylation of CaV1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca2+ entry into the cell, which leads to more forceful contraction. In vitro reconstitution studies and in vivo proteomics analysis have revealed that Ser-1700 is a key site of phosphorylation mediating this effect, but the functional role of this amino acid residue in regulation in vivo has remained uncertain. Here we have studied the regulation of calcium current and cell contraction of cardiomyocytes in vitro and cardiac function and homeostasis in vivo in a mouse line expressing the mutation Ser-1700–Ala in the CaV1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type CaV1.2 current in both neonatal and adult cardiomyocytes. In addition, the incremental increase elicited by isoproterenol was abolished in neonatal cardiomyocytes and was substantially reduced in young adult myocytes. In contrast, cellular contractility was only moderately reduced compared with wild type, suggesting a greater reserve of contractile function and/or recruitment of compensatory mechanisms. Mutant mice develop cardiac hypertrophy by the age of 3–4 mo, and maximal stress-induced exercise tolerance is reduced, indicating impaired physiological regulation in the Fight-or-Flight Response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca2+ current and regulation by β-adrenergic activation. As a consequence, blocking PKA phosphorylation at this site impairs cardiovascular physiology in vivo, leading to reduced exercise capacity in the Fight-or-Flight Response and development of cardiac hypertrophy.
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phosphorylation sites required for regulation of cardiac calcium channels in the fight or flight Response
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Ruth E Westenbroek, Todd Scheuer, William A. CatterallAbstract:L-type Ca2+ currents conducted by CaV1.2 channels initiate excitation–contraction coupling in the heart. Their activity is increased by β-adrenergic/cAMP signaling via phosphorylation by PKA in the Fight-or-Flight Response, but the sites of regulation are unknown. We describe the functional role of phosphorylation of Ser1700 and Thr1704—sites of phosphorylation by PKA and casein kinase II at the interface between the proximal and distal C-terminal regulatory domains. Mutation of both residues to Ala in STAA mice reduced basal L-type Ca2+ currents, due to a small decrease in expression and a substantial decrease in functional activity. The increase in L-type Ca2+ current caused by isoproterenol was markedly reduced at physiological levels of stimulation (3–10 nM). Maximal increases in calcium current at nearly saturating concentrations of isoproterenol (100 nM) were also significantly reduced, but the mutation effects were smaller, suggesting that alternative regulatory mechanisms are engaged at maximal levels of stimulation. The β-adrenergic increase in cell contraction was also diminished. STAA ventricular myocytes exhibited arrhythmic contractions in Response to isoproterenol, and up to 20% of STAA cells failed to sustain contractions when stimulated at 1 Hz. STAA mice have reduced exercise capacity, and cardiac hypertrophy is evident at 3 mo. We conclude that phosphorylation of Ser1700 and Thr1704 is essential for regulation of basal activity of CaV1.2 channels and for up-regulation by β-adrenergic signaling at physiological levels of stimulation. Disruption of phosphorylation at those sites leads to impaired cardiac function in vivo, as indicated by reduced exercise capacity and cardiac hypertrophy.
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Calcium Channel Regulation in the Fight-or-Flight Response
Biophysical Journal, 2013Co-Authors: William A. CatterallAbstract:Voltage-gated calcium channel type 1.2 (Cav1.2) is activated in Response to cardiac action potentials and conducts calcium entry that initiates excitation-contraction coupling. Cav1.2 activity is increased by stimulation of the beta-adrenergic receptor/cAMP-dependent protein kinase (PKA) signaling pathway in the Fight-or-Flight Response. Regulation of Cav1.2 channels by PKA requires formation of a noncovalent autoinhibitory signaling complex consisting of the body of the Cav1.2 channel, its proteolytically processed distal C-terminal domain, an A Kinase Anchoring Protein (AKAP), and PKA. The distal C-terminus serves as an autoinhibitor of channel activity, and PKA phosphorylation of sites at the interface between the distal and proximal halves of the C-terminus relieves this autoinhibition in Response to beta-adrenergic stimulation. Mice with gene deletions or mutations that prevent these regulatory events have impaired beta-adrenergic Response, altered exercise behavior, and heart failure. Overall, our results give new insights into the molecular mechanisms that control calcium channel function and cardiovascular physiology in stress, exercise and heart failure.
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molecular mechanism of calcium channel regulation in the fight or flight Response
Biophysical Journal, 2011Co-Authors: Matthew D Fuller, Martin Sadilek, Michelle A. Emrick, Todd Scheuer, William A. CatterallAbstract:During the Fight-or-Flight Response, the sympathetic nervous system stimulates L-type calcium currents in the heart conducted by Cav1.2 channels through activation of β-adrenergic receptors, adenylyl cyclase, and phosphorylation by cAMP-dependent protein kinase (PKA), thereby increasing cardiac contractility and beat rate. The channel α1 subunit C-terminus contains binding sites for multiple regulatory proteins including the PKA/A kinase anchoring protein 15 (AKAP15) complex. The C-terminus is proteolytically cleaved but remains associated non-covalently with the truncated channel and acts as a potent autoinhibitor of channel activity. Relief of this autoinhibition provides an attractive mechanism for cellular regulatory signals to produce the large increases in calcium current observed physiologically. We reconstituted regulation of Cav1.2 channels in non-muscle tsA-201 cells by forming an autoregulatory signaling complex composed of the Cav1.2Δ1800 channel, the noncovalently-associated distal C-terminal domain, the auxiliary α2δ1 and β2b subunits, and AKAP15. During whole-cell recordings of channel activity we observed a 3.6-fold range of Cav1.2 activity from a minimum in the presence of protein kinase inhibitors to a maximum with activation of adenylyl cyclase with forskolin. Equivalent modulation was not observed for the full-length (untruncated) channel or the truncated channel without the distal C-terminus. Basal channel activity in unstimulated cells was regulated by phosphorylation of two novel sites at Ser1700 in a PKA consensus sequence and Thr1704 in a casein kinase 2 consensus sequence, both strategically located at the interface between the distal and proximal C-terminal regulatory domains. Further stimulation of channel activity via PKA signaling required only phosphorylation of Ser1700. Phosphorylation at Ser1928 did not significantly alter channel activity. These results define the signaling complex required for Cav1.2 channel regulation and elucidate the sites of phosphorylation that regulate channel activity.Supported by NIH grants R01 HL085372, T32 HL007312-31 and AHA fellowship 09POST2080270
Donald M. Bers - One of the best experts on this subject based on the ideXlab platform.
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different paths same destination divergent action potential Responses produce conserved cardiac fight or flight Response in mouse and rabbit hearts
The Journal of Physiology, 2019Co-Authors: Lianguo Wang, Donald M. Bers, Stefano Morotti, Srinivas Tapa, Samantha Francis D Stuart, Yanyan Jiang, Zhen Wang, Rachel C Myles, Kieran E Brack, Eleonora GrandiAbstract:KEY POINTS Cardiac electrophysiology and Ca2+ handling change rapidly during the Fight-or-Flight Response to meet physiological demands. Despite dramatic differences in cardiac electrophysiology, the cardiac Fight-or-Flight Response is highly conserved across species. In this study, we performed physiological sympathetic nerve stimulation (SNS) while optically mapping cardiac action potentials and intracellular Ca2+ transients in innervated mouse and rabbit hearts. Despite similar heart rate and Ca2+ handling Responses between mouse and rabbit hearts, we found notable species differences in spatio-temporal repolarization dynamics during SNS. Species-specific computational models revealed that these electrophysiological differences allowed for enhanced Ca2+ handling (i.e. enhanced inotropy) in each species, suggesting that electrophysiological Responses are fine-tuned across species to produce optimal cardiac Fight-or-Flight Responses. ABSTRACT Sympathetic activation of the heart results in positive chronotropy and inotropy, which together rapidly increase cardiac output. The precise mechanisms that produce the electrophysiological and Ca2+ handling changes underlying chronotropic and inotropic Responses have been studied in detail in isolated cardiac myocytes. However, few studies have examined the dynamic effects of physiological sympathetic nerve activation on cardiac action potentials (APs) and intracellular Ca2+ transients (CaTs) in the intact heart. Here, we performed bilateral sympathetic nerve stimulation (SNS) in fully innervated, Langendorff-perfused rabbit and mouse hearts. Dual optical mapping with voltage- and Ca2+ -sensitive dyes allowed for analysis of spatio-temporal AP and CaT dynamics. The rabbit heart responded to SNS with a monotonic increase in heart rate (HR), monotonic decreases in AP and CaT duration (APD, CaTD), and a monotonic increase in CaT amplitude. The mouse heart had similar HR and CaT Responses; however, a pronounced biphasic APD Response occurred, with initial prolongation (50.9 ± 5.1 ms at t = 0 s vs. 60.6 ± 4.1 ms at t = 15 s, P < 0.05) followed by shortening (46.5 ± 9.1 ms at t = 60 s, P = NS vs. t = 0). We determined the biphasic APD Response in mouse was partly due to dynamic changes in HR during SNS and was exacerbated by β-adrenergic activation. Simulations with species-specific cardiac models revealed that transient APD prolongation in mouse allowed for greater and more rapid CaT Responses, suggesting more rapid increases in contractility; conversely, the rabbit heart requires APD shortening to produce optimal inotropic Responses. Thus, while the cardiac Fight-or-Flight Response is highly conserved between species, the underlying mechanisms orchestrating these effects differ significantly.
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Different paths, same destination: divergent action potential Responses produce conserved cardiac Fight-or-Flight Response in mouse and rabbit hearts.
The Journal of physiology, 2019Co-Authors: Lianguo Wang, Stefano Morotti, Srinivas Tapa, Yanyan Jiang, Zhen Wang, Rachel C Myles, Kieran E Brack, Samantha D. Francis Stuart, Donald M. BersAbstract:KEY POINTS Cardiac electrophysiology and Ca2+ handling change rapidly during the Fight-or-Flight Response to meet physiological demands. Despite dramatic differences in cardiac electrophysiology, the cardiac Fight-or-Flight Response is highly conserved across species. In this study, we performed physiological sympathetic nerve stimulation (SNS) while optically mapping cardiac action potentials and intracellular Ca2+ transients in innervated mouse and rabbit hearts. Despite similar heart rate and Ca2+ handling Responses between mouse and rabbit hearts, we found notable species differences in spatio-temporal repolarization dynamics during SNS. Species-specific computational models revealed that these electrophysiological differences allowed for enhanced Ca2+ handling (i.e. enhanced inotropy) in each species, suggesting that electrophysiological Responses are fine-tuned across species to produce optimal cardiac Fight-or-Flight Responses. ABSTRACT Sympathetic activation of the heart results in positive chronotropy and inotropy, which together rapidly increase cardiac output. The precise mechanisms that produce the electrophysiological and Ca2+ handling changes underlying chronotropic and inotropic Responses have been studied in detail in isolated cardiac myocytes. However, few studies have examined the dynamic effects of physiological sympathetic nerve activation on cardiac action potentials (APs) and intracellular Ca2+ transients (CaTs) in the intact heart. Here, we performed bilateral sympathetic nerve stimulation (SNS) in fully innervated, Langendorff-perfused rabbit and mouse hearts. Dual optical mapping with voltage- and Ca2+ -sensitive dyes allowed for analysis of spatio-temporal AP and CaT dynamics. The rabbit heart responded to SNS with a monotonic increase in heart rate (HR), monotonic decreases in AP and CaT duration (APD, CaTD), and a monotonic increase in CaT amplitude. The mouse heart had similar HR and CaT Responses; however, a pronounced biphasic APD Response occurred, with initial prolongation (50.9 ± 5.1 ms at t = 0 s vs. 60.6 ± 4.1 ms at t = 15 s, P
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the mitochondrial calcium uniporter selectively matches metabolic output to acute contractile stress in the heart
Cell Reports, 2015Co-Authors: Jennifer Q Kwong, Donald M. Bers, Robert N Correll, Jennifer A Schwanekamp, Ronald J Vagnozzi, Michelle A Sargent, Allen J York, Jianyi Zhang, Jeffery D MolkentinAbstract:In the heart, augmented Ca(2+) fluxing drives contractility and ATP generation through mitochondrial Ca(2+) loading. Pathologic mitochondrial Ca(2+) overload with ischemic injury triggers mitochondrial permeability transition pore (MPTP) opening and cardiomyocyte death. Mitochondrial Ca(2+) uptake is primarily mediated by the mitochondrial Ca(2+) uniporter (MCU). Here, we generated mice with adult and cardiomyocyte-specific deletion of Mcu, which produced mitochondria refractory to acute Ca(2+) uptake, with impaired ATP production, and inhibited MPTP opening upon acute Ca(2+) challenge. Mice lacking Mcu in the adult heart were also protected from acute ischemia-reperfusion injury. However, resting/basal mitochondrial Ca(2+) levels were normal in hearts of Mcu-deleted mice, and mitochondria lacking MCU eventually loaded with Ca(2+) after stress stimulation. Indeed, Mcu-deleted mice were unable to immediately sprint on a treadmill unless warmed up for 30 min. Hence, MCU is a dedicated regulator of short-term mitochondrial Ca(2+) loading underlying a "Fight-or-Flight" Response that acutely matches cardiac workload with ATP production.
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Na/K-ATPase--an integral player in the adrenergic Fight-or-Flight Response.
Trends in cardiovascular medicine, 2009Co-Authors: Donald M. Bers, Sanda DespaAbstract:During activation of the sympathetic nervous system, cardiac performance is increased as part of the Fight-or-Flight stress Response. The increase in contractility with sympathetic stimulation is an orchestrated combination of intrinsic inotropic, lusitropic, and chronotropic effects, mediated in part by activation of β -adrenergic receptors and protein kinase A. This causes phosphorylation of several Ca cycling proteins in cardiac myocytes (increasing Ca entry via L-type Ca channels, sarcoplasmic reticulum Ca pumping, and the dissociation rate of Ca from the myofilaments). Here, we discuss how stimulation of the Na/K-ATPase, mediated by phosphorylation of phospholemman (a small sarcolemmal protein that associates with and modulates Na/K-ATPase), is an additional important player in the sympathetic Fight-or-Flight Response. Enhancement of Na/K- ATPase activity limits the rise in [Na] i caused by the higher level of Na influx and by doing so limits the rise in cellular and sarcoplasmic reticulum Ca load by favoring Ca extrusion via the Na/Ca exchanger. Thus, phospholemman-mediated activation of the Na/K-ATPase may prevent Ca overload and triggered arrhythmias during stress.
Charles B Nemeroff - One of the best experts on this subject based on the ideXlab platform.
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post traumatic stress disorder the neurobiological impact of psychological trauma
Dialogues in clinical neuroscience, 2011Co-Authors: Jonathan E Sherin, Charles B NemeroffAbstract:The classic Fight-or-Flight Response to perceived threat is a reflexive nervous phenomenon thai has obvious survival advantages in evolutionary terms. However, the systems that organize the constellation of reflexive survival behaviors following exposure to perceived threat can under some circumstances become dysregulated in the process. Chronic dysregulation of these systems can lead to functional impairment in certain individuals who become “psychologically traumatized” and suffer from post-traumatic stress disorder (PTSD), A body of data accumulated over several decades has demonstrated neurobiological abnormalities in PTSD patients. Some of these findings offer insight into the pathophysiology of PTSD as well as the biological vulnerability of certain populations to develop PTSD, Several pathological features found in PTSD patients overlap with features found in patients with traumatic brain injury paralleling the shared signs and symptoms of these clinical syndromes.
Tomoyuki Kuwaki - One of the best experts on this subject based on the ideXlab platform.
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Orexin (hypocretin) participates in central autonomic regulation during Fight-or-Flight Response.
Peptides, 2021Co-Authors: Tomoyuki KuwakiAbstract:Our daily life does not only involve a calm resting state but is rather full of perturbations that induce active states such as moving, eating, and communicating. During such active conditions, cardiorespiratory regulation should be adjusted according to bodily demand, which differs from that during the resting state, by modulating or resetting the operating point. To explore neural mechanisms in the state-dependent adjustment of central autonomic regulation, my research group has recently focused on the Fight-or-Flight Response because the stressor induces not only cognitive, emotional, and behavioral changes but also autonomic changes. In this brief review, I will summarize our discovery using orexin knockout mice and orexin neuron-ablated mice for the possible contribution of orexin, a hypothalamic neuropeptide, to the state-dependent adjustment of the central autonomic regulation. In addition, I will introduce some recent discovery using optogenetic manipulation of the orexin and related systems. The diversity of synaptic control of the cardiovascular and respiratory neurons appears necessary for animals to adapt themselves to ever-changing life circumstances and behavioral states. The orexin system is likely to function as one of the essential modulators for coordinating the circuits controlling autonomic functions and behaviors.
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Orexin neurons and emotional stress.
Vitamins and hormones, 2012Co-Authors: Tomoyuki Kuwaki, Wei ZhangAbstract:Stress increases cardiac function, ventilation, and body temperature and induces analgesia. These changes, which result in an increase in metabolic rate, oxygen supply, and the conduction velocity of nerve impulses, prepare the body for a Fight-or-Flight Response. A part of the hypothalamus called the defense area has long been known to play a key role in these Responses, but the precise mechanisms are largely unknown. Our recent findings suggest that orexin (hypocretin) neurons act as a master switch of the Fight-or-Flight Response. In addition, our results, as well as those from other researchers, suggest that orexin neurons do not modulate specific behaviors such as the Fight-or-Flight Responses but rather integrate the autonomic functions and behaviors in a broad sense or in a vigilance state-dependent manner. The orexin system seems to be a pivotal link between the subconscious and the conscious brain functions.
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Orexin neurons are indispensable for stress‐induced thermogenesis in mice
The Journal of Physiology, 2010Co-Authors: Wei Zhang, Jinko Sunanaga, Yoshiko Takahashi, Taketsugu Mori, Takeshi Sakurai, Yuichi Kanmura, Tomoyuki KuwakiAbstract:Orexin neurons contribute to cardiovascular, respiratory and analgesic components of the Fight-or-Flight Response against stressors. Here, we examined whether the same is true for stress-induced hyperthermia. We used prepro-orexin knockout mice (ORX-KO) and orexin neuron-ablated mice (ORX-AB) in which the latter lack not only orexin, but also other putative neurotransmitter/modulators contained in the orexin neurons. In Response to repetitive insertion of a temperature probe into their rectum (handling stress), ORX-KO mice showed a normal temperature change as compared to that of wild-type littermates (WT) while ORX-AB showed an attenuated Response. Stress-induced expression of uncoupling protein-1, a key molecule in non-shivering thermogenesis in the brown adipose tissue (BAT), was also blunted in ORX-AB but not in ORX-KO. When the BAT was directly activated by a β3 adrenergic agonist, there was no difference in the resultant BAT temperature among the groups, indicating that BAT per se was normal in ORX-AB. In WT and ORX-KO, handling stress activated orexin neurons (as revealed by increased expression of c-Fos) and the resultant hyperthermia was largely blunted by pre-treatment with a β3 antagonist. This observation further supports the notion that attenuated stress-induced hyperthermia in ORX-AB mice was caused by a loss of orexin neurons and abnormal BAT regulation. This study pointed out, for the first time, the possible importance of co-existent neurotransmitter/modulators in the orexin neurons for stress-induced hyperthermia and the importance of integrity of the orexin neurons for full expression of multiple facets of the Fight-or-Flight Response.
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Orexin links emotional stress to autonomic functions.
Autonomic neuroscience : basic & clinical, 2010Co-Authors: Tomoyuki KuwakiAbstract:We studied autonomic functions in orexin-deficient mice and found abnormalities in the emotional state-dependent adjustment of the central autonomic regulation on circulation and respiration. These are summarized as follows. 1) Orexin-deficient mice exposed to a stressor exhibited an attenuated Fight-or-Flight Response, including increases in respiration and blood pressure and stress-induced analgesia. 2) Stimulation to the amygdala (AMG) or the bed nucleus of the stria terminalis (BNST), both of which are implicated in the stress-induced autonomic Responses, induced long-lasting cardiorespiratory excitation in wild-type mice but not in the orexin neuron-ablated mice. Hence, it is likely that the orexin system is one of the essential modulators required for orchestrating the neural circuits controlling autonomic functions and emotional behaviors.
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Orexin neurons are indispensable for stress-induced thermogenesis in mice.
The Journal of physiology, 2010Co-Authors: Wei Zhang, Jinko Sunanaga, Yoshiko Takahashi, Taketsugu Mori, Takeshi Sakurai, Yuichi Kanmura, Tomoyuki KuwakiAbstract:Orexin neurons contribute to cardiovascular, respiratory and analgesic components of the Fight-or-Flight Response against stressors. Here, we examined whether the same is true for stress-induced hyperthermia. We used prepro-orexin knockout mice (ORX-KO) and orexin neuron-ablated mice (ORX-AB) in which the latter lack not only orexin, but also other putative neurotransmitter/modulators contained in the orexin neurons. In Response to repetitive insertion of a temperature probe into their rectum (handling stress), ORX-KO mice showed a normal temperature change as compared to that of wild-type littermates (WT) while ORX-AB showed an attenuated Response. Stress-induced expression of uncoupling protein-1, a key molecule in non-shivering thermogenesis in the brown adipose tissue (BAT), was also blunted in ORX-AB but not in ORX-KO. When the BAT was directly activated by a β3 adrenergic agonist, there was no difference in the resultant BAT temperature among the groups, indicating that BAT per se was normal in ORX-AB. In WT and ORX-KO, handling stress activated orexin neurons (as revealed by increased expression of c-Fos) and the resultant hyperthermia was largely blunted by pre-treatment with a β3 antagonist. This observation further supports the notion that attenuated stress-induced hyperthermia in ORX-AB mice was caused by a loss of orexin neurons and abnormal BAT regulation. This study pointed out, for the first time, the possible importance of co-existent neurotransmitter/modulators in the orexin neurons for stress-induced hyperthermia and the importance of integrity of the orexin neurons for full expression of multiple facets of the Fight-or-Flight Response.
Michelle A. Emrick - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanism of calcium channel regulation in the fight or flight Response
Biophysical Journal, 2011Co-Authors: Matthew D Fuller, Martin Sadilek, Michelle A. Emrick, Todd Scheuer, William A. CatterallAbstract:During the Fight-or-Flight Response, the sympathetic nervous system stimulates L-type calcium currents in the heart conducted by Cav1.2 channels through activation of β-adrenergic receptors, adenylyl cyclase, and phosphorylation by cAMP-dependent protein kinase (PKA), thereby increasing cardiac contractility and beat rate. The channel α1 subunit C-terminus contains binding sites for multiple regulatory proteins including the PKA/A kinase anchoring protein 15 (AKAP15) complex. The C-terminus is proteolytically cleaved but remains associated non-covalently with the truncated channel and acts as a potent autoinhibitor of channel activity. Relief of this autoinhibition provides an attractive mechanism for cellular regulatory signals to produce the large increases in calcium current observed physiologically. We reconstituted regulation of Cav1.2 channels in non-muscle tsA-201 cells by forming an autoregulatory signaling complex composed of the Cav1.2Δ1800 channel, the noncovalently-associated distal C-terminal domain, the auxiliary α2δ1 and β2b subunits, and AKAP15. During whole-cell recordings of channel activity we observed a 3.6-fold range of Cav1.2 activity from a minimum in the presence of protein kinase inhibitors to a maximum with activation of adenylyl cyclase with forskolin. Equivalent modulation was not observed for the full-length (untruncated) channel or the truncated channel without the distal C-terminus. Basal channel activity in unstimulated cells was regulated by phosphorylation of two novel sites at Ser1700 in a PKA consensus sequence and Thr1704 in a casein kinase 2 consensus sequence, both strategically located at the interface between the distal and proximal C-terminal regulatory domains. Further stimulation of channel activity via PKA signaling required only phosphorylation of Ser1700. Phosphorylation at Ser1928 did not significantly alter channel activity. These results define the signaling complex required for Cav1.2 channel regulation and elucidate the sites of phosphorylation that regulate channel activity.Supported by NIH grants R01 HL085372, T32 HL007312-31 and AHA fellowship 09POST2080270
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β adrenergic regulated phosphorylation of the skeletal muscle cav1 1 channel in the fight or flight Response
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Michelle A. Emrick, Martin Sadilek, Keiichi Konoki, William A. CatterallAbstract:CaV1 channels initiate excitation–contraction coupling in skeletal and cardiac muscle. During the Fight-or-Flight Response, epinephrine released by the adrenal medulla and norepinephrine released from sympathetic nerves increase muscle contractility by activation of the β-adrenergic receptor/cAMP-dependent protein kinase pathway and up-regulation of CaV1 channels in skeletal and cardiac muscle. Although the physiological mechanism of this pathway is well defined, the molecular mechanism and the sites of protein phosphorylation required for CaV1 channel regulation are unknown. To identify the regulatory sites of phosphorylation under physiologically relevant conditions, CaV1.1 channels were purified from skeletal muscle and sites of phosphorylation on the α1 subunit were identified by mass spectrometry. Two phosphorylation sites were identified in the proximal C-terminal domain, serine 1575 (S1575) and threonine 1579 (T1579), which are conserved in cardiac CaV1.2 channels (S1700 and T1704, respectively). In vitro phosphorylation revealed that CaV1.1-S1575 is a substrate for both cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II, whereas CaV1.1-T1579 is a substrate for casein kinase 2. Treatment of rabbits with isoproterenol to activate β-adrenergic receptors increased phosphorylation of S1575 in skeletal muscle CaV1.1 channels in vivo, and treatment with propranolol to inhibit β-adrenergic receptors reduced phosphorylation. As S1575 and T1579 in CaV1.1 channels and their homologs in CaV1.2 channels are located at a key regulatory interface between the distal and proximal C-terminal domains, it is likely that phosphorylation of these sites in skeletal and cardiac muscle is directly involved in calcium channel regulation in Response to the sympathetic nervous system in the Fight-or-Flight Response.
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β-Adrenergic–regulated phosphorylation of the skeletal muscle CaV1.1 channel in the Fight-or-Flight Response
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Michelle A. Emrick, Martin Sadilek, Keiichi Konoki, William A. CatterallAbstract:CaV1 channels initiate excitation–contraction coupling in skeletal and cardiac muscle. During the Fight-or-Flight Response, epinephrine released by the adrenal medulla and norepinephrine released from sympathetic nerves increase muscle contractility by activation of the β-adrenergic receptor/cAMP-dependent protein kinase pathway and up-regulation of CaV1 channels in skeletal and cardiac muscle. Although the physiological mechanism of this pathway is well defined, the molecular mechanism and the sites of protein phosphorylation required for CaV1 channel regulation are unknown. To identify the regulatory sites of phosphorylation under physiologically relevant conditions, CaV1.1 channels were purified from skeletal muscle and sites of phosphorylation on the α1 subunit were identified by mass spectrometry. Two phosphorylation sites were identified in the proximal C-terminal domain, serine 1575 (S1575) and threonine 1579 (T1579), which are conserved in cardiac CaV1.2 channels (S1700 and T1704, respectively). In vitro phosphorylation revealed that CaV1.1-S1575 is a substrate for both cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II, whereas CaV1.1-T1579 is a substrate for casein kinase 2. Treatment of rabbits with isoproterenol to activate β-adrenergic receptors increased phosphorylation of S1575 in skeletal muscle CaV1.1 channels in vivo, and treatment with propranolol to inhibit β-adrenergic receptors reduced phosphorylation. As S1575 and T1579 in CaV1.1 channels and their homologs in CaV1.2 channels are located at a key regulatory interface between the distal and proximal C-terminal domains, it is likely that phosphorylation of these sites in skeletal and cardiac muscle is directly involved in calcium channel regulation in Response to the sympathetic nervous system in the Fight-or-Flight Response.
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molecular mechanism of calcium channel regulation in the fight or flight Response
Science Signaling, 2010Co-Authors: Matthew D Fuller, Martin Sadilek, Michelle A. Emrick, Todd Scheuer, William A. CatterallAbstract:During the Fight-or-Flight Response, the sympathetic nervous system stimulates L-type calcium ion (Ca 2+ ) currents conducted by Ca V 1 channels through activation of β-adrenergic receptors, adenylyl cyclase, and phosphorylation by adenosine 3′,5′-monophosphate–dependent protein kinase [also known as protein kinase A (PKA)], increasing contractility of skeletal and cardiac muscles. We reconstituted this regulation of cardiac Ca V 1.2 channels in non-muscle cells by forming an autoinhibitory signaling complex composed of Ca V 1.2Δ1800 (a form of the channel truncated at the in vivo site of proteolytic processing), its noncovalently associated distal carboxyl-terminal domain, the auxiliary α 2 δ 1 and β 2b subunits, and A-kinase anchoring protein 15 (AKAP15). A factor of 3.6 range of Ca V 1.2 channel activity was observed from a minimum in the presence of protein kinase inhibitors to a maximum upon activation of adenylyl cyclase. Basal Ca V 1.2 channel activity in unstimulated cells was regulated by phosphorylation of serine-1700 and threonine-1704, two residues located at the interface between the distal and the proximal carboxyl-terminal regulatory domains, whereas further stimulation of channel activity through the PKA signaling pathway only required phosphorylation of serine-1700. Our results define a conceptual framework for Ca V 1.2 channel regulation and identify sites of phosphorylation that regulate channel activity.