Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac Ca
            <sub>V</sub>
            1.2 channels during β1-adrenergic regulation

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The undersigned authors wish to note, "The KefFC system of E. coli is maintained in an inactive state by the binding of glutathione (GSH) and is activated by the formation of GSH adducts (GSX), particularly those with bulky substituents. We described two crystal structures with density present in the ligand-binding domain that we interpreted as GSH and GSX. Recently, an independent, experienced crystallographer, who had viewed the structures from our study in a different context, made representations to us that cast doubt on position of the succinimido ring of GSX. We have further reviewed the density maps with the aid of an experienced crystallographer. As a consequence, we believe it is important to draw this altered interpretation of the crystal structures to the attention of readers. In both structures, the density for the backbone of GSH is clear and allows unequivocal assignment of the position of the tripeptide. In PDB coordinate set 3L9X, the density for the succinimido ring is very weak, making interpretation very speculative and the assignment rests on the identity of the ligand added to the crystallization mixture, for which there are two diastereomers in the solutiona possibility that provides some basis for weakening the density. However, in 3L9W there are two anomalies that affect the interpretation of the bound ligand. First, there is no density for the carbon atom attached to the sulfur of GSH and second, there is extra density adjacent to the position of sulfur that could be modelled as a constrained succinimido ring. However, this density could also be water or any other molecule that is trapped in the structure. Thus, while there is good evidence for the peptide, the evidence that it is in the GSH form is uncertain."There are no new data on either the structures or on the gating mechanism. However, we believe that we should be cautious in interpreting the structural data and that the field in general should be made aware of the alternative views of the electron density data. Note that the mutagenesis and spectroscopic data that were presented in the original manuscript are not affected by this alternative interpretation." Tarmo P. The authors note that the following grant should be added to the Acknowledgments: "NIH Grant AG002132." The authors note "The method used for exogenous expression of Ca V 1.2 channels in ref. 32 was incorrectly described as 'viral transduction' in the text. In fact, Yang et al. created transgenic mice with inducible, cardiomyocyte-specific expression of exogenous Ca V 1.2 channels regulated by a tetracycline-inducible promoter. When crossed with a transgenic mouse line expressing doxycycline-regulated reverse transcriptional activator under control of the α-myosin heavy chain protomer, the resulting double transgenic offspring expressed exogenous Ca V 1.2 channels in their cardiac myocytes after treatment with doxycycline. The authors regret the error in describing these methods."www.pnas.org/cgi

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Phosphorylation sites required for regulation of cardiac calcium channels in the fight-or-flight response

Westenbroek

Scheuer

et al. 2013

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“…Evidently, phosphorylation of Ser-1700 is a primary event in cardiovascular regulation. example, PKA-dependent phosphorylation of S1928 is prominent in transfected cells and cardiomyocytes (10,24), but its phosphorylation has little or no effect on β-adrenergic up-regulation of cardiac Ca V 1.2 channel activity in transfected cells or cardiomyocytes (13,25,26). Two sites in the C terminus of the skeletal muscle Ca V 1.1 channel are phosphorylated in vivo as assessed by mass spectrometry (S1575 and T1579), and phosphorylation of S1575 is increased by β-adrenergic stimulation (27).…”

Section: Significancementioning

confidence: 99%

Basal and β-adrenergic regulation of the cardiac calcium channel Ca _V 1.2 requires phosphorylation of serine 1700

Westenbroek

Scheuer

et al. 2014

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L-type calcium (Ca 2+ ) currents conducted by voltage-gated Ca 2+ channel Ca V 1.2 initiate excitation-contraction coupling in cardiomyocytes. Upon activation of β-adrenergic receptors, phosphorylation of Ca V 1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca 2+ 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 Ca V 1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type Ca V 1.2 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 fightor-flight response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca 2+ 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.U pon membrane depolarization, Ca V 1.2 channels conduct L-type calcium (Ca 2+ ) current into cardiomyocytes and initiate excitation-contraction coupling (1, 2). Ca 2+ influx through Cav1.2 channels activates Ca 2+ release from the sarcoplasmic reticulum, which leads to contraction of myofilaments. As the initiator of excitation-contraction coupling, Ca 2+ influx via Ca V 1.2 channels is tightly regulated. Under conditions of fear, stress, and exercise, the sympathetic nervous system activates the fight-or-flight response, in which the marked increase in contractile force of the heart is caused by epinephrine and norepinephrine acting through β-adrenergic receptors, activation of adenylyl cyclase, increased cAMP, activation of cAMP-dependent protein kinase (PKA), and phosphorylation of the Ca V 1.2 channel (1, 3). Phosphorylation of the Ca V 1.2 channel leads to a threefold to fourfold increase in peak current amplitude in mammalian cardiomyocytes. Regulation of the Ca V 1.2 channel by the cAMP signaling pathway is altered in cardiac hypertrophy and heart failure (4-6). Under those pathological conditions, responsiveness of Ca V 1.2 channel activit...

“…We have previously proposed that the autoinhibitory complex of the Ca V 1.2 channel with its proteolytically processed dCT noncovalently bound is the natural substrate for regulation by the β-adrenergic pathway, which relieves the autoinhibition by the dCT (24). Unfortunately, the only confirmed site of cAMP-dependent phosphorylation of the Ca V 1.2 channel [S1928 in the dCT (19,32)] is not conserved in Ca V 1.1 and is not required for regulation of Ca V 1.2 channels in vivo (33,34). Therefore, we set out to identify additional sites of cAMPdependent phosphorylation that may be responsible for regulation of Ca V 1.1 and Ca V 1.2 channels by the β-adrenergic pathway.…”

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confidence: 99%

β-Adrenergic–regulated phosphorylation of the skeletal muscle Ca _V 1.1 channel in the fight-or-flight response

Emrick¹,

Sadı́lek

Konoki³

et al. 2010

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Ca V 1 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 Ca V 1 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 Ca V 1 channel regulation are unknown. To identify the regulatory sites of phosphorylation under physiologically relevant conditions, Ca V 1.1 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 Ca V 1.2 channels (S1700 and T1704, respectively). In vitro phosphorylation revealed that Ca V 1.1-S1575 is a substrate for both cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II, whereas Ca V 1.1-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 Ca V 1.1 channels in vivo, and treatment with propranolol to inhibit β-adrenergic receptors reduced phosphorylation. As S1575 and T1579 in Ca V 1.1 channels and their homologs in Ca V 1.2 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-orflight response.adrenalin | calcium channels | cyclic AMP | mass spectometry | protein kinase A T he voltage-gated calcium channels Ca V 1.1 and Ca V 1.2 initiate excitation-contraction coupling in skeletal and cardiac muscle, respectively (1-4), and up-regulation of L-type calcium currents through these channels by epinephrine and norepinephrine via activation of β-adrenergic receptors, G proteins, adenylyl cyclase, and cAMP-dependent protein kinase (PKA) phosphorylation increases contractility in the fight-or-flight response (1, 5-8). In skeletal muscle, Ca V 1.1 channels initiate excitation-contraction coupling by direct protein-protein interactions with the ryanodine-sensitive calcium release channels in the sarcoplasmic reticulum (3). Ca V 1.1 channels also conduct slowly activated, sustained calcium currents (9), which do not participate directly in initiation of excitationcontraction coupling in single muscle twitches (10). However, tetanic stimulation of skeletal muscle is required for development of substantial contractile force (11), and slow calcium currents through Ca V 1.1 channels are necessary for the increase in contractile force with increasing frequency of stimulation of...