Unchanged β-Adrenergic Stimulation of Cardiac L-type Calcium Channels in Cav1.2 Phosphorylation Site S1928A Mutant Mice

Lemke, Toni; Welling, Andrea; Christel, Carl J.; Blaich, Anne; Bernhard, Detlef; Lenhardt, Peter; Hofmann, Franz; Moosmang, Sven

doi:10.1074/jbc.m804981200

Cited by 117 publications

(138 citation statements)

References 27 publications

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“…However, none of these sites has been shown to be required for regulation of Ca V 1.2 channels in vivo. For example, Ser1928 has been well characterized as a PKA phosphorylation site both in vitro and in vivo (8,13,21), but its phosphorylation is not required for PKA-dependent up-regulation of channel activity in transfected nonmuscle cells, virally transduced ventricular myocytes, or acutely dissociated ventricular myocytes from genetically modified mice (10,25,26). Exhaustive analysis of endogenous phosphorylation of Ca V 1.1 channels by mass spectrometry identified two previously undetected phosphorylation sites: Ser1575 and Thr1579 (27).…”

mentioning

confidence: 99%

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

Westenbroek

Scheuer

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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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

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confidence: 99%

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

Westenbroek

Scheuer

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…This finding implicates one or more PKA sites other than S1700 in the regulation of Ca V 1.2 channels. A well-characterized site that is phosphorylated in an Iso-and PKA-dependent fashion is S1928 (10,23,24); however, no evidence has emerged for a strong effect of phosphorylation of this site in the regulation of cardiac Ca V 1.2 channels in transfected cells (13) or in cardiomyocytes dissociated from mutant mice (25,26). It is possible that regulation via phosphorylation of S1928 is unmasked in vivo by mutation of S1700 or that additional, unidentified PKA phosphorylation site(s) are responsible for the remaining regulation of Ca V 1.2 channels in STAA and SA mice.…”

Section: Discussionmentioning

confidence: 99%

“…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

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…1904 (17) and ␣ 1C 1796 knockin mice could be due to multiple factors, including deficits in macromolecular complex formation (17,18 (9, 38 -45), is not required for ␤-adrenergic receptor stimulation of Ca V 1.2, as shown in cardiomyocytes infected with adenovirus expressing a DHP-resistant S1928A-␣ 1C (35) and in an ␣ 1C S1928A knockin mouse (46). The role for Ca 2ϩ channels in mediating cAMP-response element-binding protein signaling in the heart has not been delineated.…”

Section: Discussionmentioning

confidence: 99%

The PDZ Motif of the α1C Subunit Is Not Required for Surface Trafficking and Adrenergic Modulation of CaV1.2 Channel in the Heart

Yang

Katchman

Weinberg

et al. 2015

Journal of Biological Chemistry

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Background:The mechanisms responsible for Ca V 1.2 regulation by the ␣ 1C C terminus are unknown. Results: Trafficking, basal function, and adrenergic modulation of Ca V 1.2 were not altered in cardiomyocytes of transgenic mice expressing PDZ-deleted ␣ 1C . Conclusion: PDZ-mediated interactions are not required for Ca V 1.2 trafficking and function in the heart. Significance: The regulation of Ca V 1.2 by auxiliary proteins does not depend on the PDZ ligand motif in the heart.

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Unchanged β-Adrenergic Stimulation of Cardiac L-type Calcium Channels in Cav1.2 Phosphorylation Site S1928A Mutant Mice

Cited by 117 publications

References 27 publications

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

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

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

The PDZ Motif of the α1C Subunit Is Not Required for Surface Trafficking and Adrenergic Modulation of CaV1.2 Channel in the Heart

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Unchanged β-Adrenergic Stimulation of Cardiac L-type Calcium Channels in Cav1.2 Phosphorylation Site S1928A Mutant Mice

Cited by 117 publications

References 27 publications

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

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

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

The PDZ Motif of the α1C Subunit Is Not Required for Surface Trafficking and Adrenergic Modulation of CaV1.2 Channel in the Heart

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