Immunolocalization of triadin, DHP receptors, and ryanodine receptors in adult and developing skeletal muscle of rats

Sl, Carl; Felix, K; Ah, Caswell; Nr, Brandt; Jp, Brunschwig; Meissner, Gerhard; Dg, Ferguson

doi:10.1002/mus.880181104

Cited by 32 publications

(21 citation statements)

References 29 publications

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“…Studies localizing C subunit and the L-type Ca 2ϩ channel in both wild-type and RII␣ knockout cultured skeletal muscle cells support the conclusion that PKA is anchored near the L-type Ca 2ϩ channel in skeletal muscle. In developing myotubes that are not cross-striated, the C subunit and L-type Ca 2ϩ channel are diffusely distributed, similar to the pattern of the ␣ 1 subunit of the L-type Ca 2ϩ channel described previously (29,30). In longer term cultures of differentiated myotubes containing cross-striations, the C subunit and the L-type Ca 2ϩ channel appear to be preferentially localized to the transverse tubules ( Fig.…”

Section: Discussionsupporting

confidence: 81%

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca ²⁺ channel activity by cAMP-dependent protein kinase

Burton

Johnson

Hausken

et al. 1997

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

124

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Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid͞kainate receptor, the L-type Ca 2؉ channel, and the K Ca channel. We hypothesized that the targeted disruption of the gene for the ubiquitously expressed RII␣ subunit would reveal those tissues and signaling events that require anchored PKA. RII␣ knockout mice appear normal and healthy. In adult skeletal muscle, RI␣ protein levels increased to partially compensate for the loss of RII␣. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity was observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca 2؉ channel in RII␣ knockout skeletal muscle was unchanged compared with wild type although it was more sensitive to inhibitors of PKA-AKAP interactions. The C subunit colocalized with the L-type Ca 2؉ channel in transverse tubules in wild-type skeletal muscle and retained this localization in knockout muscle. The RI␣ subunit was shown to bind AKAPs, although with a 500-fold lower affinity than the RII␣ subunit. The potentiation of the L-type Ca 2؉ channel in RII␣ knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RI␣ is capable of physiologically relevant anchoring interactions.Following the hormonal elevation of cAMP, phosphorylation of protein substrates by cAMP-dependent protein kinase (PKA) influences many physiological processes, including cellular differentiation, metabolism, and ion channel activity. PKA is a holoenzyme consisting of two regulatory (R) and two catalytic (C) subunits. Multiple subunits have been identified for R (RI␣, RI␤, RII␣, and RII␤) and C (C␣, C␤, and C␥) subunits. In general, the ␣ isoforms are constitutively expressed in most tissues, whereas the ␤ isoforms are highly expressed in the brain with a lower and more selective expression in other tissues (1). Despite the ubiquitous expression of PKA, cells are capable of specific responses to hormones and neurotransmitters. It has been proposed that the selective phosphorylation of specific proteins by PKA is achieved in part by the subcellular compartmentalization of PKA (2).The RII subunits (RII␣ and RII␤) are postulated to anchor PKA holoenzyme to subcellular compartments thereby positioning PKA in close proximity to its substrates (3). In support of this, RII subunits have been shown to bind to a family of A-kinase anchor proteins (AKAPs) (4, 5). These AKAPs contain a conserved amphipathic helix that binds to the amino terminus of the RII subunit (5, 6). In addition to binding to the RII subunit of PKA, AKAP 79 interacts with the calcium and calmodulin-dependent protein phosphatase 2B (calcineurin...

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Section: Discussionsupporting

confidence: 81%

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca ²⁺ channel activity by cAMP-dependent protein kinase

Burton

Johnson

Hausken

et al. 1997

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

124

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“…Staining with anti-CH2 produced a striated pattern, consistent with staining of the junctions between the transverse (tϪ) tubules and sarcoplasmic reticulum ( Fig. 2 A and B), where Ca V 1.2a channels are located in high density (23)(24)(25). Staining intensity was similar across the width of the myocyte and was unchanged by treatment with Iso ( Fig.…”

Section: Resultsmentioning

confidence: 58%

Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac Ca _V 1.2 channels during β1-adrenergic regulation

Hulme

Westenbroek

Scheuer

et al. 2006

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

139

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During the fight-or-flight response, epinephrine and norepinephrine released by the sympathetic nervous system increase L-type calcium currents conducted by Ca V1.2a channels in the heart, which contributes to enhanced cardiac performance. Activation of ␤-adrenergic receptors increases channel activity via phosphorylation by cAMP-dependent protein kinase (PKA) tethered to the distal C-terminal domain of the ␣1 subunit via an A-kinase anchoring protein (AKAP15). Here we measure phosphorylation of S1928 in dissociated rat ventricular myocytes in response to ␤-adrenergic receptor stimulation by using a phosphospecific antibody. Isoproterenol treatment increased phosphorylation of S1928 in the distal C-terminal domain, and a similar increase was observed with a direct activator of adenylyl cyclase, forskolin, confirming that the cAMP and PKA are responsible. Pretreatment with selective ␤1-and ␤2-adrenergic antagonists reduced the increase in phosphorylation by 79% and 42%, respectively, and pretreatment with both agents completely blocked it. In contrast, treatment with these agents in the presence of 1,2-bis(2-aminophenoxy)ethane-N,Ntetraacetic acid (BAPTA)-acetoxymethyl ester to buffer intracellular calcium results in only ␤1-stimulated phosphorylation of S1928. Whole-cell patch clamp studies with intracellular BAPTA demonstrated that 98% of the increase in calcium current was attributable to ␤1-adrenergic receptors. Thus, ␤-adrenergic stimulation results in phosphorylation of S1928 on the Ca V1.2 ␣1 subunit in intact ventricular myocytes via both ␤1-and ␤2-adrenergic receptor pathways, but the ␤2-dependent increase in phosphorylation depends on elevated intracellular calcium and does not contribute to regulation of whole-cell calcium currents at basal calcium levels. Our results correlate phosphorylation of S1928 with ␤1-adrenergic functional up-regulation of cardiac calcium channels in the presence of BAPTA in intact ventricular myocytes.calcium channel ͉ cAMP ͉ heart ͉ protein kinase ͉ sympathetic regulation T he primary calcium current in cardiac myocytes is conducted by Ca v 1.2a channels (1, 2). Calcium entering through these channels triggers release of calcium from the sarcoplasmic reticulum, which in turn activates contraction. Modulation of calcium entry through the Ca V 1.2 channel is a key point of regulation of cardiac contraction by the sympathetic nervous system, primarily through epinephrine and norepinephrine acting at ␤-adrenergic receptors (1, 3). Activation of ␤-adrenergic signaling leads to 3-to 4-fold increases in calcium current (2), mediated by protein kinase A (PKA) phosphorylation (1-3). Increases in calcium current are produced by activation of adenylyl cyclase with forskolin, inhibition of phosphodiesterases, and activation of PKA with cyclic nucleotide analogs (4-6).Cardiac calcium channels are heteromeric protein complexes (7). The pore-forming ␣1 subunit consists of four homologous domains surrounding an ion conduction pore and contains the gating machinery and the receptor sites for...

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“…Triadin was first identified in skeletal muscle as a 94-to 95-kDa transmembrane protein (7,8) that is abundantly expressed on the junctional sarcoplasmic reticulum (jSR), were it colocalizes with RyR1 and DHPR (9).…”

Section: H]ryanodine Binding Studies Of Skeletal Muscle Sr Vesicles Dmentioning

confidence: 99%

Triadins Modulate Intracellular Ca2+ Homeostasis but Are Not Essential for Excitation-Contraction Coupling in Skeletal Muscle

Shen

Franzini‐Armstrong

López

et al. 2007

Journal of Biological Chemistry

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To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca 2؉ imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca 2؉ transients. release mediated by these two channels (for review see Refs. 1-4). These proteins, including calsequestrin (Csq), calmodulin, triadin, junctin, Junctophilins 1 and 2, MG-29, FKBP12, and others yet to be discovered, along with the RyR and DHPR make up the so-called calcium release units (CRUs) (5).Triadins, a multimember family of proteins that are the product of alternative splicing from a single gene and expressed almost exclusively in striated muscle (3, 6), have generated significant attention in recent years for their involvement in a variety of cellular events in muscle cells, but their precise role in muscle function is mostly unknown. Triadin was first identified in skeletal muscle as a 94-to 95-kDa transmembrane protein (7,8) that is abundantly expressed on the junctional sarcoplasmic reticulum (jSR), were it colocalizes with RyR1 and DHPR (9).Early studies of binding assays of solubilized SR proteins showed that triadin could not only be coimmunoprecipitated with other triadic proteins (10) but also could associate into macromolecular complexes with both the DHPR and RyR1 (7,11,12). Based in this association triadin was proposed as the key molecular linker mediating the DHPR/RyR1 communication during muscle contraction. Although functional interactions between triadin and the DHPR in skeletal muscle have proven difficult to confirm, functional and structural interactions between triadin and RyR1 have been documented by several investigators. In vitro studies have shown that the SR luminal domain of triadin not only interacts with RyR1 but appears to anchor Csq to it, mediating the functional coupling between these two proteins via specific domains (13-17).Several studies have suggested a major role for triadin 95 in modulating RyR channel properties. Both an anti-triadin anti-* This work was supported by American Heart Association Grant 0530250N (to C. F. P.) and National Institute of Health Grant PO1AR47605 (to P. D. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Together these studies suggest a negative regulatory role for triadin on RyR...

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Immunolocalization of triadin, DHP receptors, and ryanodine receptors in adult and developing skeletal muscle of rats

Cited by 32 publications

References 29 publications

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca ²⁺ channel activity by cAMP-dependent protein kinase

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca ²⁺ channel activity by cAMP-dependent protein kinase

Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac Ca _V 1.2 channels during β1-adrenergic regulation

Triadins Modulate Intracellular Ca2+ Homeostasis but Are Not Essential for Excitation-Contraction Coupling in Skeletal Muscle

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Immunolocalization of triadin, DHP receptors, and ryanodine receptors in adult and developing skeletal muscle of rats

Cited by 32 publications

References 29 publications

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca 2+ channel activity by cAMP-dependent protein kinase

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca 2+ channel activity by cAMP-dependent protein kinase

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

Triadins Modulate Intracellular Ca2+ Homeostasis but Are Not Essential for Excitation-Contraction Coupling in Skeletal Muscle

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Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca ²⁺ channel activity by cAMP-dependent protein kinase

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca ²⁺ channel activity by cAMP-dependent protein kinase

Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac Ca _V 1.2 channels during β1-adrenergic regulation