Search

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on [beta]-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage-gated calcium channels ([Ca.sub.v]) acting through the [Ca.sub.v][beta] subunit. Here, we investigate how Rad couples phosphorylation to augmented [Ca.sup.2+] influx and increased cardiac contraction. We show that reversal required phosphorylation of [Ser.sup.272] and [Ser.sup.300] within Rad's polybasic, hydrophobic C- terminal domain (CTD). Phosphorylation of [Ser.sup.25] and [Ser.sup.38] in Rad's N-terminal domain (NTD) alone was ineffective. Phosphorylation of [Ser.sup.272] and [Ser.sup.300] or the addition of 4 Asp residues to the CTD reduced Rad's association with the negatively charged, cytoplasmic plasmalemmal surface and with [Ca.sub.v][beta], even in the absence of [Ca.sub.v][alpha], measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad's C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from [Ca.sub.v][beta], is sufficient for sympathetic upregulation of [Ca.sup.2+] currents., Introduction Calcium influx through voltage-gated L-type [Ca.sup.2+] channels ([Ca.sub.v]1.2) is an essential link in the chain of signals that trigger the contraction of every heartbeat. Norepinephrine released from sympathetic neurons [...]

L-type voltage-gated CaV1.2 channels crucially regulate cardiac muscle contraction. Activation of β-adrenergic receptors (β-AR) augments contraction via protein kinase A (PKA)-induced increase of calcium influx through CaV1.2 channels. To date, the full β-AR cascade has never been heterologously reconstituted. A recent study identified Rad, a CaV1.2 inhibitory protein, as essential for PKA regulation of CaV1.2. We corroborated this finding and reconstituted the complete pathway with agonist activation of β1-AR or β2-AR in Xenopus oocytes. We found, and distinguished between, two distinct pathways of PKA modulation of CaV1.2: Rad dependent (~80% of total) and Rad independent. The reconstituted system reproduces the known features of β-AR regulation in cardiomyocytes and reveals several aspects: the differential regulation of posttranslationally modified CaV1.2 variants and the distinct features of β1-AR versus β2-AR activity. This system allows for the addressing of central unresolved issues in the β-AR-CaV1.2 cascade and will facilitate the development of therapies for catecholamine-induced cardiac pathologies. [ABSTRACT FROM AUTHOR]

Typical 2-Cys peroxiredoxins are required to remove hydrogen peroxide from several different cellular compartments. Their activity can be regulated by hyperoxidation and consequent inactivation of the active-site peroxidatic cysteine. Here we developed a simple assay to quantify the hyperoxidation of peroxiredoxins. Hyperoxidation of peroxiredoxins can only occur efficiently in the presence of a recycling system, usually involving thioredoxin and thioredoxin reductase. We demonstrate that there is a marked difference in the sensitivity of the endoplasmic reticulum-localized peroxiredoxin to hyperoxidation compared with either the cytosolic or mitochondrial enzymes. Each enzyme is equally sensitive to hyperoxidation in the presence of a robust recycling system. Our results demonstrate that peroxiredoxin IV recycling in the endoplasmic reticulum is much less efficient than in the cytosol or mitochondria, leading to the protection of peroxiredoxin IV from hyperoxidation.