Phosphorylation-dependent regulation of ryanodine receptors: a novel role for leucine/isoleucine zippers.

Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function.

Insight towards the identification of cytosolic Ca2+ -binding sites in ryanodine receptors from skeletal and cardiac muscle.

Ca2+ plays a critical role in several processes involved in skeletal and cardiac muscle contraction. One key step in cardiac excitation-contraction (E-C) coupling is the activation of the cardiac ryanodine receptor (RYR2) by cytosolic Ca2+ elevations. Although this process is not critical for skeletal E-C coupling, the activation and inhibition of the skeletal ryanodine receptor (RYR1) seem to be important for overall muscle function. The RYR1 and RYR2 channels fall within the large category of Ca2+ -binding proteins that harbour highly selective Ca2+ -binding sites to receive and translate the various Ca2+ signals into specific functional responses. However, little is known about the precise localization of these sites within the cytosolic assembly of both RYR isoforms, although several experimental lines of evidence have highlighted their EF-hand nature. EF-hand proteins share a common helix-loop-helix structural motif with highly conserved residues involved in Ca2+ coordination. The first step in predicting EF-hand positive regions is to compare the primary protein structure with the EF-hand motif by employing available bioinformatics tools. Although this simple method narrows down search regions, it does not provide solid evidence regarding which regions bind Ca2+ in both RYR isoforms. In this review, we seek to highlight the key findings and experimental approaches that should strengthen our future efforts to identify the cytosolic Ca2+ -binding sites responsible for activation and inhibition in the RYR1 channel, as much less work has been conducted on the RYR2 channel.

Coupled gating between cardiac calcium release channels (ryanodine receptors).

Excitation-contraction coupling in heart muscle requires the activation of Ca(2+)-release channels/type 2 ryanodine receptors (RyR2s) by Ca(2+) influx. RyR2s are arranged on the sarcoplasmic reticular membrane in closely packed arrays such that their large cytoplasmic domains contact one another. We now show that multiple RyR2s can be isolated under conditions such that they remain physically coupled to one another. When these coupled channels are examined in planar lipid bilayers, multiple channels exhibit simultaneous gating, termed "coupled gating." Removal of the regulatory subunit, the FK506 binding protein (FKBP12.6), functionally but not physically uncouples multiple RyR2 channels. Coupled gating between RyR2 channels may be an important regulatory mechanism in excitation-contraction coupling as well as in other signaling pathways involving intracellular Ca(2+) release.

Dilated cardiomyopathy and sudden death resulting from constitutive activation of protein kinase a.

beta-Adrenergic receptor (betaAR) signaling, which elevates intracellular cAMP and enhances cardiac contractility, is severely impaired in the failing heart. Protein kinase A (PKA) is activated by cAMP, but the long-term physiological effect of PKA activation on cardiac function is unclear. To investigate the consequences of chronic cardiac PKA activation in the absence of upstream events associated with betaAR signaling, we generated transgenic mice that expressed the catalytic subunit of PKA in the heart. These mice developed dilated cardiomyopathy with reduced cardiac contractility, arrhythmias, and susceptibility to sudden death. As seen in human heart failure, these abnormalities correlated with PKA-mediated hyperphosphorylation of the cardiac ryanodine receptor/Ca(2+)-release channel, which enhances Ca(2+) release from the sarcoplasmic reticulum, and phospholamban, which regulates the sarcoplasmic reticulum Ca(2+)-ATPase. These findings demonstrate a specific role for PKA in the pathogenesis of heart failure, independent of more proximal events in betaAR signaling, and support the notion that PKA activity is involved in the adverse effects of chronic betaAR signaling.

Properties of a new calcium-permeable single channel from tracheal microsomes.

After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel.

beta-adrenergic receptor blockers restore cardiac calcium release channel (ryanodine receptor) structure and function in heart failure.

BACKGROUND: beta-Adrenergic receptor blockade is one of the most effective treatments for heart failure, a leading cause of mortality worldwide. The use of beta-adrenergic receptor blockers in patients with heart failure is counterintuitive, however, because they are known to decrease contractility in normal hearts. The ryanodine receptor (RyR2) on cardiac sarcoplasmic reticulum is the key calcium release channel required for excitation-contraction coupling. In failing hearts, the stoichiometry and function of the RyR2 macromolecular complex is altered. Decreased levels of phosphatases (PP1 and PP2A) and hyperphosphorylation by protein kinase A result in dissociation of the regulatory protein FKBP12.6 and channels with increased open probability. METHODS AND RESULTS: Here, we show that systemic oral administration of a beta-adrenergic receptor blocker reverses protein kinase A hyperphosphorylation of RyR2, restores the stoichiometry of the RyR2 macromolecular complex, and normalizes single-channel function in a canine model of heart failure. CONCLUSIONS: These results may, in part, explain the improved cardiac function observed in heart failure patients treated with beta-adrenergic receptor blockers.

FKBP12 modulates gating of the ryanodine receptor/calcium release channel.

Excitation-contraction (EC) coupling in muscle requires the activation of intracellular calcium release channels (CRC). Four type 1 ryanodine receptor (RyR1) molecules form each tetrameric CRC. Each RyR1 contains a binding site for the FK506 binding protein (FKBP12), a cis-trans peptidyl-prolyl isomerase that is required for coordinated gating of the four RyR1 subunits comprising the channel. When FKBP12 is bound to RyR1, it stabilizes the four subunits that form each CRC. We propose that binding of one FKBP12 to each RyR1 lowers the energy of twisted-amide peptidyl-prolyl bonds and stabilizes RyR1 in a conformation that permits coordinated gating of the four RyR1 subunits.

Effect of luminal Ca2+ on the stability of coupled gating between ryanodine receptors from the rat heart.

AIM: Two or more RYR2 channels reconstituted into a bilayer lipid membrane (BLM) can open and close either independently (single gating) or simultaneously (coupled gating). The coupled gating phenomenon has been suggested as an attractive candidate for a termination mechanism of Ca2+ release from the sarcoplasmic reticulum, required for periodic contraction and relaxation of cardiac muscle. METHODS: Using the method of reconstitution of a channel into the BLM, we investigated the potential effect of luminal Ca2+on the stability of the interaction between coupled RYR2 channels isolated from the rat heart. We introduced a new parameter - the coupling stability - for each detected simultaneous opening and closing and further averaged values for experiments performed under identical conditions. RESULTS: We found that the coupling stability during simultaneous opening of RYR2 channels was significantly lower in comparison with the simultaneous closing under the same experimental conditions. Furthermore, high concentration of luminal Ca2+ (53 mmol L(-1)) as well as the absence of luminal Ca2+ noticeably destabilized functional coupling between coupled RYR2 channels during opening, in contrast to lower tested concentrations (8-20 mmol L(-1)). CONCLUSIONS: We provide experimental evidence that the strength of interaction between coupled RYR2 channels depends on the functional state of the channels. Furthermore, we show, for the first time, the regulation role of luminal Ca2+ in the inter-RYR2 functional coupling in the rat heart.

FKBP12 binding modulates ryanodine receptor channel gating.

The ryanodine receptor (RyR1)/calcium release channel on the sarcoplasmic reticulum of skeletal muscle is comprised of four 565,000-dalton RyR1s, each of which binds one FK506 binding protein (FKBP12). RyR1 is required for excitation-contraction coupling in skeletal muscle. FKBP12, a cis-trans peptidyl-prolyl isomerase, is required for the normal gating of the RyR1 channel. In the absence of FKBP12, RyR1 channels exhibit increased gating frequency, suggesting that FKBP12 "stabilizes" the channel in the open and closed states. We now show that substitution of a Gly, Glu, or Ile for Val2461 in RyR1 prevents FKBP12 binding to RyR1, resulting in channels with increased gating frequency. In the case of the V2461I mutant RyR1, normal channel function can be restored by adding FKBP12.6, an isoform of FKBP12. These data identify Val2461 as a critical residue required for FKBP12 binding to RyR1 and demonstrate the functional role for FKBP12 in the RyR1 channel complex.