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.

Coupled gating between individual skeletal muscle Ca2+ release channels (ryanodine receptors)

Excitation-contraction coupling in skeletal muscle requires the release of intracellular calcium ions (Ca2+) through ryanodine receptor (RyR1) channels in the sarcoplasmic reticulum. Half of the RyR1 channels are activated by voltage-dependent Ca2+ channels in the plasma membrane. In planar lipid bilayers, RyR1 channels exhibited simultaneous openings and closings, termed "coupled gating." Addition of the channel accessory protein FKBP12 induced coupled gating, and removal of FKBP12 uncoupled channels. Coupled gating provides a mechanism by which RyR1 channels that are not associated with voltage-dependent Ca2+ channels can be regulated.

Scaffolding proteins in cardiac myocytes.

Post-translational modification, such as protein phosphorylation, plays a critical role to reversibly amplify and modulate signaling pathways. Since kinases and phosphatases have broad substrate recognition motifs, compartmentalization and localization of signaling complexes are required to achieve specific signals. Scaffolds are proteins that associate with two or more binding partners and function to enhance the efficiency and/or specificity of cellular signaling pathways. The identification of scaffolding proteins that control the tempo and/or spatial organization of signaling pathways in cells has benefited enormously from recent technological advances that allow for the detection of protein-protein interactions, including in vivo in intact cells. This review will focus on scaffolding proteins that nucleate multi-protein complexes (and could represent novel entry points into signaling pathways that might be amenable to therapeutic manipulation) in cardiomyocytes.

Rapamycin inhibits vascular smooth muscle cell migration.

Abnormal vascular smooth muscle cell (SMC) proliferation and migration contribute to the development of restenosis after percutaneous transluminal coronary angioplasty and accelerated arteriopathy after cardiac transplantation. Previously, we reported that the macrolide antibiotic rapamycin, but not the related compound FK506, inhibits both human and rat aortic SMC proliferation in vitro by inhibiting cell cycle-dependent kinases and delaying phosphorylation of retinoblastoma protein (Marx, S.O., T. Jayaraman, L.O. Go, and A.R. Marks. 1995. Circ. Res. 362:801). In the present study the effects of rapamycin on SMC migration were assayed in vitro using a modified Boyden chamber and in vivo using a porcine aortic SMC explant model. Pretreatment with rapamycin (2 ng/ml) for 48 h inhibited PDGF-induced migration (PDGF BB homodimer; 20 ng/ml) in cultured rat and human SMC (n = 10; P < 0.0001), whereas FK506 had no significant effect on migration. Rapamycin administered orally (1 mg/kg per d for 7 d) significantly inhibited porcine aortic SMC migration compared with control (n = 15; P < 0.0001). Thus, in addition to being a potent immunosuppressant and antiproliferative, rapamycin also inhibits SMC migration.

Cell cycle progression and proliferation despite 4BP-1 dephosphorylation.

Proliferation and cell cycle progression in response to growth factors require de novo protein synthesis. It has been proposed that binding of the eukaryotic translation initiation factor 4E (eIF-4E) to the inhibitory protein 4BP-1 blocks translation by preventing access of eIF-4G to the 5' cap of the mRNA. The signal for translation initiation is thought to involve phosphorylation of 4BP-1, which causes it to dissociate from eIF-4E and allows eIF-4G to localize to the 5' cap. It has been suggested that the ability of the macrolide antibiotic rapamycin to inhibit 4BP-1 phosphorylation is responsible for the potent antiproliferative property of this drug. We now show that rapamycin-resistant cells exhibited normal proliferation despite dephosphorylation of 4BP-1 that allows it to bind to eIF-4E. Moreover, despite rapamycin-induced dephosphorylation of 4BP-1, eIF-4E-eIF-4G complexes (eIF-4F) were still detected. In contrast, amino acid withdrawal, which caused a similar degree of 4BP-1 dephosphorylation, resulted in dissociation of the eIF-4E-eIF-4G complex. Thus, 4BP-1 dephosphorylation is not equivalent to eIF-4E inactivation and does not explain the antiproliferative property of rapamycin.

Rapamycin resistance tied to defective regulation of p27Kip1.

The potent antiproliferative activity of the macrolide antibiotic rapamycin is known to involve binding of the drug to its cytosolic receptor, FKBP12, and subsequent interaction with targets of rapamycin, resulting in inhibition of p70 S6 kinase (p70S6K). However, the downstream events that lead to inhibition of cell cycle progression remain to be elucidated. The antiproliferative effects of rapamycin are associated with prevention of mitogen-induced downregulation of the cyclin-dependent kinase inhibitor p27Kip1, suggesting that the latter may play an important role in the growth pathway targeted by rapamycin. Murine BC3H1 cells, selected for resistance to growth inhibition by rapamycin, exhibited an intact p70S6K pathway but had abnormally low p27 levels that were no longer responsive to mitogens or rapamycin. Fibroblasts and T lymphocytes from mice with a targeted disruption of the p27Kip1 gene had impaired growth-inhibitory responses to rapamycin. These results suggest that the ability to regulate p27Kip1 levels is important for rapamycin to exert its antiproliferative effects.

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.

AKAPs as antiarrhythmic targets?

Phosphorylation of ion channels plays a critical role in the modulation and amplification of biophysical signals. Kinases and phosphatases have broad substrate recognition sequences. Therefore, the targeting of kinases and phosphatases to specific sites enhances the regulation of diverse signaling events. Ion channel macromolecular complexes can be formed by the association of A-kinase anchoring proteins (AKAPs) or other adaptor proteins directly with the channel. The discovery that leucine/isoleucine zippers play an important role in the recruitment of phosphorylation-modulatory proteins to certain ion channels has permitted the elucidation of specific ion channel macromolecular complexes. Disruption of signaling complexes by genetic defects can lead to abnormal physiological function. This chapter will focus on evidence supporting the concept that ion channel macromolecular complex formation plays an important role in regulating channel function in normal and diseased states. Moreover, we demonstrate that abnormal complex formation may directly lead to abnormal channel regulation by cellular signaling pathways, potentially leading to arrhythmogenesis and cardiac dysfunction.

Role for p27(Kip1) in Vascular Smooth Muscle Cell Migration.

BACKGROUND: Rapamycin is a potent inhibitor of smooth muscle cell (SMC) proliferation and migration. Rapamycin-mediated inhibition of SMC proliferation is associated with upregulation of the cyclin-dependent kinase inhibitor p27(Kip1). Previously, we showed that mixed embryonic fibroblasts obtained from p27(Kip1)(-/-) mice were relatively rapamycin-resistant, suggesting that p27(Kip1) plays an integral role in modulating the antiproliferative effects of rapamycin. We hypothesized that the antimigratory effect of rapamycin may also be mediated by p27(Kip1). METHODS AND RESULTS: Rapamycin (1 to 10 nmol/L) inhibited basic fibroblast growth factor-induced migration of wild-type (WT) but not p27(Kip1)(-/-) SMCs in a dose-dependent manner (P<0.05) in a modified Boyden chamber. The effects of rapamycin on aortic SMC explant migration were also studied with WT, p27(+/-), and p27(-/-) mice. Rapamycin 4 mg. kg(-1). d(-1) IP for 5 days inhibited SMC migration by 90% in the WT and p27(Kip1)(+/-) (P<0.05) but not p27(Kip1)(-/-) animals. CONCLUSIONS: Lack of p27(Kip1) reduces rapamycin-mediated inhibition of SMC migration. These novel findings suggest a role for p27(Kip1) in the signaling pathway(s) that regulates SMC migration.

Leucine/isoleucine zipper coordination of ion channel macromolecular signaling complexes in the heart. Roles in inherited arrhythmias.

The sympathetic nervous system controls the force and rate of contraction of the heart. The rapid response to stress and exercise mediated by increased sympathetic nervous system (SNS) activity requires the coordinated regulation of several ion channels in response to activation of beta-adrenergic receptors. The microenvironment of target channels is mediated by the assembly of macromolecular signaling complexes in which targeting proteins recruit phosphatases and kinases and in turn bind directly to the channel protein via highly conserved leucine/isoleucine zippers (LIZs). Disruption of local signaling by disease-associated LIZ mutations unbalances the physiologic response to SNS stimulation and increases the risk of arrhythmia in mutation carriers.

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.

Outward currents in isolated ventral cochlear nucleus neurons.

Neurons of the ventral cochlear nucleus (VCN) perform diverse information processing tasks on incoming activity from the auditory nerve. We have investigated the cellular basis for functional diversity in VCN cells by characterizing the outward membrane conductances of acutely isolated cells using whole-cell, tight-seal, current- and voltage-clamp techniques. The electrical responses of isolated cells fall into two broad categories. Type 1 cells respond to small depolarizations with a regular train of action potentials. Under voltage clamp, these cells exhibit a noninactivating outward current for voltage steps positive to -35 mV. Analysis of tail currents reveals two exponentially decaying components with slightly different voltage dependence. These currents reverse at -73 mV, near the potassium equilibrium potential of -84 mV, and are blocked by tetraethylammonium (TEA). The major outward current in Type I cells thus appears to be mediated by potassium channels. In contrast to Type I cells, Type II cells respond to small depolarizations with only one to three short-latency action potentials and exhibit strong rectification around -70 mV. Under voltage clamp, these cells exhibit a noninactivating outward current with a threshold near -70 mV. Analysis of tail currents reveals two components with different voltage sensitivity and kinetics. A low-threshold current with slow kinetics is partly activated at rest. This current reverses at -77 mV and is blocked by 4-aminopyridine (4-AP) but is only partly affected by TEA. The other component is a high-threshold current activated by steps positive to -35 mV. This current is blocked by TEA, but not by 4-AP. A simple model based on the voltage dependence and kinetics of the slow low-threshold outward current in Type II cells was developed. The model produces current- and voltage-clamp responses that resemble those recorded experimentally. Our results indicate that the two major classes of acoustic response properties of VCN neurons are in part attributable to the types of outward (potassium) conductances present in these cells. The low-threshold conductance in the Type II (bushy) cells probably plays a role in the preservation of information about the acoustic stimulus phase from the auditory nerve to central auditory nuclei involved in low-frequency sound localization.

Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells.

Multiple growth factors can stimulate quiescent vascular smooth muscle cells to exit from G0 and reenter the cell cycle. The macrolide antibiotic rapamycin, bound to its cytosolic receptor FKBP, is an immunosuppressant and a potent inhibitor of cellular proliferation. In the present study, the antiproliferative effects of rapamycin on human and rat vascular smooth muscle cells were examined and compared with the effects of a related immunosuppressant, FK520. In vascular smooth muscle cells, rapamycin, at concentrations as low as 1 ng/mL, inhibited DNA synthesis and cell growth. FK520, an analogue of the immunosuppressant FK506, is structurally related to rapamycin and binds to FKBP but did not inhibit vascular smooth muscle cell growth. Molar excesses of FK520 blocked the antiproliferative effects of rapamycin, indicating that the effects of rapamycin required binding to FKBP. Rapamycin-FKBP inhibited retinoblastoma protein phosphorylation at the G1/S transition. This inhibition of retinoblastoma protein phosphorylation was associated with a decrease in p33cdk2 kinase activity. These observations suggest that rapamycin, but not FK520, inhibits vascular smooth muscle cell proliferation by reducing cell-cycle kinase activity.

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.

Variable effects of adenosine on retrograde conduction in patients with atrioventricular nodal reentry tachycardia.

Adenosine has been demonstrated to reliably produce transient block of atrioventricular nodal (AVN) conduction, and has been advocated as a method of differentiating retrograde conduction via the atrioventricular node from accessory pathway conduction. However, the response of retrograde AVN to adenosine in patients with typical atrioventricular nodal reentry tachycardia (AVNRT) remains unclear. We evaluated 13 patients (mean age 45 +/- 20 years) with typical AVNRT prior to AVN modification. During right ventricular pacing, a rapid bolus of adenosine (0.2 mg/kg; maximum 18 mg) was administered. Adenosine sensitivity, defined by transient ventriculoatrial block, was observed in six patients, while in seven patients ventriculoatrial conduction was unaffected. An adenosine bolus administered during sinus rhythm or atrial pacing resulted in antegrade atrioventricular block in all the adenosine resistant patients in whom this was performed (n = 6). Comparisons of AVN electrophysiological characteristics between the adenosine sensitive and adenosine resistant patients were performed. There was no difference with respect to ventriculoatrial effective refractory period, ventriculoatrial Wenckebach, AVNRT cycle length, and His to atrial echo interval in AVNRT. However, there was a trend toward a longer antegrade fast pathway ERP in the adenosine sensitive group (P = 0.07). Electrophysiological properties do not predict retrograde AVN adenosine sensitivity. Adenosine does not cause retrograde AVN block in all patients with AVNRT, and therefore cannot reliably distinguish between retrograde conduction via the AVN or an accessory pathway.

PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts.

The ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is the major source of calcium (Ca2+) required for cardiac muscle excitation-contraction (EC) coupling. The channel is a tetramer comprised of four type 2 RyR polypeptides (RyR2) and four FK506 binding proteins (FKBP12.6). We show that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (Po). Using cosedimentation and coimmunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein, mAKAP. In failing human hearts, RyR2 is PKA hyperphosphorylated, resulting in defective channel function due to increased sensitivity to Ca2+-induced activation.

Uncommon atrial flutter: characteristics, mechanisms, and results of ablative therapy.

Thirty-seven patients with atrial flutter were studied with catheter mapping and radiofrequency ablation. Uncommon atrial flutter occurred in 20 out of 37 (54%) patients. Atrial endocardial mapping showed two types of uncommon atrial flutter. In 15 patients (group I) it was characterized by a single clockwise circuit whereas in 5 patients (Group II) it was characterized by the presence of more than one circuit and/or localized atrial fibrillation. RFA ablation was acutely successful in 14 out of 15 patients (93%) in Group I and in 2 out of 5 (40%) patients in Group II. On long-term follow-up a significantly larger number of patients in Group I versus Group II (86% vs 20%) remained free of atrial flutter recurrence. We conclude that uncommon atrial flutter is a heterogeneous entity involving one or more reentrant circuits. Uncommon atrial flutter with multiple circuits may not be suitable for RFA.