Abstract-Excitation-contraction coupling in heart muscle requires the activation of Ca 2ϩ -release channels/type 2 ryanodine receptors (RyR2s) by Ca 2ϩ 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 2ϩ release.

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.

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation–contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are “leaky.” RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF.

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 Val 2461 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 Val 2461 as a critical residue required for FKBP12 binding to RyR1 and demonstrate the functional role for FKBP12 in the RyR1 channel complex.

Background-␤-Adrenergic receptor blockade is one of the most effective treatments for heart failure, a leading cause of mortality worldwide. The use of ␤-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 ␤-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 ␤-adrenergic receptor blockers. Key Words: heart failure Ⅲ calcium Ⅲ sarcoplasmic reticulum Ⅲ ion channels Ⅲ receptors, adrenergic, beta C linical trials have shown that ␤-adrenergic receptor blocker therapy results in a 20% to 66% reduction in mortality at 12 months in patients with heart failure (HF). [1][2][3] It is not intuitively obvious, however, how ␤-adrenergic receptor blockers can improve cardiac function in failing hearts, because they are known to decrease contractility in normal hearts. 4 -6 HF is a complex disease 7 that is characterized by a hyperadrenergic state. 8 However, ␤ 1 -adrenergic receptors are downregulated and uncoupled from G proteins in failing hearts. 9,10 Nevertheless, we recently showed that ryanodine receptor (RyR2) is protein kinase A (PKA)-hyperphosphorylated in HF, 11 indicating that the net effect of ␤-adrenergic signaling is upregulated in HF with respect to RyR2 as a substrate for PKA phosphorylation. It is likely that the PKA hyperphosphorylation of RyR2 is a maladaptive response, 12 because it results in the depletion of the regulatory subunit FKBP12.6, 11,13 yielding channels that are pathologically sensitive to Ca 2ϩ -induced Ca 2ϩ release from the sarcoplasmic reticulum (SR). 11 RyR2 is a macromolecular complex that includes FKBP12.6 as well as PKA and 2 phosphatases (PP1 and PP2A) that are bound to the cytoplasmic domain of the channel via targeting proteins. 11,14 In failing hearts, the RyR2 macromolecular complex undergoes remodeling characterized by a reduction in the amounts of PP1, PP2A, and FKBP12.6 that are bound to the cytoplasmic domain of the channel. 11 In the present study, we used a well-characterized canine model of pacing-induced HF to show that ␤-adrenergic receptor blockade both restores the normal stoichiometry of the RyR2 macromolecular complex and normalizes the function of the channel. Methods H...