Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-05-10
2002-12-03
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007210, C435S007320, C435S007310, C435S007700, C436S501000
Reexamination Certificate
active
06489125
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application, various publications are referenced in parentheses by author and year. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
The contraction of striated muscle is initiated when calcium (Ca
2+
) is released from tubules within the muscle cell known as the sarcoplasmic reticulum (SR). Calcium release channels (ryanodine receptors) on the sarcoplasmic reticulum are required for excitation-contraction (EC) coupling. The type 2 ryanodine receptor (RyR2) is found in the heart, while the type 1 ryanodine receptor (RyR1) is found in skeletal muscle. The RyR2 receptor is a tetramer comprised of four 565,000 dalton RyR2 polypeptides and four 12,000 dalton FK-506 binding proteins (FKBP12.6). FKBP12s are regulatory subunits that stabilize RyR channel function (Brillantes et al., 1994) and facilitate coupled gating between neighboring RyR channels (Marx et al., 1998) which are packed into dense arrays. in specialized regions of the sarcoplasmic reticulum that release intracellular stores of Ca
2+
triggering muscle contraction.
RyRs are ligand activated channels, and Ca
2+
is the important physiological ligand that activates the channels in cardiac muscle during excitation-contraction coupling. The Ca
2+
-dependence of RyR channel activity is biphasic such that low cytosolic Ca
2+
concentration (&mgr;M) activates the channels and high Ca
2+
concentration (mM) inactivates the channels (Bezprozvanny et al., 1991). One FKBP12 molecule is bound to each RyR subunit. Dissociation of FKBP12 significantly alters the biophysical properties of the channels resulting in the appearance of subconductance states, and increased open probability (Po) due to an increased sensitivity to Ca
2+
-dependent activation (Brillantes et al., 1994; Kaftan et al., 1996). In addition, dissociation of FKBP12 from RyR channels inhibits coupled gating of RyR channels resulting in channels that gate stochastically rather than as an ensemble (Marx et al., 1998). Coupled gating of arrays of RyR channels is thought to be important for efficient excitation-contraction coupling that regulates muscle contraction (Marx et al., 1998).
FKBPs are cis-trans peptidyl-prolyl isomerases that are widely expressed and subserve a variety of cellular functions (Marks, 1996). FKBP12s are tightly bound to and regulate the function of the skeletal (RyR1) (Brillantes et al., 1994; Jayaraman et al., 1992) and cardiac (RyR2) (Kaftan et al., 1996) muscle Ca
2+
release channels, as well as a related intracellular Ca
2+
release channel known as the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) (Cameron et al., 1997), and the type I transforming growth factor &bgr; (TGF&bgr;) receptor (T&bgr;RI) (Chen et al., 1997).
The present application discloses the following. Protein kinase A (PKA) phosphorylation regulates the binding of FKBP12.6 to the RyR channel both in vitro and in vivo. PKA phosphorylation of the cardiac Ca
2+
release channel (RyR2) on the sarcoplasmic reticulum dissociates the regulatory subunit FKBP12.6 from the RyR2 channel resulting in altered channel function manifested as an increased open probability, increased sensitivity to Ca
2+
-induced activation, and destabilization of the RyR2 channel resulting in subconductance states. RyR2 PKA phosphorylation is physiologically regulated in vivo. RyR2 channels from failing hearts were PKA hyperphosphorylated and exhibited decreased binding of the FKBP12.6 regulatory subunit resulting in the same severe defects in single channel properties observed in in vitro PKA hyperphosphorylated RyR2 channels. The RyR2 channel comprises a macromolecular complex that includes the regulatory subunit FKBP12.6, protein kinase A (PKA), the PKA regulatory subunit RII, protein phosphatase 2A (PP2A), protein phosphatase 1 (PP1), and muscle A kinase anchoring protein (mAKAP). Taken together these data demonstrate that local regulation of the RyR2 channel via PKA phosphorylation is a potent mechanism for modulating Ca
2+
release from the cardiac sarcoplasmic reticulum. Dysregulation of this control mechanism occurs in failing hearts and can explain the observed defects in excitation-contraction coupling that contribute to cardiac dysfunction. Methods for treating heart disease are disclosed, as are methods for screening for compounds that alleviate heart disease.
SUMMARY OF THE INVENTION
This invention is directed to a method of regulating contraction of a subject's heart by administering to the subject a compound which regulates protein kinase A (PKA) phosphorylation of a type 2 ryanodine (RyR2) receptor of the subject's heart.
This invention provides a method of treating a subject's heart failure by administering to the subject a compound which decreases protein kinase A (PKA) phosphorylation of a type 2 ryanodine (RyR2) receptor of the subject's heart, thereby alleviating the subject's heart failure. This invention also provides a method of treating a subject's heart failure by administering to the subject a compound which decreases dissociation of a FKBP12.6 binding protein from a type 2 ryanodine (RyR2) receptor of the subject's heart, thereby alleviating the subject's heart failure. This invention in addition provides a method of treating a subject's heart failure by administering to the subject a compound which mimics binding of a FKBP12.6 binding protein to a type 2 ryanodine (RyR2) receptor of the subject's heart, thereby alleviating the subject's heart failure.
This invention provides a method of treating a subject's cardiac arrhythmia by administering to the subject a compound which decreases protein kinase A (PKA) phosphorylation of a type 2 ryanodine (RyR2) receptor of the subject's heart, thereby alleviating the subject's cardiac arrhythmia. This invention also provides a method of treating a subject's cardiac arrhythmia by administering to the subject a compound which decreases dissociation of a FKBP12.6 binding protein from a type 2 ryanodine (RyR2) receptor of the subject's heart, thereby alleviating the subject's cardiac arrhythmia. This invention in addition provides a method of treating a subject's cardiac arrhythmia by administering to the subject a compound which mimics binding of a FKBP12.6 binding protein to a type 2 ryanodine (RyR2) receptor of the subject's heart, thereby alleviating the subject's cardiac arrhythmia.
This invention provides a method for identifying a chemical compound which specifically binds to a type 2 ryanodine (RyR2) receptor, which comprises contacting cells expressing the RyR2 receptor with the chemical compound under conditions suitable for binding and detecting specific binding of the chemical compound to the RyR2 receptor. This invention also provides a method for identifying a chemical compound which specifically binds to a type 2 ryanodine (RyR2) receptor, which comprises contacting a fraction containing sacroplasmic reticulum or endoplasmic reticulum from a cell extract of cells expressing the RyR2 receptor, with the chemical compound under conditions suitable for binding and detecting specific binding of the chemical compound to the RyR2 receptor.
This invention provides a method involving competitive binding for identifying a chemical compound which specifically binds to a type 2 ryanodine (RyR2) receptor, which comprises separately contacting cells expressing the RyR2 receptor, with both the chemical compound and a second chemical compound known to bind to the RyR2 receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the RyR2 receptor, a decrease in binding of
Marks Andrew R.
Marx Steven O.
Cooper & Dunham LLP
Kemmerer Elizabeth
Li Ruixiang
The Trustees of Columbia University In The City of New York
White John P.
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