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RationaleCalmodulin (CaM) associates with cardiac ryanodine receptor type-2 (RyR2) as an important regulator. Defective CaM-RyR2 interaction may occur in heart failure, cardiac hypertrophy, and catecholaminergic polymorphic ventricular tachycardia. However, the in situ binding properties for CaM-RyR2 are unknown.ObjectiveWe sought to measure the in situ binding affinity and kinetics for CaM-RyR2 in normal and heart failure ventricular myocytes, estimate the percentage of Z-line-localized CaM that is RyR2-bound, and test cellular function of defective CaM-RyR2 interaction.Methods and resultsUsing fluorescence resonance energy transfer in permeabilized myocytes, we specifically resolved RyR2-bound CaM from other potential binding targets and measured CaM-RyR2 binding affinity in situ (Kd=10-20 nmol/L). Using RyR2(ADA/+) knock-in mice, in which half of the CaM-RyR2 binding is suppressed, we estimated that >90% of Z-line CaM is RyR2-bound. Functional tests indicated a higher propensity for Ca2+ wave production and stress-induced ventricular arrhythmia in RyR2(ADA/+) mice. In a post-myocardial infarction rat heart failure model, we detected a decrease in the CaM-RyR2 binding affinity (Kd≈51 nmol/L; ≈3-fold increase) and unaltered RyR2 affinity for the FK506-binding protein FKBP12.6 (Kd~0.8 nmol/L).ConclusionsCaM binds to RyR2 with high affinity in cardiac myocytes. Physiologically, CaM is bound to >70% of RyR2 monomers and inhibits sarcoplasmic reticulum Ca2+ release. RyR2 is the major binding site for CaM along the Z-line in cardiomyocytes, and dissociating CaM from RyR2 can cause severe ventricular arrhythmia. In heart failure, RyR2 shows decreased CaM affinity, but unaltered FKBP 12.6 affinity.

Studies with isolated membrane fractions have shown that calmodulin (CaM) inhibits the activity of cardiac muscle cell [Ca.sup.2+] release channel ryanodine receptor 2 (RyR2). To determine the physiological importance of [...]

Monobromobimane (MBB) is a lipophilic reagent that selectively modifies free cysteine residues in proteins. Because of its lipophilic character, MBB is capable of labeling cysteine residues in membrane proteins under native conditions. Reaction of MBB with the sulfhydryl groups of free cysteines leads to formation of highly fluorescent derivatives. Here we describe a procedure for the detection and relative quantitation of MBB-labeled cysteines using fluorescence and mass spectrometric analyses, which allow determination of free cysteine content and unambiguous identification of MBB-modified cysteine residues. We have applied this approach to the analysis of the free and redox-sensitive cysteine residues of a large membrane protein, the sarcoplasmic reticulum [Ca.sup.2+] release channel with a molecular mass of 2.2 million Da. Labeling was performed under physiologic conditions where the channel complex is in its native environment and is functionally active. The purified MBB-labeled channel complex was enzymatically digested, and the resulting peptides were separated by reversed-phase high-performance chromatography. MBB-labeled peptides were detected by fluorescence and identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Under MALDI conditions, partial photolytic fragmentation of the MBB--peptide bound occurred, thus allowing convenient screening for the MBB-modified peptides in the MS spectrum by detection of the specific mass increment of 190.07 Da for MBB-modified cysteine residues. Modification of the peptides was further confirmed by tandem mass spectrometric analysis, utilizing sequencing information and the presence of the specific immonium ion for the MBB-modified cysteine residues at m/z 266.6. Quantitative information was obtained by comparison of both fluorescence and MS signal intensities of MBB-modified peptides. Combination of fluorescence with MS detection and analysis of MBB-labeled peptides supported by a customized software program provides a convenient method for identifying and quantifying redox-sensitive cysteines in membrane proteins of native biological systems. Identification of one redox-sensitive cysteine (2327) in the native membrane-bound sarcoplasmic reticulum [Ca.sup.2+] release channel is described.

Ryanodine receptors (RyRs) are presented as a family of intracellular Ca(super 2)(super plus) channels that are regulated by calmodulin (CaM), which is sensitive to redox regulations. It demonstrates that corresponding amino acid substitutions in the CaM binding site differentially affect CaM binding and CaM regulation of RyR3 and those of the two other isoforms.

Ion channels subserve diverse cellular functions. Reactive oxygen and nitrogen species modulate ion channel function by a number of mechanisms including 1) transcriptional regulation of gene expression, 2) posttranslational modifications of channel proteins, i.e. nitrosylation, nitration, and oxidation of key amino acid residues, 3) by altering the gain in other signaling pathways that may in turn lead to changes in channel activity or channel gene expression, and 4) by modulating trafficking or turnover of channel proteins, as typified by oxygen radical activation of NF-kB, with subsequent changes in proteasomal degradation of channel degradation. Regardless of the mechanism, as was discussed in a symposium at the 2003 Experimental Biology Meeting in San Diego, CA, changes in the cellular level of reactive oxygen and nitrogen species can have profound effects on the activity of ion channels and cellular function. calcium channels; potassium channels; sodium channels; chloride channels; nitric oxide; cystic fibrosis transmembrane conductance regulator

The dynamic behavior of both surface and bulk of mixed solutions of surfactants and polymers of opposite electrical charges are described. It was found that in the bulk, the surfactant not only screens the polymer charges but is likely to start binding to the polymer chains.

Ryanodine receptors (RyRs) are Ca(super ++) channels that control cellular functions. An estimation of the RyR2 ion diffusion constants and electrostatic interactions, in a self-consistent coherent formulation, is provided by the extended PNP.

Calmodulin is a ubiquitous [Ca.sup.2+] binding protein that modulates the in vitro activity of the skeletal muscle ryanodine receptor (RyR1). Residues 3614-3643 of RyR1 comprise the CaM binding domain and mutations within this region result in a loss of both high-affinity [Ca.sup.2+]-bound calmodulin (CaCaM) and [Ca.sup.2+]-free CaM (apoCaM) binding (L3624D) or only CaCaM binding (W3620A). To investigate the functional role of CaM binding to this region of RyR1 in intact skeletal muscle, we compared the ability of RyR1, L3624D, and W3620A to restore excitation-contraction (EC) coupling after expression in RyR1-deficient (dyspedic) myotubes. W3620A-expressing cells responded normally to 10 mM caffeine and 500 [micro]M 4-chloro-m-cresol (4-cmc). Interestingly, L3624D-expressing cells displayed a bimodal response to caffeine, with a large proportion of cells (~44%) showing a greatly attenuated response to caffeine. However, high and low caffeine-responsive L3624D-expressing myotubes exhibited [Ca.sup.2+] transients of similar magnitude after activation by 4-cmc (500 [micro]M) and electrical stimulation. Expression of either L3624D or W3620A in dyspedic myotubes restored both L-type [Ca.sup.2+] currents (retrograde coupling) and voltage-gated SR [Ca.sup.2+] release (orthograde coupling) to a similar degree as that observed for wildtype RyR1, although L-current density was somewhat larger and activated at more hyperpolarized potentials in W3620A-expressing myotubes. The results indicate that CaM binding to the 3614-3643 region of RyR1 is not essential for voltage sensor activation of RyR1. KEY WORDS: ryanodine receptor * muscle * calcium channel * caffeine * sarcoplasmic reticulum

Results show that the redox state of the skeletal muscle calcium release channel/ryanodine receptor is controlled by physiological oxygen tension and that it involves thiol groups and a response to nitric oxide. Data further reveal that nitric acid activates the calcium channel by S-nitrosylation of cysteine residue.

Ryanodine receptor 1 (RyR1) is an intracellular calcium ion (Ca2+) release channel required for skeletal muscle contraction. Although cryo‐electron microscopy identified binding sites of three coactivators Ca2+, ATP, and caffeine (CFF), the mechanism of co‐regulation and synergy of these activators is unknown. Here, we report allosteric connections among the three ligand‐binding sites and pore region in (i) Ca2+ bound‐closed, (ii) ATP/CFF bound‐closed, (iii) Ca2+/ATP/CFF bound‐closed, and (iv) Ca2+/ATP/CFF bound‐open RyR1 states. We identified two dominant networks of interactions that mediate communication between the Ca2+‐binding site and pore region in Ca2+ bound‐closed state, which partially overlapped with the pore communications in ATP/CFF bound‐closed RyR1 state. In Ca2+/ATP/CFF bound‐closed and ‐open RyR1 states, co‐regulatory interactions were analogous to communications in the Ca2+ bound‐closed and ATP/CFF bound‐closed states. Both ATP‐ and CFF‐binding sites mediate communication between the Ca2+‐binding site and the pore region in Ca2+/ATP/CFF bound—open RyR1 structure. We conclude that Ca2+, ATP, and CFF propagate their effects to the pore region through a network of overlapping interactions that mediate allosteric control and molecular synergy in channel regulation. [ABSTRACT FROM AUTHOR]

The effects of imperatoxin activator, a 33 amino acid peptide isolated from the venom of the African scorpion, on rabbit skeletal and canine cardiac muscle Ca2+ release channels (CRCs) were investigated. Addition of imperatoxin activator stimulated voltage- and concentration-dependent subconductance states in both skeletal and cardiac muscles CRCs. Moreover, the activator also influenced [3H] ryanodine binding to skeletal and cardiac sarcoplasmic reticulum vesicles depending on the level of channel activation.

The gene coding for the ryanodine receptor, the skeletal muscle calcium channel of the lobster, was partially cloned and sequenced. A bacterial heterologous expression system was used to synthesized an engineered channel consisting of the amino terminal of mammalian ryanodine receptor and the carboxy terminal of that of lobsters. The deduced amino acid sequence of the carboxy terminal of the lobster ryanodine receptor reveal that there is a cytoplasmic domain that binds calcium ions. The engineered proteins were used to investigate calcium binding affinity.