Peptide Probe of Ryanodine Receptor Function IMPERATOXIN A, A PEPTIDE FROM THE VENOM OF THE SCORPION PANDINUS IMPERATOR, SELECTIVELY ACTIVATES SKELETAL-TYPE RYANODINE RECEPTOR ISOFORMS

El-Hayek, Roque; Lokuta, Andrew J.; Arévalo, Carolina; Valdivia, Héctor H.

doi:10.1074/jbc.270.48.28696

Cited by 82 publications

(82 citation statements)

References 44 publications

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“…The effect of CaM (1 M) on radioligand binding was determined under identical assay conditions. Assays were carried out in duplicate according to established protocols (38), and each datum point was obtained by averaging the duplicates.…”

Section: Methodsmentioning

confidence: 99%

Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

Kobayashi

Bannister

Gangopadhyay

et al. 2005

Journal of Biological Chemistry

120

111

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Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca 2؉ channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The SternVolmer quenching constant (K Q ) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22°C and was unaffected by calmodulin. [ 3 H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca 2؉ release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37°C, yet required the presence of calmodulin at 22°C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca 2؉ channel opening rather than the domain switch itself.Dantrolene (hydrated 1-(((5-(4-nitrophenyl)-2-furanyl)methylene)amino)-2,4-imidazolidinedione sodium salt) is an intracellularly acting skeletal muscle relaxant used for the treatment of malignant hyperthermia (MH). 1 MH is a potentially deadly, pharmacogenetically mediated, hypermetabolic response to volatile anesthetics that results from unregulated intramyoplasmic Ca 2ϩ release (1). The drug is known to inhibit excitation-contraction coupling of skeletal muscle (2) by suppressing depolarization-induced sarcoplasmic reticulum (SR) Ca 2ϩ release in normal and MH-susceptible skeletal muscle without affecting voltage sensor activation (3). In MH, the voltage dependence of contractile activation is shifted to lower voltages (4), whereas in the presence of clinical concentrations of dantrolene, i.e. 10 M (5), the voltage dependence of contractile activation is shifted to higher voltages (6, 7). Normalization of the voltage dependence of contractile activation may therefore be one of the important components of the therapeutic action of dantrolene. Dantrolene also confers a normal Mg 2ϩ sensitivity to MH-susceptible muscle fibers, which would otherwise show a considerably reduced sensitivity to the normal inhibitory action of myoplasmic Mg 2ϩ on the SR Ca 2ϩ release mechanism (...

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Section: Methodsmentioning

confidence: 99%

Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

Kobayashi

Bannister

Gangopadhyay

et al. 2005

Journal of Biological Chemistry

120

111

View full text Add to dashboard Cite

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“…5B), suggesting that both toxins act on RyR1 based on a direct protein-protein interaction mechanism. This property is analogous to that of IpTx a , which also activates RyR1 by a direct interaction (14,22) (Fig. 5B).…”

Section: Fig 4 Effect Of Bjtx-1 and Bjtx-2 On [ 3 H]ryanodine Bindimentioning

confidence: 83%

“…In addition, the fraction of Ca 2ϩ released by low caffeine concentrations was increased (32). (22). Downward arrows indicate the addition of 10 nmols of Ca 2ϩ to the 1-ml reaction medium.…”

Section: Discussionmentioning

confidence: 99%

“…The recording solution contained ϳ5 M free Ca 2ϩ , as assessed by a calibration curve, which was sufficient to activate RyRs. Different concentrations of B. judaicus toxins were added to the cis chamber, which corresponded to the cytosolic side of the channel (22). Signals were digitized at 4 kHz and analyzed after filtering with a low-pass 8-pole Bessel filter at a sampling frequency of 1.5 kHz.…”

Section: Materials-[mentioning

confidence: 99%

“…[ Planar Lipid Bilayer Technique-Pig skeletal RyRs were reconstituted into Muller-Rudin planar lipid bilayers as described previously (22). Single channel data were collected at steady voltages (ϩ35 mV, cis chamber grounded) for 2 min in symmetrical 300 mM cesium methanesulfonate and 10 mM Na-HEPES (pH 7.2).…”

Section: Materials-[mentioning

confidence: 99%

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