Abstract
Maurocalcine (MCa) isolated from Scorpio maurus palmatus venom shares 82% sequence identity with imperatoxin A. Both scorpion toxins are putative mimics of the II-III loop peptide (termed peptide A (pA)) of alpha(1s)-dihydropyridine receptor and are thought to act at a common site on ryanodine receptor type 1 (RyR1) important for skeletal muscle EC coupling. The relationship between the actions of synthetic MCa (sMCa) and pA on RyR1 were examined. sMCa released Ca(2+) from SR vesicles (EC(50) = 17.5 nm) in a manner inhibited by micromolar ryanodine or ruthenium red. pA (0.5-40 microm) failed to induce SR Ca(2+) release. Rather, pA enhanced Ca(2+) loading into SR and fully inhibited Ca(2+)-, caffeine-, and sMCa-induced Ca(2+) release. The two peptides modified single channel gating behavior in distinct ways. With Cs(+)-carrying current, 10 nm to 1 microm sMCa induced long lived subconductances having 48% of the characteristic full open state and occasional transitions to 29% at either positive or negative holding potentials. In contrast, pA stabilized long lived channel closures with occasional burst transitions to 65% (s1) and 86% (s2) of the full conductance. The actions of pA and sMCa were observed in tandem. sMCa stabilized additional subconductance states proportional to pA-induced subconductances (i.e. 43% of pA-modified s1 and s2 substates), revealing a proportional gating mechanism. [(3)H]Ryanodine binding and surface plasmon resonance analyses indicated that the peptides did not interact by simple competition for a single class of mutually exclusive sites on RyR1 to produce proportional gating. The actions of sMCa were also observed with ryanodine-modified channels and channels deficient in immunophilin 12-kDa FK506-binding protein. These results provide evidence that sMCa and pA stabilize distinct RyR1 channel states through distinct mechanisms that allosterically stabilize gating states having proportional conductance.
Highlights
Excitation-contraction (EC)1 coupling in muscle cells is the signaling process by which electrical stimuli arriving at the Expression of cDNAs encoding cardiac/skeletal muscle chimeric ␣1-dihydropyridine receptors (DHPRs) in dysgenic myotubes, which lack endogenous ␣1s-DHPR, identified the a site within the cytoplasmic loop between repeats II and III essential for the physical coupling with ryanodine receptors (RyRs) and skeletal EC coupling [9, 10]
We find that synthetic MCa (sMCa) and peptide A (pA) stabilize distinct subconductance states through an interaction with the ryanodine receptor type 1 (RyR1) channel complex that cannot be explained by simple competition at a common effector site
MCa, Imperatoxin A (IpTxa), and pA, showing a common basic domain terminating with an amino acid possessing a hydroxyl-containing side chain that has been proposed to contribute essential structure for activating RyR1 [26]
Summary
Materials—[3H]Ryanodine was obtained from PerkinElmer Life Sciences with specific activity of 57 Ci/mmol and purity of Ͼ90%. Protocol A used a buffer composed of 3 nM [3H]ryanodine, 200 mM KCl, 10 mM HEPES, ϳ7 M free Ca2ϩ, pH 7.2, and incubation was performed at 36 °C for 1.5 h. Before SPR experiments, purified RyR1 was dialyzed overnight at 4 °C in a buffer containing 150 mM NaCl, 10 mM HEPES, pH 7.4, 0.005% polysorbate in order to reduce the sucrose and NaCl concentration. In these conditions, RyR1 has been characterized by an apparent sedimentation coefficient of 30 S that corresponds to a homotetramer of ϳ565-kDa subunits [36, 37]. Arithmetic average time is used for analyzing the long subconductance states induced by sMCa and the long closed time induced by pA, because there are not enough events to fit with a reasonable biexponential function
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