The cGMP phosphodiesterase (PDE) of cattie retinal rod outer segments comprises three types of subunits: the two heavy catalytic ones, PDEa and PDE.8, each around 85 kDa, and the light inhibitory one, PDEy or I (11 kDa). The relative stoichiometry is usually assumed to be 1:1:1. PDE activation in the visual transduction cascade results from removal of the inhibitor by the a subunit of transducin (Ta). The stoichiometric complex Ta-I, separated from activated PDE, has been isolated and characterized. Analyzing now the activated PDE, we find that it still contains some inhibitor and is resolvable into two species, one with 50% of the inhibitor content of the native enzyme and the other totally devoid of it. The same two species are observed upon activation of PDE by very short tryptic proteolysis, which specifically degrades the inhibitor. This leads us to conclude that the composition of the native enzyme is PDEa8-I2. (14) and preserved at -80'C. The thawed pellets were homogenized, illuminated, and then washed in medium salt buffer, to eliminate some minor proteins not relevant to this study. Extraction and "crude" purification were then carried out using the light, nucleotide, and ionic-strength dependence of binding of the species (14). Crude PDE was obtained by low ionic strength extraction of the illuminated membrane pellet and crude transducin was obtained by subsequent extraction of the same pellet after addition of 100 ,uM GTP[yS]. Total extract was obtained by direct low ionic strength extraction of an illuminated membrane pellet in the presence of GTP [yS]. Low salt buffer was 5 mM Hepes/1 mM dithiothreitol, pH 7.5; medium salt buffer was 20 mM Hepes/120 mM KCl/1 mM dithiothreitol, pH 7.5. Rhodopsin concentration in all extraction procedures was 2 mg/ml. GTP [yS] was used at 100 ,uM.Protein Chromatography. Separation and purification of transducin subunits, native PDE, and the various PDE units were performed as described (15), on an ion exchange column (Polyanion SI from Pharmacia) (unfortunately this very efficient column is not commercially available anymore, but we have a stock of them). Elution was obtained by Na2SO4 gradients (from 0 to 600 mM) in a buffer containing 20 mM Hepes, pH 7.5/1 mM MgSO4/5 mM 2-mercaptoethanol. Various gradient programs were used to optimize the separation of the different PDE species (see figure legends). Proteins were eluted from the column (0.5 ml/min) followed by UV absorption. Fractions (250 ,ul) 2424The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Membrane vesicles enriched in both ryanodine receptor and dihydropyridine receptor were obtained from rabbit skeletal muscle and solubilized with 3- [(3-cholamidopropyl) (14,17,18).Three different mechanisms have been proposed for excitation-contraction coupling in skeletal muscle: Ca2 -induced Ca2+ release, inositol 1,4,5-trisphosphate-induced Ca2W release, and direct physical coupling (for review see refs. 19 and 20). Current evidence favors a model in which a voltagedriven conformational change ofthe al subunit ofthe DHP-R activates Ca2+ release by the RyR through direct physical interaction (11,21,22
In the present report we studied the interaction between the skeletal muscle ryanodine receptor and the ubiquitous S100A1 Ca2+ binding protein. S100A1 did not affect equilibrium [3H]ryanodine binding to purified rabbit skeletal muscle terminal cisternae at 100 microM free [Ca2+]. At nanomolar free [Ca2+], however, S100A1 activated by 40 +/- 6.7% (mean +/- SE, n = 5) the [3H]ryanodine binding activity; the half-maximal concentration for stimulation of [3H]ryanodine binding was approximately 70 nM, a value well below the estimated S100A1 concentration in skeletal muscle fibers. Scatchard analysis of [3H]ryanodine binding performed in the presence of 100 microM EGTA indicates that S100A1 increases the apparent affinity of the receptor for ryanodine (Kd = 191 vs 383 nM in the presence and in the absence of 100 nM S100A1, respectively). The effect of S100A1 was also tested on the single-channel gating properties of the purified ryanodine receptor after reconstitution into a lipid planar bilayer. Currents carried by purified ryanodine receptor channels were modulated by both cis Ca2+ and ruthenium red. In the presence of nanomolar [Ca2+], S100A1 activated the channel by increasing (6.0 +/- 2.8)-fold (mean +/- SE, n = 3) the normalized open probability. The interaction between S100A1 and the purified RYR was verified using the optical biosensor BIAcore: we show that the two proteins interact directly both at millimolar and at nanomolar calcium concentrations. We next mapped the regions of the skeletal muscle RYR involved in the interaction with S100A1 by performing ligand overlays on a panel RYR of fusion proteins in the presence of 100 nM S100A1. Our results indicate that the skeletal muscle RYR contains three potential S100A1 binding domains. Binding of S100A1 to the RYR fusion proteins occurred at both nanomolar and millimolar free [Ca2+]. S100A1 binding domain 1 binds the ligand in the presence of 1 mM free [Ca2+] or 1 mM EGTA. Maximal binding to S100A1#2 was achieved in the presence of 1 mM free [Ca2+]. The S100A1#3 domain, which overlaps with calcium-dependent calmodulin binding domain 3 (CaM 3), exhibits weak and strong S100A1 binding activity in the presence of either millimolar or nanomolar Ca2+, respectively. The interaction between S100A1 and the purified RYR complex was also investigated by affinity chromatography: in the presence of nanomolar Ca2+, we observed binding of native RYR complex to S100A1-conjugated Sepharose. This interaction could be inhibited by the presence of RYR polypeptides encompassing S100A1 binding sites S100A1#1, S100A1#2, and S100A1#3.
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