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.