We have defined regions of the skeletal muscle ryanodine receptor (RyR1) essential for bidirectional signaling with dihydropyridine receptors (DHPRs) and for the organization of DHPR into tetrad arrays by expressing RyR1-RyR3 chimerae in dyspedic myotubes. RyR1-RyR3 constructs bearing RyR1 residues 1-1681 restored wildtype DHPR tetrad arrays and, in part, skeletal-type excitationcontraction (EC) coupling (orthograde signaling) but failed to enhance DHPR Ca 2؉ currents (retrograde signaling) to WT RyR1 levels. Within this region, the D2 domain (amino acids 1272-1455), although ineffective on its own, dramatically enhanced the formation of tetrads and EC coupling rescue by constructs that otherwise are only partially effective. These findings suggest that the orthograde signal and DHPR tetrad formation require the contributions of numerous RyR regions. Surprisingly, we found that RyR3, although incapable of supporting EC coupling or tetrad formation, restored a significant level of Ca 2؉ current, revealing a functional interaction with the skeletal muscle DHPR. Thus, our data support the hypotheses that (i) the structural/functional link between RyR1 and the skeletal muscle DHPR requires multiple interacting regions, (ii) the D2 domain of RyR1 plays a key role in stabilizing this interaction, and (iii) a form of retrograde signaling from RyR3 to the DHPR occurs in the absence of direct proteinprotein interactions.calcium release ͉ freeze-fracture ͉ myotubes ͉ voltage clamp T he basis for excitation-contraction (EC) coupling in skeletal muscle is a direct functional interaction between the dihydropyridine receptor (DHPR) in the plasmalemma/T tubules and ryanodine receptor (RyR)1 of the sarcoplasmic reticulum. The interaction requires an appropriate intermolecular link between the two skeletal muscle channels and depends on a specific positioning of the DHPR relative to the RyR. Three convenient experimental assays are available for assessing this structure-function correlation. The first approach is a functional assay that detects orthograde signaling, by which DHPRs control the functional state of RyRs (1-3) and produces depolarization-induced sarcoplasmic reticulum Ca 2ϩ release in the absence of Ca 2ϩ permeation through DHPRs. The second method measures the retrograde signal by which RyRs control the gating of DHPRs (4-6) as reflected in the size of DHPR Ca 2ϩ currents via whole-cell voltage clamp. The third approach is a structural assay based on the technique of freeze-fracture for electron microscopy, which permits the study of the intermolecular interaction at the basis of the DHPR-RyR conformational coupling (7-10). In images from freeze-fracture replicas, DHPRs arrange themselves in tetrads in a RyR1-dependent manner.In previous studies, various 12) and several skeletal-cardiac and skeletal-insect DHPR chimerae (13-15) have been tested to assess the contributions of various domains to DHPR-RyR1 coupling. Although some details are not fully agreed on, it is clear that a specific segment of the ␣ 1S DHPR sub...