DEFECTION AND, FUNCTIONAL-CHARACTERIZATION OF RYANODINE RECEPTORS FROM SEA-URCHIN EGGS

1. Immunoblot analysis, [H-3]ryanodine binding, and planar lipid bilay er techniques were used to identify and characterize the functional pr operties of ryanodine receptors (RyRs) from Lytechinus pictus and Stro ngylocentrotus purpuratus sea urchin eggs. 2. An antibody against mamm alian skeletal RyRs identified an similar to 400 kDa band in the corti cal microsomes of sea urchin eggs while a cardiac-specific RyR antibod y failed to recognize this protein. [H-3]Ryanodine binding to cortical microsomes revealed the presence of a high-affinity (K-d = 13 nM), sa turable (maximal density of receptor sites, B-max = 1.56 pmol (mg prot ein)(-1)) binding site that exhibited a biphasic response to Ca2+. 3. Upon reconstitution of cortical microsomes into lipid bilayers, only s parse and unstable openings of a high-conductance cation channel were detected. Addition of crude sea urchin egg homogenate to the cytosolic (cis side) of the channel increased the frequency of openings and sta bilized channel activity. The homogenate-activated channels were Ca2sensitive, selective for Ca2+ over Cs+, and driven by ryanodine into a long-lived subconductance state that represented similar to 40 % of th e full conductance level. Homogenate dialysed in membranes with a mole cular weight cut-off less than or equal to 2000 lacked the capacity to increase the frequency of RyR openings and to stabilize channel activ ity. 4. Direct application of cyclic adenosine diphosphoribose (cADPR) or photolysis of NPE-cADPR ('caged' cADPR) by ultraviolet laser pulse s produced transient activation of sea urchin egg RyRs. Calmodulin (Ca M) failed to activate reconstituted RyRs; however, channel activity wa s inhibited by the CaM blocker trifluoroperazine, suggesting that CaM was necessary but not sufficient to sustain RyR activity. 5. These fin dings suggest that a functional Ca2+ release unit in sea urchin eggs i s a complex of several molecules, one of which corresponds to a protei n functionally similar to mammalian RyRs.