Detection and functional characterization of ryanodine receptors from sea urchin eggs.
1. Immunoblot analysis, [3H]ryanodine binding, and planar lipid bilayer techniques were used to identify and characterize the functional properties of ryanodine receptors (RyRs) from Lytechinus pictus and Strongylocentrotus purpuratus sea urchin eggs. 2. An antibody against mammalian skeletal RyRs identified an approximately 400 kDa band in the cortical microsomes of sea urchin eggs while a cardiac-specific RyR antibody failed to recognize this protein. [3H]Ryanodine binding to cortical microsomes revealed the presence of a high-affinity (Kd = 13 nM), saturable (maximal density of receptor sites, Bmax = 1.56 pmol (mg protein)-1) binding site that exhibited a biphasic response to Ca2+. 3. Upon reconstitution of cortical microsomes into lipid bilayers, only sparse 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 stabilized channel activity. The homogenate-activated channels were Ca2+ sensitive, selective for Ca2+ over Cs+, and driven by ryanodine into a long-lived subconductance state that represented approximately 40 % of the full conductance level. Homogenate dialysed in membranes with a molecular weight cut-off <= 2000 lacked the capacity to increase the frequency of RyR openings and to stabilize channel activity. 4. Direct application of cyclic adenosine diphosphoribose (cADPR) or photolysis of NPE-cADPR ('caged' cADPR) by ultraviolet laser pulses produced transient activation of sea urchin egg RyRs. Calmodulin (CaM) failed to activate reconstituted RyRs; however, channel activity was inhibited by the CaM blocker trifluoroperazine, suggesting that CaM was necessary but not sufficient to sustain RyR activity. 5. These findings suggest that a functional Ca2+ release unit in sea urchin eggs is a complex of several molecules, one of which corresponds to a protein functionally similar to mammalian RyRs.
Lokuta
A J
AJ
Department of Physiology, University of Wisconsin Medical School, Madison, WI 53706, USA.
Darszon
A
A
Beltrán
C
C
Valdivia
H H
HH
D036541:ADP-ribosyl Cyclase; D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors; D004553:Electric Conductivity; D015151:Immunoblotting; D008861:Microsomes / Q000378:metabolism; D010063:Ovum / Q000378:metabolism*; D019761:Phosphorus-Oxygen Lyases / Q000494:pharmacology; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D012617:Sea Urchins / Q000378:metabolism*; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D014268:Trifluoperazine; D036541:ADP-ribosyl Cyclase; D019761:Phosphorus-Oxygen Lyases