Novel ryanodine-binding properties in mammalian retina.
The ryanodine receptor (RyR)/Ca2+ release channel mobilizes Ca2+ from internal calcium stores to support a variety of neuronal functions. To investigate the presence of such a protein in mammalian retina, we applied ryanodine binding, PCR and antibodies against known RyRs. Surprisingly, ryanodine-binding properties of retinal endoplasmic reticulum-enriched membrane fraction were vastly different from those of skeletal and cardiac muscles ryanodine-binding proteins. In common with the skeletal and cardiac muscle, ryanodine bound with high-affinity to two or more types of binding site (Kd1 = 20.6 and Kd2 = 114 nM); binding was strongly stimulated by high concentrations of NaCl; it was inhibited by tetracaine and the protein appeared to possess an ATP-binding site. Unlike cardiac and skeletal muscle, RyRs in retina binding was Ca2+-independent; inhibited by caffeine and dantrolene; less sensitive to ruthenium red; and unaffected by La3+. Also, in retina, ryanodine rapidly associated to and dissociated from its binding sites. Furthermore, although the protein bound the ATP analog BzATP, retinal ryanodine binding was not stimulated by nucleotides. Immunostaining of bovine retinal sections with anti-RyR2 showed a strong staining of amacrine, horizontal and ganglion cells. Finally, using RT-PCR, the three known RyR isoforms were identified in retina. However, consistent with the novel binding properties, the peptide maps yielded by trypsin treatment and Western blotting demonstrate different patterns. Together, the results suggest that retina expresses a novel ryanodine-binding protein, likely to be a ryanodine receptor. Its presence in retina suggests that this protein might play a role in controlling intracellular Ca2+ concentration.
Shoshan-Barmatz
Varda
V
Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel. vardasb@bgumail.bgu.ac.il
Orr
Irit
I
Martin
Cecile
C
Vardi
Noga
N
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D012433:Ryanodine / Q000378:metabolism*; D013696:Temperature
D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D012433:Ryanodine; D002118:Calcium