Functional expression of recombinant type 1 ryanodine receptor in insect sf21 cells

We have investigated the biochemical properties of the rabbit ryanodine rec eptor type 1 (RyR1) from skeletal muscle functionally expressed in insect s f21 cells infected with recombinant baculovirus. Equilibrium [H-3]ryanodine binding assays applied to total membrane fractions from sf21 cells express ing recombinant RyR1 showed a nonhyperbolic saturation curve (Hill coeffici ent=2.1). The [H-3]ryanodine binding was enhanced by 1 mM AMP-PCP and 10 mM caffeine, whereas 10 mM Mg2+ and 5 muM ruthenium red reduced the specific binding. The dependence of [H-3]ryanodine binding on ionic strength showed positive cooperativity (Hill coefficient = 2.2) with a plateau at 1 M KCl. The recombinant RyR1 showed a bell-shaped [H-3]ryanodine binding curve when free [Ca2+] was increased, with an optimal concentration around 100 muM. Confocal microscopy studies using the Ca2+ ATPase selective inhibitor, thap sigargin coupled to fluorescein and ryanodine coupled to Texas red demonstr ated that the recombinant RyR1 and the Ca2+ ATPase co-localize to the same intracellular membrane. No significant RyR1 fluorescence was observed at th e plasma membrane. Fluo-4-loaded sf21 cells expressing recombinant RyR1 responded to activatin g-low ryanodine concentrations (100 nM) or caffeine (10 mM) with a sharp ri se in intracellular Ca2+ followed by a sustained phase, in contrast, sf21 c ells expressing the human bradykinin type 2 receptor did not respond to rya nodine or caffeine. These results demonstrate the expression of recombinant RyR1 in sf21 cells with functional properties similar to what has been previously reported for native RyR1 in mammalian tissues, however, some differences were observed in [H-3]ryanodine binding assays compared to native rabbit RyR1. Hence, the baculovirus expression system provides a generous source of protein to acc omplish structure-function studies and an excellent model to assess functio nal properties of wild type and mutant RyR1. (C) 2001 Harcourt Publishers L td.