FUNCTIONAL-CHARACTERIZATION OF THE RECOMBINANT TYPE-3 CA2+ RELEASE CHANNEL (RYANODINE RECEPTOR) EXPRESSED IN HEK293 CELLS

To investigate the channel properties of the mammalian type 3 ryanodin e receptor (RyR3), we have cloned the RyR3 cDNA from rabbit uterus by reverse transcriptase-polymerase chain reaction and expressed! the cDN A inn HEK293 cells, Immunoblotting studies showed that the cloned RyR3 was indistinguishable from the native mammalian RyR3 in molecular siz e and immunoreactivity. Ca2+ release measurements using the fluorescen ce Ca2+ indicator fluo 3 revealed that the cloned RyR3 functioned as a caffeine-and ryanodine-sensitive Ca2+ release channel in HEK293 cells , Functional properties of the cloned RyR3 were further characterized by using single channel recordings in lipid bilayers. The cloned RyR3 channel exhibited a K+ conductance of 777 picosiemens in 250 mM KCl an d a Ca2+ conductance of 137 picosiemens inn 250 mM CaCl2 and displayed a pCa(2+)/pK(+) ratio of 6.3 and an open time constant of about 1.16 ms, The response of the cloned RyR3 to cytoplasmic Ca2+ concentrations was biphasic. The channel was activated by Ca2+ at about 100 nM and i nactivated at about 10 mM. Ca2+ alone was able to activate the cloned RyR3 fully, Calmodulin activated the cloned RyR3 at low Ca2(+) concent rations but inhibited the channel at low Ca2+ concentrations. The clon ed RyR3 was activated by ATP, caffeine, and perchlorate, inhibited by Mg2+ and ruthenium red, and modified by ryanodine, cyclic ADP-ribose d id not seem to affect single channel activity of the cloned RyR3. The most prominent differences of the cloned RyR3 from the rabbit skeletal muscle ryanodine receptor were in the gating kinetics, extent of maxi mal activation by Ca2+, and sensitivity to Ca2+ inactivation. Results of the present study provide initial insights into the single channel properties of the mammalian RyR3.