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 ryanodine receptor (RyR3), we have cloned the RyR3 cDNA from rabbit uterus by reverse transcriptase-polymerase chain reaction and expressed the cDNA in HEK293 cells. Immunoblotting studies showed that the cloned RyR3 was indistinguishable from the native mammalian RyR3 in molecular size and immunoreactivity. Ca2+ release measurements using the fluorescence 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 and a Ca2+ conductance of 137 picosiemens in 250 mM CaCl2 and displayed a pCa2+/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 inactivated at about 10 mM. Ca2+ alone was able to activate the cloned RyR3 fully. Calmodulin activated the cloned RyR3 at low Ca2+ concentrations but inhibited the channel at high Ca2+ concentrations. The cloned RyR3 was activated by ATP, caffeine, and perchlorate, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Cyclic ADP-ribose did 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 maximal 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.
Chen
S R
SR
Cardiovascular Research Group, Department of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada T2N 4N1. swchen@cvr.ucalgary.ca
Li
X
X
Ebisawa
K
K
Zhang
L
L
D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D036563:Cyclic ADP-Ribose; D018076:DNA, Complementary; D006801:Humans; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D010472:Perchlorates / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D017670:Sodium Compounds / Q000494:pharmacology; D014162:Transfection
D015220:Calcium Channels; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D018076:DNA, Complementary; D008051:Lipid Bilayers; D009124:Muscle Proteins; D010472:Perchlorates; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D017670:Sodium Compounds; D036563:Cyclic ADP-Ribose; D000246:Adenosine Diphosphate Ribose; D000255:Adenosine Triphosphate; C031068:sodium perchlorate