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Volume 272, Number 39, Issue of September 26, 1997 pp. 24234-24246
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional Characterization of the Recombinant Type 3 Ca²⁺ Release Channel (Ryanodine Receptor) Expressed in HEK293 Cells

(Received for publication, April 30, 1997, and in revised form, July 3, 1997)

From the Cardiovascular Research Group, Department of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada T2N 4N1

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.

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