Abstract
Type 1 ryanodine receptor (RyR1) is a large intracellular Ca2+ release channels in the sarcoplasmic reticulum of skeletal muscle and plays an essential role in excitation-contraction coupling. Although the RyR1 channel is believed to be opened via some physical interaction with dihydropyridine receptor of the T-tubule, the detailed mechanism remains still unclear. Fluorescence resonance energy transfer (FRET) is a powerful tool for detection of conformational changes of the protein, and should be applicable to direct monitoring of the RyR1 channel gating. We here generated the functional fluorescent RyR1 channels by randomly inserting GFP variants using transposon-based mutagenesis. The Tn5 transposon encompassing the kanamycin-resistant gene (Kanr) was randomly inserted into full-length RyR1 cDNA via in vitro transposon reaction. Clones with a transposon were selected by kanamycin resistance, and Kanr was replaced by genes encoding GFP variants. The resultant clones were transfected into HEK293 cells to screen GFP fluorescence. So far, several tens of fluorescent RyR1 clones were obtained. We are currently testing the channel activity by caffeine-induced Ca2+ release. [J Physiol Sci. 2008;58 Suppl:S74]
