Molecular Cloning and Expression of Cdna Encoding the Ryanodine Receptor Type 2 from Rattus Norvegicus Cerebral Artery Smooth Muscle

Ryanodine receptors (RyRs) are a family of Ca2+ release channels found in intracellular organelles. RyR channels are ubiquitously expressed in many cell types and participate in a wide variety of physiological processes (e.g., neurotransmission, secretion, regulation of myogenic tone, cardiac contractility). We have cloned and sequenced cDNA encoding the Ca2+ release channel isoform 2 of the ryanodine receptor (RyR2) from smooth muscle cells of Rattus norvegicus cerebral arteries and determined RyR2 protein expression. The RyR2 cDNA is 14,850 bp in length, resulting in a protein product of 4,950 amino acids with a theoretical pI/MW of 5.81/561,699.41 Da. We used total RNA from endothelium free cerebral arteries and conducted reverse transcription. We divided the RyR2 cDNA in two equal parts because of its huge size; N-terminal part: 1-7,486 bp and C-terminal part: 7,487-14,862 bp. We used gene specific primers to get both N and C-terminal parts of the cDNA sequence. Primers for the N-terminal part: forward 5' - CG ACG CGT ATG GCT GAT GCG GGC GAA G - 3'; reverse 5' - GA CGC GTC GAC CAG CCG TGT CTA GAG AG - 3'; for C-terminal part: forward 5' - GGA CGC GTC GAC CAC TAA GTG CTA CAG AC - 3'; reverse 5' - GAT AAG AAT GCG GCC GCT TAA TTT AAC TGG TCC - 3'. After cloning the cDNAs into the mammalian vector pIRESneo, we characterized the clones by restriction analysis and confirmed the resulting cDNA sequence by automated analysis. Using immunohistochemistry, Western blotting and surface biotinylation assays, we evaluated the cytosolic and membrane surface expression of RyR2 in HEK cells. Our results demonstrate that RyR2 protein effectively expresses in both plasma and internal membranes. Our study demonstrates protein expression from the newly cloned RyR2 from cerebral artery smooth muscle. Functional characterization of this RyR2 is underway. Support: HL104631, R37AA11560 (AMD).