Abstract
An oligonucleotide probe to a conserved 3' region within the three identified ryanodine receptor-calcium release channel isoforms hybridized to a single clone from a rabbit kidney cDNA library. The kidney clone encoded the carboxyl-terminal 338 amino acids within the putative transmembrane domain of the type 2 ryanodine receptor sequence. Reverse transcriptase-polymerase chain reaction with isoform-specific oligonucleotide primers demonstrated the presence of the type 2 ryanodine receptor transcript in rabbit kidney, as well as in a non-excitable cell line, LLC-RK1, derived from rabbit kidney epithelial cells. Amplification by rapid amplification of 5' cDNA ends indicated the kidney type 2 ryanodine receptor transcript extended >7000 base pairs from the stop codon and is therefore not homologous to the short RyR-1 transcript of approximately 2500 base pairs previously observed in rabbit brain. [3H]Ryanodine binding and immunoblot analysis with a type 2 ryanodine receptor-specific antibody demonstrated that the native type 2 ryanodine receptor protein is expressed in the kidney. These observations suggest that the type 2 ryanodine receptor isoform may play a functional role in regulating intracellular calcium homeostasis in non-excitable cells.
Highlights
Two distinct classes of calcium channel are known to be involved in the release of calcium ions from intracellular stores [1]
Rabbit Kidney RyR cDNA Clone, RK5—Examination of the optimally aligned rabbit RyR-1, -2, and -3 isoform nucleotide sequences revealed the highest sequence identity in the putative channel-forming transmembrane domain located at the carboxyl terminus of the protein
The observation of a single RyR-2 cDNA clone with a probe that can hybridize to all three isoforms suggests the specific expression of this RyR isoform in rabbit kidney
Summary
Materials—The rabbit kidney cortex cDNA library was obtained from Stratagene, the TriClone System from Invitrogen, and the 5ЈRACE system from Life Technologies, Inc. Isolation of Messenger RNA and RT-PCR—Isolation of mRNA, cDNA synthesis, and cloning of PCR products into the pCRII vector were all performed using the TriClone system (Invitrogen). RT-PCR to detect a ϳ700 –900-bp sequence within the region covered by rabbit kidney cDNA clone RK5 was performed using ϳ1 g of isolated mRNA and the appropriate pair of isoform-specific primers. First strand synthesis was primed with oligo(dT) and PCR performed with either RyR-2 (PR1 and PR2), RyR-1 (PR3 and PR4), or RyR-3 (PR5 and PR6) isoform-specific oligonucleotide primers. RyR-2 Antiserum Production and Purification—A 24-amino acid peptide corresponding to residues 4676 – 4699 of the rabbit RyR-2, present within the region covered by rabbit kidney clone RK5, was synthesized on an Applied Biosystems model 430A peptide synthesizer and coupled via an additional COOH-terminal cysteine to keyhole limpet hemocyanin using the bifunctional reagent sulfo-SMCC (Pierce). Nonspecific binding was assessed in the presence of 100 M unlabeled ryanodine and deducted from the final calculation of specific ryanodine binding
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