cDNA cloning reveals a tissue specific expression of alternatively spliced transcripts of the ryanodine receptor type 3 (RyR3) calcium release channel

160

171

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. Ca 2؉ release measurements using the fluorescence Ca 2؉ indicator fluo 3 revealed that the cloned RyR3 functioned as a caffeine-and ryanodine-sensitive Ca 2؉ Ryanodine receptors are a family of intracellular Ca 2ϩ release channels that were originally identified in the sarcoplasmic reticulum (SR) 1 of striated muscles. To date, three members of this family have been identified in mammalian tissues, namely the skeletal muscle (RyR1), the cardiac muscle (RyR2), and the brain (RyR3) ryanodine receptor. These proteins are the products of different genes and share 66 -70% amino acid sequence identity (1-5). Earlier studies using RNA blot analysis revealed that the expression patterns of these isoforms were very different (6). RyR1 was predominantly expressed in skeletal muscle, whereas RyR2 was mainly expressed in heart and brain. The expression of RyR3 was detected in smooth muscle tissues and certain regions of the brain. However, results of recent ribonuclease protection assay demonstrate that all three RyR isoforms are widely and differentially expressed (7). These studies also indicate that most tissues express more than one RyR isoform. For example, skeletal muscles express both RyR1 and RyR3, although RyR3 is expressed at a much lower level than that of RyR1.

Section: Discussionmentioning

confidence: 79%

Section: Rt-pcr Cloning Of the Rabbit Uterusmentioning

confidence: 99%

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

Chen

Ebisawa

et al. 1997

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171

“…Analysis of its mRNA showed that there were several tissuespecific alternative splicing variants of RyR3, especially between brain and peripheral tissues (11,21). These alternatively spliced variants might generate potential heterogeneity in the function of RyR3 among tissues.…”

Section: Ryanodine Receptor (Ryr)mentioning

confidence: 99%

Characterization of Type 3 Ryanodine Receptor (RyR3) of Sarcoplasmic Reticulum from Rabbit Skeletal Muscles

Murayama

Ogawa

1997

We investigated type 3 isoform (RyR3) of ryanodine receptor in rabbit skeletal muscles using an antibody specific for RyR3. By Western blot analysis and by immunoprecipitation, a single polypeptide for RyR3 was detected in sarcoplasmic reticulum vesicles from rabbit diaphragm but not in those from back muscle. The molecular mass was slightly smaller than that of RyR1, the major isoform in skeletal muscles. Each of RyR1 and RyR3 formed a homotetramer in rabbit diaphragm.

“…A third isoform named RyR3 has been found to be present at low levels in almost all tissues (11)(12)(13), but so far no convincing evidence for a preferential association with a specific cell type or function has been obtained.…”

mentioning

confidence: 99%

Expression of the Ryanodine Receptor Type 3 Calcium Release Channel during Development and Differentiation of Mammalian Skeletal Muscle Cells

Tarroni¹,

Rossi²,

Conti³

et al. 1997

Self Cite

In vertebrate skeletal muscles, the type 1 isoform of ryanodine receptor (RyR1) is essential in triggering contraction by releasing Ca 2؉ from the sarcoplasmic reticulum in response to plasma membrane depolarisation. Recently, the presence of another RyR isoform, RyR3, has been detected in mammalian skeletal muscle cells, raising the question of the eventual relevance of RyR3 for muscle cell physiology. The expression of RyR3 was investigated during differentiation of skeletal muscle cells. Using antibodies able to distinguish the different RyR isoforms and Western blot analysis, the RyR3 protein was detected in the microsomal fractions of differentiated skeletal muscle cells but not of undifferentiated cells. Accordingly, blocking muscle differentiation by the addition of either transforming growth factor-␤ or basic fibroblast growth factor prevented the expression of the RyR3 protein. In differentiated skeletal muscle cells, RyR3 was expressed independent of cell fusion and myotube formation. The expression of RyR3 was also investigated during development of the diaphragm muscle. The RyR3 content in the diaphragm muscle increased between the late stage of fetal development and the first postnatal days. However, at variance with RyR1, which reached maximum levels of expression 2-3 weeks after birth, the expression of RyR3 was found to be higher in the neonatal phase of the diaphragm muscle development (2-15 days after birth) than in the same muscle from adult mice. The differential content of RyR3 in adult skeletal muscles was found not to be mediated by neurotrophic factors or electrical activity. These findings indicate that RyR3 is preferentially expressed in differentiated skeletal muscle cells. In addition, during skeletal muscle development, its expression is regulated differently from that of RyR1.