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Expression of cyclic-nucleotide-sensitive and -insensitive isoforms of the plasma membrane Ca2+ pump in smooth muscle
1Department of Biomedical Sciences, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada.
Abstract:
cDNA clones encoding the plasma membrane Ca2+ pump isoform PMCA1 were obtained from rabbit stomach smooth muscle. The PMCA1 gene has a 154 base exon which can be alternatively spliced. In splices containing 0, 87 or 114 bases of this exon, the mRNA downstream from this position encodes a protein containing the peptide sequence Lys-Arg-Asn-Ser-Ser (KRNSS), which can be phosphorylated by cyclic-nucleotide-sensitive protein kinase. However, in those splices containing 154 bases, the mRNA encodes a protein that does not contain this sequence. The cDNA clone obtained in this study did not contain the latter exon, and thus it coded for KRNSS. The presence of the various splices of PMCA1 was determined in stomach smooth muscle and other tissues by reverse transcription followed by a polymerase chain reaction. Percentage of transcripts encoding the potentially cyclic-nucleotide-sensitive isoform in various tissues were as follows: liver, 100%; stomach mucosa, 100%; heart, 100%; stomach smooth muscle, 86%; aorta, 83%; brain, 55%. Thus brain was the only tissue which expressed a very high proportion of the isoform of PMCA1 that is insensitive to cyclic-nucleotide-dependent protein kinases.
Insights
Researchers identified alternative splicing in the plasma membrane Ca2+ pump (PMCA1) gene. Most tissues express a splice variant sensitive to cyclic-nucleotide-dependent protein kinases, except for the brain, which primarily expresses an insensitive isoform.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Physiology
Background:
- The plasma membrane Ca2+ pump (PMCA) is crucial for calcium homeostasis.
- PMCA1 is a key isoform involved in regulating intracellular calcium levels.
- Alternative splicing of PMCA1 can generate functionally distinct protein variants.
Purpose of the Study:
- To investigate the alternative splicing of the PMCA1 gene in rabbit stomach smooth muscle.
- To determine the expression patterns of different PMCA1 splice variants across various tissues.
- To identify PMCA1 isoforms potentially regulated by cyclic-nucleotide-sensitive protein kinases.
Main Methods:
- cDNA cloning of PMCA1 from rabbit stomach smooth muscle.
- Reverse transcription coupled with polymerase chain reaction (RT-PCR) to detect PMCA1 splice variants.
- Analysis of alternative exon inclusion in PMCA1 transcripts.
Main Results:
- A cDNA clone for rabbit stomach smooth muscle PMCA1 was isolated, encoding a splice variant with the KRNSS sequence.
- The KRNSS sequence, which is phosphorylatable by cyclic-nucleotide-sensitive protein kinase, is present in PMCA1 isoforms derived from splices including 0, 87, or 114 bases of a specific exon.
- Transcripts encoding the potentially cyclic-nucleotide-sensitive PMCA1 isoform were highly abundant in liver (100%), stomach mucosa (100%), and heart (100%), with lower proportions in stomach smooth muscle (86%), aorta (83%), and brain (55%).
Conclusions:
- The PMCA1 gene exhibits alternative splicing, leading to isoforms with and without a cyclic-nucleotide-sensitive phosphorylation site.
- Most examined tissues predominantly express the PMCA1 isoform containing the KRNSS phosphorylation motif.
- The brain uniquely shows a significant proportion of PMCA1 transcripts lacking this motif, suggesting tissue-specific regulation of calcium signaling pathways.