Molecular basis of cardiac troponin T isoform heterogeneity in rabbit heart.

In the rabbit heart, multiple isoforms of cardiac troponin T (cTnT1 through cTnT5, from largest in size to smallest), a protein essential for calcium-regulated myofibrillar ATPase activity, have been identified, and a correlation has been found between these isoforms and myofilament sensitivity to calcium. We have sought to establish the molecular basis of this diversity. Restriction-digest analysis of genomic DNA has indicated that the rabbit cTnT gene is a single-copy gene. cTnT cDNA clones were isolated from cDNA libraries, yielding a consensus sequence for the protein. Newborn rabbit heart cDNAs, obtained using the reverse-transcriptase polymerase chain reaction (RT-PCR), were amplified using primers derived from this cDNA. Three full-length cDNAs that differed by the inclusion or exclusion of three short nucleotide sequences within the cDNAs were obtained. Amplification in the 5' half of the cDNAs confirmed that multiple cTnT products arose because of the variable inclusion of an 18- and a 30-nt sequence. The 30-nt sequence has homology with previously described alternatively spliced exons in rat and chicken cTnT, whereas the 18-nt sequence has not been described previously. RT-PCR in the 3' half of the cDNAs confirmed an additional region of heterogeneity: the presence, in part or in full, or absence of a 9-nt region, which matches the alternatively spliced exon 12 described for rat cTnT. In vitro transcription and translation of four cDNA clones containing both the 18- and 30-nt sequences, the 30-nt sequence, the 18-nt sequence, or neither generated protein isoforms that comigrated with cTnT1, cTnT2, cTnT3, and cTnT4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A two-motif isoform of the major calcium channel subunit in skeletal muscle.

Evidence is presented that two isoforms of the voltage-dependent, dihydropyridine-sensitive calcium channel alpha 1 subunit are present in newborn and adult skeletal muscle and that expression of these isoforms is developmentally regulated. A voltage-dependent calcium channel alpha 1 cDNA from newborn muscle was cloned and found to be identical to that published from the adult, except that it was 2 kb shorter owing to an internal deletion. Nucleotide sequences, Northern blots, reverse-transcriptase PCR experiments, and sequencing of the PCR product confirmed that a segment corresponding to the inner two repeats of the structural prototype four homologous motifs is missing from the immature isoform. Immunological studies using antisera raised against synthetic peptides that correspond to sequences in the two isoforms show that the abbreviated transcript is predominant in newborn muscle, whereas the four-repeat isoform is the major species in the adult.