Mutually exclusive exon splicing of the cardiac calcium channel alpha 1 subunit gene generates developmentally regulated isoforms in the rat heart.

Abstract

Several clones were isolated from a rat genonic library in order to further characterize a region of variability within the third membrane-spanning region of the fourth motif (IVS3) of the L-type voltage-dependent calcium channel. We report here that this diversity arises from alternative splicing of a primary transcript containing a single pair of adjacent exons each encoding a unique sequence for the IVS3 region. Definitive proof of a mutually exclusive splicing mechanism was obtained by genomic mapping of flanking upstream and downstream exons and by extensive sequence analysis of the relevant exon/intron boundaries. S1 nuclease protection experiments revealed that both variant forms of the IVS3 were equally expressed in newborn and fetal rat heart, whereas only a single isoform predominated in adult rat heart. The results demonstrate the existence of an important developmentally regulated switch mediated by alternatively spliced exons in cardiac tissue at a time when major changes in excitation occur.Voltage-dependent calcium channels (VDCCs) are a diverse class of molecules found in all excitable cells (1-4). These channels are essential for many cellular functions such as muscle contraction, propagation of action potentials, maintenance of electrical activity, excitation-secretion coupling, and neurotransmitter regulation (2, 3). The L-type VDCC from rabbit skeletal muscle has been purified and extensively characterized biochemically. The channel exists as a pentameric macromolecule consisting of a1, a<-8, 3,, and y subunits, all of which have been cloned and sequenced (4-7).Functionally, the a1 subunit alone can act as a VDCC (8, 9) and contains binding sites for calcium antagonists (10, 11), although native gating characteristics are lacking (9). The a2-8 and, in some cases, P subunits significantly increase current amplitude displayed by expressed a1 subunits (8,(12)(13)(14). More importantly, the 83 subunit modulates gating properties of the expressed skeletal muscle a1 subunit in mammalian L-cells (14). In Xenopus oocytes, the expression of the skeletal muscle 'y subunit determines the inactivation properties of the coexpressed cardiac a1 subunit (15). Thus, a normal functioning calcium channel probably requires the presence of all the other subunits.The a1 subunits from rabbit cardiac muscle and lung (8, 13, 16), rat aorta (17), and carp skeletal muscle (18) have been cloned and their primary structures have been described.Like the skeletal muscle a1 subunit, the cardiac and smooth muscle isoforms are large hydrophobic polypeptides comprising four repeating motifs (I-IV), each containing six putative transmembrane segments (S1-S6), flanked by Nand C-terminal cytoplasmic domains. Sequence analysis suggests that the cardiac and smooth muscle a1 subunits are encoded by the same gene, which is distinct from that encoding the skeletal muscle channel (9,12,17).In the cloning of the rat aorta a1 subunit, we described the existence of two cDNA species that were identical in all but the third membrane...

The landscape of human mutually exclusive splicing

“…This modulation suggests crucial spatio‐temporal functional roles for MXEs and can in many cases not be observed at the gene level, as gene counts can remain largely invariant. A well‐known case for similar expression of MXEs in newborn heart but expression of only one MXE variant in adult heart is the ion channel CACNA1C (Diebold et al , 1992), an example for the switch of expression are the MXEs of the SLC25A3 gene (Wang et al , 2008). We surmise that the observed specificity in combination with a generally lower expression could also explain the discovery of 654 (358) novel exons that have so far eluded annotation efforts (Fig 1A, Appendix Fig S23).…”

Tissue-specific expression of calcium channels

“…No major differences have been observed in basic electrophysiologic and pharmacologic properties of the two isoforms, including the amplitude of inward current, steady-state activation and inactivation, and DHP sensitivity (Welling et al 1992b). However, Northern-blot and polymerase chain reaction (PCR) analyses show that both splice variants are differentially expressed in heart and smooth muscle ) and during cardiac development (Diebold et al 1992).…”

Molecular cloning of a murine N‐type calcium channel α1 subunit

“…2B). Alternate splicing of L-type Ca*' channels was shown to be tissue-specific and development-dependent [23] and it might hence be a mechanism to fine tune Ca'+ channel function between tissues and during development.…”