Molecular heterogeneity of calcium channel β-subunits in canine and human heart: evidence for differential subcellular localization

Abstract

Multiple Ca2+ channel beta-subunit (Ca(v)beta) isoforms are known to differentially regulate the functional properties and membrane trafficking of high-voltage-activated Ca2+ channels, but the precise isoform expression pattern of Ca(v)beta subunits in ventricular muscle has not been fully characterized. Using sequence data from the Human Genome Project to define the intron/exon structure of the four known Ca(v)beta genes, we designed a systematic RT-PCR strategy to screen human and canine left ventricular myocardial samples for all known Ca(v)beta isoforms. A total of 18 different Ca(v)beta isoforms were detected in both canine and human ventricles including splice variants from all four Ca(v)beta genes. Six of these isoforms have not previously been described. Western blots of ventricular membrane fractions and immunocytochemistry demonstrated that all four Ca(v)beta subunit genes are expressed at the protein level, and the Ca(v)beta subunits show differential subcellular localization with Ca(v)beta1b, Ca(v)beta2, and Ca(v)beta3 predominantly localized to the T-tubule sarcolemma, whereas Ca(v)beta1a and Ca(v)beta4 are more prevalent in the surface sarcolemma. Coexpression of the novel Ca(v)beta2c subunits (Ca(v)beta(2cN1), Ca(v)beta(2cN2), Ca(v)beta(2cN4)) with the pore-forming alpha1C (Ca(v)1.2) and Ca(v)alpha2delta subunits in HEK 293 cells resulted in a marked increase in ionic current and Ca(v)beta2c isoform-specific modulation of voltage-dependent activation. These results demonstrate a previously unappreciated heterogeneity of Ca(v)beta subunit isoforms in ventricular myocytes and suggest the presence of different subcellular populations of Ca2+ channels with distinct functional properties.

Diversity and Developmental Expression of L-type Calcium Channel β2 Proteins and Their Influence on Calcium Current in Murine Heart

“…Whereas numerous studies described cloning of Ca 2ϩ channel subunits and Ca 2ϩ channel composition in the human, guinea pig, and rat heart (5,7,25,29,39,40,41) the molecular "make-up" of the mouse cardiac LTCC is much less defined, although several mouse lines have been created with Ca 2ϩ channel subunit transgenes as preclinical models for heart diseases (22,23,24,42,43).…”

“…Most groups (29,30,37,44) used strategies aimed at the specific amplification of a DNA fragment to obtain CaV␤2 variants, which is critically determined by the primer pair used for PCR. In contrast, the screening of cDNA libraries represents a rather unbiased approach to identify which mRNA is expressed.…”

“…The six 5Ј exons 1A, 1B, 2A, 2B, 2C, and 2D (nomenclature according to the human Cacnb2 gene by Foell et al (29)) encode for alternate V1 regions and are scattered among the 5Ј ϳ270 kbp of the gene (Fig. 2A).…”

“…2, A and B). According to the nomenclature of Foell et al (29), these clones are of the CaV␤2a type (a for the A-type exon 7). Differences were only observed within the V1 domain which gave rise to N termini of type N1 (exons 1A plus 2A), N3 (exon 2B), N4 (exon 2C), and N5 (exon 2D) (Fig.…”

“…22,23,24) carrying a rat CaV␤2 splice variant ("rat CaV␤2a") (25) expressed in rat and rabbit brain (26), but not in rabbit heart (26), have only escalated this requirement, because it has never been shown that the mouse orthologue of this variant is endogenously expressed in the mouse heart. So far, five CaV␤2 variants varying only in the V1 domain have been identified from different species (25,27,28) and in human heart these variants have been obtained mainly by RT-PCR approaches (29,30). In contrast, there is little information on the CaV␤ proteins present in mouse heart, their respective splice variants, and expression ratios.…”