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

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Abstract
Multiple Ca2+channel β-subunit (Cavβ) 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 Cavβ 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 Cavβ genes, we designed a systematic RT-PCR strategy to screen human and canine left ventricular myocardial samples for all known Cavβ isoforms. A total of 18 different Cavβ isoforms were detected in both canine and human ventricles including splice variants from all four Cavβ genes. Six of these isoforms have not previously been described. Western blots of ventricular membrane fractions and immunocytochemistry demonstrated that all four Cavβ subunit genes are expressed at the protein level, and the Cavβ subunits show differential subcellular localization with Cavβ1b, Cavβ2, and Cavβ3predominantly localized to the T-tubule sarcolemma, whereas Cavβ1aand Cavβ4are more prevalent in the surface sarcolemma. Coexpression of the novel Cavβ2csubunits (Cavβ2cN1, Cavβ2cN2, Cavβ2cN4) with the pore-forming α1C(Cav1.2) and Cavα2δ subunits in HEK 293 cells resulted in a marked increase in ionic current and Cavβ2cisoform-specific modulation of voltage-dependent activation. These results demonstrate a previously unappreciated heterogeneity of Cavβ subunit isoforms in ventricular myocytes and suggest the presence of different subcellular populations of Ca2+channels with distinct functional properties.