title: Manipulating L-type calcium channels in cardiomyocytes using split-intein protein transsplicing.
abstract: Manipulating expression of large genes (>6 kb) in adult cardiomyocytes is challenging because these cells are only efficiently transduced by viral vectors with a 4-7 kb packaging capacity. This limitation impedes understanding structure-function mechanisms of important proteins in heart. L-type calcium channels (LTCCs) regulate diverse facets of cardiac physiology including excitation-contraction coupling, excitability, and gene expression. Many important questions about how LTCCs mediate such multidimensional signaling are best resolved by manipulating expression of the 6.6 kb pore-forming α1C-subunit in adult cardiomyocytes. Here, we use split-intein-mediated protein transsplicing to reconstitute LTCC α1C-subunit from two distinct halves, overcoming the difficulty of expressing full-length α1C in cardiomyocytes. Split-intein-tagged α1C fragments encoding dihydropyridine-resistant channels were incorporated into adenovirus and reconstituted in cardiomyocytes. Similar to endogenous LTCCs, recombinant channels targeted to dyads, triggered Ca(2+) transients, associated with caveolin-3, and supported β-adrenergic regulation of excitation-contraction coupling. This approach lowers a longstanding technical hurdle to manipulating large proteins in cardiomyocytes.
authors.affiliation: Department of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032.
authors.forename: Prakash
authors.initials: P
authors.lastname: Subramanyam
authors.forename: Donald D
authors.initials: DD
authors.lastname: Chang
authors.forename: Kun
authors.initials: K
authors.lastname: Fang
authors.forename: Wenjun
authors.initials: W
authors.lastname: Xie
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Marks
authors.forename: Henry M
authors.initials: HM
authors.lastname: Colecraft
mesh_terms: D000256:Adenoviridae; D000704:Analysis of Variance; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D047668:Inteins / Q000235:genetics*; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000235:genetics; D019154:Protein Splicing / Q000502:physiology*; D045663:Quantum Dots
chemical_list: D020746:Calcium Channels, L-Type
keywords: CaV1.2; gene transfer; protein splicing; ventricular myocytes