Engineering the Composition of L-Type (Cav1.2) Channels in Heart Cells using Split Intein-Mediated Protein Trans-Splicing

Abstract

Ca2+ influx through L-type (CaV1.2) channels in heart regulates excitation-contraction (EC) coupling, action potential duration, and gene expression. This versatility of L-type channels in heart is hypothesized to be mediated in part by differential sub-cellular compartmentalization and hormonal modulation of distinct CaV1.2 channel pools in cardiac myocytes. Such prevailing hypotheses are best tested directly in cardiac myocytes, since their unique cyto-architecture and signaling environment cannot be replicated in heterologous cells. There are two major hurdles: first, the large size of pore-forming α1C subunits exceeds the packaging capacity of viral vectors, necessary to express exogenous CaV1.2 channels in adult cardiac myocytes; second, endogenous channels are a source of confounding contaminating signals. To overcome these limitations, we focused on functionally reconstituting two separately expressed moieties of the channel using a novel split-intein-mediated protein splicing approach. In HEK 293 cells, split-intein fragments were trans spliced to generate full-length α1C as determined by Western blot. When co-expressed with auxiliary β subunits, trans-spliced α1C trafficked normally to the cell surface and yielded robust whole-cell currents (ICa). The split-intein fragments were readily incorporated into adenoviral vectors which when used to infect adult myocytes yielded trans-spliced α1C that was detected at the cell surface. To isolate exogenous ICa, we introduced mutations that reduced dihydropyridine sensitivity of trans-spliced α1C (α1CDHP-). In the presence of 10 μM nifedipine, myocytes expressing trans-spliced α1CDHP- yielded significantly larger currents than controls expressing trans-spliced α1C. The results demonstrate a novel approach to robustly engineer the pore-forming α1C subunit composition of CaV1.2 channels in cardiac myocytes, removing a longstanding technical obstacle to L-type channel structure-function studies in heart.