부산대학교 도서관

Introduction: The Ca2+ channel CaV1.2 is an essential part of excitation contraction coupling, neurotransmission and vascular tone. Identifying protein partners of CaV1.2 is critical to make mechanistic insights into these fundamental processes. Prior CaV1.2 proximity proteomes used APEX labeling, which labels other amino acids, introduces oxidative stress and is catalyzed ex vivo.Hypothesis: α1C-TurboID knockin rats can generate tissue specific interactomes in vivo, in cells with intact cell and matrix contacts, which may differ substantially from interactomes made from ex vivo peroxidase catalyzed labeling.Methods: CRISPR/Cas9 homology directed repair (HDR) was applied to rat spermatogonial stem cells with a targeting vector that includes cacna1c exon 44, coding the C-terminus of CaV1.2, and birA*, which codes TurboID, cloned 3’ to the stop codon (Fig A). Figures were made with Biorender.Results: Streptavidin blotting of brain and heart lysates demonstrated robust biotinylation in knockin rats (Fig B). In paced heart myocytes, α1C-TurboID produces Ca2+ transients and responds appropriately to adrenergic stimulation (Fig C), indicating proper localization to the sarcolemma. Expression of α1C-TurboID does not alter systolic function (Fig D). Mass spectrometry of heart lysates affinity purified on streptavidin beads identified 629 proteins, of which 454 were significantly enriched in knockin rats. These include known sarcolemmal proteins and interactors of CaV1.2 (Fig E), which was confirmed by antibody detection (Fig F). 73% of these 454 proteins were not significantly enriched in the APEX-α1C proteome.Conclusion: TurboID fusion to CaV1.2 generates a reliable and substantially different proteome compared to other techniques. Successful in vivo characterization of the channel interactome confirms α1C-TurboID rats will be a powerful tool for identifying changing protein networks across developmental stages, tissues, and disease models.