Protein-protein interactions are critically important for cellular functions, including regulation of ion channels. Ion channels are typically part of large macromolecular complexes that impact their function. These complexes have traditionally been elucidated via standard biochemical techniques including immunoprecipitation, pull-down assays and mass spectrometry. Recently, several methods have been developed to provide a more complete depiction of the microenvironment or "neighborhood" of proteins of interest. These new methods, which fall broadly under the category of proximity-dependent labeling techniques, aim to overcome the limitations imposed by antibody-based techniques and mass spectrometry. In this chapter, we describe the use of proximity labeling to elucidate the cardiac CaV1.2 macromolecular complex under basal conditions and after β-adrenergic stimulation. Using these methodologies, we have identified the mechanism underlying adrenergic stimulation of the Ca2+ current in the heart.
Keywords: APEX; Calcium channels; Interactome; Mass spectrometry; Proximity-labeling; Sympathetic nervous system.
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