Proteomic Analysis Identifies MaxiK Channel Intracellular Partners from Human Coronary Artery

Abstract

The large conductance voltage and calcium-activated potassium (MaxiK) channel, a key modulator of vascular tone, interacts with several types of proteins (e.g. regulatory subunits, G-protein coupled receptors, signaling kinases). In this study, we searched for MaxiK channel partners in human coronary artery using a proteomic approach. A MaxiK polyclonal antibody and 3 coronary arteries from explanted hearts were used to immunoprecipitate MaxiK. IgG was used as negative control. Immunoprecipitated proteins were separated by 1-D SDS-PAGE; subsequently, gel bands were excised for trypsin digestion and LC/MS/MS analysis. As positive control, we searched for MaxiK peptides and found 7 peptides (93 amino acids) from MaxiK C-terminus. No peptides of MaxiK were found in IgG negative controls (n=3). Various groups of intracellular proteins that form complex with MaxiK channels were identified. A group of proteins are cytoskeleton proteins, which include coronin-1A, PACN2, tubulin beta-3 chain and beta-centractin. PACN2 plays a role in the formation of flask-shaped caveolae at the plasma membrane and coronin-1A is involved in cell locomotion. Another group of proteins are linked to transcription and mRNA-splicing. These proteins are TFIID subunit 9B, DDX1, and pre-mRNA-splicing factor SYF1. Mitochondria metabolic proteins were also identified as MaxiK partners including ADP/ATP translocase and PCCB (Propionyl-CoA carboxylase beta chain). In addition, we found that Hsp90 co-chaperone CDC37 (CDC37), WASL and WASL-interacting protein family member 1 (WIPF1) associate with MaxiK. These three proteins also form complex with Hsp90. WASL and WIPF1 are involved in the formation of actin filament and cell motility. Hsp90 binds to WASL regulating actin polymerization. CDC37 and Hsp90 assemble to stabilize protein kinases. In summary, proteomic analysis identified intracellular proteins forming complex with MaxiK channel and the underlying functions for these interactions need further investigation. Supported by NIH.