Phospholamban – Characterization of the Potential Interaction Partner SLMAP
by Nina Zaremba
Date of Examination:2024-04-02
Date of issue:2024-10-10
Advisor:Prof. Dr. Stephan E. Lehnart
Referee:Prof. Dr. Stephan E. Lehnart
Referee:Prof. Dr. Peter Rehling
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Abstract
English
The identification of novel PLN interaction partners is of high importance to understand how it is regulated on a molecular level and to develop new therapeutic strategies for cardiomyopathy. SLMAP, a novel putative PLN interaction partner was identified initially with complexome profiling of cardiac membrane from mouse tissue. It was shown that SERCA2a, PLN and SLMAP may occur in potential super complexes. To characterize the direct interaction of PLN and SLMAP, co immunoprecipitation experiments in transfected HEK293A cells and a yeast two-hybrid were performed. The direct putative interaction of SLMAP and PLN could not be confirmed with these approaches. To further test, if SLMAP interacts indirectly with PLN mediated by SERCA2a, it was tried to pull down SLMAP from ventricular cardiac mouse tissue with purified PLN and co-immunoprecipitation. Therefore, experiments in transfected SLMAP /- knockout hiPSC derived cardiomyocytes were performed. The indirect interaction of SLMAP and PLN was not confirmed, and the starting hypothesis that PLN is a potential SLMAP interaction partner was rejected. Since SLMAP gene variants were found in patients with Brugada syndrome, the electrophysiological function, namely sodium current and the action potential, of SLMAP was analyzed. With automated robotic patch clamp, it was shown that the sodium current is reduced in the SLMAP-/- knockout hiPSC derived cardiomyocytes compared to the wildtype. Furthermore, the action potential duration was significantly prolonged. Together, this points towards an important role of SLMAP in cardiomyocytes for action potential regulation under physiological and pathophysiological conditions. Furthermore, it was biochemically analyzed if SLMAP influences the sodium currents at the plasma membrane via transcriptome and proteome studies. Sodium handling proteins, such as Na¬v1.5 and Na,K-ATPase, were significantly reduced in the SLMAP-/- hiPSC derived cardiomyocytes compared to the wildtype. However, no changes could be detected on the mRNA level. Due to these findings, it was hypothesized that SLMAP has an influence on the protein trafficking of the sodium handling protein complexes. To address this question, protein trafficking was analyzed via a surface biotinylation assay. Thereby, it was shown that Na¬v1.5 and Na,K-ATPase levels are reduced in the plasma membrane in hiPSC derived cardiomyocytes. To identify hypothetical interaction partner of SLMAP, the complexome of SLMAP was analyzed in hiPSC derived cardiomyocytes. Here, members of the TMED family, which are involved in the protein trafficking to the plasma membrane, were identified as hypothetical interaction partners.
Keywords: PLN; SLMAP; Phospholamban; Calcium release unit