BPS2026 – Elucidation of the membrane binding properties of Junctophilin

Various signaling events require proximity between the plasma membrane (PM) and the membrane of the endoplasmic (ER) or sarcoplasmic reticulum (SR). This is particularly the case in excitation-contraction coupling (EC-coupling) of cardiac and skeletal muscle, where such junctions require physical proximity between Ryanodine receptors and L-type calcium channels. Junctophilins (JPHs) have been found to promote ER/PM and SR/PM junctions directly, and can also interact with ion channels involved in the process. However, how exactly JPH associates with membranes has remained enigmatic. Membrane occupation and recognition nexus (MORN) motifs of JPHs have previously been proposed to mediate PM interactions, but the role of MORN repeats in binding membranes has been questioned. Using coarse-grain simulations and nanodisc-based binding assays, we identify a conserved cluster of positively charged JPH residues that may mediate binding to PI (4,5) P2. Six substitutions of these basic residues obliterate a mutant JPH’s ability to bind nanodiscs containing 5% PI (4,5) P2. This finding is observed for both muscle and neuronal JPH isoforms. Microscopy and FRET assays indicate that the mutant fails to robustly localize at the PM in mammalian cells. Our results show that the MORN repeats themselves should not be considered as membrane binding motifs, but JPH has obtained membrane binding through a specific cluster of positive charges.