Non-canonical regulation of the plasma membrane copper transporter CTR1 through modulation of membrane mechanical properties

We describe a non-canonical, membrane receptor-like regulation of the human copper transporter CTR1 in response to copper stimuli. CTR1 is the sole high-affinity trimeric plasma-membrane copper-importing channel that self-regulates by undergoing endocytosis to limit copper uptake. We observed that preceding copper-induced endocytosis, CTR1 forms clusters on the plasma membrane, a phenomenon that is typically observed in membrane receptors. We deciphered the mechanism of CTR1 clustering and studied its ramifications on plasma membrane physical properties harboring the clusters that favors endocytosis. Membrane tension and fluctuation are fundamental regulators of pre- and post-endocytic events. Using coarse-grain MD simulation and coupled Interference Reflection Microscopy-Total Internal Reflection Fluorescence Microscopy we demonstrated that CTR1 clusters induce positive membrane curvature, increase in local membrane tension and decrease in local membrane fluctuation; alterations that favors formation of endocytic pits. Clustering is facilitated by copper-sequestering Methionine rich extracellular amino-terminus of CTR1. MD-simulations and IRM-TIRF imaging revealed that CTR1 clustering is facilitated by membrane cholesterol, depletion of which delays CTR1 endocytosis. CTR1 clustering promotes clathrin-coated pit formation that engages recruitment of adapter protein AP-2. To summarize, we report a hitherto unknown 'pre-endocytic' 'receptor-like' phenomenon of ligand-induced clustering of a metal channel, that in-turn regulates self-endocytosis by modulating membrane properties.