BPS2026 – Specific interaction of cholesterol inhibits the transport activity of the MgtE magnesium channel

Despite Mg 2+ being the most abundant divalent cation in the cell, the Mg 2+ transport mechanisms and homeostasis are poorly understood. It has been shown that the prokaryotic MgtE Mg 2+ channel can functionally compensate TRPM7 deficiency in B cells, which prompted us to investigate whether MgtE can be influenced by the membrane composition of the mammalian cell. Our sequence analysis results of MgtE reveals the presence of cholesterol recognition motifs (CRAC and CARC), suggesting the possibility of direct cholesterol binding to MgtE. Importantly, utilizing the fluorescence-based liposome flux assay, we demonstrate that cholesterol, an important mammalian cell membrane lipid implicated in health and disease, inhibits the Mg 2+ transport activity of MgtE by ∼40% even at low concentration. We also show that cholesterol binds to both closed and open states of MgtE with an apparent K d of ∼12–16 μM, probably at one of the CARC motifs in the transmembrane region. This is confirmed by the fluorescent analog of cholesterol, 25-NBD cholesterol, in both micelles and membranes. Interestingly, experiments with other sterols suggest a specific interaction of cholesterol with MgtE. Gating-related structural dynamics results utilizing Trp fluorescence of MgtE show that increasing membrane cholesterol decreases the EC 50 values associated with the changes in the emission maximum, suggesting that cholesterol might facilitate the channel closing, which is in agreement with our functional studies. Further, independent of the functional state of MgtE, we observe a significant reduction in the “average” rotational dynamics of Trp residues, which is accompanied by increased hydration dynamics in cholesterol-containing membranes. Overall, our work constitutes the first report that demonstrates the inhibitory effect of cholesterol in Mg 2+ channels in general, and MgtE in particular, by direct mode of binding and are relevant for understanding lipid-driven ion channel regulation.