Gating-related Structural Dynamics of the MgtE Magnesium Channel in Membrane-Mimetics Utilizing Site-Directed...

Magnesium is the most abundant divalent cation present in the cell, and an abnormal Mg²⁺ homeostasis is associated with several diseases in humans. However, among ion channels, the mechanisms of intracellular regulation and transport of Mg²⁺ are poorly understood. MgtE is a homodimeric Mg²⁺-selective channel and is negatively regulated by high intracellular Mg²⁺ concentration where the cytoplasmic domain of MgtE acts as a Mg²⁺ sensor. Most of the previous biophysical studies on MgtE have been carried out in detergent micelles and the information regarding gating-related structural dynamics of MgtE in physiologically-relevant membrane environment is scarce. In this work, we monitored the changes in gating-related structural dynamics, hydration dynamics and conformational heterogeneity of MgtE in micelles and membranes using the intrinsic site-directed Trp fluorescence. For this purpose, we have engineered six single-Trp mutants in the functional Trp-less background of MgtE to obtain site-specific information on the gating-related structural dynamics of MgtE in membrane-mimetic systems. Our results indicate that Mg²⁺-induced gating might involve the possibility of a 'conformational wave' from the cytosolic N-domain to transmembrane domain of MgtE. Although MgtE is responsive to Mg²⁺-induced gating in both micelles and membranes, the organization and dynamics of MgtE is substantially altered in physiologically important phospholipid membranes compared to micelles. This is accompanied by significant changes in hydration dynamics and conformational heterogeneity. Overall, our results highlight the importance of lipid-protein interactions and are relevant for understanding gating mechanism of magnesium channels in general, and MgtE in particular.