Stereospecific Properties and Intracellular Transport\nof Novel Intrinsically Fluorescent Neurosteroids

Allopregnanolone\n(AlloP) is an example of neuroactive steroids\n(NAS), which is a potent allosteric activator of the γ-aminobutyric\nacid A (GABA<sub><i>A</i></sub>) receptor. The mechanisms\nunderlying the biological activity of AlloP and other NAS are only\npartially understood. Here, we present intrinsically fluorescent analogs\nof AlloP (MQ-323) and its 3β-epimer, epi-allopregnanolone (E-AlloP)\n(YX-11), and show, by a combination of spectroscopic and computational\nstudies, that these analogs mimic the membrane properties of AlloP\nand E-AlloP very well. We found stereospecific differences in the\norientation and dynamics of the NAS as well as in their impact on\nmembrane permeability. However, all NAS are unable to condense the\nlipid bilayer, in stark contrast to cholesterol. Using Förster\nresonance energy transfer (FRET) and electrophysiological measurements,\nwe show that MQ-323 but not YX-11 binds at the intersubunit site of\nthe ELICα<sub>1</sub>GABA<sub><i>A</i></sub> receptor\nand potentiates GABA-induced receptor currents. In aqueous solvents,\nYX-11 forms aggregates at much lower concentrations than MQ-323, and\nloading both analogs onto cyclodextrin allows for their uptake by\nhuman astrocytes, where they become enriched in lipid droplets (LDs),\nas shown by quantitative fluorescence microscopy. Trafficking of the\nNAS analogs is stereospecific, as uptake and lipid droplet targeting\nis more pronounced for YX-11 compared to MQ-323. In summary, we present\nnovel minimally modified analogs of AlloP and E-AlloP, which enable\nus to reveal stereospecific membrane properties, allosteric receptor\nactivation, and intracellular transport of these neurosteroids. Our\nfluorescence design strategy will be very useful for the analysis\nof other NAS in the future.