Research Gallery > Project 4: Fluorescent Anesthetics: Screening, In Vivo Imaging, Mechanistic Studies
 
This project is principally concerned with defining structure-binding relationships of natural anesthetic binding proteins, and discovering novel anesthetic targets and ligands.  This work is being conducted in collaboration with Prof. Roderic Eckenhoff, an anesthesiologist at the UPenn Medical School.  The putative neuronal targets of anesthetics, the ligand-gated, cysteine-loop receptor/ion channels, have not yielded to high-resolution structural characterization, nor are they sufficiently plentiful to conduct biochemical binding experiments. The GABAA receptor is potentiated by anesthetics by an unknown molecular mechanism.

Horse spleen apoferritin (HSAF, iron removed) has yielded high-resolution structures of the anesthetic complex.  These structures represent the highest resolution data for an anesthetic protein complex yet deposited in the PDB (1zx1), and the only dataset for isoflurane (1xz3).  The anesthetic binding site is at an intersubunit, interhelical cavity. Thus, ferritin exhibits similarities to the superfamily of ligand-gated channels (Figure 9).

Figure 9

Figure 9. Conservation of secondary, tertiary, and quaternary structures in anesthetic binding.  Panel (a) shows the ferritin homodimer in which the anesthetic pocket is occupied (in this case by propofol, shown in red). Panel (b) shows the transmembrane region of the homology model for the GABAA receptor; an alpha/beta heterodimer is shown (alpha yellow, beta blue).  Residues that have been shown by mutagenesis to modulate anesthetic activity are green, and residues photolabeled by azietomidate are red.
We have recently discovered that a fluorescent probe binds HSAF with high affinity (Kd = 150 μM) and showed that it is a useful anesthetic probe because of its solvachromatic fluorescence properties, small molecular volume, and ability to induce anesthesia in Xenopus and zebrafish. We can quantify isoflurane binding within the ferritin “anesthetic binding cavity” by measuring the decrease in the probe's fluorescence upon displacement.  We have begun using HSAF as a screening assay for identifying new anesthetic candidates in large molecular libraries.  The fluorophore appears to be the first fluorescent general anesthetic molecule, where the dye can be soaked into animals and is readily excited at 488 nm, and observed with a fluorescein emission filter (Figure 10).  Most interestingly, the molecule is observed to be localized in the CNS of a living tadpole. Near-term goals are to conduct fluorescence screening assays, perform high-resolution imaging in neuronal tissues and animal models, and exploit the fluorescence of the fluorophore to perform pull-down assays and isolate molecular targets of this anesthetic molecule. 

Figure 10

Figure 10. Confocal micrograph of living tadpole that was soaked in 33 μM fluorophore solution.