Ethylene is an important regulator of plant growth. We identified an Arabidopsis mutant, responsive-to-antagonist1 (ran1), that shows ethylene phenotypes in response to treatment with trans-cyclooctene, a potent receptor antagonist. Genetic epistasis studies revealed an early requirement for RAN1 in the ethylene pathway. RAN1 was cloned and found to encode a protein with similarity to copper-transporting P-type ATPases, including the human Menkes/Wilson proteins and yeast Ccc2p. Expression of RAN1 complemented the defects of a ccc2delta mutant, demonstrating its function as a copper transporter. Transgenic CaMV 35S::RAN1 plants showed constitutive expression of ethylene responses, due to cosuppression of RAN1. These results provide an in planta demonstration that ethylene signaling requires copper and reveal that RAN1 acts by delivering copper to create functional hormone receptors.

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Background: Propofol binding to GABA A R sites of uncertain location potentiates receptor function and produces anesthesia in vivo. Results: A photoreactive propofol analog identifies propofol-binding sites in ␣1␤3 GABA A Rs. Conclusion: Propofol binds to each class of intersubunit sites in the GABA A R transmembrane domain. Significance: This study demonstrates that propofol binds to the same sites in a GABA A R as etomidate and barbiturates.

Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABA A receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABA A receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABA A receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels.Most general anesthetics alter the activity of ligand-gated ion channels, and electrophysiology, photolabeling, and transgenic animal experiments imply that this effect contributes to the mechanism of anesthesia (1-9). Although the molecular mechanism for this effect is not yet clear, photolabeling studies indicate that anesthetics bind within the transmembrane regions of Cys-loop ligand-gated ion channels such as the nicotinic acetylcholine and the ␥-aminobutyric acid (GABA) 2 type A receptors (2, 9 -11). Practical difficulties associated with overexpression, purification, and crystallization of ion channels have thus far stymied investigation of the structural and energetic bases underlying anesthetic recognition. However, general anesthetics also bind specifically to sites in soluble proteins, including firefly luciferase, human serum albumin (HSA), and horse spleen apoferritin (HSAF) (12)(13)(14), and x-ray crystal structures have been determined for complexes of these proteins with several general anesthetics (14 -16). In particular, HSAF is an attractive model for studying anesthetic-protein interactions because it has the highest affinity for anesthetics of any protein studied to date, has a unique anesthetic binding site, and is a multimer of 4-helix bundles, much like the putative anesthetic binding regions in ligand-gated channels. In addition, apoferritin is commercially available and crystallizes readily. Most importantly, however, the affinity of HSAF f...

Propofol is the most commonly used sedative-hypnotic drug for noxious procedures, yet the molecular targets underlying either its beneficial or toxic effects remain uncertain. In order to determine targets and thereby mechanisms of propofol, we have synthesized a photoactivateable analogue by substituting an alkyldiazirinyl moiety for one of the isopropyl arms but in the meta position. m-Azipropofol retains the physical, biochemical, GABAA receptor modulatory, and in vivo activity of propofol and photoadducts to amino acid residues in known propofol binding sites in natural proteins. Using either mass spectrometry or radiolabeling, this reagent may be used to reveal sites and targets that underlie the mechanism of both the desirable and undesirable actions of this important clinical compound.