Identification of a General Anesthetic Binding Site in the Diacylglycerol-binding Domain of Protein Kinase Cδ

Abstract

Protein kinase C (PKC) is an important signal transduction protein that has been proposed to interact with general anesthetics at its cysteine-rich diacylglycerol/ phorbol ester-binding domain C1, a tandem repeat of C1A and C1B subdomains. To test this hypothesis, we expressed, purified, and characterized the high affinity phorbol-binding subdomain, C1B, of mouse protein kinase C␦, and studied its interaction with general anesthetic alcohols. When the fluorescent phorbol ester, sapintoxin-D, bound to PKC␦ C1B in buffer at a molar ratio of 1:2, its fluorescence emission maximum, max , shifted from 437 to 425 nm. The general anesthetic alcohols, butanol and octanol, further shifted max of the PKC␦ C1B-bound sapintoxin-D in a concentration-dependent, saturable manner to ϳ415 nm, suggesting that alcohols interact at a discrete allosteric binding site. To identify this site, PKC␦ C1B was photolabeled with three photoactivable diazirine alcohol analogs, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry showed photoincorporation of all three alcohols in PKC␦ C1B at a stoichiometry of 1:1 in the labeled fraction. The photolabeled PKC␦ C1B was subjected to tryptic digest, the fragments were separated by online chromatography and sequenced by mass spectrometry. Each azialcohol photoincorporated at Tyr-236. Inspection of the known structure of PKC␦ C1B shows that this residue is situated adjacent to the phorbol ester binding pocket, and within ϳ10 Å of the bound phorbol ester. The present results provide direct evidence for an allosteric anesthetic site on protein kinase C.

Identification of a GABAAReceptor Anesthetic Binding Site at Subunit Interfaces by Photolabeling with an Etomidate Analog

“…␣Met-236 and ␤Met-286 were labeled at similar levels (330 and 120 cpm/pmol) and with similar pharmacological specificity; i.e., enhancement by GABA and inhibition by ϳ90% in the presence of 200 M etomidate, consistent with the IC 50 value of 30 M seen for etomidate inhibition at the subunit level. In addition, azietomidate and other aliphatic diazirines preferentially label reactive side chains (Glu, Asp, and Tyr) in the nAChR and in soluble proteins (Pratt et al, 2000;Das et al, 2004;Ziebell et al, 2004). The lack of labeling of those side chains in ␣M1 (Tyr-225 and Tyr-231) and ␤M3 (Asp-282, Tyr-284, and Glu-298) provides evidence of the structural specificity of labeling and identifies regions in the TMD that do not contribute to the etomidate binding site.…”

In Vivo Activation of Azipropofol Prolongs Anesthesia and Reveals Synaptic Targets

“…These probes share physicochemical properties with their parent molecules, retain anesthetic activity, and undergo photolysis under long wave ultraviolet light (UVA) (315-400 nm), a feature that limits damage to cellular macromolecules upon irradiation following equilibration with the ligands. With these compounds, anesthetic binding sites have been mapped on integrin lymphocyte function-associated antigen (3,6), Torpedo nicotinic receptors (7,8), ␤-tubulin (9), PKC (10), and GABA A receptors (11,12), among others. Direct identification of anesthetic substrates from complex homogenates has proceeded with a neurosteroid analog (2) and halothane (13), the latter an unaltered general anesthetic containing a carbon-bromine bond broken by shorter UV wavelengths to create reactive carbon-centered radicals (14).…”

An alcohol binding site on the neural cell adhesion molecule L1

“…Azialcohols, bearing a photolabile diazirine moiety on one carbon, are stable general anesthetics with normal potencies that obey the MeyerOverton rule. Since their introduction (23), azialcohols have been used to locate binding sites on the nicotinic acetylcholine receptor (24), protein kinase C (25), and adenylate kinase (26).…”