Anaesthetic actions on other targets: protein kinase C and guanine nucleotide-binding proteins  M.J. Rebecchi, S.N. Pentyala  British Journal of Anaesthesia  Volume 89, Issue 1, Pages 62-78 (July 2002) DOI: 10.1093/bja/aef160 Copyright © 2002 British Journal of Anaesthesia Terms and Conditions

Fig 1 PKC isoforms. British Journal of Anaesthesia 2002 89, 62-78DOI: (10.1093/bja/aef160) Copyright © 2002 British Journal of Anaesthesia Terms and Conditions

Fig 2 Metabotropic receptor signalling: this scheme represents the regulatory mechanisms of two key signalling molecules, G-protein and protein kinase C (PKC). Both interact with heptahelical transmembrane receptors directly. G-protein transduces the signal from the receptor to downstream channels (Na+, Ca2+, K+) and effectors (phosholipase C [PLC], phospholipase A [PLA], phospholipase D [PLD], adenylyl cyclase [AC]) and the feedback regulation is under the control of regulators of G-protein subunits (RGS) proteins and PKC, which negatively modulates the receptor. PKC interacts with diacylglycerol (DAG), calcium (Ca2+) and phosphatidyl serine (PS) and also transduces the signal downstream to effectors and channels. British Journal of Anaesthesia 2002 89, 62-78DOI: (10.1093/bja/aef160) Copyright © 2002 British Journal of Anaesthesia Terms and Conditions

Fig 3 Control of protein kinase C (PKC) activity (conventional): PKC isozymes interact with several partners in transducing signals. Where diacylglycerol (DAG), calcium (Ca2+), phosphatidyl serine (PS) and phosphatase act as positive modulators of PKC, 3-phosphatidylinositol-dependent (3-PI dependent) kinase exerts negative regulation. Inter actions with downstream receptors for activated C kinases (RACK) bind PKC, laterally organizing the enzymes with their substrates. British Journal of Anaesthesia 2002 89, 62-78DOI: (10.1093/bja/aef160) Copyright © 2002 British Journal of Anaesthesia Terms and Conditions

Fig 4 Halothane promotes the interaction of α with βγ subunits: Gβγ subunits were incorporated into artificial phosphatidylcholine membranes and unbound protein was separated from the bound form by gel filtration. The membranes incorporated with Gβγ were transferred to assay tubes containing non-myristoylated, coumarin-labelled Gαi and the interaction assay performed with and without halothane. The samples were incubated for 30 min at 30°C and the complex was centrifuged at 100 000 g for 45 min. Gαi, bound to βγ (pelleted as membrane fraction) was separated from free Gαi (supernatant). The pellet was resuspended in the buffer and the fluorescence of the sample was read at excitation and emission wavelengths of 350 nm and 470 nm, respectively, in an ISS spectrofluorometer. Binding is represented as normalized fluorescence values. The enhanced affinity is independent of the guanine nucleotide (GDP or GTP) bound to the α subunits; normally GTP-charged α subunits have low affinity for their βγ-binding partners. British Journal of Anaesthesia 2002 89, 62-78DOI: (10.1093/bja/aef160) Copyright © 2002 British Journal of Anaesthesia Terms and Conditions

Fig 5 Structure of Gαi in the GTP and GDP bound state. This figure shows ribbon diagrams of the αi1 subunit charged with a non-hydrolysable GTP analogue or GDP (based on x-ray crystallographic structures).127 The α-helical and ras-like domains, and the guanine nucleotide binding and switch regions are indicated. In the GDP state, a micro-domain is formed from the disordered N- and C-terminal regions that do not appear in the GTP state. Other changes in loop conformations are also induced, creating a new binding surface for receptor, Gβγ subunit and effector. A large cavity, unique to the GTP state, is shown in purple and is of sufficient volume to bind volatile anaesthetics. This cavity is lost in the GDP state. Anaesthetic binding to this site may affect binding of guanine nucleotide and prevent the normal coupling of the switch region and the N/C-terminal micro-domain conformations. British Journal of Anaesthesia 2002 89, 62-78DOI: (10.1093/bja/aef160) Copyright © 2002 British Journal of Anaesthesia Terms and Conditions