Ann E. Remmers

Membrane Microviscosity Modulates μ‐Opioid Receptor Conformational Transitions and Agonist Efficacy

Abstract: The influence of membrane microviscosity on μ‐opioid agonist and antagonist binding, as well as agonist efficacy, was examined in membranes prepared from SH‐SY5Y cells and from a C6 glioma cell line stably expressing the rat μ‐opioid receptor (C6μ). Addition of cholesteryl hemisuccinate (CHS) to cell membranes increased membrane microviscosity and reduced the inhibitory effect of sodium and guanine nucleotides on the affinity of the full agonists sufentanil and [D‐Ala2,N‐MePhe4,Gly‐ol5]enkephalin (DAMGO) for the μ‐opioid receptor. Binding of the antagonists [3H]naltrexone and [3H]diprenorphine and the partial agonist nalbuphine was unaffected by CHS. The effect of CHS on agonist binding was reversed by subsequent addition of cis‐vaccenic acid, suggesting that the effect of CHS is the result of increased membrane microviscosity and not a specific sterol—receptor interaction. CHS addition increased the potency of DAMGO to stimulate guanosine‐5′‐O‐(3‐[35S]thio)triphosphate binding by fourfold, whereas the potency of nalbuphine was unaffected. However, nalbuphine efficacy relative to that of the full agonist DAMGO was strongly increased in CHS‐treated membranes compared with that in control membranes. Membrane rigidification also resulted in an increased efficacy for the partial agonists meperidine, profadol, and butorphanol relative to that of DAMGO as measured by agonist‐stimulated GTPase activity in control and CHS‐modified membranes. These findings support a regulatory role for membrane microviscosity in receptor‐mediated G protein activation.

Modulation of Opioid Receptor Binding by Cis and Trans Fatty Acids

Abstract: In synaptosomal brain membranes, the addition of oleic acid (cis), elaidic acid (trans), and the cis and trans isomers of vaccenic acid, at a concentration of 0.87 μmol of lipid/mg of protein, strongly reduced the Bmax and, to a lesser degree, the binding affinity of the μ‐selective opioid [3H]Tyr‐D‐Ala‐Gly‐(Me)Phe‐Gly‐ol ([3H]DAMGO). At comparable membrane content, the cis isomers of the fatty acids were more potent than their trans counterparts in inhibiting ligand binding and in decreasing membrane microviscosity, both at the membrane surface and in the core. However, trans‐vaccenic acid affected opioid receptor binding in spite of just marginally altering membrane microviscosity. If the receptors were uncoupled from guanine nucleotide regulatory protein, an altered inhibition profile was obtained: the impairment of KD by the fatty acids was enhanced and that of Bmax reduced. Receptor interaction of the δ‐opioid [3H](D‐Pen2,D‐Pen5)enkephalin was modulated by lipids to a greater extent than that of [3H]DAMGO: saturable binding was abolished by both oleic and elaidic acids. The binding of [3H]naltrexone was less susceptible to inhibition by the fatty acids, particularly in the presence of sodium. In the absence of this cation, however, cis‐vaccenic acid abolished the low‐affinity binding component of [3H]naltrexone. These findings support the membrane model of opioid receptor sequestration depicting different ionic environments for the μ‐ and δ‐binding sites. The results of this work show distinct modulation of different types and molecular states of opioid receptor by fatty acids through mechanisms involving membrane fluidity and specific interactions with membrane constituents.

Rapid kinetics of G protein subunit association: A rate‐limiting conformational change?

G protein subunit association and dissociation are thought to play an important role in signal transduction. We measured αβγ heterocomplex formation using resonance energy transfer. Fluorescein‐labelled α(F‐α) emission was quenched ∼ 10% on mixing with eosin‐labelled βγ(E‐βγ). Unlabelled βγ did not quench F‐α fluorescence. Stopped‐flow kinetics showed a t ½ ranging from 2.5 s to 0.25 s for 50 nM to 1200 nM E‐βγ. The rate saturated at high E‐βγ concentrations consistent with a two‐step mechanism. We report the first rapid‐mix studies of G protein subunit association kinetics which suggest that α and βγ combine by a two‐step process with a maximal rate of 4.1 ± 0.4 s−1.

[9] α2-adrenergic receptor coupling to G proteins

Publisher Summary This chapter focuses on methods utilizing G protein subunit antibodies and receptor sequence peptides to probe α 2 AR-G protein interactions. Activation of the α 2 AR inhibits adenylylcyclase through the activation of a pertussis toxin-sensitive guanine nucleotide-binding protein G i (22). As all three G i subtypes, along with G o , are inactivated by pertussis toxin, a method to discriminate among the G i subtypes is desirable in order to determine which G protein(s) are essential for α 2 AR inhibition of adenylylcyclase. Use of antisera directed against the C-terminal region of G il /G i2 or G i3 proteins in a high-affinity α 2 AR agonist-binding assay and adenylylcyclase assay demonstrated that both G i2 and G i3 transduce α 2 AR inhibition of adenylylcyclase. Various approaches are applicable to examining pertussis toxin-sensitive receptor signal transduction, are discussed. The G-protein-coupled receptors, including the α 2 AR, have a similar topographical arrangement in the membrane. These receptors contain seven membrane-spanning domains connected by three intracellular and three extracellular loops, with the C terminus intracellular.