High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated, Low-Density Plasma Membrane Fragments.
RESULTS
HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye.


CONCLUSIONS
Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.
Department of Biomathematics, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic.
Lenka
L
Roubalova
Department of Biomathematics, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic.
Miroslava
M
Vosahlikova
Department of Biomathematics, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic.
Jana
J
Brejchova
Department of Biophysical Chemistry, J. Heyrovsky Institute of Physical Chemistry of the Czech Academy of Sciences, Prague, Czech Republic.
Jan
J
Sykora
Department of Neurochemistry, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic.
Vladimir
V
Rudajev
Department of Biomathematics, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic.
Petr
P
Svoboda
D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D002592:Cetomacrogol / Q000494:pharmacology*; D003902:Detergents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D037342:Pertussis Toxin / Q000633:toxicity; D017465:Receptors, Opioid, delta / Q000378:metabolism*; D013696:Temperature
D003902:Detergents; D017465:Receptors, Opioid, delta; D002592:Cetomacrogol; D037342:Pertussis Toxin