title: High- and low-affinity sites for sodium in δ-OR-Gi1α (Cys (351)-Ile (351)) fusion protein stably expressed in HEK293 cells; functional significance and correlation with biophysical state of plasma membrane.
abstract: The effect of sodium, potassium, and lithium on δ-opioid receptor ligand binding parameters and coupling with the cognate G proteins was compared in model HEK293 cell line stably expressing PTX-insensitive δ-OR-Gi1α (Cys(351)-Ile(351)) fusion protein. Agonist [(3)H]DADLE binding was decreased in the order Na(+) ≫ Li(+) > K(+) > (+)NMDG. When plotted as a function of increasing NaCl concentrations, the binding was best-fitted with a two-phase exponential decay considering two Na(+)-responsive sites (r (2) = 0.99). High-affinity Na(+)-sites were characterized by Kd = 7.9 mM and represented 25 % of the basal level determined in the absence of ions. The remaining 75 % represented the low-affinity sites (Kd = 463 mM). Inhibition of [(3)H]DADLE binding by lithium, potassium, and (+)-NMDG proceeded in low-affinity manner only. Surprisingly, the affinity/potency of DADLE-stimulated [(35)S]GTPγS binding was increased in a reverse order: Na(+) < K(+) < Li(+). This result was demonstrated in PTX-treated as well as PTX-untreated cells. Therefore, it is not restricted to Gi1α(Cys(351)-Ile(351)) within the δ-OR-Gi1α fusion protein, but is also valid for stimulation of endogenous G proteins of Gi/Go family in HEK293 cells. Biophysical studies of interaction of ions with polar head-group region of lipids using Laurdan generalized polarization indicated the low-affinity type of interaction only proceeding in the order: Cs(+) < K(+) < Na(+) < Li(+). The results are discussed in terms of interaction of Na(+), K(+) and Li(+) with the high- and low-affinity sites located in water-accessible part of δ-OR binding pocket. We also consider the role of negatively charged Cl(-), Br(-), and I(-) counter anions in inhibition of both [(3)H]DADLE and [(35)S]GTPγS binding.
authors.affiliation: Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Vídeňská 1083, 14220, Prague 4, Czech Republic.
authors.forename: Miroslava
authors.initials: M
authors.lastname: Vošahlíková
authors.forename: Piotr
authors.initials: P
authors.lastname: Jurkiewicz
authors.forename: Lenka
authors.initials: L
authors.lastname: Roubalová
authors.forename: Martin
authors.initials: M
authors.lastname: Hof
authors.forename: Petr
authors.initials: P
authors.lastname: Svoboda
mesh_terms: D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D016308:Enkephalin, Leucine-2-Alanine / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008051:Lipid Bilayers / Q000378:metabolism; D008094:Lithium / Q000494:pharmacology; D017465:Receptors, Opioid, delta / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*
chemical_list: D008051:Lipid Bilayers; D017465:Receptors, Opioid, delta; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D016308:Enkephalin, Leucine-2-Alanine; D008094:Lithium; D012964:Sodium; D019206:GTP-Binding Protein alpha Subunits, Gi-Go