Interaction of GABA Receptor in the Fish Brain with Ro7-1986/1 and Fipronil by Fluorescent-labeled Ligand†

doi:10.7503/cjcu20140942

Abstract

Abstract:

Two kinds of γ-aminobutyric acid(GABA) receptor ligands, Ro7-1986/1 and Fipronil, were explored for preparing fluorescein conjugates including FITC-Ro7-1986/1 and FITC-Fipronil. Fourier transform infrared spectroscopy(FTIR) and ¹H NMR were used to characterize the structures of the synthesized products. The interactions between GABA receptor in hypophthalmichthys nobilis brain and both ligands were studied by fluorescence-labeling method. In addition, the effects of GABA on the binding of Ro7-1986/1 and GABA receptors were also investigated. The results showed that the equilibrium dissociation constants(K_d) and the maximum number of binding sites [RT] between Ro7-1986/1 and receptor were (67±5) nmol/L and (13.8±1.8) pmol/mg protein, respectively. The values of K_d and [RT] between Fipronil and receptor were (346±6) nmol/L and (40.6±3.5) pmol/mg protein, respectively. Meanwhile, GABA was able to significantly elevate the binding of between ligand and receptor, which indicated the pharmacological properties of GABA receptor in fish brain are remarkably similar to those in mammalian brain. Moreover, Fipronil exhibited more excellent affinities to fish by comparison to mammals.

Key words: Ro7-1986/1, Fipronil, Fluorescence-labeled method, γ-Aminobutyric acid receptor

CLC Number:

O657

TrendMD:

YANG Shan, ZHANG Bo, ZHANG Yuan, REN Tianrui. Interaction of GABA Receptor in the Fish Brain with Ro7-1986/1 and Fipronil by Fluorescent-labeled Ligand^†[J]. Chem. J. Chinese Universities, 2015, 36(3): 456.

Figures/Tables 7

Fig.1 Structures of FITC-Ro7(A) and FITC-Fipronil(B)

Fig.2 FTIR spectra of FITC(a), Ro7-1986/1(b), FITC-Ro7(c), Fipronil(d) and FITC-Fipronil(e)

Fig.3 Excitation(a) and emission(b) spectra of FITC(A), FITC-Ro7(B) and FITC-Fipronil(C)

Fig.4 Fluorescence intensity vs. FITC-Ro7(A) and FITC-Fipronil(B) concentration a. In 50% methanol; b. in tissue suspension; c. in buffer.

Fig.5 Saturability of FITC-Ro7(A), FITC-Fipronil(B) and Scatchard analysis of FITC-Ro7(C),FITC-Fipronil(D) binding to cortical membranes by different methods Supernatant(S1), tissue suspension(P1), supernatant after dissociation(S2) in the binding assay of FITC-Ro7. Supernatant(S1'), tissue suspension(P1'), supernatant after dissociation(S2') in the binding assay of FITC- Fipronil.

Table 1 Equilibrium binding properties of FITC-Ro7 and FITC-Fipronil binding to GABA receptor

Comp.	K_d/(nmol·L^-1)	[RT]/(pmol·mg^-1 protein)	Comp.	K_d/(nmol·L^-1)	[RT]/(pmol·mg^-1 protein)
FITC-Ro7(S₁ )	109±9	21.3±2.5	FITC-Fipronil(S₁')	502±8	56.2±5.0
FITC-Ro7(P₁)	86±7	16.0±2.2	FITC-Fipronil(P₁')	377±7	39.3±3.7
FITC-Ro7(S₂)	67±5	13.8±1.8	FITC-Fipronil(S₂')	346±6	40.6±3.5

Fig.6 GABA enhancement of FITC-Ro7 binding to hypophthalmichthys nobilis brain membranes Each point represents the ratio of binding in the presence and absence of GABA. The data shown are representative of three independent experiments, each point in triplicate.

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