Membrane organization of the serotonin 1A receptor monitored by a detergent-free approach

Abstract

: 1. Insolubility of membrane constituents in nonionic detergents such as Triton X-100 has been a widely used biochemical criterion to indicate their localization in membrane domains. However, concerns on the possibility of membrane perturbation in the presence of detergents have led to the development of detergent-free approaches.2. We have explored the organization of the serotonin(1A) receptor, an important G-protein coupled receptor, from bovine hippocampus and CHO cells using a detergent-free approach in order to address the points of agreement with our previous results using Triton X-100.3. A significant fraction of the serotonin(1A) receptor has been found to be localized in a heavy density fraction obtained using a detergent-free approach to isolate membrane domains. In addition, we have characterized the membrane fractions isolated in terms of their lipid composition and membrane physical properties.4. The results obtained on the membrane localization of the serotonin(1A) receptor from the present experiments using a detergent-free approach correlate well with our earlier findings obtained using a detergent-based method (Kalipatnapu, S., and Chattopadhyay, A., FEBS Lett. 576:455-460, 2004). These results provide important information on the membrane organization of the hippocampal serotonin(1A) receptor and are relevant in view of the concerns on the use of detergent in determination of membrane organization of constituent proteins and lipids.

Fig. 1.

(A) Typical pattern of isolation of light, heavy, and extra heavy membrane fractions from hippocampal membranes on a sucrose density gradient using the detergent-free method of Luria et al. (2002). The membrane fraction at 10–22.5% sucrose interface is designated as “light,” at 22.5–35% sucrose interface as “heavy,” and a faintly visible fraction at 35–40% sucrose interface as “extra heavy.” (B) Comparison of total protein content of light, heavy, and extra heavy membrane fractions. Values are expressed as a percentage of the total protein content recovered from all the three membrane fractions isolated. The data points represent means ± SD of duplicate points from three independent experiments. See Materials and Methods section for other details. (C) Comparison of ligand binding to serotonin_1A receptors from the light and heavy membrane fractions isolated using a detergent-free method from native hippocampal membranes. The white bars represent the binding of the agonist [³H]8-OH-DPAT and the shaded bars that of the antagonist [³H]p-MPPF. Values are expressed as a percentage of the total recovered ligand binding obtained from the light and heavy membrane fractions. The data points represent means ± SD of duplicate points from three independent experiments. See Materials and Methods section for other details. (D) Comparison of ligand binding to serotonin_1A receptors from the light and heavy membrane fractions isolated using a detergent-free method from CHO-5-HT_1AR-EYFP cell membranes. The white bars represent the binding of the agonist [³H]8-OH-DPAT and the shaded bars that of the antagonist [³H]p-MPPF. Values are expressed as a percentage of the total recovered ligand binding obtained from the light and heavy membrane fractions. The data points represent means ± SD of duplicate points from two independent experiments. See Materials and Methods section for other details.

Fig. 2.

Fluorescence polarization of DPH incorporated into native hippocampal membranes (shown as native), light and heavy membrane fractions. Fluorescence polarization experiments were carried out with membranes containing 50 nmol of total phospholipid at a probe to phospholipid ratio of 1:100 (mol/mol) at room temperature (23°C). Values represent means ± SE of three independent experiments. Fluorescence polarization values for the light and heavy membrane fractions are significantly different (P < 0.05). See Materials and Methods section for other details.