Solubilization of high affinity G-protein-coupled serotonin 1A receptors from bovine hippocampus using pre-micellar CHAPS at low concentration

Abstract

The serotonin1A (5-HT1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioural and cognitive functions. This paper reports an efficient strategy to solubilize 5-HT1A receptors from bovine hippocampal membranes using the zwitterionic detergent CHAPS which is mild and non-denaturing. Since high concentration of CHAPS has earlier been shown to induce dissociation and depletion of G-protein sub-units, a low (pre-micellar) concentration of CHAPS was used for solubilizing 5-HT1A receptors in the presence of NaCl followed by PEG precipitation. This results in solubilization of 5-HT1A receptors with a high degree of efficiency and gives rise to high affinity, functionally active G-protein-sensitive solubilized receptors. Optimal solubilization of the receptor from the native source with high ligand binding affinity and intact signal transduction components may constitute the first step in the molecular characterization of the 5-HT1A receptor in particular, and G-protein-coupled receptors in general.

Solubilization of Serotonin1AReceptors Heterologously Expressed in Chinese Hamster Ovary Cells

“…The CMC is an important parameter for a given detergent since at this concentration the detergent starts to accumulate in the membrane. The change in solubilization efficiency of CHAPS with NaCl concentration, observed earlier by us in case of native hippocampal membranes (Chattopadhyay et al, 2002) and in our present results with T-CHO membranes, could be related to the dependence of its CMC with NaCl concentration.…”

“…Thus, the CMC of CHAPS decreases from 6.41 mM in absence of any salt to 4.10 mM in presence of 1.5 M NaCl which amounts to a reduction of 36% (Chattopadhyay et al, 2002;Chattopadhyay and Harikumar, 1996).…”

“…The 5-HT 1A receptor therefore represents an important member of the seven transmembrane domain G-protein-coupled receptor family. We have earlier partially purified and solubilized the 5-HT 1A receptor from bovine hippocampus in a functionally active form (Chattopadhyay et al, 2002;Chattopadhyay and Harikumar, 1996) and have shown modulation of ligand binding by metal ions, guanine nucleotides, alcohols, and covalent modifications of the disulfides and sulfhydryl groups Harikumar and Chattopadhyay, 1998a,b, 1999). …”

“…These factors have led to extensive use of CHAPS in solubilization of membrane proteins and receptors (Banerjee et al, 1995;Ofri et al, 1992). We have previously used CHAPS at low concentration to solubilize 5-HT 1A receptors from bovine hippocampus in a functional form (Chattopadhyay et al, 2002;Chattopadhyay and Harikumar, 1996).…”

Interaction of Serotonin1AReceptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

“…Furthermore, it was the first serotonin receptor for which polyclonal antibodies were obtained (Fargin et al, 1988) allowing their visualization at the subcellular level in various regions of the brain. We have earlier partially purified and solubilized the 5-HT 1A receptor from bovine hippocampus in a functionally active form (Chattopadhyay et al, 2002;Chattopadhyay and Harikumar, 1996) and reported, for the first time, solubilization of 5-HT 1A receptors stably expressed in Chinese Hamster Ovary (CHO) cells (Chattopadhyay et al, 2004). We have shown modulation of ligand binding by metal ions, guanine nucleotides, alcohols, and covalent modifications of the disulfides and sulfhydryl groups Harikumar and Chattopadhyay, 1998a,b, 1999.…”