Serotonin Type 3 Receptors and Anesthetics
(supported by NIH grantP01GM58448)
While heteromeric GABAA receptors are likely targets for many general anesthetics like etomidate, propofol, volatile anesthetics, and alcohols, it is difficult to purify large quantities of GABAA receptors for biochemical studies and photolabeling. Serotonin type 3 (5HT3) receptors in the CNS are modulated by alcohols and anesthetics and probably play physiological roles in post-operative nausea, pain, and addictive behaviors. Furthermore, cell lines expressing cloned 5HT3A receptors have been reported to produce sufficient quantities of these for photolabeling experiments. In collaboration with Professor Keith Miller at Mass General, we are producing cell lines that express either murine or human 5HT3A receptors. The genes encoding 5HT3A receptor protein has also been modified to include affinity purification tags. Our cell lines have improved on published results for 5HT3A by utilizing suspension growth-enabled HEK293S cells as well as inducible expression vector. In this system, cells can grow quickly to high density without expending energy producing 5HT3A receptors, because the gene for this protein is suppressed by a Tet operon genetic element and constitutive expression of the TetR repression protein. Addition of tetracycline turns on 5HT3A receptor expression in all the cells in a coordinated fashion, enabling us to optimize protein production. Biochemical purification can then proceed based on the affinity tags.
The only other eukaryotic ligand-gated ion channel for which photolabeling and analysis at the amino-acid level is routine is the Torpedo nicotinic acetylcholine receptor (nAChR). This protein represents a structural and functional model for other members of the ion channel superfamily. It also displays pharmacologic sensitivity to anesthetics that is similar to other ligand-gated ion channels. However, its utility as a model is limited, because it is not a CNS receptor, and mutations cannot be incorporated into Torpedo rays.
With our 5HT3A receptor expression system, we can incorporate mutations and even improve protein analysis by incorporating new protease sites or charged groups that will improve our ability to utilize MS techniques to analyze protein fragments for sequencing and photolabel incorporation.
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