EXPRESSION OF RECOMBINANT HOMOOLIGOMERIC 5-HYDROXYTRYPTAMINE(3) RECEPTORS PROVIDES NEW INSIGHTS INTO THEIR MATURATION AND STRUCTURE

A recombinant baculovirus containing a mouse 5-hydroxytryptamine(3) (5 -HT3) receptor subunit cDNA under the control of the polyhedrin promot er was shown to direct the production of large amounts of functional 5 -HT3 receptor in insect cells, as assayed by Western blotting and liga nd binding, After solubilization, the receptor was purified to homogen eity by affinity chromatography and characterized pharmacologically. T he ligand binding characteristics of the recombinant receptor were ess entially identical to those of the native receptor, both before and af ter purification, Only fully glycosylated receptors bound to the ligan d affinity resin, although subsequent removal of the sugar did not aff ect ligand binding, Visualization of the purified receptor using elect ron microscopy showed that the receptor preparation contained a homoge neous population of pentameric doughnut shaped particles, The general appearance of the recombinant homooligomeric channels was indistinguis hable from that of native 5-HT3 receptors. Yields of purified receptor were of the order of 200 mu g/3 liters of original culture. The amoun t and homogeneity of the purified receptor are sufficient to begin pre liminary crystallization trials.