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Description:
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The 5 -HT3 receptor is a member of the Cys -loop family of ligand -gated ion channels (LGICs ) and mediates excitatory fast synaptic transmission in the central and peripheral nervous system . Despite the clear physiological importance of the 5 -HT3 receptor , only a small number of published studies have directly examined the structure of the receptor . The principal objective of this work is to express and affinity -purify the mouse 5 -HT3A receptor and then begin detailed structural characterization of the receptor . A mouse 5 -HT3A receptor containing a C -terminal ƒÑ -bungarotoxin (ƒÑBgTx ) pharmatope tag was constructed and stably transfected into HEK293 cells . To obtain sufficient quantities of receptor protein for affinity -purification , ƒÑBgTx -5 -HT3A receptor -HEK cells were cultured in 140 mm tissue culture dishes ( ~1000 dishes ) . Typically , cells were treated with 100 ƒÝM serotonin 24 h prior to harvesting resulting in a ~ 2 .5 fold increase in receptor expression . ƒÑBgTx -5 -HT3A receptors were affinity -purified using an ƒÑBgTx -derivatized affinity column . The lipid -protein interface and the agonist -binding site of purified 5 -HT3ARs were directly examined using photoaffinity labeling with the hydrophobic probe ([125I]TID ) and [3H]5 -HT , respectively . The preliminary results of these studies include : 1 ) [125I]TID photoincorporates into the 5 -HT3A receptor and the labeling maps to two proteolytic fragments , designated V8 -17K and V8 -8K ; 2 ) N -terminal sequencing of each rpHPLC -purified fragment revealed that V8 -17 starts at Val195 and based on its apparent molecular weight extends through the M1 , M2 , and M3 transmembrane segments . V8 -8K starts at Val424 and contains the M4 transmembrane segment ; 3 ) Within the M4 transmembrane segment , [125I]TID photoincorporated into Ser451 , which corresponds (in the aligned sequence ) to Thr422 in the lipid -exposed face of the Torpedo muscle -type nACh receptor ƒÑ1M4 segment ; 4 ) [3H]5 -HT photoincorporates into the 5 -HT3A receptor in a specific manner (MDL72222 significantly inhibits the labeling ) . Collectively , the results obtained so far are consistent with each of these two important LGICs displaying a high -degree of structural homology . Additional studies are in progress to further identify lipid exposed segments /residues in the M1 and M3 transmembrane segments as well as to probe the structure of the agonist binding site in the 5 -HT3A receptor . The results will be compared with those previously determined for the Torpedo nAChR in order to provide a more detailed structural comparison of these two important LGICs . |