1 research outputs found
Bupropion Binds to Two Sites in the <i>Torpedo</i> Nicotinic Acetylcholine Receptor Transmembrane Domain: A Photoaffinity Labeling Study with the Bupropion Analogue [<sup>125</sup>I]-SADU-3-72
Bupropion, a clinically used antidepressant and smoking-cessation
drug, acts as a noncompetitive antagonist of nicotinic acetylcholine
receptors (nAChRs). To identify its binding site(s) in nAChRs, we
developed a photoreactive bupropion analogue, (±)-2-(<i>N</i>-<i>tert</i>-butylamino)-3′-[<sup>125</sup>I]-iodo-4′-azidopropiophenone (SADU-3-72). Based on inhibition
of [<sup>125</sup>I]ÂSADU-3-72 binding, SADU-3-72 binds with high affinity
(IC<sub>50</sub> = 0.8 μM) to the <i>Torpedo</i> nAChR
in the resting (closed channel) state and in the agonist-induced desensitized
state, and bupropion binds to that site with 3-fold higher affinity
in the desensitized (IC<sub>50</sub> = 1.2 μM) than in the resting
state. Photolabeling of <i>Torpedo</i> nAChRs with [<sup>125</sup>I]ÂSADU-3-72 followed by limited <i>in-gel</i> digestion
of nAChR subunits with endoproteinase Glu-C established the presence
of [<sup>125</sup>I]ÂSADU-3-72 photoincorporation within nAChR subunit
fragments containing M1–M2–M3 helices (αV8-20K,
βV8-22/23K, and γV8-24K) or M1–M2 helices (δV8-14).
Photolabeling within βV8-22/23K, γV8-24K, and δV8-14
was reduced in the desensitized state and inhibited by ion channel
blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine
(TCP)) state, and this pharmacologically specific photolabeling was
localized to the M2-9 leucine ring (δLeu<sup>265</sup>, βLeu<sup>257</sup>)
within the ion channel. In contrast, photolabeling within the αV8-20K
was enhanced in the desensitized state and not inhibited by TCP but
was inhibited by bupropion. This agonist-enhanced photolabeling was
localized to αTyr<sup>213</sup> in αM1. These results
establish the presence of two distinct bupropion binding sites within
the <i>Torpedo</i> nAChR transmembrane domain: a high affinity
site at the middle (M2-9) of the ion channel and a second site near
the extracellular end of αM1 within a previously described halothane
(general anesthetic) binding pocket