Bupropion Binds to Two Sites in the Torpedo Nicotinic Acetylcholine Receptor Transmembrane Domain: A Photoaffinity Labeling Study with the Bupropion Analogue [¹²⁵I]-SADU-3-72

Abstract

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 analog, (±)-2-(N-tert-butylamino)-3′-[125I]-iodo-4′-azidopropiophenone (SADU-3-72). Based upon inhibition of [125I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC50 = 0.8 μM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with three-fold higher affinity in the desensitized (IC50 = 1.2 μM) than in the resting state. Photolabeling of Torpedo nAChRs with [125I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [125I]SADU-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 (δLeu265, βLeu257) 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 αTyr213 in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo 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.

Interaction of nicotinic receptors with bupropion: Structural, functional, and pre-clinical perspectives

“…These results are in agreement with the [ 3 H]imipramine binding experiments showing that BP discriminates between the desensitized and resting hα3β4 AChRs (manuscript in preparation). The competition binding results also indicated that (±)-SADU-3-72 has high affinity, even higher than (±)-BP, for desensitized and resting states [80,81] . These results propose that although (±)-BP discriminates between the desensitized and resting states better than (±)-SADU-3-72, the latter is a superior probe for the resting state.…”

“…The  subunit is removed for clarity. (C) Model of the BP binding sites based on [ 125 I]SADU-3-72 photoaffinity labeling of Torpedo AChR (modified from [81]). Residues δ-Leu265 and -Leu257 (shown in green), forming the leucine ring, were found photolabeled in the resting state, whereas 1-Tyr213 (shown in orange), located near the extracellular end of α1-M1 was photolabeled in the desensitized state.…”

“…In order to determine the pharmacologic properties of (±)-SADU-3-72 compared to that for (±)-BP at particular muscletype AChRs conformational states, binding and functional approaches were used [79][80][81] . The Ca 2+ influx results show that (±)-SADU-3-72 is 17-and 6-fold more potent than (±)-BP in inhibiting human (h)α1β1γδ and hα1β1εδ AChRs, respectively (Table 1).…”

“…125 I]SADU-3-72 [81] competition binding experiments, (±)-BP binds with 2-3 times higher affinity to the desensitized AChR compared to the resting AChR (Table 2). These results are in agreement with the [ 3 H]imipramine binding experiments showing that BP discriminates between the desensitized and resting hα3β4 AChRs (manuscript in preparation).…”

“…Based on the photoaffinity labeling results of [ 125 I]SADU-3-72, two non-overlapping binding sites for (±)-SADU-3-72 and (±)-BP in the Torpedo AChR transmembrane domain were identified [81] . One binding site was found within the ion channel, whereas a second site was found near the extracellular end of α1-M1, both depending on the receptor conformational state.…”