A hydrophobic inhibitor of the nicotinic acetylcholine receptor acts on the resting state.
3-(Trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) has recently been found to be a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAcChoR) by both photolabeling and flux assays (White et al., 1991). However, these experiments were done when TID was in equilibrium with the nAcChoR, and thus only its interactions with the resting and the desensitized states of the nAcChoR were studied. In this work we characterized the interaction between TID and nAcChoR in the open and resting states using a flux assay. When TID and acetylcholine were simultaneously mixed with the nAcChoR in native Torpedo vesicles, TID did not inhibit agonist-induced 86Rb+ flux. However, following prolonged preincubation (4 min) of TID with nAcChoR, complete inhibition was observed with a half-inhibition constant of 0.4 microM TID and a Hill coefficient of 0.9. This suggested that TID might act either on the resting or the desensitized state in preference to the open state of the nAcChoR. Preincubation of nAcChoR with TID, followed by a 7 ms agonist-induced flux assay, showed that the flux response declined exponentially with preincubation time. Assuming a pseudo-first-order process, analysis revealed the rate constant for the onset of inhibition of nAcChoR in the resting state to be in the range of (1.2-3.4) x 10(6) M-1 s-1. To test if fast desensitization was enhanced by TID under these conditions, we used a fluorescent analog of acetylcholine. Stopped-flow fluorimetry showed that the fraction of nAcChoR in the predesensitized state did not increase during preincubation with TID.(ABSTRACT TRUNCATED AT 250 WORDS)
Wu
G
G
Department of Anesthesia, Massachusetts General Hospital, Boston.
Raines
D E
DE
Miller
K W
KW
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001389:Azirines / Q000737:chemistry*; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D018733:Nicotinic Antagonists; D000442:Octanols / Q000494:pharmacology; D010777:Photochemistry; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000648:ultrastructure; D013329:Structure-Activity Relationship; D013570:Synaptic Membranes / Q000737:chemistry; D014101:Torpedo
D001389:Azirines; D018733:Nicotinic Antagonists; D000442:Octanols; D011978:Receptors, Nicotinic; D000109:Acetylcholine