Abstract
Most general anesthetics including long chain aliphatic alcohols act as noncompetitive antagonists of the nicotinic acetylcholine receptor (nAChR). To locate the sites of interaction of a long chain alcohol with the Torpedo nAChR, we have used the photoactivatible alcohol 3-[(3)H]azioctanol, which inhibits the nAChR and photoincorporates into nAChR subunits. At 1 and 275 microm, 3-[(3)H]azioctanol photoincorporated into nAChR subunits with increased incorporation in the alpha-subunit in the desensitized state. The incorporation into the alpha-subunit was mapped to two large proteolytic fragments. One fragment of approximately 20 kDa (alpha V8-20), containing the M1, M2, and M3 transmembrane segments, showed enhanced incorporation in the presence of agonist whereas the other of approximately 10 kDa (alpha V8-10), containing the M4 transmembrane segment, did not show agonist-induced incorporation of label. Within alpha V8-20, the primary site of incorporation was alpha Glu-262 at the C-terminal end of alpha M2, labeled preferentially in the desensitized state. The incorporation at alpha Glu-262 approached saturation between 1 microm, with approximately 6% labeled, and 275 microm, with approximately 30% labeled. Low level incorporation was seen in residues at the agonist binding site and the protein-lipid interface at approximately 1% of the levels in alpha Glu-262. Therefore, the primary binding site of 3-azioctanol is within the ion channel with additional lower affinity interactions within the agonist binding site and at the protein-lipid interface.
Highlights
The molecular sites of actions of general anesthetics are currently unknown
1 The abbreviations used are: GABA, ␥-aminobutyric acid; 1-AP, 1-azidopyrene; nAChR, nicotinic acetylcholine receptor; PAGE, polyacrylamide gel electrophoresis; V8 protease, S. aureus glutamyl endopeptidase; EndoLysC, endoproteinase Lys-C; ␣BgTx. ␣-bungarotoxin; PTH, phenylthiohydantoin; VDAC, voltage dependent anion channel; carb, carbamylcholine; HPLC, high pressure liquid chromatography. Receptors such as nicotinic acetylcholine receptors and serotonin 5HT3 receptor. These ion channels belong to a superfamily that is composed of five homologous subunits arranged as a pseudopentamer with each subunit composed of a large N-terminal extracellular segment and four transmembrane segments, M1-M4
Based on photoaffinity labeling and mutational analyses of the nAChR, the M2 segments of each subunit are ␣-helices arranged around the central axis and contribute to the lumen of the nAChR ion channel
Summary
Materials—nAChR-enriched membranes were isolated from Torpedo californica electric organ [16]. For the ␣V8-20 and ␣V8-10 fragments, approximately equal aliquots were subjected to either liquid scintillation counting or sequence analysis Prior to sequencing, these samples (because they contained SDS) were treated on the sequencing filters for 4 min with gas-phase trifluoroacetic acid, followed by a 5-min wash with ethyl acetate. To estimate incorporation in large subunit fragments (␣V8-20 and ␣V8-10) or in fragments isolated by HPLC, incorporation was calculated as the 3H loaded divided by three times the observed initial yield of the sequence (three times because only one-third of the PTH-amino acids was measured for mass calculations). The radioactivity released and the mass levels reflect only the sequenced material
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