AGONIST-INDUCED PHOTOINCORPORATION OF A P-BENZOYLPHENYLALANINE DERIVATIVE OF SUBSTANCE-P INTO MEMBRANE-SPANNING REGION-2 OF THE TORPEDO NICOTINIC ACETYLCHOLINE-RECEPTOR DELTA-SUBUNIT

The neuropeptide substance P acts, at micromolar concentrations, as a noncompetitive antagonist of nicotinic acetylcholine receptors (AChRs) of both neuronal and muscle subtypes. The mechanism of this inhibitio n has been shown to be most consistent with stabilization of a noncond ucting desensitized state of the AChR, via binding to a site distinct from both the agonist site and the high affinity noncompetitive antago nist site. We have used a radioiodinated photoreactive analogue of sub stance P, containing the amino acid p-benzoyl-L-phenylalanine in place of the Phe(8) residue of substance P, to identify the sites of intera ction of substance P within the Torpedo californica AChR. AChR-rich me mbrane suspensions were photolabeled in the absence or presence of the agonist carbamylcholine and/or nonradioactive substance P, and incorp oration into AChR subunits was assessed by autoradiography after sodiu m dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence o f agonist I-125 incorporation was detected in each subunit and was ins ensitive to substance P, whereas in the presence of carbamylcholine th ere was a 2-fold increase in photoincorporation into the AChR delta su bunit that was inhibited by the addition of an excess of substance P. The sites of specific photoincorporation in the delta subunit were ini tially mapped by use of Staphylococcus aureus V8 protease to a 14-kDa fragment extending from delta Ile-192 to Glu-280. Further fragmentatio n of this 14-kDa fragment with trypsin and S. aureus V8 protease estab lished that the sites of specific incorporation were restricted to the region delta Ser-253 to Glu-280, which contains the membrane-spanning region 2 that is known to form the lining of the ion channel. These r esults establish that in the presence of agonist at least a part of th e undecapeptide substance P binds within the ion channel in the desens itized state of the AChR, and it is likely that the binding of substan ce P to this site is responsible for the action of substance P as a no ncompetitive AChR antagonist.