Photoaffinity cross-linking of Alzheimer's disease amyloid fibrils reveals interstrand contact regions between assembled beta-amyloid peptide subunits.

The assembly of the beta-amyloid peptide (Abeta) into amyloid fibrils is essential to the pathogenesis of Alzheimer's disease. Detailed structural information about fibrillogenesis has remained elusive due to the highly insoluble, noncrystalline nature of the assembled peptide. X-ray fiber diffraction, infrared spectroscopy, and solid-state NMR studies performed on fibrils composed of Abeta peptides have led to conflicting models of the intermolecular alignment of beta-strands. We demonstrate here the use of photoaffinity cross-linking to determine high-resolution structural constraints on Abeta monomers within amyloid fibrils. A photoreactive Abeta(1-40) ligand was synthesized by substituting L-p-benzoylphenylalanine (Bpa) for phenylalanine at position 4 (Abeta(1-40) F4Bpa). This peptide was incorporated into synthetic amyloid fibrils and irradiated with near-UV light. SDS-PAGE of dissolved fibrils revealed the light-dependent formation of a covalent Abeta dimer. Enzymatic cleavage followed by mass spectrometric analysis demonstrated the presence of a dimer-specific ion at MH(+) = 1825.9, the predicted mass of a fragment composed of the N-terminal Abeta(1-5) F4Bpa tryptic peptide covalently attached to the C-terminal Abeta(29-40) tryptic peptide. MS/MS experiments and further chemical modifications of the cross-linked dimer led to the localization of the photo-cross-link between the ketone of the Bpa4 side chain and the delta-methyl group of the Met35 side chain. The Bpa4-Met35 intermolecular cross-link is consistent with an antiparallel alignment of Abeta peptides within amyloid fibrils.

The Alzheimer's peptide a beta adopts a collapsed coil structure in water.

The self-assembly of the soluble peptide Abeta into Alzheimer's disease amyloid is believed to involve a conformational change. Hence the solution conformation of Abeta is of significant interest. In contrast to studies in other solvents, in water Abeta is collapsed into a compact series of loops, strands, and turns and has no alpha-helical or beta-sheet structure. Conformational stabilization is primarily attributed to van der Waals and electrostatic forces. A large conspicuous uninterrupted hydrophobic patch covers approximately 25% of the surface. The compact coil structure appears meta-stable, and because fibrillization leads to formation of intermolecular beta-sheet secondary structure, a global conformational rearrangement is highly likely. A molecular hypothesis for amyloidosis includes at least two primary driving forces, changes in solvation thermodynamics during formation of amyloid deposits and relief of internal conformational stress within the soluble precursor during formation of lower-energy amyloid fibrils.

Activation barriers to structural transition determine deposition rates of Alzheimer's disease a beta amyloid.

Brain amyloid composed of the approximately 40-amino-acid human beta-amyloid peptide A beta is integral to Alzheimer's disease pathology. To probe the importance of a conformational transition in Abeta during amyloid growth, we synthesized and examined the solution conformation and amyloid deposition activity of A beta congeners designed to have similar solution structures but to vary substantially in their barriers to conformational transition. Although all these peptides adopt similar solution conformations, a covalently restricted Abeta congener designed to have a very high barrier to conformational rearrangement was inactive, while a peptide designed to have a reduced barrier to conformational transition displayed an enhanced deposition rate relative to wild-type A beta. The hyperactive peptide, which is linked to a heritable A beta amyloidosis characterized by massive amyloid deposition at an early age, displayed a reduced activation barrier to deposition consistent with a larger difference in activation entropy than in activation enthalpy relative to wild-type A beta. These results suggest that in Alzheimer's disease, as in the prion diseases, a conformational transition in the depositing peptide is essential for the conversion of soluble monomer to insoluble amyloid, and alterations in the activation barrier to this transition affect amyloidogenicity and directly contribute to human disease.

Alzheimer's disease amyloid propagation by a template-dependent dock-lock mechanism.

Amyloid plaques composed of the peptide Abeta are an integral part of Alzheimer's disease (AD) pathogenesis. We have modeled the process of amyloid plaque growth by monitoring the deposition of soluble Abeta onto amyloid in AD brain tissue or synthetic amyloid fibrils and show that it is mediated by two distinct kinetic processes. In the first phase, "dock", Abeta addition to the amyloid template is fully reversible (dissociation t(1/2) approximately 10 min), while in the second phase, "lock", the deposited peptide becomes irreversibly associated (dissociation t(1/2) >> 1000 min) with the template in a time-dependent manner. The most recently deposited peptide dissociates first while Abeta previously deposited becomes irreversibly "locked" onto the template. Thus, the transition from monomer to neurotoxic amyloid is mediated by interaction with the template, a mechanism that has also been proposed for the prion diseases. Interestingly, two Abeta peptides bearing primary sequence alterations implicated in heritable Abeta amyloidoses displayed faster lock-phase kinetics than wild-type Abeta. Inhibiting the initial weak docking interaction between depositing Abeta and the template is a viable therapeutic target to prevent the critical conformational transition in the conversion of Abeta((solution)) to Abeta((amyloid)) and thus prevent stable amyloid accumulation. While thermodynamics suggest that inhibiting amyloid assembly would be difficult, the present study illustrates that the protein misfolding diseases are kinetically vulnerable to intervention.

Deposition of monomeric, not oligomeric, Abeta mediates growth of Alzheimer's disease amyloid plaques in human brain preparations.

Senile plaques composed of the peptide Abeta contribute to the pathogenesis of Alzheimer's disease (AD), and mechanisms underlying their formation and growth may be exploitable as therapeutic targets. To examine the process of amyloid plaque growth in human brain, we have utilized size exclusion chromatography (SEC), translational diffusion measured by NMR, and in vitro models of Abeta amyloid growth to identify the oligomerization state of Abeta that is competent to add onto an existing amyloid deposit. SEC of radiolabeled and unlabeled Abeta over a concentration range of 10(-)(10)-10(-)(4) M demonstrated that the freshly dissolved peptide eluted as a single low molecular weight species, consistent with monomer or dimer. This low molecular weight Abeta species isolated by SEC was competent to deposit onto preexisting amyloid in preparations of AD cortex, with first-order kinetic dependence on soluble Abeta concentration, establishing that solution-phase oligomerization is not rate limiting. Translational diffusion measurements of the low molecular weight Abeta fraction demonstrate that the form of the peptide active in plaque deposition is a monomer. In deliberately aged (>6 weeks) Abeta solutions, a high molecular weight (>100 000 M(r)) species was detectable in the SEC column void. In contrast to the active monomer, assembled Abeta isolated from the column showed little or no focal association with AD tissue. These studies establish that, at least in vitro, Abeta exists as a monomer at physiological concentrations and that deposition of monomers, rather than of oligomeric Abeta assemblies, mediates the growth of existing amyloid in human brain preparations.

Stereochemical specificity of Alzheimer's disease beta-peptide assembly.

The formation and growth of insoluble amyloid deposits composed primarily of the human beta-amyloid peptide (A beta) in brain is an essentially invariant feature of Alzheimer's disease (AD) and is widely believed to contribute to the progressive neurodegeneration of the disorder. To probe the specificity of amyloid formation and growth, we synthesized and examined the self-assembly of D- and L-stereoisomers of A beta in vitro. While both enantiomers formed insoluble aggregates at similar rates with amyloid-like fibrillar morphology, deposition of soluble A beta peptide onto preexisting A beta aggregates was stereospecific. Although the L-peptide deposited readily onto immobilized L-A beta aggregates with first-order kinetic dependence on soluble peptide concentration, essentially no association between the D-peptide and L-template was observed. Similarly, the D-peptide deposited with first-order kinetics onto a D-A beta aggregate template but did not deposit onto a similar template composed of aggregates of the L-enantiomer. Furthermore, although the L-A beta isomer deposited onto authentic AD amyloid in preparations of unfixed AD brain, no focal association between the D-peptide and brain amyloid was detected. These results establish that deposition of soluble A beta onto preexisting amyloid template is stereospecific, likely involving direct docking interactions between peptide backbone and/or side chains rather than simple hydrophobic association.

Oligomerization of endogenous and synthetic amyloid beta-protein at nanomolar levels in cell culture and stabilization of monomer by Congo red.

Amyloid beta-proteins (A beta) are proteolytic fragments of the beta-amyloid precursor protein (beta APP) that are secreted by mammalian cells throughout life but also accumulate progressively as insoluble cerebral aggregates in Alzheimer's disease (AD). Because mounting evidence indicates that A beta aggregation and deposition are early, critical features of AD leading to neurotoxicity, many studies of A beta aggregation have been conducted using synthetic peptides under generally nonphysiological conditions and concentrations. We recently described the oligomerization of A beta peptides secreted by beta APP-expressing cells at low nanomolar (20-30 ng/mL) levels into sodium dodecyl sulfate- (SDS-) stable oligomers of 6-16 kDa. Here, we extensively characterize this in vitro system and show that the amyloid binding dye, Congo red, acts to markedly decrease oligomer/monomer ratios by stabilizing the 4 kDa A beta monomers (ID50 approximately equal to 3.4 microM). Addition of radioiodinated synthetic A beta 1-40 to the cultures or to their conditioned media at physiological concentrations (0.25-2.5 nM) reveals that it undergoes progressive aggregation into SDS-stable oligomers of 6-25 kDa during brief (approximately 4 h) incubation at 37 degrees C, and this is inhibitable by Congo red. The level of A beta oligomers can be quantitated in the Chinese hamster ovary (CHO) conditioned medium by size-exclusion chromatography as well as by SDS-polyacrylamide gel electrophoresis (PAGE), and comparison of these two methods suggests that aggregation of A beta into higher molecular weight polymers that are not detectable by SDS-PAGE occurs in the cultures. We conclude that both endogenous and synthetic A beta can assemble into stable oligomers at physiological concentrations in cell culture, providing a manipulable system for studying the mechanism of early A beta aggregation and identifying inhibitors thereof under biologically relevant conditions.

p-(4-Hydroxybenzoyl)phenylalanine: a photoreactive amino acid analog amenable to radioiodination for elucidation of peptide-protein interaction. Application to substance P receptor.

Benzoylphenylalanine, a photoreactive phenylalanine analog that can be incorporated into a peptide during solid-phase synthesis, is a useful probe for investigating the interactions of bioactive peptides with their receptors. This probe, however, lacks versatility because it is not detectable by Edman sequencing and because it cannot be labeled with radioiodine, requiring radiolabeling of the peptide ligand at a site distal to the photoreactive amino acid. The separation of the radioisotope and photoaffinity labels along the primary sequence limits identification of the photoinsertion site to a peptide fragment rather than a specific amino acid of the receptor protein. We have now synthesized p-(4-hydroxybenzoyl)phenylalanine by a synthetic route involving reaction of 4-(chloromethyl)benzoic anhydride with phenol in polyphosphoric acid to give the 4-(chloromethyl)benzoyl ester of 4-(chloromethyl)-4'-hydroxybenzophenone followed by reaction of the benzophenone derivative with ethyl acetamidocyanoacetate and subsequent hydrolysis of the product to give p-(4-hydroxybenzoyl)phenylalanine. The novel photolabile amino acid was incorporated into substance P (replacing Phe8 or Lys3) to give 11-mer peptides that bind with high (nM) affinity and specificity to the substance P receptor. Radioiodination of the substance P analogs resulted in the incorporation of 125I at the photoreactive amino acid residue, yielding probes of high (approximately 2000 Ci/mmol) specific activity. Subsequent photolysis of the radiolabeled peptides in the presence of substance P receptor caused covalent attachment of the peptide to the receptor with high photoinsertion yield (approximately 30%); photolabeling was abolished in the presence of excess unlabeled SP. p-(4-Hydroxybenzoyl)phenylalanine retains p-benzoylphenylalanine's high insertion yield and low reactivity with water, but in contrast allows placement of radioiodine and the photoactive moieties within the same residue, providing the ability to identify the specific site(s) of interaction, and identification of the residue by Edman sequencing. This novel amino acid may be useful in the elucidation of the interaction of a variety of peptides with their receptors.

A beta deposition inhibitor screen using synthetic amyloid.

The formation, growth, and maturation of brain amyloid "senile" plaques are essential pathological processes in Alzheimer's disease (AD) and key targets for therapeutic intervention. The process of in vitro deposition of A beta at physiological concentrations onto plaques in AD brain preparations has been well characterized, but is cumbersome for drug discovery. We describe here a high-through put screen for inhibitors of A beta deposition onto a synthetic template (synthaloid) of fibrillar A beta immobilized in a polymer matrix. Synthaloid is indistinguishable from plaques in AD brain (the natural template) in deposition kinetics, pH profile, and structure-activity relationships for both A beta analogs and inhibitors. Synthaloid, in contrast to current A beta aggregation screens, accurately predicted inhibitor potency for A beta deposition onto AD cortex preparations, validating its use in searching for agents that can slow the progression of AD and exposing a previously inaccessible target for drug discovery.

Amyloid beta-peptide is transported on lipoproteins and albumin in human plasma.

The amyloid beta-peptide (Abeta) is the major constituent of neuritic plaques in Alzheimer's disease and occurs as a soluble 40-42-residue peptide in cerebrospinal fluid and blood of both normal and AD subjects. It is unclear whether Abeta, once it is secreted by cells, remains free in biological fluids or is associated with other proteins and thus transported and metabolized with them. Such knowledge of the normal fate of Abeta is a prerequisite for understanding the changes that may lead to the pathological aggregation of soluble Abeta in vivo, the possible influence of certain extracellular proteins, particularly apolipoprotein E, on plaque formation, and the pharmacology of putative Abeta-lowering drugs. To address the question of Abeta distribution in human biological fluids, we incubated fresh human plasma from 38 subjects with physiological concentrations (0.5-0.7 nM) of radioiodinated Abeta1-40 and seven plasma samples with Abeta1-42. Lipoproteins and lipid-free proteins were separated and analyzed for bound iodinated Abeta1-40. We found that up to 5% of Abeta added to plasma is bound to selected lipoproteins: very low density, low density, and high density, but not lipoprotein(a). The large majority ( approximately 89%), however, is bound to albumin, and very little Abeta is free. Abeta distribution in plasma was not significantly influenced by apolipoprotein E genotype. We conclude that Abeta is normally bound to and transported by albumin and specific lipoproteins in human plasma under physiological conditions.

Intra-arterial infusion of [125I]A beta 1-40 labels amyloid deposits in the aged primate brain in vivo.

Alzheimer's disease is characterized by extracellular amyloid deposits in the brain at both vascular sites (cerebrovascular amyloid, CVA) and within the neuropil (plaques). In the present study we demonstrated that brain amyloid of aged non-human primates is efficiently detected by [125I]A beta in vitro, and assessed the detection of that amyloid in vivo by intravascular infusion of [125I]A beta. Aged squirrel monkeys (Saimiri sciureus) were anesthetized and infused intra-arterially with [125I]A beta, and sacrificed 2 h later. Analysis of the anterior frontal and temporal cortices by autoradiography demonstrated that [125I]A beta was deposited on CVA and that essentially every amyloid deposit which could be detected with thioflavin S or anti-A beta antibodies was also labeled by [125I]A beta. These experiments suggest that intravascular infusion of radiolabeled A beta can be used to detect and image amyloid deposits in the human AD brain.

Point substitution in the central hydrophobic cluster of a human beta-amyloid congener disrupts peptide folding and abolishes plaque competence.

Alzheimer's disease (AD) is pathologically characterized by the presence of numerous insoluble amyloid plaques in the brain composed primarily of a 40-43 amino acid peptide, the human beta-amyloid peptide (A beta). The process of A beta deposition can be modeled in vitro by deposition of physiological concentrations of radiolabeled A beta onto preexisting amyloid in preparations of unfixed AD cerebral cortex. Using this model system, it has been shown that A beta deposition is biochemically distinct from A beta aggregation and occurs readily at physiological A beta concentrations, but which regions and conformations of A beta are essential to A beta deposition is poorly understood. We report here that an active congener, A beta (10-35)-NH2, displays time dependence, pH-activity profile, and kinetic order of deposition similar to A beta (1-40), and is sufficiently soluble for NMR spectroscopy in water under conditions where it actively deposits. To examine the importance of the central hydrophobic cluster of A beta (LVFFA, residues 17-21) for in vitro A beta deposition, an A beta (10-35)-NH2 analog with a single point substitution (F19T) in this region was synthesized and examined. Unlike A beta (10-35)-NH2, the F19T analog was plaque growth incompetent, and NMR analysis indicated that the mutant peptide was significantly less folded than wild-type A beta. These results support previous studies suggesting that the plaque competence of A beta correlates with peptide folding. Since compounds that alter A beta folding may reduce amyloid deposition, the central hydrophobic cluster of A beta will be a tempting target for structure-based drug design when high-resolution structural information becomes available.

Zinc-induced aggregation of human and rat beta-amyloid peptides in vitro.

The major pathological feature of Alzheimer's disease is the presence of a high density of amyloid plaques in the brain tissue of patients. The plaques are predominantly composed of human beta-amyloid peptide (A beta), a 39-43-mer peptide the neurotoxicity of which is related to its aggregation state. Previous work has demonstrated that certain metals that have been implicated as risk factors for Alzheimer's disease (Al, Fe, and Zn) also cause substantial aggregation of A beta. In particular, we reported that zinc cations at concentrations of > 10(-4) M dramatically accelerate the rate of A beta aggregation at physiological peptide concentrations at 37 degrees C in vitro. In the present study, we investigate the effect of Zn2+ on aggregation of radiolabeled and unlabeled human and rat A beta over a wide range of peptide concentrations in the presence and absence of salt and blocking protein. Aggregation was assayed by centrifugation and filtration using amino acid analysis, immunoassay, and gamma-counting for quantification over a wide range of concentrations of Zn2+ and A beta above and below physiological values. The results of this study demonstrate the following: (a) Radioiodinated A beta accurately tracked unlabeled A beta, (b) zinc concentrations of at least 10(-4) M were required to induce significant aggregation of A beta, and (c) rat and human A beta species were cleared from aqueous solutions by similar concentrations of zinc. These results stand in significant quantitative disagreement (approximately 100-fold in zinc concentration) with one previous study that reported significant aggregation of A beta by < 1 microM Zn2+. Differences between the present study and the latter study from another laboratory appear to result from inappropriate reliance on optical density to measure A beta concentrations and nonspecific loss of A beta to plastic in the absence of blocking protein.

In vitro growth of Alzheimer's disease beta-amyloid plaques displays first-order kinetics.

A salient pathological feature of Alzheimer's disease (AD) is the presence of amyloid plaques in the brains of affected patients. The plaques are predominantly composed of human beta-amyloid peptide (A beta). Although the aggregation of synthetic A beta has been extensively studied, the mechanism of AD plaque growth is poorly understood. In order to address this question, we used an in vitro model of plaque growth to determine if assembly or aggregation of A beta is required for deposition. Labeled A beta at physiological concentrations readily deposited onto both neuritic and diffuse plaques and cerebrovascular amyloid in unfixed AD brain tissue, whereas essentially no deposition was detected in tissue without performed amyloid. Using this in vitro model of plaque growth, the kinetics of A beta deposition onto plaques was examined in two independent but complementary systems. Intact sections of unfixed AD brain cortex (analyzed by autoradiographic densitometry) allowed definitive morphological analysis of the site of deposition, while homogenates of the same tissue (analyzed by radioisotope counting) allowed precise quantitation of deposition over a wide range of conditions. Essentially identical results were obtained for both systems. Growth of preexisting tissue plaques by deposition of A beta was found to follow first-order dependence on A beta concentration and exhibited a pH optimum of 7. In sharp contrast, A beta aggregation in the absence of template follows higher order kinetics and shows a pH optimum of 5. On the basis of criteria of kinetic order, pH dependence, and structure-activity relationships, we conclude that aggregation of A beta (template-independent initial nidus formation) and deposition of A beta (template-dependent subsequent plaque growth) are fundamentally distinct biochemical processes. The process of plaque growth and maturation by A beta deposition may be an important target for therapeutic intervention to block the progression of AD.

1H NMR of A beta amyloid peptide congeners in water solution. Conformational changes correlate with plaque competence.

To begin to examine the structural basis for the deposition of soluble A beta amyloid peptide onto senile plaques in Alzheimer's disease, we have prepared A beta congeners and measured their activity in an in vitro plaque growth assay. The N-terminal fragment, A beta (1-28)-OH, was inactive at all pH values tested. While the central fragment, A beta (10-35)-NH2, and the full length peptide, A beta (1-40)-OH, were inactive below pH 4, both were active (plaque competent) between pH 5 and 9. The active and inactive fragments were studied by nuclear magnetic resonance spectroscopy in water at submillimolar concentrations at pH 2.1 and 5.6. Changes in chemical shifts, coupling constants, and nuclear Overhauser enhancements indicate a pH dependent folding transition in A beta (10-35)-NH2 as it becomes active. The conformation of the active fragment is not helical, and preliminary data indicate the presence of several turns and at least two short strands. In contrast, the inactive fragment A beta (1-28)-OH did not undergo a similar folding transition. Earlier nuclear magnetic resonance studies of amyloid peptides in fluorinated alcohols or detergent micelles at low pH described a helical conformation and proposed a helix to sheet transition in plaque formation; the present study demonstrates that no such conformations are present in water under conditions where the peptides can adhere to authentic amyloid plaques.

Mapping peptide-binding domains of the substance P (NK-1) receptor from P388D1 cells with photolabile agonists.

The tachykinin substance P (SP) is a peptide transmitter of primary afferents. Its actions on both central and peripheral targets are mediated by a G-protein-coupled receptor of known primary structure. To identify contact sites between the undecapeptide SP and its receptor, we prepared radiolabeled photoreactive analogs of SP (H-RPKPQQFFGLM-NH2) by replacing amino acids in the peptide with p-benzoyl-L-phenylalanine (BPA). SP, BPA3-SP, and BPA8-SP bind with high affinity (Kd < 3 nM) to SP receptors on the murine cell line P388D1, triggering intracellular calcium responses. Both binding and calcium responses are blocked by the specific SP receptor antagonist CP-96345. On photolysis, radioiodinated BPA3-SP, and BPA8-SP covalently label a heterogeneously glycosylated protein of about 75 kDa; labeling is abolished by excess unlabeled SP or CP-96345. The labeled receptors were digested with V8 protease and/or trypsin, and the resulting fragments were analyzed by electrophoresis, high pressure liquid chromatography, and chemical or enzymatic modification. BPA3-SP and BPA8-SP photo-incorporate into different regions of the murine SP receptor. The results establish that the third and the eighth positions of SP, respectively, interact with the NH2-terminal extracellular tail (residues 1-21) and second extracellular loop (residues 173-183) of the SP receptor. A model for the agonist peptide-binding sites of the SP receptor is proposed based on photoaffinity labeling and mutagenesis studies.

Local anesthetics inhibit substance P binding and evoked increases in intracellular Ca2+.

BACKGROUND: During spinal and epidural anesthesia, local anesthetics reach concentrations in cerebrospinal fluid and spinal cord tissues at which their actions may extend beyond the classic blockade of sodium channels. This study examines the effects of several clinical and experimental local anesthetics on the binding and actions of a peptide neurotransmitter, substance P, known to be important in nociceptive transmission in the dorsal horn. METHODS: The binding of radiolabeled (Bolton-Hunter modified) substance P was studied in chick brain membranes in the presence of local anesthetics. The increase in intracellular calcium [Ca2+]in evoked by substance P was measured by the fluorescent indicator fura-2 loaded in a murine cell line expressing substance P (NK1) receptors. Cells were preincubated with bupivacaine before and during the transient addition of substance P. RESULTS: Both substance P binding and Ca2+ increase were inhibited half-maximally by approximately 1 mM bupivacaine at pH 7.5, whereas tetracaine, lidocaine, and benzocaine were slightly less potent at inhibiting binding. Concentration-dependent substance P-binding studies showed that bupivacaine's inhibition was not competitive. Inhibition of substance P binding by bupivacaine increased with increasing pH, but the protonated species appears to have some inhibitory activity, and quaternary lidocaine also inhibited binding. There was no stereoselectively to the binding inhibition. CONCLUSIONS: Because millimolar concentrations of local anesthetics are within the range measured in spinal cord during intrathecal and epidural procedures, these results are consistent with a direct action of local anesthetics on tachykinin-mediated neurotransmission during regional anesthesia.

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 inhibition has been shown to be most consistent with stabilization of a nonconducting desensitized state of the AChR, via binding to a site distinct from both the agonist site and the high affinity noncompetitive antagonist site. We have used a radioiodinated photoreactive analogue of substance P, containing the amino acid p-benzoyl-L-phenylalanine in place of the Phe8 residue of substance P, to identify the sites of interaction of substance P within the Torpedo california AChR. AChR-rich membrane suspensions were photolabeled in the absence or presence of the agonist carbamylcholine and/or nonradioactive substance P, and incorporation into AChR subunits was assessed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of agonist 125I incorporation was detected in each subunit and was insensitive to substance P, whereas in the presence of carbamylcholine there was a 2-fold increase in photoincorporation into the AChR delta subunit that was inhibited by the addition of an excess of substance P. The sites of specific photoincorporation in the delta subunit were initially mapped by use of Staphylococcus aureus V8 protease to a 14-kDa fragment extending from delta Ile-192 to Glu-280. Further fragmentation of this 14-kDa fragment with trypsin and S. aureus V8 protease established 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 results establish that in the presence of agonist at least a part of the undecapeptide substance P binds within the ion channel in the desensitized state of the AChR, and it is likely that the binding of substance P to this site is responsible for the action of substance P as a noncompetitive AChR antagonist.