Specific N-glycans regulate an extracellular adhesion complex during somatosensory dendrite patterning.

N-glycans are molecularly diverse sugars borne by over 70% of proteins transiting the secretory pathway and have been implicated in protein folding, stability, and localization. Mutations in genes important for N-glycosylation result in congenital disorders of glycosylation that are often associated with intellectual disability. Here, we show that structurally distinct N-glycans regulate an extracellular protein complex involved in the patterning of somatosensory dendrites in Caenorhabditis elegans. Specifically, aman-2/Golgi alpha-mannosidase II, a conserved key enzyme in the biosynthesis of specific N-glycans, regulates the activity of the Menorin adhesion complex without obviously affecting the protein stability and localization of its components. AMAN-2 functions cell-autonomously to allow for decoration of the neuronal transmembrane receptor DMA-1/LRR-TM with the correct set of high-mannose/hybrid/paucimannose N-glycans. Moreover, distinct types of N-glycans on specific N-glycosylation sites regulate DMA-1/LRR-TM receptor function, which, together with three other extracellular proteins, forms the Menorin adhesion complex. In summary, specific N-glycan structures regulate dendrite patterning by coordinating the activity of an extracellular adhesion complex, suggesting that the molecular diversity of N-glycans can contribute to developmental specificity in the nervous system.

Interplay between membrane curvature and protein conformational equilibrium investigated by solid-state NMR.

Many membrane proteins sense and induce membrane curvature for function, but structural information about how proteins modulate their structures to cause membrane curvature is sparse. We review our recent solid-state NMR studies of two virus membrane proteins whose conformational equilibrium is tightly coupled to membrane curvature. The influenza M2 proton channel has a drug-binding site in the transmembrane (TM) pore. Previous chemical shift data indicated that this pore-binding site is lost in an M2 construct that contains the TM domain and a curvature-inducing amphipathic helix. We have now obtained chemical shift perturbation, protein-drug proximity, and drug orientation data that indicate that the pore-binding site is restored when the full cytoplasmic domain is present. This finding indicates that the curvature-inducing amphipathic helix distorts the TM structure to interfere with drug binding, while the cytoplasmic tail attenuates this effect. In the second example, we review our studies of a parainfluenza virus fusion protein that merges the cell membrane and the virus envelope during virus entry. Chemical shifts of two hydrophobic domains of the protein indicate that both domains have membrane-dependent backbone conformations, with the ß-strand structure dominating in negative-curvature phosphatidylethanolamine (PE) membranes. 31P NMR spectra and 1H-31P correlation spectra indicate that the ß-strand-rich conformation induces saddle-splay curvature to PE membranes and dehydrates them, thus stabilizing the hemifusion state. These results highlight the indispensable role of solid-state NMR to simultaneously determine membrane protein structures and characterize the membrane curvature in which these protein structures exist.

Synthetic Host-Guest Assembly in Cells and Tissues: Fast, Stable, and Selective Bioorthogonal Imaging via Molecular Recognition.

Bioorthogonal strategies are continuing to pave the way for new analytical tools in biology. Although a significant amount of progress has been made in developing covalent reaction based bioorthogonal strategies, balanced reactivity, and stability are often difficult to achieve from these systems. Alternatively, despite being kinetically beneficial, the development of noncovalent approaches that utilize fully synthetic and stable components remains challenging due to the lack of selectivity in conventional noncovalent interactions in the living cellular environment. Herein, we introduce a bioorthogonal assembly strategy based on a synthetic host-guest system featuring Cucurbit[7]uril (CB[7]) and adamantylamine (ADA). We demonstrate that highly selective and ultrastable host-guest interaction between CB[7] and ADA provides a noncovalent mechanism for assembling labeling agents, such as fluorophores and DNA, in cells and tissues for bioorthogonal imaging of molecular targets. Additionally, by combining with covalent reaction, we show that this CB[7]-ADA based noncovalent interaction enables simultaneous bioorthogonal labeling and multiplexed imaging in cells as well as tissue sections. Finally, we show that interaction between CB[7] and ADA fulfills the demands of specificity and stability that is required for assembling molecules in the complexities of a living cell. We demonstrate this by sensitive detection of metastatic cancer-associated cell surface protein marker as well as by showing the distribution and dynamics of F-actin in living cells.

Molecular Mechanisms for Drug Hypersensitivity Induced by the Malaria Parasite's Chloroquine Resistance Transporter.

Mutations in the Plasmodium falciparum 'chloroquine resistance transporter' (PfCRT) confer resistance to chloroquine (CQ) and related antimalarials by enabling the protein to transport these drugs away from their targets within the parasite's digestive vacuole (DV). However, CQ resistance-conferring isoforms of PfCRT (PfCRTCQR) also render the parasite hypersensitive to a subset of structurally-diverse pharmacons. Moreover, mutations in PfCRTCQR that suppress the parasite's hypersensitivity to these molecules simultaneously reinstate its sensitivity to CQ and related drugs. We sought to understand these phenomena by characterizing the functions of PfCRTCQR isoforms that cause the parasite to become hypersensitive to the antimalarial quinine or the antiviral amantadine. We achieved this by measuring the abilities of these proteins to transport CQ, quinine, and amantadine when expressed in Xenopus oocytes and complemented this work with assays that detect the drug transport activity of PfCRT in its native environment within the parasite. Here we describe two mechanistic explanations for PfCRT-induced drug hypersensitivity. First, we show that quinine, which normally accumulates inside the DV and therewithin exerts its antimalarial effect, binds extremely tightly to the substrate-binding site of certain isoforms of PfCRTCQR. By doing so it likely blocks the normal physiological function of the protein, which is essential for the parasite's survival, and the drug thereby gains an additional killing effect. In the second scenario, we show that although amantadine also sequesters within the DV, the parasite's hypersensitivity to this drug arises from the PfCRTCQR-mediated transport of amantadine from the DV into the cytosol, where it can better access its antimalarial target. In both cases, the mutations that suppress hypersensitivity also abrogate the ability of PfCRTCQR to transport CQ, thus explaining why rescue from hypersensitivity restores the parasite's sensitivity to this antimalarial. These insights provide a foundation for understanding clinically-relevant observations of inverse drug susceptibilities in the malaria parasite.

Structural Influences: Cholesterol, Drug, and Proton Binding to Full-Length Influenza A M2 Protein.

The structure and functions of the M2 protein from Influenza A are sensitive to pH, cholesterol, and the antiinfluenza drug Amantadine. This is a tetrameric membrane protein of 97 amino-acid residues that has multiple functions, among them as a proton-selective channel and facilitator of viral budding, replacing the need for the ESCRT proteins that other viruses utilize. Here, various amino-acid-specific-labeled samples of the full-length protein were prepared and mixed, so that only interresidue (13)C-(13)C cross peaks between two differently labeled proteins representing interhelical interactions are observed. This channel is activated at slightly acidic pH values in the endosome when the His(37) residues in the middle of the transmembrane domain take on a +2 or +3 charged state. Changes observed here in interhelical distances in the N-terminus can be accounted for by modest structural changes, and no significant changes in structure were detected in the C-terminal portion of the channel upon activation of the channel. Amantadine, which blocks proton conductance by binding in the aqueous pore near the N-terminus, however, significantly modifies the tetrameric structure on the opposite side of the membrane. The interactions between the juxtamembrane amphipathic helix of one monomer and its neighboring monomer observed in the absence of drug are disrupted in its presence. However, the addition of cholesterol prevents this structural disruption. In fact, strong interactions are observed between cholesterol and residues in the amphipathic helix, accounting for cholesterol binding adjacent to a native palmitoylation site and near to an interhelix crevice that is typical of cholesterol binding sites. The resultant stabilization of the amphipathic helix deep in the bilayer interface facilitates the bilayer curvature that is essential for viral budding.

Interpreting Thermodynamic Profiles of Aminoadamantane Compounds Inhibiting the M2 Proton Channel of Influenza A by Free Energy Calculations.

The development of novel anti-influenza drugs is of great importance because of the capability of influenza viruses to occasionally cross interspecies barriers and to rapidly mutate. One class of anti-influenza agents, aminoadamantanes, including the drugs amantadine and rimantadine now widely abandoned due to virus resistance, bind to and block the pore of the transmembrane domain of the M2 proton channel (M2TM) of influenza A. Here, we present one of the still rare studies that interprets thermodynamic profiles from isothermal titration calorimetry (ITC) experiments in terms of individual energy contributions to binding, calculated by the computationally inexpensive implicit solvent/implicit membrane molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach, for aminoadamantane compounds binding to M2TM of the avian "Weybridge" strain. For all eight pairs of aminoadamantane compounds considered, the trend of the predicted relative binding free energies and their individual components, effective binding energies and changes in the configurational entropy, agrees with experimental measures (ΔΔG, ΔΔH, TΔΔS) in 88, 88, and 50% of the cases. In addition, information yielded by the MM-PBSA approach about determinants of binding goes beyond that available in component quantities (ΔH, ΔS) from ITC measurements. We demonstrate how one can make use of such information to link thermodynamic profiles from ITC with structural causes on the ligand side and, ultimately, to guide decision making in lead optimization in a prospective manner, which results in an aminoadamantane derivative with improved binding affinity against M2TM(Weybridge).

Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers.

The M2 protein of influenza A virus is a membrane-spanning tetrameric proton channel targeted by the antiviral drugs amantadine and rimantadine. Resistance to these drugs has compromised their effectiveness against many influenza strains, including pandemic H1N1. A recent crystal structure of M2(22-46) showed electron densities attributed to a single amantadine in the amino-terminal half of the pore, indicating a physical occlusion mechanism for inhibition. However, a solution NMR structure of M2(18-60) showed four rimantadines bound to the carboxy-terminal lipid-facing surface of the helices, suggesting an allosteric mechanism. Here we show by solid-state NMR spectroscopy that two amantadine-binding sites exist in M2 in phospholipid bilayers. The high-affinity site, occupied by a single amantadine, is located in the N-terminal channel lumen, surrounded by residues mutated in amantadine-resistant viruses. Quantification of the protein-amantadine distances resulted in a 0.3 A-resolution structure of the high-affinity binding site. The second, low-affinity, site was observed on the C-terminal protein surface, but only when the drug reaches high concentrations in the bilayer. The orientation and dynamics of the drug are distinct in the two sites, as shown by (2)H NMR. These results indicate that amantadine physically occludes the M2 channel, thus paving the way for developing new antiviral drugs against influenza viruses. The study demonstrates the ability of solid-state NMR to elucidate small-molecule interactions with membrane proteins and determine high-resolution structures of their complexes.

Stereoselective synthesis and pharmacological evaluation of [4.3.3]propellan-8-amines as analogs of adamantanamines.

Amantadine (1) exerts its anti-Parkinson effects by inhibition of the NMDA associated cation channel and its antiviral activity by inhibition of the M2 protein channel of influenza A viruses. Herein the synthesis, NMDA receptor affinity and anti-influenza activity of analogous propellanamines 3 are reported. The key steps in the synthesis of the diastereomeric propellanamines syn-3 and anti-3 are diastereoselective reduction of the ketone 7 with L-Selectride to give anti-11, Mitsunobu inversion of the alcohol anti-13 into syn-13, and SN2 substitution of diastereomeric mesylates syn-14 and anti-14 with NaN3. The affinity of the propellanamines syn-3 and anti-3 to the PCP binding site of the NMDA receptor is similar to that of amantadine (Ki=11 µM). However, both propellanamines syn-3 and anti-3 do not exhibit activity against influenza A viruses. Compared to amantadine (1), the structurally related propellanamines syn-3 and anti-3 retain the NMDA antagonistic activity but loose the antiviral activity.

Where does amantadine bind to the influenza virus M2 proton channel?

Structures of the influenza A virus M2 proton channel in the open conformation have been determined by X-ray crystallography, and in the closed conformation by NMR. Whereas the X-ray structure shows a single inhibitor molecule in the middle of the channel, four inhibitor molecules bind the channel's outer surface in the NMR structure. In both structures, the strongest hot spots (i.e., regions that contribute substantially to the free energy of binding any potential ligand) lie inside the pore, and other hot spots are found at exterior locations. By considering all available models, we propose the primary drug binding site is inside the pore, but that exterior binding occurs under appropriate conditions.

Novel benzopolycyclic amines with NMDA receptor antagonist activity.

A new series of benzopolycyclic amines active as NMDA receptor antagonists were synthesized. Most of them exhibited increased activity compared with related analogues previously published. All the tested compounds were more potent than clinically approved amantadine and one of them displayed a lower IC50 value than memantine, an anti-Alzheimer's approved drug.

Structural basis for the function and inhibition of an influenza virus proton channel.

The M2 protein from influenza A virus is a pH-activated proton channel that mediates acidification of the interior of viral particles entrapped in endosomes. M2 is the target of the anti-influenza drugs amantadine and rimantadine; recently, resistance to these drugs in humans, birds and pigs has reached more than 90% (ref. 1). Here we describe the crystal structure of the transmembrane-spanning region of the homotetrameric protein in the presence and absence of the channel-blocking drug amantadine. pH-dependent structural changes occur near a set of conserved His and Trp residues that are involved in proton gating. The drug-binding site is lined by residues that are mutated in amantadine-resistant viruses. Binding of amantadine physically occludes the pore, and might also perturb the pK(a) of the critical His residue. The structure provides a starting point for solving the problem of resistance to M2-channel blockers.

Tackling Influenza A virus by M2 ion channel blockers: Latest progress and limitations.

Influenza outbreaks cause pandemics in millions of people. The treatment of influenza remains a challenge due to significant genetic polymorphism in the influenza virus. Also, developing vaccines to protect against seasonal and pandemic influenza infections is constantly impeded. Thus, antibiotics are the only first line of defense against antigenically distinct strains or new subtypes of influenza viruses. Among several anti-influenza targets, the M2 protein of the influenza virus performs several activities. M2 protein is an ion channel that permits proton conductance through the virion envelope and the deacidification of the Golgi apparatus. Both these functions are critical for viral replication. Thus, targeting the M2 protein of the influenza virus is an essential target. Rimantadine and amantadine are two well-known drugs that act on the M2 protein. However, these drugs acquired resistance to influenza and thus are not recommended to treat influenza infections. This review discusses an overview of anti-influenza therapy, M2 ion channel functions, and its working principle. It also discusses the M2 structure and its role, and the change in the structure leads to mutant variants of influenza A virus. We also shed light on the recently identified compounds acting against wild-type and mutated M2 proteins of influenza virus A. These scaffolds could be an alternative to M2 inhibitors and be developed as antibiotics for treating influenza infections.

Three-dimensional structure and interaction studies of hepatitis C virus p7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine by solution nuclear magnetic resonance.

Hepatitis C virus (HCV) protein p7 plays an important role in the assembly and release of mature virus particles. This small 63-residue membrane protein has been shown to induce channel activity, which may contribute to its functions. p7 is highly conserved throughout the entire range of HCV genotypes, which contributes to making p7 a potential target for antiviral drugs. The secondary structure of p7 from the J4 genotype and the tilt angles of the helices within bilayers have been previously characterized by nuclear magnetic resonance (NMR). Here we describe the three-dimensional structure of p7 in short chain phospholipid (1,2-dihexanoyl-sn-glycero-3-phosphocholine) micelles, which provide a reasonably effective membrane-mimicking environment that is compatible with solution NMR experiments. Using a combination of chemical shifts, residual dipolar couplings, and PREs, we determined the structure of p7 using an implicit membrane potential combining both CS-Rosetta decoys and Xplor-NIH refinement. The final set of structures has a backbone root-mean-square deviation of 2.18 Å. Molecular dynamics simulations in NAMD indicate that several side chain interactions might be taking place and that these could affect the dynamics of the protein. In addition to probing the dynamics of p7, we evaluated several drug-protein and protein-protein interactions. Established channel-blocking compounds such as amantadine, hexamethylene amiloride, and long alkyl chain iminosugar derivatives inhibit the ion channel activity of p7. It has also been shown that the protein interacts with HCV nonstructural protein 2 at the endoplasmic reticulum and that this interaction may be important for the infectivity of the virus. Changes in the chemical shift frequencies of solution NMR spectra identify the residues taking part in these interactions.

Host-guest interaction of adamantine with a ß-cyclodextrin-functionalized AuPd bimetallic nanoprobe for ultrasensitive electrochemical immunoassay of small molecules.

A modular labeling strategy was presented for electrochemical immunoassay via supramolecular host-guest interaction between ß-cyclodextrin (ß-CD) and adamantine (ADA). An ADA-labeled antibody (ADA-Ab) was synthesized via amidation, and the number of ADA moieties loaded on a single antibody was calculated to be ~7. The ß-CD-functionalized gold-palladium bimetallic nanoparticles (AuPd-CD) were synthesized in aqueous solution via metal-S chemistry and characterized with transmission electron microscopy and X-ray photoelectron spectra. After the ADA-Ab was bound to the antigen-modified electrode surface with a competitive immunoreaction, AuPd-CD as a signal tag was immobilized onto the immunosensor by a host-guest interaction, leading to a large loading of AuPd nanoparticles. The highly efficient electrocatalysis by AuPd nanoparticles for NaBH4 oxidation produced an ultrasensitive response to chloramphenicol as a model of a small molecule antigen. The immunoassay method showed a wide linear range from 50 pg/mL to 50 µg/mL and a detection limit of 4.6 pg/mL. The specific recognition of antigen by antibody resulted in good selectivity for the proposed method. The host-guest interaction strategy provided a universal labeling approach for the ultrasensitive detection of small molecule targets.

Computational study of drug binding to the membrane-bound tetrameric M2 peptide bundle from influenza A virus.

The M2 protein of influenza A virus performs the crucial function of transporting protons to the interior of virions enclosed in the endosome. Adamantane drugs, amantadine (AMN) and rimantidine (RMN), block the proton conduction in some strains, and have been used for the treatment and prophylaxis of influenza A infections. The structures of the transmembrane (TM) region of M2 that have been solved in micelles using NMR (residues 23-60) (Schnell and Chou, 2008) and by X-ray crystallography (residues 22-46) (Stouffer et al., 2008) suggest different drug binding sites: external and internal for RMN and AMN, respectively. We have used molecular dynamics (MD) simulations to investigate the nature of the binding site and binding mode of adamantane drugs on the membrane-bound tetrameric M2-TM peptide bundles using as initial conformations the low-pH AMN-bound crystal structure, a high-pH model derived from the drug-free crystal structure, and the high-pH NMR structure. The MD simulations indicate that under both low- and high-pH conditions, AMN is stable inside the tetrameric bundle, spanning the region between residues Val27 to Gly34. At low pH the polar group of AMN is oriented toward the His37 gate, while under high-pH conditions its orientation exhibits large fluctuations. The present MD simulations also suggest that AMN and RMN molecules do not show strong affinity to the external binding sites.

The involvement of a Na⁺- and Cl⁻-dependent transporter in the brain uptake of amantadine and rimantadine.

Despite their structural similarity, the two anti-influenza adamantane compounds amantadine (AMA) and rimantadine (RIM) exhibit strikingly different rates of blood-brain barrier (BBB) transport. However, the molecular mechanisms facilitating the higher rate of in situ BBB transport of RIM, relative to AMA, remain unclear. The aim of this study, therefore, was to determine whether differences in the extent of brain uptake between these two adamantanes also occurred in vivo, and elucidate the potential carrier protein facilitating their BBB transport using immortalized human brain endothelial cells (hCMEC/D3). Following oral administration to Swiss Outbred mice, RIM exhibited 2.4-3.0-fold higher brain-to-plasma exposure compared to AMA, which was not attributable to differences in the degree of plasma protein binding. At concentrations representative of those obtained in vivo, the hCMEC/D3 cell uptake of RIM was 4.5-15.7-fold higher than that of AMA, with Michaelis-Menten constants 6.3 and 238.4 µM, respectively. The hCMEC/D3 cellular uptake of both AMA and RIM was inhibited by various cationic transporter inhibitors (cimetidine, choline, quinine, and tetraethylammonium) and was dependent on extracellular pH, membrane depolarization and Na⁺ and Cl⁻ ions. Such findings indicated the involvement of the neutral and cationic amino acid transporter B°,⁺ (ATB°,⁺) in the uptake of AMA and RIM, which was demonstrated to be expressed (at the protein level) in the hCMEC/D3 cells. Indeed, AMA and RIM appeared to interact with this transporter, as shown by a 53-70% reduction in the hCMEC/D3 uptake of the specific ATB°,⁺ substrate ³H-glycine in their presence. These studies suggest the involvement of ATB°,⁺ in the disposition of these cationic drugs across the BBB, a transporter with the potential to be exploited for targeted drug delivery to the brain.

Conformational heterogeneity of the M2 proton channel and a structural model for channel activation.

The M2 protein of influenza virus A is a proton-selective ion channel activated by pH. Structure determination by solid-state and solution NMR and X-ray crystallography has contributed significantly to our understanding, but channel activation may involve conformations not captured by these studies. Indeed, solid-state NMR data demonstrate that the M2 protein possesses significant conformational heterogeneity. Here, we report molecular dynamics (MD) simulations of the M2 transmembrane domain (TMD) in the absence and presence of the antiviral drug amantadine. The ensembles of MD conformations for both apo and bound forms reproduced the NMR data well. The TMD helix was found to kink around Gly-34, where water molecules penetrated deeply into the backbone. The amantadine-bound form exhibited a single peak approximately 10 degrees in the distribution of helix-kink angle, but the apo form exhibited 2 peaks, approximately 0 degrees and 40 degrees . Conformations of the apo form with small and large kink angles had narrow and wide pores, respectively, around the primary gate formed by His-37 and Trp-41. We propose a structural model for channel activation, in which the small-kink conformations dominate before proton uptake by His-37 from the exterior, and proton uptake makes the large-kink conformations more favorable, thereby priming His-37 for proton release to the interior.

Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel.

Influenza A and B viruses contain proton-selective ion channels, A/M2 and BM2, respectively, and the A/M2 channel activity is inhibited by the drugs amantadine and its methyl derivative rimantadine. The structure of the pore-transmembrane domain has been determined by both x-ray crystallography [Stouffer et al. (2008) Nature 451:596-599] and by NMR methods [Schnell and Chou (2008) Nature 451:591-595]. Whereas the crystal structure indicates a single amantadine molecule in the pore of the channel, the NMR data show four rimantadine molecules bound on the outside of the helices toward the cytoplasmic side of the membrane. Drug binding includes interactions with residues 40-45 with a polar hydrogen bond between rimantadine and aspartic acid residue 44 (D44) that appears to be important. These two distinct drug-binding sites led to two incompatible drug inhibition mechanisms. We mutagenized D44 and R45 to alanine as these mutations are likely to interfere with rimantadine binding and lead to a drug insensitive channel. However, the D44A channel was found to be sensitive to amantadine when measured by electrophysiological recordings in oocytes of Xenopus laevis and in mammalian cells, and when the D44 and R45 mutations were introduced into the influenza virus genome. Furthermore, transplanting A/M2 pore residues 24-36 into BM2, yielded a pH-activated chimeric ion channel that was partially inhibited by amantadine. Thus, taken together our functional data suggest that amantadine/rimantadine binding outside of the channel pore is not the primary site associated with the pharmacological inhibition of the A/M2 ion channel.

Lipophilic cationic drugs increase the permeability of lysosomal membranes in a cell culture system.

Lysosomes accumulate many drugs several fold higher compared to their extracellular concentration. This mechanism is believed to be responsible for many pharmacological effects. So far, uptake and release kinetics are largely unknown and interactions between concomitantly administered drugs often provoke mutual interference. In this study, we addressed these questions in a cell culture model. The molecular mechanism for lysosomal uptake kinetics was analyzed by live cell fluorescence microscopy in SY5Y cells using four drugs (amantadine, amitriptyline, cinnarizine, flavoxate) with different physicochemical properties. Drugs with higher lipophilicity accumulated more extensively within lysosomes, whereas a higher pK(a) value was associated with a more rapid uptake. The drug-induced displacement of LysoTracker was neither caused by elevation of intra-lysosomal pH, nor by increased lysosomal volume. We extended our previously developed numerical single cell model by introducing a dynamic feedback mechanism. The empirical data were in good agreement with the results obtained from the numerical model. The experimental data and results from the numerical model lead to the conclusion that intra-lysosomal accumulation of lipophilic xenobiotics enhances lysosomal membrane permeability. Manipulation of lysosomal membrane permeability might be useful to overcome, for example, multi-drug resistance by altering subcellular drug distribution.

Pathway to lamellar bodies for surfactant protein A.

Alveolar surfactant protein A (SP-A) is endocytosed by type II epithelial cells through clathrin-dependent uptake and targeted to lamellar bodies for resecretion. However, the mechanism for secretion of newly synthesized SP-A, whether regulated exocytosis of lamellar bodies or constitutive secretion, is unresolved. If it is the latter, lamellar body SP-A would represent endocytosed protein. Amantadine, an inhibitor of clathrin-coated vesicle budding, was used to evaluate the role of endocytosis in accumulation of SP-A in lamellar bodies. In isolated rat lungs, amantadine (10 mM) inhibited uptake of endotracheally instilled (35)S-labeled biosynthesized surfactant proteins by >80%. To study trafficking of newly synthesized SP-A, lungs were perfused for up to 6 h with [(35)S]methionine, and surfactant was isolated from lung lavage fluid and lamellar bodies were isolated from lung homogenate. With control lungs, the mean specific activity of [(35)S]SP-A (disintegrations per minute per microgram of SP-A) increased linearly with time of perfusion: it was significantly higher in isolated lamellar bodies than in surfactant and was increased in both compartments by 50-60% in the presence of 0.1 mM 8-bromo-cAMP. These results suggest a precursor-product relationship between lamellar body and extracellular [(35)S]SP-A. Specific activities in both compartments were unaffected by addition of amantadine (10 mM) to the lung perfusate, indicating that uptake from the alveolar space was not responsible for the increase in lamellar body [(35)S]SP-A. Thus the pathway for secretion of newly synthesized SP-A is by transfer from the site of synthesis to the storage/secretory organelle prior to lamellar body exocytosis.