Polyhistidine peptide inhibitor of the Abeta calcium channel potently blocks the Abeta-induced calcium response in cells. Theoretical modeling suggests a cooperative binding process.

On the basis of the consistent demonstrations that the Abeta peptide of Alzheimer's disease forms calcium permeant channels in artificial membranes, we have proposed that the intracellular calcium increase observed in cells exposed to Abeta is initiated by calcium fluxes through Abeta channels. We have found that a small four-histidine peptide, NAHis04, potently inhibits the Abeta-induced calcium channel currents in artificial lipid membranes. Here we report that NaHis04 also potently blocks the intracellular calcium increase which is observed in cells exposed to Abeta. PC12 cells loaded with Fura-2AM show a rapid increase in fluorescence and a rapid return to baseline after Abeta is added to the medium. This fluorescence change occurs even when the medium contains nitrendipine, a voltage-gated calcium channel blocker, but fails to occur when application of Abeta is preceded by addition of NAHis04. Steep dose-response curves of the percentage of responding cells and cell viability show that NAHis04 inhibits in the micromolar range in an apparently cooperative manner. We have developed numerous models of Abeta pores in which the first part of the Abeta sequence forms a large beta-barrel ending at His 13. We have modeled how up to four NAHis04 peptides may block these types of pores by binding to side chains of Abeta residues Glu 11, His 13, and His 14.

Models of membrane-bound Alzheimer's Abeta peptide assemblies.

Although it is clear that amyloid beta (Aß) peptides play a pivotal role in the development of Alzheimer's disease, the precise molecular model of action remains unclear. Aß peptide forms assemble both in aqueous solution and in lipid membranes. It has been proposed that deleterious effects occur when the peptides interact with membranes, possibly by forming Ca(2+) permeant ion channels. In the accompanying manuscript, we propose models in which the C-terminus third of six Aß42 peptides forms a six-stranded ß-barrel in highly toxic soluble oligomers. Here we extend this hypothesis to membrane-bound assemblies. In these Aß models, the hydrophobic ß-barrel of a hexamer may either reside on the surface of the bilayer, or span the bilayer. Transmembrane pores are proposed to form between several hexamers. Once the ß-barrels of six hexamers have spanned the bilayer, they may merge to form a more stable 36-stranded ß-barrel. We favor models in which parallel ß-barrels formed by N-terminus segments comprise the lining of the pores. These types of models explain why the channels are selective for cations and how metal ions, such as Zn(2+) , synthetic peptides that contain histidines, and some small organic cations may block channels or inhibit formation of channels. Our models were developed to be consistent with microscopy studies of Aß assemblies in membranes, one of which is presented here for the first time.

Beta-barrel models of soluble amyloid beta oligomers and annular protofibrils.

Both soluble and membrane-bound prefibrillar assemblies of Abeta (Aß) peptides have been associated with Alzheimer's disease (AD). The size and nature of these assemblies vary greatly and are affected by many factors. Here, we present models of soluble hexameric assemblies of Aß42 and suggest how they can lead to larger assemblies and eventually to fibrils. The common element in most of these assemblies is a six-stranded ß-barrel formed by the last third of Aß42, which is composed of hydrophobic residues and glycines. The hydrophobic core ß-barrels of the hexameric models are shielded from water by the N-terminus and central segments. These more hydrophilic segments were modeled to have either predominantly ß or predominantly α secondary structure. Molecular dynamics simulations were performed to analyze stabilities of the models. The hexameric models were used as starting points from which larger soluble assemblies of 12 and 36 subunits were modeled. These models were developed to be consistent with numerous experimental results.

Models of voltage-dependent conformational changes in NaChBac channels.

Models of the transmembrane region of the NaChBac channel were developed in two open/inactivated and several closed conformations. Homology models of NaChBac were developed using crystal structures of Kv1.2 and a Kv1.2/2.1 chimera as templates for open conformations, and MlotiK and KcsA channels as templates for closed conformations. Multiple molecular-dynamic simulations were performed to refine and evaluate these models. A striking difference between the S4 structures of the Kv1.2-like open models and MlotiK-like closed models is the secondary structure. In the open model, the first part of S4 forms an alpha-helix, and the last part forms a 3(10) helix, whereas in the closed model, the first part of S4 forms a 3(10) helix, and the last part forms an alpha-helix. A conformational change that involves this type of transition in secondary structure should be voltage-dependent. However, this transition alone is not sufficient to account for the large gating charge movement reported for NaChBac channels and for experimental results in other voltage-gated channels. To increase the magnitude of the motion of S4, we developed another model of an open/inactivated conformation, in which S4 is displaced farther outward, and a number of closed models in which S4 is displaced farther inward. A helical screw motion for the alpha-helical part of S4 and a simple axial translation for the 3(10) portion were used to develop models of these additional conformations. In our models, four positively charged residues of S4 moved outwardly during activation, across a transition barrier formed by highly conserved hydrophobic residues on S1, S2, and S3. The S4 movement was coupled to an opening of the activation gate formed by S6 through interactions with the segment linking S4 to S5. Consistencies of our models with experimental studies of NaChBac and K(v) channels are discussed.

Models of the structure and gating mechanisms of the pore domain of the NaChBac ion channel.

The NaChBac prokaryotic sodium channel appears to be a descendent of an evolutionary link between voltage-gated K(V) and Ca(V) channels. Like K(V) channels, four identical six-transmembrane subunits comprise the NaChBac channel, but its selectivity filter possesses a signature sequence of eukaryotic Ca(V) channels. We developed structural models of the NaChBac channel in closed and open conformations, using K(+)-channel crystal structures as initial templates. Our models were also consistent with numerous experimental results and modeling criteria. This study concerns the pore domain. The major differences between our models and K(+) crystal structures involve the latter portion of the selectivity filter and the bend region in S6 of the open conformation. These NaChBac models may serve as a stepping stone between K(+) channels of known structure and Na(V), Ca(V), and TRP channels of unknown structure.

Mapping discontinuous epitopes for MRK-16, UIC2 and 4E3 antibodies to extracellular loops 1 and 4 of human P-glycoprotein.

P-glycoprotein (P-gp), an ATP-dependent efflux pump, is associated with the development of multidrug resistance in cancer cells. Antibody-mediated blockade of human P-gp activity has been shown to overcome drug resistance by re-sensitizing resistant cancer cells to anticancer drugs. Despite the potential clinical application of this finding, the epitopes of the three human P-gp-specific monoclonal antibodies MRK-16, UIC2 and 4E3, which bind to the extracellular loops (ECLs) have not yet been mapped. By generating human-mouse P-gp chimeras, we mapped the epitopes of these antibodies to ECLs 1 and 4. We then identified key amino acids in these regions by replacing mouse residues with homologous human P-gp residues to recover binding of antibodies to the mouse P-gp. We found that changing a total of ten residues, five each in ECL1 and ECL4, was sufficient to recover binding of both MRK-16 and 4E3 antibodies, suggesting a common epitope. However, recovery of the conformation-sensitive UIC2 epitope required replacement of thirteen residues in ECL1 and the same five residues replaced in the ECL4 for MRK-16 and 4E3 binding. These results demonstrate that discontinuous epitopes for MRK-16, UIC2 and 4E3 are located in the same regions of ECL1 and 4 of the multidrug transporter.

Structural model of the Ca V 1.2 pore.

Understanding the structure and functional mechanisms of voltage-gated calcium channels remains a major task in membrane biophysics. In the absence of three dimensional structures, homology modeling techniques are the method of choice, to address questions concerning the structure of these channels. We have developed models of the open Ca(V)1.2 pore, based on the crystal structure of the mammalian voltage-gated potassium channel K(V)1.2 and a model of the bacterial sodium channel NaChBac. Our models are developed to be consistent with experimental data and modeling criteria. The models highlight major differences between voltage-gated potassium and calcium channels in the P segments, as well as the inner pore helices. Molecular dynamics simulations support the hypothesis of a clockwise domain arrangement and experimental observations of asymmetric calcium channel behavior. In the accompanying paper these models were used to study structural effects of a channelopathy mutation.

Mechanotransduction through the cytoskeleton.

We constructed a model cytoskeleton to investigate the proposal that this interconnected filamentous structure can act as a mechano- and signal transducer. The model cytoskeleton is composed of rigid rods representing actin filaments, which are connected with springs representing cross-linker molecules. The entire mesh is placed in viscous cytoplasm. The model eukaryotic cell is submitted to either shock wave-like or periodic mechanical perturbations at its membrane. We calculated the efficiency of this network to transmit energy to the nuclear wall as a function of cross-linker stiffness, cytoplasmic viscosity, and external stimulation frequency. We found that the cytoskeleton behaves as a tunable band filter: for given linker molecules, energy transmission peaks in a narrow range of stimulation frequencies. Most of the normal modes of the network are spread over the same frequency range. Outside this range, signals are practically unable to reach their destination. Changing the cellular ratios of linker molecules with different elastic characteristics can control the allowable frequency range and, with it, the efficiency of mechanotransduction.

STAM: simple transmembrane alignment method.

MOTIVATION: The database of transmembrane protein (TMP) structures is still very small. At the same time, more and more TMP sequences are being determined. Molecular modeling is an interim answer that may bridge the gap between the two databases. The first step in homology modeling is to achieve a good alignment between the target sequences and the template structure. However, since most algorithms to obtain the alignments were constructed with data derived from globular proteins, they perform poorly when applied to TMPs. In our application, we automate the alignment procedure and design it specifically for TMP. We first identify segments likely to form transmembrane alpha-helices. We then apply different sets of criteria for transmembrane and non-transmembrane segments. For example, the penalty for insertion/deletions in the transmembrane segments is much higher than that of a penalty in the loop region. Different substitution matrices are used since the frequencies of occurrence of the various amino acids differ for transmembrane segments and water-soluble domains. RESULTS: This program leads to better models since it does not treat the protein as a single entity with the same properties, but accounts for the different physical properties of the various segments. STAM is the first multisequence alignment program that is directly targeted at transmembrane proteins. AVAILABILITY: Source code and installation package are available on request from the authors. Web access is currently implemented.