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ABSTRACT: A cytotoxic peptide, polytheonamide B (pTB), from marine sponge was examined for cytotoxic spectrum and specific activity to mammalian cells was demonstrated. pTB is composed of alternative D- and L-amino acid residues throughout the 48-mer peptide. This suggests the formation of a β-helix similar to gramicidin channels. Planar bilayer experiments revealed that pTB forms monovalent cation-selective channels, being compatible with the inner pore diameter of ∼4Å for a β-helical structure. pTB penetrated vectorially into the membrane, formed a channel by means of a single molecule, and remained in the membrane. These functional properties may account for specific cytotoxic activity.
FEBS letters 09/2010; 584(18):3995-9. · 3.54 Impact Factor
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ABSTRACT: The human ether-à-go-go related gene product (HERG) channel is essential for electrical activity of heart cells, and block of this channel by many drugs leads to lethal arrhythmias. Tyr(652) and Phe(656) of the sixth transmembrane helix are candidates for the drug binding site. In the tetrameric HERG channel, a drug with asymmetric structure should interact unevenly with multiple residues from different subunits. To elucidate the topology of the drug-binding site, we constructed tandem dimers of HERG channels and the aromatic Tyr(652) and Phe(656) residues were replaced by alanine singly or doubly. Eight types of HERG channels, including homotetrameric mutants, having different numbers and arrangements of aromatic residues at the blocking site, were studied. Effects of cisapride on channels expressed in Xenopus laevis oocytes were examined electrophysiologically. The inhibition constants (K(i)) were increased significantly as the diagonal Tyr(652) were deleted, whereas those for the diagonal Phe(656)-deleted mutant were not changed. These results suggest that Tyr(652) residues from adjacent subunits contributed to the binding. Two types of double mutants of tandem dimers showed significantly distinct affinities, suggesting that the coexistence of Tyr(652) and Phe(656) on a subunit in diagonal position is crucial to having a high affinity. Thermodynamic double-mutant cycle analyses revealed interactions between Tyr(652) and Phe(656) upon binding. The kinetics and voltage-dependence of blocking suggested transitions of the binding site from low to high affinity. These approaches using a set of mutant HERG channels gave a dynamic picture of the spatial arrangements of residues that contribute to the drug-channel interaction.
Molecular pharmacology 07/2008; 73(6):1643-51. · 4.53 Impact Factor
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ABSTRACT: Ion channels are signal transduction molecules that switch ion permeation pathways on and off (gating). Crystal structures of several kinds of potassium channels have revealed open and closed conformations, which provide static pictures of gating status. Here we studied KcsA potassium channels undergoing conformational changes at the single-molecule level. A KcsA channel with a gold nanocrystal attached was irradiated by white X-rays and motions of the diffraction spot from the nanocrystal were tracked in real time. Upon gating, the KcsA channels twisted around the axis of the pore. These conformational changes were prevented by an open-channel blocker, tetrabuthylammonium. Random clockwise and counterclockwise twisting in the range of several tens of degrees originated in the transmembrane domain and was transmitted to the cytoplasmic domain. This coupling suggests a mechanical interplay between the transmembrane and cytoplasmic domains.
Cell 02/2008; 132(1):67-78. · 32.40 Impact Factor
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ABSTRACT: KcsA is the first potassium channel for which the molecular structure was revealed. However, the high resolution structural information is limited to the transmembrane domain, and the dynamic picture of the full KcsA channel remains unsolved. We have developed a new approach to investigate the surface structure of proteins, and we applied this method to investigate the full length of the KcsA channel. Single-cysteine substitution was introduced into 25 sites, and specific reaction of these mutated channels to a bare surface of a flat gold plate was evaluated by surface plasmon resonance measurements. The surface plasmon resonance signals revealed the highest exposure for the mutant of the C-terminal end. When the gate of the KcsA channel is kept closed at pH 7.5, the extent of exposure showed periodic patterns for the consecutive sites located in the cytoplasmic (CP) and N-terminal domain. This suggests that these stretches take the alpha-helical structure. When the channel was actively gated at pH 4.0, many sites in the CP domain became exposed. Compared with the rigid structure in pH 7.5, these results indicate that the CP domain became loosely packed upon active gating. The C-terminal end of the M2 helix is a moving part of the gate, and it is exposed to the outer surface slightly at pH 4.0. By adding a channel blocker, tetrabutylammonium, the gate is further exposed. This suggests that in the active gating tetrabutylammonium keeps the gate open rather than being trapped in the central cavity.
Journal of Biological Chemistry 10/2006; 281(38):28379-86. · 4.77 Impact Factor
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ABSTRACT: The streaming potential (V(stream)) is a signature feature of ion channels in which permeating ions and water molecules move in a single file. V(stream) provides a quantitative measure of the ion and water flux (the water-ion coupling ratio), the knowledge of which is a prerequisite for elucidating the mechanisms of ion permeation. We have developed a method to measure V(stream) with the whole-cell patch-clamp configuration. A HEK293 cell stably expressing the HERG potassium channel was voltage clamped and exposed to hyperosmotic solutions for short periods of time (<1 s) by an ultrafast solution switching system (the osmotic pulse [quick jump-and-away] method). The reversal potentials were monitored by a series of voltage ramps before, during, and after the osmotic pulse. The shifts of the reversal potentials immediately after the osmotic jump gave V(stream). In symmetrical K+ solutions (10 mM), the V(stream)s measured at different osmolalities showed a linear relationship with a slope of -0.7 mV/DeltaOsm, from which the water-ion coupling ratio (n, the ratio of the flux of water to the flux of cations; Levitt, D.G., S.R. Elias, and J.M. Hautman. 1978. Biochim. Biophys. Acta. 512:436-451) was calculated to be 1.4. In symmetrical 100 mM K+ solutions, the coupling ratio was decreased significantly (n = 0.9), indicating that the permeation process through states with increased ion occupancy became significant. We presented a diagrammatic representation linking the water-ion coupling ratio to the mode of ion permeation and suggested that the coupling ratio of one may represent the least hydrated ion flux in the single-file pore.
The Journal of General Physiology 12/2005; 126(5):529-38. · 3.84 Impact Factor
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ABSTRACT: Cross-linking of proteins catalyzed by tissue transglutaminase has been suggested to play key roles in a variety of cellular events, including cell apoptosis and human pathogenesis (e.g. polyglutamine and Alzheimer diseases). It has often been suggested that tissue transglutaminase enhances aggregation and precipitation of damaged or pathogenic proteins. To ascertain whether this is accurate, we investigated the effects of tissue transglutaminase-catalyzed modulation on the aggregation of structurally damaged and unfolded proteins. Our results indicated that the aggregation and precipitation of some unfolded proteins were inhibited by transglutaminasecatalyzed reaction, although the effect was strongly dependent upon the target protein species. To elucidate the molecular events underlying the inhibitory effect, extensive analysis was performed with regard to reduced beta-lactoglobulin using a number of techniques, including chromatography and spectroscopy. The results indicated that cross-linking yields high molecular weight soluble polymers but inhibits the growth of insoluble aggregates. The cross-linked beta-lactoglobulin retained stable secondary structures with a hydrophobic core. We concluded that the transglutaminase-catalyzed intermolecular cross-linking did not necessarily enhance protein aggregation but could sometimes have a suppressive effect. The results of the present study suggested that tissue transglutaminase modifies aggregation and deposition of damaged or pathogenic proteins in vivo in a wide variety of manners depending on the target protein species and solution conditions.
Journal of Biological Chemistry 05/2005; 280(17):17520-5. · 4.77 Impact Factor
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ABSTRACT: The fast inactivation of the human ether-à-go-go related gene product (HERG) channel is a form of C-type inactivation and is decelerated by external tetraethylammonium (TEA) and potassium. From the time constant of inactivation, the dissociation constants of TEA (K(TEA)) and potassium (K(K)) to the inactivation-impeding site were evaluated. K(TEA) was found to exhibit unexpected voltage dependence: K(TEA) decreased with depolarization. This was opposite the voltage dependence of K(K) on inactivation, in which permeating potassium impeded closure of the inactivation gate upon binding to a site in the pore (a "foot-in-the-door" mechanism). Further experiments on inactivation revealed anomalous mole fraction effects between permeating alkali cations and TEA, while no anomalous effects were seen between permeating ion species (K+, Rb+, Cs+). The results indicate that TEA and permeating ions impede inactivation through binding to different but closely interacting sites. K(TEA) was influenced by permeating ions through their bindings in the pore. As the size of the occupied ion was increased the dissociation constant of TEA to the ion-occupied pore decreased. Thus, we conclude that an ion bound to the inactivation-impeding site in the selectivity filter is located in close proximity to TEA bound at the entrance of the filter. The order of affinity of alkali cations for the inactivation-impeding site, Rb+ > Cs+ > K+, indicated that the selectivity of the site differed significantly from permeation selectivity, K+ > Rb+ > Cs+.
The Japanese Journal of Physiology 02/2003; 53(1):25-34. · 1.04 Impact Factor
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