Journal of Membrane Biology最新文献

Vigna radiata H⁺-translocating pyrophosphatases (VrH⁺-PPases, EC 3.6.1.1) are present in various endomembranes of plants, bacteria, archaea, and certain protozoa. They transport H⁺ into the lumen by hydrolyzing pyrophosphate, which is a by-product of many essential anabolic reactions. Although the crystal structure of H⁺-PPases has been elucidated, the H⁺ translocation mechanism of H⁺-PPases in the solution state remains unclear. In this study, we used hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to investigate the dynamics of H⁺-PPases between the previously proposed R state (resting state, Apo form), I state (intermediate state, bound to a substrate analog), and T state (transient state, bound to inorganic phosphate). When hydrogen was replaced by proteins in deuterium oxide solution, the backbone hydrogen atoms, which were exchanged with deuterium, were identified through MS. Accordingly, we used deuterium uptake to examine the structural dynamics and conformational changes of H⁺-PPases in solution. In the highly conserved substrate binding and proton exit regions, HDX-MS revealed the existence of a compact conformation with deuterium exchange when H⁺-PPases were bound with a substrate analog and product. Thus, a novel working model was developed to elucidate the in situ catalytic mechanism of pyrophosphate hydrolysis and proton transport. In this model, a proton is released in the I state, and the TM5 inner wall serves as a proton piston.

For a long time, the physiological activity of TRP ion channels and the response to various stimuli have been the focus of attention, and the physiological functions mediated by ion channels have subtle links with the occurrence of various diseases. Our group has been engaged in the study of ion channels. In recent years, the report rate of TRPA1, the only member of the TRPA subfamily in the newly described TRP channel, has been very high. TRPA1 channels are not only abundantly expressed in peptidergic nociceptors but are also found in many nonneuronal cell types and tissues, and through the regulation of Ca²⁺ influx, various neuropeptides and signaling pathways are involved in the regulation of nerves, respiration, circulation, and various diseases and inflammation throughout the body. In this review, we mainly summarize the effects of TRPA1 on various systems in the body, which not only allows us to have a more systematic and comprehensive understanding of TRPA1 but also facilitates more in-depth research on it in the future.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Structural analysis of CFTR has identified a narrow, hydrophobic region close to the extracellular end of the open channel pore that may function as a selectivity filter. The present study combines comprehensive mutagenesis of hydrophobic amino-acid side-chains within the selectivity filter with functional evaluation of channel Cl^- conductance and anion selectivity. Among these hydrophobic amino-acids, one (F337) appears to play a dominant role in determining both conductance and selectivity. Anion selectivity appears to depend on both side-chain size and hydrophobicity at this position. In contrast, conductance is disrupted by all F337 mutations, suggesting that unique interactions between permeating Cl^- ions and the native phenylalanine side-chain are important for conductance. Surprisingly, a positively charged lysine side-chain can be substituted for several hydrophobic residues within the selectivity filter (including F337) with only minor changes in pore function, arguing against a crucial role for overall hydrophobicity. These results suggest that localized interactions between permeating anions and amino-acid side-chains within the selectivity filter may be more important in determining pore functional properties than are global features such as overall hydrophobicity.

Using a flexibility prediction algorithm and in silico structural modeling, we have calculated the intrinsic flexibility of several magainin derivatives. In the case of magainin-2 (Mag-2) and magainin H2 (MAG-H2) we have found that MAG-2 is more flexible than its hydrophobic analog, Mag-H2. This affects the degree of bending of both peptides, with a kink around two central residues (R10, R11), whereas, in Mag-H2, W10 stiffens the peptide. Moreover, this increases the hydrophobic moment of Mag-H2, which could explain its propensity to form pores in POPC model membranes, which exhibit near-to-zero spontaneous curvatures. Likewise, the protective effect described in DOPC membranes for this peptide regarding its facilitation in pore formation would be related to the propensity of this lipid to form membranes with negative spontaneous curvature. The flexibility of another magainin analog (MSI-78) is even greater than that of Mag-2. This facilitates the peptide to present a kind of hinge around the central F12 as well as a C-terminal end prone to be disordered. Such characteristics are key to understanding the broad-spectrum antimicrobial actions exhibited by this peptide. These data reinforce the hypothesis on the determinant role of spontaneous membrane curvature, intrinsic peptide flexibility, and specific hydrophobic moment in assessing the bioactivity of membrane-active antimicrobial peptides.

In this study a lipid bilayer membrane model was used in which the bilayer is tethered to a solid substrate with molecular tethers. Voltage-current (V-I) measurements of the tethered bilayer membranes (tBLM) and tBLM with benzyl alcohol (BZA) incorporated in their structures, were measured using triangular voltage ramps of 0-500 mV. The temperature dependence of the conductance deduced from the V-I measurements are described. An evaluation of the activation energies for electrical conductance showed that BZA decreased the activation/ Born energies for ionic conduction of tethered lipid membranes. It is concluded that BZA increased the average pore radius of the tBLM.