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The molecular structures of the various intrinsic lipids in membranes regulate lipid-protein interactions. These different lipid structures with unique volumes produce different lipid molecular packing stresses/lateral stresses in lipid membranes. Most studies examining lipid packing effects have used phosphatidylcholine and phosphatidylethanolamine (PE), which are the main phospholipids of eukaryotic cell membranes. In contrast, Gram-negative or Gram-positive bacterial membranes are composed primarily of phosphatidylglycerol (PG) and PE, and the physical and thermodynamic properties of each acyl chain in PG at the molecular level remain unresolved. In this study, we used 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG, 16:0-18:1 PG) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (PAPG, 16:0-20:4 PG) to prepare lipid bilayers (liposome) with the rod-type fluorescence probe DPH. We measured the lipid packing conditions by determining the rotational freedom of DPH in POPG or PAPG bilayers. Furthermore, we investigated the effect of different monoacyl chains on a K + channel (KcsA) structure when embedded in POPG or PAPG membranes. The results revealed that differences in the number of double bonds and carbon chain length in the monoacyl chain at sn-2 affected the physicochemical properties of the membrane and the structure and orientation of KcsA., Competing Interests: Declaration of competing interest FT (Department of Cellular Architecture Studies, Institute of Tropical Medicine, Nagasaki University) is supported by Shionogi & Co., Ltd. Shionogi & Co., Ltd., had no role in the experiments, data analysis, writing, or reviewing of this study., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Although interactions between lipids and membrane proteins (MPs) have been considered crucially important for understanding the functions of lipids, lack of useful and convincing experimental methods has hampered the analysis of the interactions. Here, we developed a surface plasmon resonance (SPR)-based concise method for quantitative analysis of lipid-MP interactions, coating the sensor chip surface with self-assembled monolayer (SAM) with C 6 -chain. To develop this method, we used bacteriorhodopsin (bR) as an MP, and examined its interaction with various types of lipids. The merits of using C 6 -SAM-modified sensor chip are as follows: (1) alkyl-chains of SAM confer a better immobilization of MPs because of the efficient preconcentration due to hydrophobic contacts; (2) SAM provides immobilized MPs with a partial membranous environment, which is important for the stabilization of MPs; and (3) a thinner C 6 -SAM layer (1 nm) compared with MP size forces the MP to bulge outward from the SAM surface, allowing extraneously injected lipids to be accessible to the hydrophobic transmembrane regions. Actually, the amount of bR immobilized on C 6 -SAM is 10 times higher than that on a hydrophilic CM5 sensor chip, and AFM observations confirmed that bR molecules are exposed on the SAM surface. Of the lipids tested, S-TGA-1, a halobacterium-derived glycolipid, had the highest specificity to bR with a nanomolar dissociation constant. This is consistent with the reported co-crystal structure that indicates the formation of several intermolecular hydrogen bonds. Therefore, we not only reproduced the specific lipid-bR recognition, but also succeeded in its quantitative evaluation, demonstrating the validity and utility of this method., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mast cell degranulating peptide (MCD) induces long-term potentiation (LTP) in the CA1 region of the hippocampus. MCD has been shown to bind to a voltage-dependent A-type potassium channel with high-affinity (less than 1 nM). However, the concentration necessary to induce LTP is more than 500 nM, suggesting that some other functions of MCD are also fundamental to LTP induction. The concentration of MCD required for LTP induction was greatly reduced by preactivating G proteins. This fact suggests that G protein activation by MCD also plays an important role in LTP induction. MCD-binding proteins were purified from rat brain. G proteins were found to exist in a non-denatured state in this affinity-purified fraction. When reconstituted into a planar lipid bilayer membrane, a potassium-selective and voltage-dependent current could be observed. This channel was blocked by MCD at a high concentration equal to the effective concentration for G protein activation. Addition of GTP-gamma-S significantly blocked the reconstituted current. Thus, we identified a pathway for LTP induction by MCD in which high concentrations of MCD activate G protein which in turns leads to blocking of a potassium channel.

To determine the purity (q) of Ca-chelating agents (such as EGTA and BAPTA) and their apparent stability constants to Ca2+ (K') using Ca(2+)-selective electrodes precisely, we have developed a new method using the double-log optimization. Free Ca2+ concentration was plotted against the ratio of the concentrations of Ca(2+)-bound to Ca(2+)-free chelator on double logarithmic co-ordinates in which a linear relationship with a slope of -1 must hold for the metal-chelator reaction with a stoichiometry of 1 to 1. Not only the q and K' values but also the selectivity coefficient of the electrode could be simultaneously estimated on the double-log plot through an optimizing method. Error analyses using a Monte Carlo simulation showed that the double-log plot is statistically more reliable and robust than the Scatchard plot and that the optimizing method is more objective and reliable than previous methods involving extrapolation and truncation procedures.

Apparent calcium stability constants and the purity of Ca-chelating agents were evaluated using Ca(2+)-selective electrodes by the double-log optimization method [Oiki S. Yamamoto T. Okada Y. (1994) Cell Calcium, 15, 199-208]. The method was amended to allow evaluation of the free Ca2+ concentrations contaminating electrolyte solutions. The value thus estimated (3.7 microM) was not significantly different from the total contaminating Ca content measured by atomic absorption spectroscopy. The purity of EGTA of different commercial brands was found to be in the range from 95.5-98.0% and was almost stable over several years of storage. The impurity of EGTA was completely eliminated by baking at 150 degrees C for 3 h. The purity of BAPTA decreased from 85.8 to 77.2% after storing for 3 months at -20 degrees C. The impurity of BAPTA was also abolished by the same drying procedure. At physiological pH (7.30), the apparent stability constants (K's) of EGTA were determined to be 7.13 and 6.97 in KCl-based solutions of 0.10 M and 0.16 M ionic strength, respectively, at 25 degrees C in the absence of Mg. At pH 7.30 and 0.20 M ionic strength K' values of BAPTA were 6.50 at 22 degrees C and 6.69 at 37 degrees C. The K' value increased with decreasing ionic strength.

By means of Ca2+- and K+-selective microelectrodes, the changes in intracellular free Ca2+ and K+ were measured during the hyperpolarizing responses induced by ATP, complement and beta-lipoprotein in mouse fibroblastic L cells. The cytoplasmic Ca2+ concentration [( Ca]i) was about 0.4 microM in the resting state. The hyperpolarizing responses always coincided with a phasic increase in [Ca]i. ATP or beta-lipoprotein induced about a 2-fold rise in [Ca]i, and complement did up to 3-fold. Both the hyperpolarizing responses and [Ca]i increases were prevented by removal of external Ca2+ or by application of a Ca-channel blocker, nifedipine. Quinine, a Ca-activated K-channel inhibitor, suppressed the hyperpolarizing responses but not the [Ca]i increases. During the hyperpolarizing response, the intracellular free K+ concentration gradually decreased from about 120 to 110 mM. Thus, it is concluded that ATP, complement and beta-lipoprotein caused a transient elevation of cytoplasmic free Ca2+ due to Ca2+ influxes, thereby inducing electrical membrane responses through activation of Ca-dependent K-channels in the fibroblasts.