David G. Rhodes

Interaction of 1,4 dihydropyridine calcium channel antagonists with biological membranes: Lipid bilayer partitioning could occur before drug binding to receptors ☆

The binding of dihydropyridine calcium channel agonists and antagonists to receptors in cardiac sarcolemmal membranes is a complex reaction that may involve an interaction with the lipid bilayer matrix of the sarcolemma. Membrane/buffer partition coefficients (lambda) for three dihydropyridine calcium channel antagonists were measured directly in the sarcolemma and sarcoplasmic reticulum membranes and found to be in the range of 5,000 to 150,000. These drugs interact primarily with the membrane bilayer component of these membranes but may also bind to non-receptor proteins. The intrinsic forward rate constants for dihydropyridine binding to sarcolemmal calcium channel receptors were apparently not strongly dependent on their membrane partition coefficients. For example, nimodipine (lambda = 6300) had a forward rate constant of 6.8 +/- 0.6 x 10(6)/M/S, whereas the forward rate constant for Bay P 8857 (lambda = 149,000) was 1.4 +/- 0.8 x 10(7)/M/S. Model calculations for this binding reaction demonstrated that since these drugs are highly lipid soluble, the dependence of these rates on lipid solubility would probably not be reflected in the experimental forward rate constants. In addition, the intrinsic forward rate constant for nimodipine binding to sarcolemmal calcium channel receptors was found not to be linearly dependent on the viscosity of the buffer medium over a five-fold range. The rate of nonspecific (non-receptor protein) drug binding to highly purified sarcoplasmic reticulum membranes essentially devoid of specific receptors for these drugs appears to be extremely fast, at least 10(3) times faster than specific drug binding to the receptor in the sarcolemma. Thus, it appears that partitioning into the lipid bilayer matrix of the sarcolemma could be a general property of dihydropyridine calcium channel antagonists and may be a prerequisite for their binding to sarcolemmal membrane receptors.

Structure and location of amiodarone in a membrane bilayer as determined by molecular mechanics and quantitative x-ray diffraction.

Amiodarone is a drug used in the treatment of cardiac arrhythmias and is believed to have a persistent interaction with cellular membranes. This study sought to examine the structure and location of amiodarone in a membrane bilayer. Amiodarone has a high membrane partition coefficient on the order of 10(6). Small angle x-ray diffraction was used to determine the position of the iodine atoms of amiodarone in dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under conditions of low temperature and hydration where the DPPC bilayer is in the gel state. The time-averaged position of the iodine atoms was determined to be approximately 6 A from the center (terminal methyl region) of the lipid bilayer. A dielectric constant of kappa = 2, which approximates that of the bilayer hydrocarbon core region, was used in calculating a minimum energy structure for membrane-bound amiodarone. This calculated structure when compared with the crystal structure of amiodarone demonstrated that amiodarone could assume a conformation in the bilayer significantly different from that in the crystal. The results reported here are an attempt to correlate the position of a membrane-active drug in a lipid bilayer with its time-averaged conformation. This type of analysis promises to be of great use in the design of drugs with greater potency and higher specificity.

Structure of polymerizable lipid bilayers. I—1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine, a tubule-forming phosphatidylcholine

This report presents the first X-ray diffraction data on diacetylenic phospholipids. The tubule-forming polymerizable lipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC), was studied by low angle X-ray diffraction from partially dehydrated oriented multibilayers in both polymerized and unpolymerized form. Bilayers of this material were found to be highly ordered, yielding as many as 16 orders of lamellar diffraction, in both the polymerized and unpolymerized states. The unit cell dimension was very small for a lipid of this size. In addition to the features usually observed in the electron density profile structure of phospholipid bilayers, the electron-dense diacetylenic portions of the fatty acyl chain produced electron density maxima at two well-defined levels on each side of the bilayer approximately 15 A and 9 A from the bilayer midplane. A model molecular conformation deduced from the one-dimensional electron density map features all-trans acyl chains tilted at approximately 28 degrees from the bilayer normal that are interdigitated with chains of the opposing monolayer by approximately two carbons at the bilayer center. The linear diacetylene moieties on beta- and gamma-chains appear at different positions along the bilayer normal axis and are roughly parallel to the bilayer surface. This model is discussed in terms of a polymerization mechanism.

The mechanism of formation of lipid tubules from liposomes

Abstract Certain diacetylenic phospholipids from liposomes in water above their chain melting transitions, which, if slowly cooled, quantitatively convert to hollow tubular structures about 1 μm in diameter and as long as hundreds of micrometers. To elucidate the nature of the conversion process, freeze fracture electron microscopy was utilized to examine samples that were rapidly quenched during tubule formation. Many transitional structures were observed, typically liposomes partially wrapped around nascent tubules. This is consistent with real-time imaging by optical microscopy indicating tubule growth by continuous transfer of lipid bilayers from liposomes by a rolling-up process. The mechanism of the conversion process, combined with preliminary X-ray scattering data indicating unusual packing of the lipid molecules, suggests an explanation for the efficiency of the conversion process and why the tubule is a favorable microstructure for the crystalline lipid.

Structure of polymerizable lipid bilayers. IV, Mixtures of long chain diacetylenic and short chain saturated phosphatidylcholines and analogous asymmetric isomers

Polymerization of 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) is enhanced by addition of short-chain saturated phosphatidylcholines such as 1,2-dinonanoyl-sn-glycero-3- phosphocholine (DNPC). Because of well established constraints on the topochemical polymerisation process, we undertook structure-based experiments to determine the nature of this effect. Two hypotheses were tested: (a) that the DNPC crystalized the proximal (m) and disordered the distal (n) methylene segments of DC8,9PC, thus providing flexibility to accommodate the conformational change upon polymer formation, or (b) that the DNPC forced lateral displacement of DC8,9PC, which would then allow interdigitation of these segments with those of the opposing monolayer and potentially more crystalline alignment of the diacetylene. Low angle X-ray diffraction studies do not support the interdigiated chain model. However, these measurements also indicate that the two lipid species may be phase separated under many conditions. An analogous structure, 1-(tricosa-10,12-diynoyl)2-nonanoyl-sn-glycero-3- phosphocholine (C8,9NPC) did not polymerize, and low angle X-ray diffraction studies indicate that bilayers of this lipid were interdigitated such that the terminal methyl group of the tricosadiynoyl chain on each lipid in the bilayer was adjacent to the diacetylenic moiety of a lipid on the opposing monolayer. Implications of these findings pertinent to identifying significant factors in polymerization of diacetylenic phospholipid bilayers are discussed.

Order in Diacetylenic Microstructures

Abstract Polymerizable diacetylenic lipids form a long, hollow tubular bilayer microstructure that has been characterized by microscopy, spectroscopy and x-ray diffraction. The lipid monomers are highly ordered at the molecular level, and at high density the microstructures themselves align giving macroscopic order.

Structure of polymerizable lipid bilayers. III, Two heptacosadiynoyl phosphatidylcholine isomers

Abstract The structures of hydrated bilayers of 1,2-bis(10,12-heptacosadiynoyl)-sn-glycero-3-phosphocholine (DC8, 13PC) and 1,2-bis-(11,13-heptacosadiynoyl)-sn-glycero-3-phosphocholine (DC9, 12PC) were investigated using low-angle X-ray diffraction. Multilayer stacks of these diacetylenic phosphatidylcholine derivatives were highly ordered, typically yielding up to 16 orders of lamellar diffraction. The unit cell repeat was smaller than expected for lipids of this size. The electron density profiles of both 27 carbon lipid bilayers contained peaks at 10 A and 16 A from the bilayer center due to the diacetylene moieties. The electron density profile for DC8, 13PC was similar to that of the previously studied polymerizable lipid. DC8, 9PC, except that in the DC8, 13PC profile, the distance between diacetylene electron density maxima on opposing sides of the bilayer was ∼4 A further apart. The electron density profile of DC9, 12PC showed that the peaks corresponding to the diacetylenic regions were at approximately the same positions as in the DC8, 13PC bilayers, but were less well resolved. Acyl chains in model structures inferred from the electron density data are tilted relative to the bilayer normal and may not be as well ordered as previously believed.

Structure of polymerizable lipid bilayers: water profile of a diacetylenic lipid bilayer using elastic neutron scattering

Elastic neutron scattering experiments have been used to study the hydration of multibilayers of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC). Previously published FTIR spectroscopic data had suggested, based on shifts in the carbonyl (C = O) stretch frequencies, that the phosphocholine headgroup in these polymerizable lipid bilayers was much less hydrated than that of saturated phosphatidylcholines. Our results demonstrate that the DC8,9PC headgroup is at least as well hydrated as that of dipalmitoylphosphatidylcholine (DPPC), a saturated lipid, under the same conditions.