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The transport of oxygen and other nonelectrolytes across lipid membranes is known to depend on both diffusion and solubility in the bilayer, and to be affected by changes in the physical state and by the lipid composition, especially the content of cholesterol and unsaturated fatty acids. However, it is not known how these factors affect diffusion and solubility separately. Herein we measured the partition coefficient of oxygen in liposome membranes of dilauroyl-, dimiristoyl- and dipalmitoylphosphatidylcholine in buffer at different temperatures using the equilibrium-shift method with electrochemical detection. The apparent diffusion coefficient was measured following the fluorescence quenching of 1-pyrenedodecanoate inserted in the liposome bilayers under the same conditions. The partition coefficient varied with the temperature and the physical state of the membrane, from below 1 in the gel state to above 2.8 in the liquid-crystalline state in DMPC and DPPC membranes. The partition coefficient was directly proportional to the partial molar volume and was then associated to the increase in free-volume in the membrane as a function of temperature. The apparent diffusion coefficients were corrected by the partition coefficients and found to be nearly the same, with a null dependence on viscosity and physical state of the membrane, probably because the pyrene is disturbing the surrounding lipids and thus becoming insensitive to changes in membrane viscosity. Combining our results with those of others, it is apparent that both solubility and diffusion increase when increasing the temperature or when comparing a membrane in the gel to one in the fluid state. [ABSTRACT FROM AUTHOR]

The key events in regulating cardiac muscle contraction involve Ca2+ binding to and release from cTnC (troponin C) and structural changes in cTnC and other thin filament proteins triggered by Ca2+ movement. Single mutations L29Q and G159D in human cTnC have been reported to associate with familial hypertrophic and dilated cardiomyopathy, respectively. We have examined the effects of these individual mutations on structural transitions in the regulatory N-domain of cTnC triggered by Ca2+ binding and dissociation. This study was carried out with a double mutant or triple mutants of cTnC, reconstituted into troponin with tryptophanless cTnI and cTnT. The double mutant, cTnC(L12W/N51C) labeled with 1,5-IAEDANS at Cys-51, served as a control to monitor Ca2+-induced opening and closing of the N-domain by Förster resonance energy transfer (FRET). The triple mutants contained both L12W and N51C labeled with 1,5-IAEDANS, and either L29Q or G159D. Both mutations had minimal effects on the equilibrium distance between Trp-12 and Cys-51-AEDANS in the absence or presence of bound Ca2+. L29Q had no effect on the closing rate of the N-domain triggered by release of Ca2+, but reduced the Ca2+-induced opening rate. G159D reduced both the closing and opening rates. Previous results showed that the closing rate of cTnC N-domain triggered by Ca2+ dissociation was substantially enhanced by PKA phosphorylation of cTnI. This rate enhancement was abolished by L29Q or G159D. These mutations alter the kinetics of structural transitions in the regulatory N-domain of cTnC that are involved in either activation (L29Q) or deactivation (G159D). Both mutations appear to be antagonistic toward phosphorylation signaling between cTnI and cTnC. [ABSTRACT FROM AUTHOR]

Upon Ca[sup 2+] activation of cardiac muscle, several structural changes occur in the troponin subunits. These changes include the opening of the cardiac troponin C (cTnC) N-domain, the change of secondary structure of the inhibitory region of cardiac troponin I (cTnI), and the change in the separation between these two proteins in the cTnC-cTnI interface. We have used Förster resonance energy transfer in Ca[sup 2+] titration and stoppedflow experiments to delineate these transitions using a reconstituted cardiac troponin. Energy transfer results were quantified to yield time-dependent profiles of changes in intersite distances during Ca[sup 2+] dissociation. The closing of the cTnC N-domain induced by release of regulatory Ca[sup 2+] from cTnC occurs in one step (t[sub ½] ∼ 5 ms), and this transition is not affected by Ca[sup 2+] release from the C-domain. The other two transitions triggered by Ca[sup 2+] dissociation are biphasic with the fast phase (t½ ∼ 5 ms) correlated with Ca[sup 2+] release from the cTnC N-domain. These transitions are slower than the release of bound regulatory Ca[sup 2+] (t[sub ½] 3.6 ms) and are coupled to one another in a cooperative manner in restoring their conformations in the deactivated state. The kinetic results define the magnitudes of structural changes relevant in Ca[sup 2+] switching between activation and deactivation of cardiac muscle contraction. [ABSTRACT FROM AUTHOR]

Focuses on the role of calcium ion-induced conformational transition in the inhibitory and regulatory regions of cardiac troponin I. Initiation of the cardiac muscle activation by the binding of calcium to the single N domain; Changes caused by binding to cardiac muscle troponin I; Use of Forster resonance energy transfer to measure intra- cardiac muscle troponin I.