Calcium binding to mixed cardiolipin-phosphatidylcholine bilayers as studied by deuterium nuclear magnetic resonance

Abstract

Calcium binding to bilayer membranes containing cardiolipin (CDL) mixed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated by using phosphorus-31 and deuterium nuclear magnetic resonance (NMR) spectroscopy. The destabilizing effect of Ca2+ on CDL bilayers, including the formation of hexagonal H11 and isotropic phases, was eliminated when CDL was mixed with sufficiently large proportion of POPC. Thus, for the mixture CDL-POPC (1:9 M/M), 31P NMR spectra retained a line shape typical of fluid bilayer lipids even in the presence of 1.0 M Ca2+. Specifically head-group-deuteriated CDL or POPC showed in this mixture 2H NMR spectra indicating that both lipids remained in a fluidlike bilayer at Ca2+ concentrations up to 1.0 M. Any phase separation of Ca2-CDL clusters could be excluded. The residence time of Ca2+ at an individual head group binding site was shorter and 10-6 s. The deuterium quadrupole splitting, .DELTA..nu.Q, of POPC deuteriated at the .alpha.-methylene segment of the choline head group was found to be linearly related to the number of bound calcium ions, X2, for the CDL-POPC (1:9 M/M) mixture. The effective surface charge density, .sigma., could be determined from the measured amount of bound Ca2+. Subsequently, the surface potential, .psi.o, and the concentration of free Ca2+ ions at the plane of ion binding were calculated by employing the Gouy-Chapman theory. Various possible models of the equilibrium binding of Ca2+ could then be tested. The Langmuir adsorption isotherm with a Ca2+ binding constant of 15.5 M-1 gave the best fit to the experimental data. Sodium binding was comparatively weak with a binding constant of 0.75 M-1. A comparison of Ca2+ binding constants for different membrane lipid with a binding constant of 0.75 M-1. A comparison of Ca2+ binding constants for different membrane lipid compositions revealed that the increase in Ca2+ binding observed in the presence of negatively charged lipids was predominantly an electrostatic effect rather than being due to differences in the intrinsic Ca2+ affinity. Ca2+ was able to reduce the surface potential by binding and neutralizing negative surface charges in addition to having a screening effect.