Cations induce shape remodeling of negatively charged phospholipid membranes.

The divalent cation Ca2+ is a key component in many cell signaling and membrane trafficking pathways. Ca2+ signal transduction commonly occurs through interaction with protein partners. However, in this study we show a novel mechanism by which Ca2+ may impact membrane structure. We find an asymmetric concentration of Ca2+ across the membrane triggers deformation of membranes containing negatively charged lipids such as phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Membrane invaginations in vesicles were observed forming away from the leaflet with higher Ca2+ concentration, showing that Ca2+ induces negative curvature. We hypothesize that the negative curvature is produced by Ca2+-induced clustering of PS and PI(4,5)P2. In support of this notion, we find that Ca2+-induced membrane deformation is stronger for membranes containing PI(4,5)P2, which is known to more readily cluster in the presence of Ca2+. The observed Ca2+-induced membrane deformation is strongly influenced by Na+ ions. A high symmetric [Na+] across the membrane reduces Ca2+ binding by electrostatic shielding, inhibiting Ca2+-induced membrane deformation. An asymmetric [Na+] across the membrane, however, can either oppose or support Ca2+-induced deformation, depending on the direction of the gradient in [Na+]. At a sufficiently high asymmetric Na+ concentration it can impact membrane structure in the absence of Ca2+. We propose that Ca2+ works in concert with curvature generating proteins to modulate membrane curvature and shape transitions. This novel structural impact of Ca2+ could be important for Ca2+-dependent cellular processes that involve the creation of membrane curvature, including exocytosis, invadopodia, and cell motility.

Magic angle spinning 31P NMR spectroscopy reveals two essentially identical ionization states for the cardiolipin phosphates in phospholipid liposomes.

Specific membrane lipid composition is crucial for optimized structural and functional organization of biological membranes. Cardiolipin is a unique phospholipid and important component of the inner mitochondrial membrane. It is involved in energy metabolism, inner mitochondrial membrane transport, regulation of multiple metabolic reactions and apoptotic cell death. The physico-chemical properties of cardiolipin have been studied extensively but despite all these efforts there is still lingering controversy regarding the ionization of the two phosphate groups of cardiolipin. Results obtained in the 1990s and early 2000s suggested that cardiolipin has two disparate pKa values where one of the protons was proposed to be stabilized by an intramolecular hydrogen bond. This has led to extensive speculations on the roles of these two putative ionization states of cardiolipin in mitochondria. More recently the notion of two pKa values has been challenged and rejected by several groups. These studies relied on external measurements of proton adsorption or electrophoretic mobility of membranes but did not take into account the low pH phase behavior and chemical stability of cardiolipin. Here we used 31P NMR to show that in the physiologically relevant membrane phospholipid environment, cardiolipin carries two negative charges at physiological pH. We additionally demonstrate the pH dependent phase behavior and chemical stability of cardiolipin containing membranes.