In the presence of external influences such as transmembrane peptides or various types of proteins, when the surface of the membrane is disturbed, these types of membrane defects occur more often, increasing the rate of pore formation[111].
Mechanical or electrical stress applied on the membrane surface also influences the rate with which water pores appear[112].
The energy requirements for lipid flip–flops can also be reduced by other means.
The following example is related to the modification of the membrane lipids. Oxida- tive stress is a chemical disturbance in the body where toxic reactive oxygen species are generated in excess resulting in extensive oxidative damage to lipids and proteins in a cellular environment[113]. This oxidative damage includes lipid peroxidation, often truncating the tail of a lipid with a double bond, fundamentally changing the characteristics of the lipid within the bilayer. Its effect on the free energy of lipid translocation has been studied, and the free energy barrier of POPS flip–flop in a pure POPC bilayer was found to reduce by 20 kJ/mol when 20 mol-% of the POPC molecules were substituted with oxidiced variants[114]. Such an energy reduction meant a 103– 104-fold increase in the flip–flop rates of POPS. Another recent study revealed a correlation between the decrease in the free energy of POPS flip–flop with increased concentrations of peroxidiced POPC molecules in a similar system[115].
Oxidative stress has implications in many neurogenerative diseases[116], or apop- tosis, where rapid PS translocation to the cellular surface is the trigger[117].
spontaneous flip–flop
P-type flippase
ABC
flippase phospholipid scramblase extracellular
or lumen
cytosol
ATP ADP ATP ADP
Figure 4.1 Four different types of transbilayer lipid motion. Spontaneous flip–flop is slow, bidirectional and requires no external energy. P-type flippases facilitate inward movement of phospholipids with external energy obtained from ATP. Likewise, ABC flippases facilitate outward movement of phospholipids by the use of ATP. Scramblase proteins are bidirectional and require no external energy sources, but can be activated by calcium binding. Figure adapted from [123].
involved in transferring excessive PS away from the cellular side of the membrane in order to prevent unwanted apoptosis[120, 121]. ABC transporters function in the opposite direction, transferring a large variety of lipids from the inner leaflet to the extracellular side[122].
The third subgroup of flip–flop-inducing proteins are referred to as scramblases, due to their role of non-selectively scrambling the lipid composition between the two membrane leaflets. They are a group of elusive membrane proteins mainly located in the plasma membrane surrounding the cell, where they participate in various cellular activities, or in the endoplasmic reticulum, where they ensure that lipids synthesized on the lumenal leaflet are efficiently transferred to the cytosolic side. They are able to function without external energy from ATP and are able to flip lipids of all types in both directions[13].
Scramblase activity is mainly initiated as a response from external signals that call for cell activation, blood coagulation or apoptosis[124]. It may involve a mass influx of Ca2+-ions into the cell through the many ion channel proteins on the cellular surface, which in turn switches the scramblase proteins on and subsequently destroys the lipid asymmetry of the plasma membrane[125]. Binding of calcium-ions to a
protein interface is proposed to change the conformation of the scramblase protein in a way that the surrounding lipids are able to access a translocation pathway with greater ease. However, a number of scramblase proteins, especially in the ER, are proposed to function without calcium[126].
Despite the fact that the existence of scramblase proteins have been known for a few decades, their structures and mechanisms are still relatively unknown. The first x-ray structure of a known scramblase protein, TMEM16, was only reported several years ago[16]. A significant breakthrough was also achieved in the last decade when opsin, a protein of the GPCR family, was reported to exhibit scramblase activities in reconstituted vesicles along with its holo-form rhodopsin [12]. With no clear calcium-binding motifs, it was proposed that the scramblase activities of opsin were due to its intrinsic properties. This led to the suggestion that all GPCRs, having very similar central structures, could have scramblase properties in addition to their main roles as signaling proteins. Later, three other GPCRs, β1-adrenergic recep- tor,β2-adrenergic receptor, and adenosine A2A receptors were shown to scramble phospholipids, supporting the suggestion[44].
A major point of interest regarding scramblase activity is the mechanism with which they translocate lipids from one leaflet to the other. As of now, these mech- anisms are relatively unknown, but significant progress has been made by studying GPCR-mediated lipid flip–flop[5]. Over the years, several different mechanisms have been suggested on how lipids might cross the hydrophobic membrane interior with the aid of transmembrane proteins. The first involves the lipid headgroup glid- ing across the membrane along the protein surface with the transmembrane helices and a transient water pore reducing the free energy barrier to an acceptable level[4].
The second is a so-called credit card model where the lipid headgroup enters the hy- drophilic transmembrane channel of the scramblase, possibly via a conformational change in the protein, and exits on the other leaflet[127]. A third option involves the lipid fully submerging into the transmembrane channel and emerging out on the other side[4], though unlikely as a general mechanism due to the non-selectivity of scramblase proteins. It has also been suggested that transient disturbances caused by transmembrane helices may cause the lipid headgroup to slip into the bilayer inte- rior in a hydrated state near the lipid-helix interface, from where it will eventually pop out onto the other side[128].
Figure 4.2 Visualization of the crystal structure of rhodopsin. The seven transmembrane alpha-helices are colored in purple. The retinal ligand is colored orange. The two palmitate chains attached to the cytosolic cysteine residues are also pictured in cyan.