Mapping the electrostatic profiles of cellular membranes

Anionic phospholipids can confer a net negative charge on biological membranes. This surface charge generates an electric field that serves to recruit extrinsic cationic proteins, can alter the disposition of transmembrane proteins and causes the local accumulation of soluble counterions, altering the local pH and the concentration of physiologically important ions such as calcium. Because the phospholipid compositions of the different organellar membranes vary, their surface charges are similarly expected to diverge. Yet, despite the important functional implications, remarkably little is known about the electrostatic properties of the individual organellar membranes. We therefore designed and implemented approaches to estimate the surface charges of the cytosolic membranes of various organelles in situ in intact cells. Our data indicate that the inner leaflet of the plasma membrane is most negative, with a surface potential of approximately –35 mV, followed by the Golgi complex > lysosomes > mitochondria ≈ peroxisomes > endoplasmic reticulum, in decreasing order.

Lipids and (glyco)proteins are the main constituents of biological membranes. Sugar moieties of glycoproteins, glycolipids, and adherent glycocalyx components such as hyaluronic acid can bear ionizable groups that confer a net negative charge on the outer surface of the plasma membrane. The aggregate surface charge of the outer membrane has been estimated indirectly by measuring the ζ potential—the potential at the slipping plane—by electrophoretic means (e.g., Tippe, 1981; Silva Filho et al., 1987) or by measuring streaming potentials (Vandrangi et al., 2012). The plasma membrane, however, is highly asymmetric; its inner (cytosolic) aspect is virtually devoid of carbohydrate moieties. Nevertheless, the cytosolic leaflet is also thought to be negatively charged, due primarily to the accumulation of anionic phospholipids, namely phosphoinositides and phosphatidylserine (PtdSer). Based on biochemical determinations of its lipid composition, the net negative charge of the plasmalemmal inner leaflet is estimated to generate an electrical field of 10⁵ V/cm (Olivotto et al., 1996). The membranes of intracellular organelles can also contain anionic lipids, but their precise lipid composition and topology have been difficult to assess and hence their surface charge has not been estimated.The surface potentials of biological membranes have important functional implications: they can alter the disposition of charged regions of transmembrane proteins, cause local accumulation of soluble counterions in the vicinity—altering the local pH as well as the concentration of physiologically important ions such as calcium—and serve to recruit extrinsic cationic proteins (McLaughlin, 1989). It is therefore important to determine the electrostatic properties of each of the organellar membranes. In principle, this could be accomplished by measuring the ζ potentials of isolated organelles. However, the purity of such preparations is imperfect, changes in lipid composition (particularly phosphoinositide degradation) and sidedness cannot be avoided, and loosely adherent components that may alter the surface charge can be removed during the isolation process. Alternative approaches to estimating the surface potential are therefore required.Here we used recombinant and synthetic polycationic peptides to obtain a quantitative estimate of the surface potential of the inner leaflet of the plasma membrane and to establish a hierarchical map of the potentials of the cytosolic surfaces of the major intracellular organelles in live cells.