18] Lipid coumarin dye as a probe of interfacial electrical potential in biomembranes

Abstract

The coumarin method has been applied for the determination of surface potentials in a few biological membranes, such as thylakoids,25 electroplax,26 vesicular stomatitis virus,27 and mycoplasms.28 Furthermore, it has been used in the reconstituted system of the ATP/ADP transport protein of mitochondria.17 As shown in the last example the method provides best values of surface potential in relation to the Gouy-Chapman model. With respect to a general application of the method in biomembranes, there are two basic obstacles: (1) The location of the probe in a inhomogeneous membrane made of lipids and proteins is unknown. (2) The insight with respect to an actual effect of the average surface potential on membrane transport, pore gating, and membrane contact [Eq. (1)] is limited at the present time. The practical problems are hardly more involved than for other techniques: (1) In some systems the blue fluorescence may be superimposed on some background. (2) The pKmw in negative membranes is rather high. Up to now it has been impossible to design other lipid pH indicators (carbonylcoumarin,29 dansyl,30,31 acridine,31 chinin,31 and naphthoic acid32) with pKi = 6–7 and red fluorescence, conserving the crucial feature of the coumarin and its insensitivity to details of the membrane surface. The main success of the method up to now has been in the field of pure colloidal systems. Surface potential and charge density have been characterized in insoluble monolayers,4,5 in micelles,6,21 in lipid vesicles,8,15,18 and in foam films.7,33 These results are the basis for studies of native and of reconstituted biomembranes and of the formation of membrane junctions. Some problems of the method remain to be solved: (1) The validity of the thermodynamic cycle has to be checked experimentally by relating the probed potentials with macroscopically determined electrical potentials in insoluble monolayers and in solvent-free bilayers. (2) The interaction of the probe with the lipid matrix has to be studied in more detail by NMR techniques and by theoretical simulation. (3) The polarization of a mixed matrix and the interaction with membrane proteins has to be investigated. (4) The effect of neutral interfaces on the pK has to be characterized such that for every charged system the appropriate pKi is chosen. The lipid coumarin is a probe of interfacial polarity as emphasized previously4,6 and as illustrated recently for a glycosurfactant.34 Finally, it should be noted that the coumarin may be used not only as an analytical tool for probing surface potentials but as a synthetic tool for the design of sensors probing interfacial effects induced by agonists, enzymes, or cells, with direct transduction of the fluorescence signal to a semiconductor.35