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Paul OShea,
Cell Biophysics Group, School of Biology, University of Nottingham, University Park, Nottingham, United Kingdom
doi: 10.1002/9780470048672.wecb310
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Biological Background Physical Chemistry Background of Membrane Potentials Chemical Tools & Techniques Spatial Imaging of Membrane Potentials Future Studies of Biological Roles and of Technologies for Measurement
Membrane potentials are a ubiquitous feature of all living cells and play different roles in manifold cellular processes. Three different membrane potentials are known to exist that include the transmembrane potential difference, the surface electrostatic potential and the membrane dipole potential. Each of these are discussed in terms of their different physical origins and characteristics together with representative examples of how they feature in cell biology as well as some examples in physiology. Techniques for the quantitative measurement of each of these potentials are described in both model systems as well as with living systems. As many of these practices involve the use of spectroscopic technologies (particularly uorescence) they lend themselves to spatial (single cell) imaging applications and examples of the biological roles that spatial variation or localisation of each of these potentials are also outlined.
Biological Background
Membranes act as one of the major macromolecular assemblies of living cells (both prokaryotic and eukaryotic) but also feature as physical envelopes in viruses such as inuenza and HIV. Typically cell membranes act as semi-permeable structures that compartmentalise cellular processes and behave as 2D uids, rapidly annealing any fractures by 2D molecular ow. Membranes, however, undertake many more biological roles than these simple generalizations imply as they also act as platforms for many important biochemical processes. Similarly, it has recently been appreciated that membranes exhibit localised structures in the form of microdomains (ie. so called rafts) as shown in Fig. 1. These act as structures upon and within which, additional physico-chemical mechanisms may operate. The following sections outline the biological features of the trinity of membrane potentials (1) and their physico-chemical origins. Practical methodologies for their respective measurements are then described that exploit these different molecular origins.
Membrane Raft;
Figure 1 Schematic of a contemporary view of a cell membrane.
solute (particularly electrolyte) movement. Thus ions that are actively transported across the membrane by membrane proteins are not easily able to return to their former compartment. This leads to a thermodynamic gradient that is utilised by cells in two ways. The rst, takes advantage of the ionic concentration gradient of a specied ion across the membrane. The second due to a small electrical capacity of the membrane (ca. 1 F cm2 ) may lead to an electrical gradient across the membrane. Thus chemical or light energy that is spent transporting the ions across the membrane is partially conserved as the ionic gradient across the membrane (e.g. as illustrated in Fig. 2). Two enormously important biological examples featuring these processes are worth emphasising, the rst example is that the conservation of light or chemical (oxidation-reduction) 1
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commonly understood membrane potential and is described very fully in Reference 6).
Ionic Source Ionic Sink
The membrane surface potential: roles in modulating ion channels and cellcell interactions
The surface potential is now understood to play important roles in many physiological processes. These range from the simplest involving coulombic repulsion between adjacent membranes that prevent cells sticking to each other. Thus sialic acid as a sugar moiety to the membrane protein glycophorin present on the cell surface of many cells and particularly red blood cells appears to prevents cell-cell aggregation. Under circumstances of uncontrolled-diabetes, however the nature of the sugars on the erythrocyte cell surface may change with sialic acid replaced by uncharged sugars. This reduces the coulombic repulsion between the red cells allowing thrombosis to occur (7, 8). The contribution to the cell membrane surface potential by sialic acid is also implicated in gating the voltage dependence of Na+ Channels (9). This kind of electrostatic effect on ion pump/channel loading/unloading (with other contributors to the surface potential such as phosphatidylserine), however, has been considered for many years. McLaughlin as long ago as 1971, for example, suggested that surface potential changes were responsible for negative or positive shifts of the current-voltage relationships of neural tissue as embodied in the Hodgkin-Huxley equations (5). This kind of process is the result of the effect of the surface potential on the activity of ions on a membrane surface and leads to changes of e.g. the pK of membrane surface-located acid-base groups (10, 11). We have exploited this latter phenomenon to develop a panel of uorescent phospholipids as real-time measures of the membrane surface potential (12). By measuring small changes of the membrane surface potential in this manner due to the interaction of charged molecules, it proved possible to study the kinetics and thermodynamics of intermolecular interactions with membranes (13).
cytoplasm
Figure 2 Schematic of an ionic circuit in a living cell or bacterium. Ions transported across the membrane by the action of energy-dependent pumping systems (ionic source) leads to a transmembrane potential difference and/or an ionic concentration gradient according to equations 2 & 3. This gradient may then equilibrate through via the ionic sinks yielding energy to manufacture ATP or drive other transport systems. Alternatively the ionic sources and sinks may be part of the same membrane protein structure and be positioned such that a travelling wave of ionic current is used as a long range signaling system (e.g. nervous conduction).
energy as an ionic/electric gradient is an essential part of the process of oxidative or photosynthetic phosphorylation. The ionic gradient in these latter cases resides in a proton gradient across the mitochondrial or chloroplast membranes respectively (2). Interestingly, in the case of the mitochondria, the proton gradient manifests mostly as an electrical gradient (3) due as mentioned above, to the relatively small electrical capacity of the mitochondrial membrane with only a small pH gradient. Whereas due to the relatively easy movement of Mg2+ ions across the chloroplast membrane, the electrical capacity is effectively much increased and the proton gradient then manifests mostly as a pH gradient with a small electrical gradient. Both these manifestations of the electrochemical potential difference of the proton across the membrane however, are equivalent and are utilised to manufacture ATP directed through the membrane ATPases. In fact, this electrochemical gradient is utilized essentially as a power source for many additional utilities such as coupled solute transport, to drive the agellum movement of some bacteria as well as for protein import across prokaryotic and eukaryotic membranes (4). The second general biological example is that the electrical gradient so generated can be utilised as a signaling mechanism such as in nervous transmission in neural cells or as a driving force for ionic currents that mediate cell signaling. These signaling mechanisms are both complex and ubiquitous with ion channels activated through soluble or membrane-bound ligands or 2nd messengers that upon opening permit e.g. calcium ion movements driven by the membrane potential that then elicits further cell signaling cascades for neurotransduction, muscle contraction and nuclear translocation of molecular switches to turn genes on or off. Typically there are spatial elements in these signaling elements and thus there are also many measurement strategies that include imaging modalities as part of the experimental goals. Finally, in the case of the role of membrane potential in nervous conduction, a traveling wave of an electrical potential gradient (voltage) is used to relay signals over relatively long distances along the cell membrane ie as in axonal linkages between cells (see e.g. 5). This latter manifestation of a membrane potential as part of the nerve impulse is perhaps the most 2
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partners resident in the uid membrane. Both these processes, however are conceptually analogous. We considered that membrane microdomains may exhibit a vastly different membrane dipole potential to that of the uid phase membrane due to their different lipid packing and complement of sterols and lipids (etc). We later demonstrated this was indeed a function of the various lipids present (15) and also showed that this parameter had a signicant effect on membrane protein conformation (16). This work demonstrated with representative ligand-receptor systems that this behaviour may alter the function of such receptor systems depending on whether they were resident in the rafts or in the uid phase regions of the membrane (15, 17). Following this a number of other laboratories also observed modulation of membrane behaviour via the agency of the dipole potential (18). Thus the membrane dipole potential appears to be an additional tool for which membrane protein function can be controlled in particular localities. The explicit molecular mechanism by which this may take place is discussed in the next section.
distance signaling. Thus, the transport of net charges across the insulator offered by the membrane will establish an electrical potential difference (m ) across the membrane. In terms of an analogy with the charging of a capacitor, this well known latter phenomenon is described by the following expressions:
V = q/C = = Em or Vm (= m )
(1)
Where, V is the electrical potential difference across the membrane ie. Vm or Em and , C = the capacitance of the membrane (dielectric) and q is the number of charges transported across the membrane. The term, m is included as this represents a more consistent nomenclature, and would be better recognised by disciplines outside biology (e.g. Electrochemists), , is the form utilised by biochemists (2) and Vm or Em is the form utilised by Physiologists (6). The transport of electrical charge across a membrane may take the form of cation, anion (both inorganic and organic) or electron transport all requiring an energetic input. It can be passive due to membrane leaks or mediated by ionophores such as valinomycin. All other things being equal it is possible to utilise equation 1 to calculate a trans-membrane (ie trans-dielectric) potential difference (19). Thus, under these circumstances there is a simple and direct equivalence of the voltage across the membrane (ie m ) to the Gibbs Free energy (ie. G). In biological membranes, however, the transport is frequently coupled either to the movements of other ions due to co- or anti-porting enzymes, or to chemical reactions as in the case of oxidative phosphorylation or hydrolysis of ATP. The value of m established in such complex systems is governed by the relation i Zi Ji = 0, where the sum has to include all ows Ji of charged species (Zi) across the membrane (1). When this relation is satised, charging of the membrane capacity has ceased, and a (pseudo-)steady state with a (approximately) constant m is then attained. Its value depends on the difference in chemical potential (i ) of all transported species and on the afnities of coupled chemical reactions. The pertinent relations are usually transcendental and cannot be solved explicitly except for some special cases. In particular, if only one species permeates through the membrane, a true equilibrium state is reached and the resulting transmembrane electrical potential difference is described by what is known as the Nernst equation (below). This equivalence, however, becomes more complicated because biological membranes are not totally impermeable to ions (i.e. there is leakage back across the membrane e.g. Reference 20), the concentration gradients must also be included in the Gibbs free energy of the expression and nally there is usually a coupling of the transport mechanisms to the movements of other ions as well as to the energy input (as in oxidative phosphorylation or the hydrolysis of ATP see Reference 2). Nevertheless, ion gradients are established across many membranes whereby the ion leakage is balanced by further energy-linked ion transport. Biological membranes, therefore, exhibit quasi-equilibrium transmembrane concentration gradients of charge based on selective transport of the ions found in physiological electrolytes. This is described by the well-known Nernst equation which incorporates the transmembrane concentration gradient of electric 3
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charge as follows:
(m ) = (/Zi ) log(Ci
(2)
In which is an abbreviation of KT/e = RT/F : With F = faraday constant, R gas constant and Ci indicate the concentration of the respective ionic species in the aqueous phases separated by the membrane Thus, the Gibbs Free energy expression for such an ionic gradient across the membrane consisting of an positively charged species C, would be of the form of the following expression:
the thermal motion/diffusion of the ions away from the interface to the bulk medium. Later, it was suggested that water molecules may adopt a specic orientation upon the charged surface due to their permanent dipoles (this latter feature has some bearing on an understanding of the dipole potential described below). A number of diffuse phases or layers as illustrated in Fig. 3a, have been identied which are thought to exist adjacent to the surfaces of membranes. These include the so-called Stern layer (Fig. 3a) which represents contact-adsorbed counter-ions (either totally or partially dehydrated) and oriented water molecules. The plane running through the centre of the contact adsorbed ions is referred to as the Inner -Helmholtz plane. The rst layer of hydrated ions is then referred to as the Outer -Helmholtz plane. The Gouy-Chapman-Stern model was formalised by considering the adsorption of counter-ions only, the process being approximated by a Langmuir-type adsorption isotherm (1, 21). Surface potentials at the electrode-solution interface have been described by a number of formalisms. The most successful of these was offered originally by Gouy and Chapman with subsequent elaborations from Chapman, Stern, Bockris etc. (outlined in ref 1 & 21). McLaughlin (22) and others (outlined in 1) suggested that a combination of the Poisson and Boltzmann equations best describes the state of affairs in the space between the membrane surface and the bulk phase aqueous solution ie. the electrode-water interface. The Poisson-Boltzmann equation, with dened boundary conditions can be solved analytically (1, 22) to yield an expression for the surface potential as follows:
(4)
with o as in Eqn. 4 and the abbreviation = /(r 0 ) whereby represents the surface charge density, and is known as the Debye length. At 25 C (r 0 kT/(2e2 NA ))1/2 = 0.304 nm M1/2 for an aqueous phase with r 80. Thus the potential prole in the diffuse layer shown in Fig. 3a is given by:
(x) = o ln {(1 + tanh(s /2o ) exp(x/))/ (1 tanh(s /2o ) exp(x/))} for x 0 (5)
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(a)
p25 addition
260
250
Percent saturation
100 80 60 40 20 0 0 2 4 6 8 10 p25 (m M) 12 14
240
230 0 50 100
250
300
350
Figure 3 Fluorescent sensors of Surface and Dipole potential in membranes. Fig. 3a (LHS) indicates the position in the a single bilayer leaet of uorescent indicators of the membrane dipole potential (upper chemical structure) and the surface potential (lower chemical structure). The RHS prole indicates how the prole of surface potential varies with distance from the membrane surface. Fig. 3b indicates the use of FPE as a surface potential indicator that responds to the addition of a charged peptide (P25) as it interacts with simple membranes (see 25 for more details).
the external aqueous phases, and d has a magnitude of several hundred millivolts (typically about 300 mV see Fig. 4). Symbolically formalising the measured molecular arrangements in order to undertake modelling of the membrane dipole potential is also not as straightforward as it may seem. It is possible to begin by identifying a vector drawn from the point of the negative charge Q to the positive charge +Q of any dipole which is the familiar electric dipole moment p. The magnitude is Q a , with a dening the distance between the centres of the charge density. The potential at a given point with a position vector r with respect to the centre of the dipole can be expressed as:
a (6)
where is the angle between r and p. Higher order terms become inuential if r a is not fullled. This expression, however, only deals with a single molecular dipole and as many such dipoles would be required to describe the overall membrane dipole potential but as a mean-eld expression, this term is practical and culmulatively offers the approximation of the estimated dipolar organisation shown in Fig. 4. A further complication, however, involves the solvent environment and this too is also often dealt with as a mean eld or in a continuum manner. But the relative permittivity (or dielectric constant) (r ) cannot be considered to possess the same value throughout the multiphase system represented by a membrane in an aqueous medium. The permittivity prole has been measured to vary from about 78.5 in the bulk aqueous 5
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of the changes of potential resulting from cellular signaling or metabolic activity. Other electrochromic membrane probes of note include, merocyanine 540 used to study the transmembrane potential in liver mitochondria although some problems are evident (5, 30, 31). Overall, measurements of transmembrane potential differences in living cells are well established with the methods fairly reliable and robust. Many of these membrane potential measurement strategies are generic and hold for plasma membranes of neurones and other excitable tissues (6).
phase to 2030 in the diffuse layer at the membrane-water interface (25) to around 2 in the membrane interior.
(7)
that when introduced into the logarithmic form of the Henderson-Hasselbalch equation, upon rearranging yields:
(8)
Thus pH = log(cH,b ), and cB and cHB , the concentrations of the dissociated and protonated species of an acid-base pair, respectively (1, 12, 25). The quantity pK Fs /(RT ln10) can be considered as an apparent pK for proton binding of an acid-base pair on the membrane surface. Eqn. 8 shows that the protonation state cB /cHB of the probe is altered if s changes at constant pH, which results in a change in the uorescence yield ie the protonated form is much less uorescent than the deprotonated form. This phenomenon has been utilised to measure the time course of the interactions of just about any charged molecules such as ions e.g. Ca2+ , peptides, and proteins that may interact with either synthetic and biological membranes with great sensitivity in real time. An example of this is shown in Fig. 3b. It has also proved possible to monitor the early events during the interactions of macromolecules with articial membrane systems (25) and with living cells (12) to obtain thermodynamic information of the intermolecular interactions (13).
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(28). Measurements that include a spatial element have become extremely important in addressing biological problems as living cells exhibit enormous heterogeneity in the spatial disposition of these potentials. Lateral gradients of each of the membrane potentials, therefore, are implicated in manifold biological processes. In our laboratories for example we have developed the concepts (outlined above) relating to the interesting possibility that elevated dipole elds within membrane microdomains (15) modulate protein structure and that this modulates signaling activity (1, 17) or the activity of ion channels (18). We developed the use of di-8-ANEPPS to determine how the membrane dipole potential may be utilised to reveal macro-molecular interactions with membranes (15, 16). By illuminating spatial variations of this parameter and particularly how it varies in membrane rafts it led us to propose that the value of d is quite different in membrane rafts as compared to uid mosaic membranes (1, 17). Figure 6 illustrates this striking heterogeneity of the d about the cell surface with measures currently being taken to correlate this or co-locate such signals that are emanating from the raft microdomains within membranes. The latter is a significant issue as there remains much confusion as to the in vivo existence of membrane rafts (33). Similar approaches utilising such indicators as FPE to visualise the membrane surface potential s are also routinely employed in our laboratories (17). By correlating the change of the uorescence and hence surface potential with the addition of net electric charges from the macromolecule that becomes bound, it is possible to quantitate on the basis of the poisson-boltzmann equation above, the number of molecules that become bound. This allows us for example to determine localised molecular interactions on the membrane surface (17, 34).
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120 100 Fluorescence 80 60 40 20 0 360 400 440 480 Wavelength (nm) (a) 3 520
16 Fluorescence difference Fluorescence 12 8 4 0 4 8 12 360 400 440 480 Wavelength (nm) 520
2 350
400
550
8 Fluoresoence difference PC-PH 4 0 4 8 360 400 440 480 Wavelength (nm) 520 0 (b)
Figure 5 Spectroscopic tools for identifying the membrane dipole potential Fig. 5a indicates the excitation spectrum of different membranes labeled with di-8-anepps with the emission collected at 580nm. Spectra are shown for membranes made up of 100% phosphatidylcholine (), membranes made up of phosphatidylcholine and 15mol% 6-Ketocholestanol ( ) and membranes made up of 15 mol% phloretin (). These excitation spectra are better visualised as the difference spectra shown in 5b in which membrane made up of different lipid micstures are compared to a reference spectra, typically these are normalized and compared to phosphatidylcholine and shown as LHS upper (phloretin) and lower (6-ketocholestanol) spectra. The spectra upper RHS indicates the spectral shift of a membrane following interaction with a peptide and the lower plot illustrates the time course of the interactions in which the upper and lower limbs of the spectra are ratioed and plotted against time.
R (460/520)
PC
PC-KC 0.004
10
15 20 Time (s)
25
30
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Figure 6 Fluorescence imaging of the membrane dipole potential in single cells. The RHS uorescence image of a broblast was obtained following excitation of di-8anepps (see Fig. 5) using a laser-scanning confocal microscope. Di-8-anepps labels all the intracellular membranes as well as the plasma membrane and thus indicates these membranes also possess very localized regions of elevated dipole potentials indicative of intracellular membrane microdomains. The LHS image indicates a bright eld image of the same cell.
References
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Further Reading
Cladera J, OShea P. Generic techniques for uorescence measurements of protein-ligand interactions; real-time kinetics and spatial imaging. In: Protein-Ligand Interactions. Harding SE, Chowdery BZ, eds. 2001. Oxford University Press, UK. pp. 169200. Loew LM. Spectroscopic Membrane Probes, Volumes 13. 1988. CRC Press, Boca Raton, FL. OShea P. Membrane potentials and membrane probes. In: Chemical Biology., eds. 2007. John Wiley & Sons, Ltd, New York. pp. 6784. Walz D., Teissi e J., Milazzo G., (Eds.) Bioelectrochemistry of Membranes. 2005. Birkhauser Verlag, Switzerland.
See Also
Membrane Assembly in Living Systems Bioenergetics and Oxidative Metabolism Lipid Rafts Ion Transport Imaging Techniques: Overview of Applications in Chemical Biology
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