Distinct electric potentials in soma and neurite membranes

Neuron,

Vol. 13, 1187-1193,

November,

1994, Copyright

@ 1994 by Cell Press

Distinct Electric Potentials in Soma and Neurite Membranes Richard S. Bedlack, Jr., Mei-de Wei, Stephen Eitan Gross, and Leslie M. Loew Department University Farmington,

of

of Physiology Connecticut Connecticut

Health 06030

H. Fox,

Center

Summary Structurally similar voltage-dependent ion channels may behave differently in different locations along the surface of a neuron. A possible reason could be that channels experience nonuniform electrical potentials along the plasmalemma. Here, we map the electrical potentials along the membrane of differentiated N 1 E-l 15 neuroblastoma cells with a potential-sensitive dye. We find that the intramembrane potential gradient is indeed more positive in the membranes of neurites than in the membranes of somata. This is not attributable to differences in ion conductances or surface charge densities between the membranes of neurites and somata; instead, it can be explained by differences in lipid composition. The spatial variation in intramembrane electrical potential may help account for electrophysiological and functional differences between neurites and somata. Introduction The ways in which voltage-dependent channels differ from one another has been explored on a variety of levels in an attempt to explain the functional diversity of excitable cells. On the most basic level, structural differences between ion channels have been documented. These account for divergent ion selectivity, modes of secondary regulation, and pharmacology (Catterall, 1988; Tsien et al., 1988; Miller, 1991; Hille, 1992). Thus, we know that different cells can respond differently to the same stimulus, depending on the types of ion channels each expresses. There are also differences in channel distribution along the membrane. Channel clustering into hot spots is especially well documented, as seen with sodium channels at the nodes of Ranvier, and differences in the surface densities of channels between somata and neuronal processes have been described recently (Llinis and Sugimori, 1980a, 1980b; Bolsover and Spector, 1986; Thompson and Coombs, 1988; Streit and Lux, 1989; Garcia et al., 1990; Westenbroek et al., 1990; Masukawa et al., 1991; Meyers, 1993; Midtgaard et al., 1993). These latter observations help explain how different regions of a cell can respond differently to the same stimulus. At a still more subtle but potentially very significant level, the behavior of a given channel can be different in different regions of a neuron. Thompson and Coombs (1988) report thatwithin the soma membrane of a molluscan neuron, rates of calcium channel inac-

tivation increase toward the axon hillock; concomitantly, the threshold potential for activation becomes less depolarized. Despite these regional variations, the authors conclude that only one species of calcium channel is involved, speculating that the differences could be due to varying intracellular calcium buffering capacities or cytoplasmic regulatory factors. A similar conclusion is reached by Streit and Lux (1989) in their study of the distribution of calcium currents in differentiating PC12 cells. In comparing calcium currents in the somata with those in growth cones, they find that the latter show significantly more inactivation during pulses of 400 ms. Belardetti et al. (1986) point out that sodium channels in invertebrate axons and growth cones inactivate much faster than sodium channels in somata. Upon severing the neurite of a crustacean peptidergic neuron, Meyers (1993) displays current-voltage curves for both sodium and calciumcurrentsthatappearto beshifted to lessdepolarized voltage in the isolated lamellipodium relative to the soma. In a series of thorough studies of calcium transients in Purkinje cells, Llinis and his coworkers have shown that calcium influx occurs almost exclusively in the dendrites (Llinds and Sugimori, 1980a, 1980b; Tank et al., 1988; Sugimori and Llinis, 1990), even though the P-type channels responsible for this current can also be found in somata (Usowicz et al., 1992). This group found subtle differences in the electrophysiological properties of the P-type channels in these two regions of Purkinje cells, although additional data from these technically difficult experiments are required before a definitive statistical distinction can be established (Usowicz et al., 1992). Taken as a whole, these observations are important because they suggest that different regions of a neuron could respond differently to the same stimulus without differences in channel distribution. The mechanism underlying such regional variations in channel function remains unclear. Although channels are usually described in terms of voltage dependences, it is important to emphasize that the regulatory elements within them (e.g., gating charges) are, fundamentally, responding to the potential gradient at their specific location in the plasmalemma-the intramembrane electric field. Therefore, structurally similar ion channels might behave differently along the surface of a neuron if the endogenous intramembrane electric field they are experiencing is nonuniform. As a first step toward testing this hypothesis, we have developed a method for mapping the intramembrane electric field along the surface of a cell by dual wavelength ratiometric imaging of a membranestaining potentiometric fluorescent dye, di-8-ANEPPS (Montana et al., 1989; Bedlack et al., 1992). In most of the systems that have been investigated, the dye response can be understood in terms of an electrochromic mechanism (Loew et al., 1978,1979) in which

Neuron 1188

INTRAMEMBRANE ACROSS

ELECTRIC FIELDS Nl E-l 15 CELLS

AS LEFT,

AFTER

SOLUTION

CHANGE

5.0 7

-1 .o -4 A

0

C

D

E

LOC. Figure

1. Measurement

of lntramembrane

Lot. Electric

Fields

along

Neurons

Reveals

Regional

Variations

The color image at the top is a map od the intramembrane electric field along the surface of a single NlE-115 neuroblastoma cell. The colors are calibrated in units of mV/A as indicated on the scale at the right. Each cell was divided into five equally sized locations. Location A always encompassed only membrane from the soma; B included membrane from the soma, axon hillock, and proximal axon; C included only axonal membrane; D included membrane from the distal axon and proximal growth cone; E included only distal growth cone membrane. lntramembrane electric field variations were referenced to the average in location A, taken as 0. The bar graph at the bottom left shows the average intramembrane electric field in each location as a mean of 20 different NlE-115 cells (error bars indicate SEM). Note that the locations appear to fall into two distinct populations, one from the soma and a separate one from the neurite; statistical testing (described in the text) confirmed this. A control exchange of salt solution (bottom right) did not change the absolute intramembrane electric field in any location; this control is relevant to Figure 3 and Figure 4.

Electrical 1189

Profiles

along

Neuron

Surface

its absorbance and fluorescence spectra are shifted in the presence of an electric field, Since ratiometric measurements eliminate artifacts due to uneven dye concentration (Tsien and Poenie, 1986), the probe can be used to measure regional variations in intramembrane electric field. An exampleofthe utility of the method can befound in a recent study in which we documented the intracellular events involved in electric field-induced neurite outgrowth (Bedlack et al., 1992). In the presence of an extracellular field, di-8-ANEPPS allowed us to directly visualize electrostatically induced cathodelocalized plasma membrane depolarizations. These served as the initial step in a cascade involving focal calcium influx and biased filopodial activity and culminating in directed neurite growth. Within the same study, di-8-ANEPPS ratioing allowed us to note that the potential of growth cones appeared more positive than the potential of somata even before the external DC field was applied. This preliminary finding suggested that the membrane of the growth cone might intrinsically have a different sensitivity to external chemical and electrical guidance cues than the membrane of the soma. Here, we confirm the presence of an endogenous nonuniformity in the intramembrane electric field along the surface of a neuronal cell. We also provide data that can explain its molecular origin. Our results imply that the process of differentiation may program a self-sustaining growth and pathfinding capability into the developing neurite membrane.

a)

K+ K+ K+

Na+

b)

Results Dual wavelength ratiometric imaging of di-B-ANEPPS in the membrane of differentiating NIE-115 mouse neuroblastomacells provided us with astriking result: the intramembrane electric field along the surface of these cells was not uniform. Statistical analysis of 20 ratio images demonstrated that these variations were significant. When the cells were divided into five equally sized regions of membrane, as shown in Figure 1, analysis of variance testing confirmed that there was a difference (p < .OOl) in intramembrane electric field between the regions (Figure 1). Bonferroni t tests refined the analysis to establish that there were two distinct populations within the regions, a population corresponding to the somata (regions A and B were not significantly different; p > .05) and a population corresponding to the neurites (C, D, and E were not significantly different from each other [p > .05], but all were different from A and B [p < .05]). Defining the positive field direction to be from the cytosolic to the external face of the membrane, the mean intramembrane electric field associated with the neurites was 1.9 + 0.4 (+ SEM) mV/A more positive than the mean intramembrane electric field associated with the somata. lntramembrane electric fields have three components (Figure 2): transmembrane potentials (arising from differences in ion concentrations between the

Figure

2. The Components

of lntramembrane

Electric

Fields

Thethreemolecularoriginsof endogenous intramembraneelectric fields and their associated potential profiles are shown. The approximate position of the sensor portion of the potentiometric dye diSANEPPS is indicated in each illustration by a rectangle. The low dielectric, high resistance region of the membrane is depicted as a stippled area with potential profiles stretching through it from each water phase. The intramembrane electric field is depicted as an arrow pointing in the field direction and with a thickness representing the field intensity. (a) The transmembrane potential originates primarily from ion concentration gradients coupled to selective membrane permeability; we presume the membrane here to be selectively permeable to potassium. This potential difference between aqueous compartments can be measured with electrodes and results in an electric field that spans the high resistance region within the membrane. (b) Charges at the interface between the low dielectric region of the membrane and the surrounding aqueous phase are the molecular origin of the surface potential. If the charge densities at the two interfaces are different, a difference in surface potential results that produces an intramembrane electric field. (c)The oriented dipoles of lipid molecules or immobilized water molecules are the molecular origin of thedipole potential within the membrane. This produces an intense intramembrane electric field spanning only the first few angstroms within the membrane. The location of the dye is appropriate to measure all the components of intramembrane electric fields.

NE!UKNl 1190

extracellular and intracellular fluids; Hille, 1992), surface potentials (arising from differences in charge density along the surface of the outer and inner faces of the membrane; McLaughlin, 1977,1989), and dipole potentials (arising from the aligned molecular dipoles of lipid ester groups or the water molecules adjacent to them; Hladky and Haydon, 1973; Flewelling and Hubbell, 1986; Honig et al., 1986; Franklin et al., 1993). All of these can influence ion movement across lipid bilayers (Szabo, 1974; McLaughlin, 1989; Hille, 1992), yet only transmembrane potentials are amenable to measurement using the standard electrophysiological methods employed for studying ion transport across cell membranes. Furthermore, the possibility of variations in any of these potentials between regions of cells (which might lead to nonuniform signal transduction sensitivity) remains untested, as electrodes measure only the total transmembrane potential drop averaged over large regions of a cell. In contrast, dual wavelength excitation of di-8-ANEPPS can be analyzed ratiometrically (Montana et al., 1989) to measure a local sum of the intramembrane electric fields associated with transmembrane potentials, surface potentials, and dipole potentials (Gross et al., 1994). The approximate location of the sensor portion of the dye is indicated in Figure 2 and defines the transverse position within the membrane at which diS-ANEPPS measures an electric field. Initially, we considered that the intramembrane electric field variation might be due to transmembrane potential differences between the neurites and the somata (Figu re 2a). It has been observed, for example, that ion channels are not uniformly distributed over the neuronal plasmalemma; indeed, in NIE-115 cells, L-type calcium channels are much more prevalent in neurites than in somata (Silver et al., 1990). An uneven distribution of channels could lead to uneven ion conductances and nonuniform transmembrane potentials, resulting in a nonuniform intramembrane electric field. We tested this hypothesis by adding valinomycin, a potassium ionophore, to our cells (Figure3)Thisshould havesetthetransmembranepotential equal to the potassium equilibrium potential over all regions of the cell membrane, thereby overwhelming any intrinsic heterogeneities in ion channel distribution. The overall intramembrane electric field was offset by varying concentrations of KCI in the presenceof valinomycin; indeed, thiswas howthefluorescence ratio was calibrated. However, the regional variation in intramembrane electric field remained, despite such normalization of the transmembrane potential. Thus, the measured nonuniformity could not have been caused by unequal ion conductances between neurites and somata. Next, we attempted to determine whether the intramembrane electric field variation could be explained by surface potential differences between neurites and somata. An uneven distribution of charged lipids could be associated with the nonuniformity of integral membrane proteins described above. Thus, it is

INTRAMEMBRANE ELECTRIC FIELDS ACROSS NIE-115 CELLS IN PRESENCE OF VALINOMYCIN

SOMA

mm

AS LEFT, AFTER ELEVATlON OF [KCLj

s9.m

Figure 3. Nonuniform lntramembrane Electric Fields Explained by Transmembrane Potential Differences Neurites and Somata

MURm

-.

Cannot Be between

The bar graph on the left shows the intramembrane electric field profile averaged across ten different NIE-115 cells bathed in a balanced salt solution supplemented with 0.3 PM valinomycin. As described in the text, this manipulation should eliminate any intrinsic differences in transmembrane potential between neurites and somata. Cells weredivided into onlytwo locations (neurite and soma) based on the statistical analysis described for Figure 1. Student’s t test revealed a difference in intramembrane electric field between the neurites and somata (p < .OOl), thus confirmingthattheobservedasymmetryjnintramembraneelectric field is not explainable by a difference in transmembrane potential between neurites and somata. The bar graph on the right shows results from the same ceils after a solution exchange in which the [KCI] is increased from 5.5 to 35.5 mM and the [NaCI] is reduced from 130 to 100 mM, still in the presence of valinomycin. Isotonically increasing the concentration of KCI depolarizes the ceil and changes the intramembrane electric field equally in both the neurites and somata. This control assured that valinomycin was acting to clamp the transmembrane potential equally in neurites and somaia.

possible to envision unequal surface charge densities between the neurite and the soma leading to unequal surface potentials (see Figure 2b) and a nonuniform intramembrane electric field. However, an extremely large difference in surface charge would be required to explain fully the measured intramembrane electric field variation. Assuming a40 A membrane thickness, the gradient in surface potential would have to be 8090 mV; using the Cuoy-Chapman theory elaborated by McLaughlin for lipid bilayer membranes (McLaughlin, 1977), this means that the neurite would need a mole fraction of negative lipids near 100% on its outer surface, with the soma and the entire inner surface being neutral. Furthermore,wedirectlytestedthesurface potential hypothesis by increasing the concentration of calcium in the extracellular fluid (Figure 4). This manipulation should screen surface charge along the outer surface of the neuron, thereby decreasing the intramembrane electric field; in fact, Figure 4 shows that the intramembrane electric field all over the cell did slightly decrease with calcium elevation. However, if a greater density of negative charge on

Electrical

Profiles

along Neuron

Surface

1191

INTRAMEMBRANE ELECTRIC FIELDS ACROSS NIE-115 CELLS

AS LEFT, AFTER ELEVAXION OF EXTRACELLULAR [Ca++]

planation for the variation in intramembrane electric field illustrated in Figure 1. Such differences might be sustained by a barrier to lipid migration identified in the axon hillock (Kobayashi et al., 1992). Discussion

-0.5-1 0 T

SOW

La

Figure 4. Nonuniform lntramembrane Electric Fields Cannot Be Explained by Surface Potential Differences between Neurites and Somata On the left, ratiometric images of ten cells in normal balanced salt solution were analyzed for differences in intramembrane electric field between the soma and neurite. On the right, these same cells were analyzed after a solution exchange that increased [CaC12] from 1.8 to 11.8 mM and decreased the [NaCI] from 130 to 120 mM. As described in the text, this manipulation should screen, and thus normalize, charges along the outer surface of the NIE-115 cells. Increasing the concentration of CaClz in thesaltsolution madetheintramembraneelectricfieldslightly more negative in both the neurites and somata of these same ten cells, indicating that this manipulation was screening negative charges on the outer surfaces of their membranes. However, since the regional intramembrane electric field difference persisted in the presence of high extracellular calcium (p < .05 with Student’s t test), it could not be attributed to differences in surface charge between the neurites and the somata.

the outer surface of neurites compared with somata was the origin of the nonuniform intramembrane electric field, high extracellular calcium should have normalized it. Since the intramembrane electric field remained significantly different in the neurite compared with the soma after elevation of calcium, the nonuniformity could not have been caused by unequal surface charge distribution between these regions. A final possibility was that the source of the nonuniform intramembrane electric fields was a difference in dipole potential (see Figure 2c) between the neurites and the somata. Though it can sense all the potentials depicted in Figure 2, the location of di-8-ANEPPS (Fluhler et al., 1985) places it near the steepest portion of the dipole potential gradient on the outer leaflet of the membrane bilayer. It follows thatthe di-8-ANEPPS fluorescence ratio is exquisitely sensitive to this source of intramembrane electric field (Gross et al., 1994). Furthermore, since dipole potential is highly dependent on the cholesterol-to-phospholipid ratio in the membrane (Szabo, 1974), recently described differences in cholesterol-to-phospholipid ratio between the membranes of neurites and the membranes of somata (Igarashi et al., 1990) provide a compelling ex-

The regional variations in endogenous intramembrane electric fields described in this work are important because they suggest that neurites and somata will have intrinsically different electrophysiological properties. In particular, voltage-gated channels residing in the neurite will experience a more positive intramembrane electric field in the outer leaflet of the bilayer than the same channel molecules residing in the soma. As a result, channels in the neurite may have a higher probability of opening spontaneously and may be more likely to respond to extracellular signals that cause small depolarizations of the neuronal plasmalemma. Of course, this prediction assumes that the gating charges in the channel protein can interact with the intense electric field associated with the dipole potential. This field can be 50 times stronger than the intramembrane electric field produced by the transmembrane potential but is confined to an approximately 5 8\ thick region within the membrane just below each aqueous interface at the level of the lipid ester groups (Figure 2) (Gross et al., 1994). In the only study to directly address this issue in a cell membrane, a known modulator of dipole potential did affect the potassium current in the squid giant axon (Strichartz et al., 1980). Indeed, though the ability of increased membrane cholesterol to increase calcium currents through voltage dependent channels has been established for several cell types (Lecher et al., 1984; Ma and Coronado, 1988; Bialecki and Tulenko, 1989; Bialecki et al., 1991; Gleason et al., 1991; Zhou et al., 1991; Sen et al., 1992), the connection to dipole potential and intramembrane electric field was not considered. Links among cholesterol-to-phospholipid ratio, intramembrane electric field, and electrophysiology may prompt a reexamination of how cholesterol influences the biology of cells. An especially pertinent example involves differentiation of the NIE-115 neuroblastoma cells used in this study. NIE-115 cells increase their cholesterol-to-phospholipid ratio concomitant with developing neurites, and preventing this increase (with inhibitors of cholesterol synthesis) can block the formation of neurites (de Laat et al., 1984). This has been ascribed to the effect of cholesterol on membrane fluidity. However, an alternate explanation may involve the effect of cholesterol on dipole potential and intramembrane electric field. Depolarizing the transmembrane potential across NIE115 plasmalemmae by increasing extracellular potassium promotes neurite outgrowth and initiation, presumably by activating voltage-gated calcium channels (Anglister et al., 1982). Further, we have shown that localized depolarizations of the membrane of

NelJtCHl 1192

NIE-115cellsarelinked todirected neuriteoutgrowth via localized calcium influx (Bedlack et al., 1992); other papers in which a localized depolarization is the putative first step in a cascade of calcium-dependent events leading to directed neurite growth have also appeared recently (Davenport and Kater, 1992; Zheng et al., 19%). It is important to appreciate that the channel moities that sense these depolarizations are really responding to local intramembrane electric field. An increased cholesterol content would affect the intramembrane electric field within the outer leaflet of the bilayer in the same direction as the described depolarizations, sensitizing, or perhaps even initiating, the cascade that culminates in enhanced neurite growth. Examination of the image in Figure 1 shows that the intramembrane electric field may be heterogeneous on a still more microscopic level than the statistically significant differences between somata and neurites that we have been able to show in this study. Some regions of the growth cone are particularly irregular, with excursions toward both polarized (green) and depolarized (red) potentials within membrane patches of just a few hundred nanometers. We have not established any statistical significance for this more focal variability, but it could correspond to localized electric fields set up by charged residues within clusters of membrane proteins. Indeed, this could, in principle, be a source for variations in intramembrane electric field on a molecular scale both laterally along the surface and within the thickness of a cell membrane. The latter could be explored with ratiometric indicators targeted to different depths within the membrane. In summary, we have shown that the intramembrane electric field in neurons that regulates several components of signal transduction is intrinsically different in neurites and somata.The most Iikelyexplanation for this phenomenon is a difference in dipole potential, perhaps stemming from a difference in cholesterol-to-phospholipid ratio, between neurites and somata. Thus, thedistribution of lipidswithin the plasmalemma may influence the process of differentiation and the ability of the cell to sustain spatially distinct structures and functions. Experimental NlE-115 NIE-115 scribed

graphs

(using

Image,

Microsoft

Excel,

and CricketGraph

soft-

ware). Initially, each cell was divided into five equally sized regions (see Figure 1). Region A always encompassed only membrane from the soma; region B included membrane from the soma, axon hillock, and proximal axon; region C included only axonal membrane; region D included membrane from the distal axon and proximal growth cone; region E included only distal growth cone membrane. Subsequent statistical testing (described in the text) justified dividing cells into only two regions, a soma and a neurite (see Figure 3 and Figure 4).

Acknowledgments We are grateful to John Carson, David Gross, Barbara Ehriich, and Alan Fein for helpful discussions of the manuscript. E. Gross was partially supported through a Rothschild Postdoctoral Feilowship. This research was supported by grants CM35063 and ES05973 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 soiely to indicate this fact. Received

April

19,1994;

revised

June

21, 1994

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Procedures

Cell Culture cells were cultured previously (Bedlack

solution. Exposure times were 0.3-0.5 s for 440 nm light and O.S1.5 s for 530 nm light; IO%-70% neutral density filters were used as needed to limit exposure to light and prevent saturation of the camera. Binning of CCD pixels (2 x 2) was also used as needed to limit exposure to light and thus reduce photobleaching. The signal-to-background ratio (comparison of di-8-ANEPPS membrane fluorescence versus extracellular fluorescence) was always >5 in both neurites and somata. Autofluorescence was negligible compared with the di-8-ANEPPS signal at both excitation wavelengths. Image processing, performed with Perceptics Biovision and NIH Image software, was used to subtract backgrounds (images of cell-free areas adjacent to the targeted cell), segment the contours of the plasma membrane using the bright ring-stain outline of the cell, generate 440 nm/530 nm ratios, and compensate for flat-field irregularities. Di-8-ANEPPS ratios were calibrated against valinomycin-mediated potassium diffusion potentials (Montana et al., 2989; Bedlack et al., 1992); potential was converted to intramembrane electric field by dividing by4OA, which was taken as the thickness of the high resistance region of the membrane. To facilitate comparisons, intramembrane electric field variations were mapped relative to the soma, which was set to 0. lntramembrane electric field comparisons among cells having different orientations and morphologies were achieved by converting di-8-ANEPPS ratios (see Figure 1) to spreadsheets and

and induced et al., 1992).

to differentiate

as de-

Measuring lntramembrane Electric Fields Differentiating NIE-115 cells on glass coverslips were stained with di-8-ANEPPS, mounted in a thermostatted chamber, and visualized using a Nikon diaphot microscope with a 63x 1.4 NA objective as described previously (Bedlack et al., 1992). For experiments, ceils were bathed in a balanced salt solution (1.8 mM CaCI,, 5.5 mM KCI, 1.0 mM MgCI,, 130 mM NaCI, 25 mM glucose, 20 mM HEPES [pH 7.41). Multiple (5-8) fields of view, each containing a single cell, were selected on a given coverslip. Dual wavelength image pairs (excitation at 440 and 530 nm; emission >570 nm) of thesewere acquired with a Photometrics 14bit cooled CCD camera before and after an exchange of the salt

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