Phorochrrrrisrrj ond Phorohiologj. Vol. 28. pp. 219-226. 0 Pergamon Press Ltd.. 1978. Printed in Great Britain
003 1-8655/78/0801-0219SO2.00/0
KINETIC FACTORS GOVERNING SENSITIZED PHOTOOXIDATION OF EXCITABLE CELL MEMBRANES J. P. POOLER and DENNISP. VALENZENO Department of Physiology, Emory University, Atlanta, GA 30322, U.S.A. (Received 28 October 1977; accepted 14 February 1978)
Abstract-The kinetic factors which determine the rate at which Na+ channels in nerve membranes become photochemically modified were studied on giant axons from lobsters using the double sucrose gap voltage clamp technique. Axons were bathed in artificial sea water containing sensitizing dyes and illuminated from a Xe arc source with light in the visible region while being repetitively step depolarized. Successive values of peak Na+ current and time-to-peak were monitored and rate constants for their change served as the assay for magnitude of modification. Action spectra for four sensitizers in the fluorescein series exhibited red shifts of roughly 17 nm demonstrating that sensitizing species are not simply free in solution. Eosin Y diffuses to its sensitization sites with a half time of 70s indicating the existence of a major diffusion barrier which may mean that dye must penetrate to the interior of the membrane to be effective. Eosin Y is removed from sensitization sites by rinse with the same half time but shows two fractions: a faster fraction comprising 80%of sensitizing effectiveness and a slower fraction comprising 20%. The concentration dependence for Eosin Y is linear below 10 pA4 and shows a progressive saturation at higher values, where the relationship is difficult to determine because of shielding. Different sensitizers vary in their ability to sensitize block of channels vs disruption of inactivation, demonstrating separate processes for the two modifications. It is suggested that both modifications proceed from single photon absorption events by individual sensitizer molecules bound or located close to the modification sites on the channels.
INTRODUCI’ION
Photochemical modification is a useful tool to study structure-function relationships of macromolecules in solution and is also a process which can alter the functional behavior of living cells (see, e.g. Jori, 1975). Since the functional behavior of cells resides in the collective properties of their macromolecules it seems feasible to study structure-function relationships in living cells using the photo-chemical modification process. This approach has been initiated in several cell types, including muscle (Kondo and Kasai, 1974), erythrocytes (DeGoeij et al., 1976), and nerve cells (Pooler, 1968, 1972). Nerve cells seem particularly amenable to this approach because their functional behavior can be monitored precisely during the modification process without the possible complication of secondary effects which can develop in post-irradiation assays. Excitable cell behavior
The functional behavior of nerve axons consists of the propagation of nerve impulses, or action potentials. The generation of propagating action potentials resides in the opening and closing of surface membrane permeability pathways through which inorganic ions such as Na+ and K + enter and leave the cell. These membrane permeability pathways are often called pores, or more commonly, channels. For some nerve cells the behavioral properties of channels have been described with considerable precision, yet their molecular nature remains essentially unknown. General reasoning suggests that channels are pro219
teinaceous but there is little solid evidence to support even this mild conjecture. A common feature of mature neuronal membranes is the existence of “fast” sodium channels, so named because the kinetics of permeability change due to channel opening and closing is faster than for other channel types, and because these channels are more permeable to sodium than to any other normally present ion species. Opening and closing of N a + channels, or gating, is controlled by the membrane potential. From a state of rest, when almost all sodium channels are closed, a large depolarization triggers the opening of many channels (activation). A return of the potential to a polarized resting value closes them again (removal of activation), while maintaining the depolarization leads to a second kind of closing with somewhat slower kinetics called inactivation. This behavior is illustrated in Fig. 1. The behavior of Na’ channels is usually studied by means of the voltage clamp method whereby the level of membrane potential is controlled by electronic feedback. By controlling the potential the electrochemical gradient for ion flux is fixed. Changes in ion flux through the membrane (conveniently measured as an electric current) then reflect changes in the cumulative permeability of all N a + channels in the region of membrane under study. Previous investigations
Many classical studies on excitable tissues from a variety of species have demonstrated the susceptibility of excitable cells to sensitized photochemical modification (Lippay, 1929; Supniewski, 1927; Auger and
220 membrane potential
J. P. POOLER and DENNIS P. VALENZENO
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tlnw
c
Figure I. Sodium channel behavior in voltage clamp. At A a large depolarization opens many sodium channels (activation). At B the membrane potential is returned to its original value causing the opened channels to close (removal of activation). At C a longer lasting depolarization leads to activation and then a spontaneous closure of
opened channels (inactivation). Fessard, 1933; Lyudkovskaya and Kayushin, 1960; Chalazanitis, 1964). Non-pigmented nerve cells, such as the lobster axons studied in the present experiments, are totally unresponsive to light in the absence of sensitizers. Most of the classical studies were carried out prior to, or without reference to, the channel hypothesis of excitability. A frequent finding has been light-induced depolarization, firing, and prolongation of electrically stimulated action potentials. The lightinduced firing rarely occurs unless the preparation is previously made hyperexcitable by bathing it in low Ca2+ solutions (see Pooler and Oxford, 1973). Voltage clamp studies have revealed certain characteristic modifications in channel behavior which are compatible with the earlier literature. These modifications include block of Na+ and K' channels and a complex disruption of sodium inactivation (Pooler, 1968, 1972; Oxford et al., 1977). The block of Na' channels and the modification of inactivation in unblocked channels occur in parallel during photooxidation (Pooler, 1972) while the activation component of gating is not changed (Pooler, 1972; and unpublished experiments). Shifts in the m and h conductance parameters of the Hodgkin-Huxley model do not occur (Oxford et a/., 1977; and unpublished experiments) suggesting that alterations in membrane surface charges are not involved. Block of sodium channels could occur by locking the gating components in the closed position, changing the ionic selectivity filter so that it is no longer permeable, or altering some other component so that ions cannot reach the gates or filter. Selectivity has not been studied carefully, but appears not to be changed (Pooler, 1972; Oxford et al., 1977). The time courses for the development of block and change in gating both proceed exponentially and all Na' channels, whether with modified gating or not, become blocked at long illumination times. This suggests that block and modification of the gating component are separate processes. The physical location of the separate functional components which can be modified is not known, however. General models of the Na' channel place the selectivity filter external to the gates
(Hille, 1975, 1976). Useful structure-function information could be obtained by establishing the location of sensitization sites within the three-dimensional architecture of the membrane. Knowing where sensitizing molecules must be in relation to components of the channel might reveal structural information on the functional components. Little is known of the chemical steps underlying excitable cell modification or of the physical factors governing its rate. Many workers have demonstrated an oxygen dependence (e.g. Lippay, 1930; Lillie et al., 1935; Kohli and Bryant, 1964; Ross et al., 1977), but the specific involvement of singlet oxygen has not been assessed. The possible need for binding of the sensitizer is not known. In order to get at these many unanswered questions it is necessary to quantify the factors which govern the magnitude of modification. These factors include dependence on sensitizer concentration, durations of exposure and rinse of the sensitizer, and intensity and wavelength of the illumination source. This information is particularly important for future work aimed at revealing the chemical events subsequent to photon absorption, and should be of utility to biologists using dyes as optical probes of membrane potential (see, e.g. Ross et al., 1977). An attempt was made previously to determine some of these factors (Pooler, 1972). Unfortunately the results were flawed because shielding by dye in the external bath distorted the results. Therefore an action spectrum for Eosin Y and an exposure time relation reported in the previous publication have been re-assessed here using a new chamber system and dye concentrations which obviate shielding artifacts. The new results show that our previous measure of action spectrum red shift was overestimated and that the time required for dye to penetrate into effective sites was underestimated. With the above background in mind the present experiments were initiated with the purpose of learning more about the basic kinetic factors which control photochemical modification of Na' channels, particularly with respect to the location and environment of the sensitizing molecules. MATERIALS AND METHODS
Experiments were performed on giant axons isolated from the circumesophageal connective nerve in lobster (Hornarus americanus) using a double sucrose gap and voltage clamp system for measurement and control of membrane current and voltage (Julian et al., 1962a, b). Axons were normally bathed in an artificial sea water (ASW)containing ions in the following millimolar concentrations: Na+ 428, K + 10, Caz+ 50, Mg2+ 8, C1- 546, SO:- 4, HEPES buffer 4. The pH was adjusted to 7.8 at the normal temperature of 2-3°C. Dyes for photosensitizing the axons (Chem Service Inc., West Chester, PA; Fisher Scientific Co., Pittsburg, PA) were used as supplied and dissolved in ASW. The illumination system is the same as the one described previously (Pooler, 1972). Briefly, the output of a lo00 W Xe arc was passed through glass filters to remove UV and IR and further filtered and attenuated with neutral density or bandpass interference filters. The beam was focused on a region of the nerve chamber covering the
Photooxidation of excitable membranes dye or rinse
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expressed as average values of these relative rate constants. Absorption spectra of sensitizers were measured with a double beam spectrophotometer (Varian Associates, model 635). RESULTS
Concentration dependence
Figure 2. Schematic view of middle portion of sucrose gap chamber. Flowing isosmotic sucrose (hatched areas) separates the Row of dye or ASW rinse solutions (dotted area) in the narrow central pool (diameter = 0.64mm) from wide side pools containing ASW and isosmotic KCI respectively. The distance between side pools is 2.75 mm. Drains (not shown) maintain constant volumes in each pool. The area of intense illumination completely covers the portion of axon in the central pool and part of that under sucrose. Only that portion of membrane between the sucrose flows in the central pool contributes to the measured current.
area of axon exposed to dye in the central pool. Figure 2 shows a schematic view of the chamber system in which a short segment of axon in the area between the sucrose streams is exposed to dye. Many experiments were performed on each axon by sequentially translating the axon from left to right in short steps, thereby bringing new nondye exposed regions of membrane into the central pool each time. For these experiments a chamber of new design with a short fixed-length light path through the dye solution was used and low concentrations (except where noted in the concentration dependence study) were employed to obviate shielding artifacts. Thirty-five mm film images of oscilloscope traces of membrane current and voltage were projected onto the platen of a Hewlett-Packard digitizer, digitized and analyzed using a calculator-plotter system (models 9864A. 9810. and 9862A. Hewlett-Packard Co., Palo Alto, CA). To separate sodium current from total membrane current each depolarizing clamp test pulse was preceded by a 30 mV depolarizing prepulse. Currents during the prepulse were linearly extrapolated to the potential of the test pulse and subtracted from total current to yield Na+ current. The holding potential was - 100 mV and test pulses were to a region of the positive limb of the Na+ current-voltage relation, normally about - 5 mV. A t this potential very little K' current flows until well past the peak in Na+ current so that variations in K' current have negligible effect on measured peak Na' currents. To assay the modification in Na' channel behavior. rate constants for changes in values of peak current and timeto-peak current were measured during illumination by applying six identical depolarizations during Illumination periods of 2.5-7.5 s. The successive decreases in peak current and increases in time-to-peak were subjected to a log regression analysis. The method is illustrated in Fig. 3. lllumination intensities were chosen so that normally at least 757; of the original current remained following illumination. There is considcrable variation in photochemical sensitivity between separate regions of an axon and between different axons. Axonal sensitivity as a function of rostra]caudal position was examined, but no consistent correlation could be discerned. To pool data from several axons with different sensitivities each rate constant from a given axon was scaled relative to the mean value obtained under a standard condition on the same axon. Normally every third trial was a standard condition. Most of the data is
The magnitude of sensitized photochemical modification as a function of sensitizer concentration was measured for Eosin Y (EY).Determining this relationship is somewhat difficult because of shielding by dye in the external bath at the highest concentration. A rough estimate of shielding based on the source intensity, transmission through a solution path length of 0.32 mm (one half the central pool diameter), and an action spectrum red shifted 17 nm relative to the free solution absorption spectrum (see section on action spectra) yields a value of no more than a 3% reduction in effective absorption at concentrations of 10pM and below, but a 24% reduction at l 0 0 p M , the highest used in this study. To reduce shielding a brief pre-illumination rinse can be used but this adds uncertainty to the value for concentration. Therefore, several experimental protocols were followed, some with and some without a pre-illumination rinse period. The results are presented in Fig. 4. Because the estimates of shielding are only approximate we did not attempt to correct the results for this factor. For concentrations up to 10 p M the magnitude of photochemical modification is linear with concentration. Above 10pM a progressive saturation occurs with a half maximum level in the vicinity of 50 p M . The complications mentioned above plus scatter in the data make it difficult to define precisely the relationship at the higher levels but linearity at
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Figure 3. Assays for magnitude of photochemical modification. During illumination the membrane is repeatedly step depolarized for durations of several milliseconds each time as shown in the top traces. Each depolarization gives rise to a characteristic activation and inactivation of sodium current as shown in the second row of traces. This characteristic pattern is progressively altered during illumination. Successive values of peak current (A,B,C) and times-to-peak current (a, b, c) are then plotted as functions of illumination time as shown in the bottom of the figure. Rate constants are calculated from'each plot and serve as the assay.
J. P. POOLER and DENNIS P. VALENZENO
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I
3.5 min dye
block
0
20
40
60
dye concentration
80
100
(UM)
Figure 4. Concentration dependence for Eosin Y as sensitizer for block of sodium channels. Four separate experiments are shown with dye exposure and rinse protocols indicated in the figure. Points are means f S.E.M. of 8 to 15 values each. the lower levels, where shielding is not a problem, is clear. The linearity implies that excited sensitizer molecules lead to membrane alteration independent of each other rather than in a cooperative interaction in which the quantum yield is dependent on concentration.
wise at the switching valve the concentration in the central pool changed exponentially (time constant = 17 s), preceded by a 20 s absolute delay. A step change at the switching valve is therefore equivalent to a step change in the central pool delayed by 37 s.* In these
I
Ill '
Dye exposure time and rinse time dependence When axons are exposed to a given concentration of sensitizer the sensitizing effectiveness does not develop immediately, but instead rises along a quasiexponential time course. Similarly, when dye-exposed axons are rinsed prior to illumination the effectiveness is removed asymptotically. In dye exposure time experiments axons were subjected to a step rise in sensitizer concentration by quick-pulling new areas of membrane into the central pool which contained 5 pM EY. Illumination then followed after various time periods in the dark. The results are shown in Fig. 5 (upper). The sensitizing effectiveness rose to a plateau level, with a half time of about 70s. This time course presumably reflects the penetration of sensitizer through a diffusion barrier between the external bath and sensitization sites on or in the axon membrane. In rinse. time experiments a step fall in sensitizer concentration could not be attained because of the time required to rinse dye from the central pool. When sensitizer concentrations were changed step*At exposure times of a minute or less and concentrations of 10pM or less the sensitizing effect is proportional to the concentration-time product (Le., the time integral of concentration). Exposure to an exponentially falling concentration for a period of several time constants (theoretically infinity) is equivalent to exposure to the original concentration held constant for a period of one time constant. [: Ce-'"')dt = Cr = fi Cdt. Therefore the sensitizing effect of a 20 s exposure at constant concentration followed by exposure to an exponentially falling concentration with a 17 s time constant is the same as exposure to a constant concentration for 37 s.
rate of channel block
'
EXPOSURE TIME
0
2
8
4 6 time (min)
A
0
2
4 6 time (rnin)
8
Figure 5. Exposure time and rinse time dependence for sensitization of sodium channel block by 5pcM Eosin Y. In exposure time measurements untreated regions of axon were pulled into the central pool containing dye and illuminated for 2.5s after exposure for the times shown on the abscissa. In rinse time measurements regions of axon were exposed to dye in the central pool for 2min. The input to the central pool was then switched to a dyefree rinse solution and the axons were illuminated for 2.5 s after times shown on the abscissa. Dead space delay is equivalent to 37 s additional dye exposure and then a step drop to a zero concentration. Points are means f S.E.M. of 9 to 56 values each.
Photooxidation of excitable membranes
I
DBFL
4 50
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ERB
500
wavelength inmi
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500
1 %"
SSO
RB
Figure 6. Action spectra for sodium channel block and absorption spectra in free solution. Regions of axons were bathed in 5 ptM dye for 2 min and illuminated without rinse using bandpass filters with peak wavelengths shown on the abscissa. Points are means f S.E.M.of 6 to 63 values each. Absorption spectra were measured on the same solutions used to bathe the axons. All curves are scaled to the same height. Dyes used are Eosin Y (EY), Rose Bengal (RB), Erythrosin B (ERB) and Dibromofluorescein (DBFL). experiments axons were exposed to S p l 4 EY for 2min. The switching valve was then changed to a sensitizer-free solution and the axons were illuminated after various rinse times. The results are shown in Fig. 5 (lower). After correcting for dead space delay the time to remove half the sensitizing effectiveness was about 70s, the same as the half time for rise , effectiveness. At longer rinse times not all of the sensitizing effectiveness was removed, however, indicating that some of the dye was prevented from leaving the sensitization sites as fast as it diffused in. This suggests that sensitizing dye exists in two fractions. A larger fraction comprising about 80% diffuses out with the same ease with which it diffuses in while the remaining 20% is possibly bound after diffusing in, thus preventing its free diffusion back out. A smaller number of experiments using 4min dye exposures rather than 2 min dye exposures gave effectively the same results.
.
Action spectra
Action spectra for four sensitizers of the fluorescein series were determined using bandpass interference filters (Bausch and Lomb, Rochester, NY,series 78-44) to isolate wavelengths spaced every IOnm. The half width of the filters is approximately 8nm. The data was corrected for intensity variations arising from the arc lamp spectrum and filter characteristics by using a spectroradiometer (Instrumentation Specialities Co., Lincoln, NE, Model SR) to measure the total energy passing through the optical system when each filter was in place. Rate constants were divided by the appropriate intensity factor. The action spectra are shown in Fig. 6, along with the absorption spectra for the dyes in the solutions which bathed the axons. For all four dyes shown the action spectra are redshifted relative to the absorption spectra by roughly 17 nm. The shifts are not simple translations, however, since the long wavelength portions of the curves
Table 1. Differentialdye effectson channel block and disruption of inactivation. Sensitizer
k (time-to-peak)* k (channel block)
Correlation coefficient
Number of points
Dibromo-fluorescein Eosin Y Erythrosin B Rose Bengal
0.36 0.38 0.28 0.16
0.92 0.92 0.91 0.91
132 250 209 286
*Rate constants for increase in time-to-peak sodium current were plotted against rate constants for channel block and subjected to a linear regression analysis. The table lists the ratio of rate constants (slope of regression line), correlation coefficient, and number of points for each sensitizer.
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are shifted more than the short wavelength portions in some cases. These results indicate that the sensitizing molecules are probably not free in the bathing solution but exist in an altered environment within the matrix of the membrane and/or bound to a membrane site. Two modification processej
For all sensitizers used in these experiments the rate at which channels were blocked was highly correlated with the rate at which inactivation in unblocked channels was disrupted (assessed as an increase in time-to-peak current). However, the amount of increase in time-to-peak associated with a given fall in peak current varied from one sensitizer to another. To quantify this, rate constants for increase in timeto-peak were plotted against rate constants for fall in peak current for each sensitizer and subjected to a linear regression analysis. The results are presented in Table 1. Correlation coefficients between the two rate constants were greater than 0.9 for all sensitizers but the slope of the relationship varied from 0.38 to 0.12. With Eosin Y a photooxidation of a duration sufficient to block 50% of the channels led to an increase in time-to-peak of 30%, but for Rose Bengal the same 50% block led to only a 12% increase in time-to-peak. The differential ability of sensitizers to modify these two aspects of channel function, plus the fact that modified inactivation can only be detected in channels which are not blocked, means that separate physical events lead to the two alterations. DISCUSSION
tion peak is red shifted 9 nm when dissolved in decane containing monoolein. On the other hand the red shift may be a result of binding. The binding of Eosin Y to serum albumin red shifts its absorption by 15 nm (Youtsey and Grossweiner, 1967). In the present work the sensitizing species may become bound to an external component of the membrane. A third possibility is binding, but to a site within the membrane matrix away from the aqueous medium such that the environment of the bound dye is .largely non-polar. Sensitizing effectiveness increased linearly with dye concentration. Evidence of saturation at the highest levels appears, although uncertainties in shielding obscure this point. Saturation, if present, could mean that only dye molecules attached to saturable binding sites lead to sensitization or that aggregation or some other cooperative interaction reduces the quantum yield at high concentrations. A peak and then fall in quantum yield as a function of Eosin Y concentration was observed in the photoinactivation of tryp sin (Glad and Spikes, 1966), but shielding by peripheral dye at the higher concentrations was a problem in that and several other studies which showed a similar effect (e.g. Weil and Mayer, 1950; Weil and Buchert, 1951; Welsh and Adams, 1954; Simon and Van Vunakis, 1964). The EY washout experiments indicate the existence of two fractions of sensitizing molecules. The slow washout of the smaller (=20%) fraction suggests that this fraction of molecules is bound, while the larger and faster fraction either does not bind or is at least not rate limited by unbinding. If there are two fractions then the action spectrum must represent a composite of these two fractions. Attempts to numerically synthesize a composite action spectrum assuming that 20% of the effect is contributed by a red-shifted fraction and 80% by a non-shifted fraction do not yield a shape similar to the action spectrum measured here. The large fraction must be shifted to get a reasonable match. The apparent bound state of the smaller fraction and the requirement for a red shift of the larger fraction shows, as stated previously, that the molecules which sensitize are not simply free in aqueous solution.
The key findings in these experiments are the following: (1) The sensitizing molecules are probably bound and/or removed from water in a non-polar environment. (2) Sensitizers must cross a major diffusion barrier to reach the sites of sensitization. (3) The two aspects of Na' channel function which are modified probably involve independent physical processes. (4) The linearity of sensitizing effectiveness with concentration at low values implies that modification proceeds without interactions between sensitizer molLocation of the sensitization sites ecules. The dye exposure time experiments show that a The state oj the sensitizer significant diffusion barrier exists between the bathing The red shift of the action spectra relative to the medium and the regions which dye molecules must free solution absorption spectra for all four sensitizers reach in order to sensitize. The half time for maxiindicates clearly that most of the sensitizing species mum effectiveness is greater than I min. An imporare in a state other than free in aqueous solution. tant question to ask is whether this diffusion barrier There are several possible reasons for the shift. The is a component of the axon membrane or merely the dye fraction which actually sensitizes may be dis- Schwann cell layer outside. Lobster axons (diasolved in the hydrophobic lipid interior of the mem- meter = 100pm) are surrounded by a Schwann cell brane leading to a shift in the absorption spectrum layer (thickness = 5pm) and dye peripheral to the of this dissolved fraction to longer wavelengths. Dye Schwann cells in unlikely to sensitize. If the diffusion absorption spectra in general are sensitive to solvent barrier is the membrane itself than the sensitization polarity (see, e.g. Williamson and Corwin, 1972). Our site must be within the bulk of the membrane or at own measurement with EY indicates that its absorp- the inner surface. Numerous tubular pathways of
Photooxidation of excitable membranes 15-70nm diameter cross the Schwann cell layer between the external medium and the Schwann cellaxon space (Govind and Lang, 1976; Holtzman et al., 1971) which provide a direct aqueous path for molecules as large as the marker protein peroxidase. Sodium ions can diffuse across this barrier with a half time no greater than 1 s (unpublished experiments) and the pharmacologic agent 2,4,6-trinitrophenol (mol wt = 228) can diffuse to its site of action within this time span (Oxford and Pooler, 1975). From this information one would predict that dyes should be able to reach the external membrane surface within a few seconds, and certainly far faster than the measured speed for the development of sensitizing effectiveness. The slow time course suggests the presence of an additional diffusion barrier inside the Schwann cell space. This additional barrier could be part of the membrane. This interpretation is clouded by data on the kinetics of the action of tetrodotoxin, a neurotoxin (mol wt = 319) known to act at the external membrane surface (Narahashi et al., 1966). On lobster axons tetrodotoxin exerts its effect with a half time of several minutes (unpublished experiments). This slow speed has been interpreted on other preparations as uptake of toxin by non-specific binding sites which slows the rise in concentration at active sites (Colquhoun and Ritchie, 1972). With sensitizers significant binding to sites ineffective in sensitizing the membrane could similarly slow the rise in concentration at effective locations. Experiments with squid giant axons in which dyes were applied either externally, as in the present work, or intracellularly showed that sensitizing effectiveness was much greater with intracellular application (Oxford et al., 1977). These results on squid axons imply that the sensitization sites are nearer the internal membrane surface. If the sites are nearer the internal surface than externally applied dye of low permeability would not reach the same concentration as internally applied dye, since the cytoplasm would represent a large sink tending to remove dye from the sensitization sites as soon as it arrived. Permeability values for Eosin Y are not available but a study of dye uptake by several types of cells in culture led Allison and Young (1964) to conclude that Eosin Y is “excluded from living cells altogether.” Even though too little dye reaches the cytoplasm to color intracellular organelles it is still quite possible that Eosin Y can penetrate to the interior of excitable membranes sufficientlywell to sensitize channel modification. From all of the above considerations of dye penetration it is not possible to conclude with confidence just where the sensitization sites for channel block are located, but it seems likely that they are nearer the inside of the membrane than the outside. Do these sites correspond to a functional component of the sodium channel? Present day models of sodium channel structure place the selectivity filter near the outside and the gating components nearer the inside sur-
225
face (Hille, 1975, 1976). While gating is affected in both lobster and squid axons the reversal potential for sodium ions is not changed by photooxidation, suggesting no change in selectivity properties (Pooler, 1972; Oxford et al., 1977). Possibly the peripherally located selectivity component is not involved while the internally located gating components are the target. Excitable membranes in general possess surface charges on both the inside and outside which contribute to the electric field within the membrane (e.g. see Hille et al., 1975). Alteration of these surface charges changes the relation between conductance parameters and membrane potential. Since photooxidation does not appear to cause a shift of conductance parameters along the voltage axis (Oxford et al., 1977; and unpublished experiments) it does not Seem likely that‘ channel modification is brought about simply by altering surface charges. Modification of two components of channel function
Channel block and disruption of inactivation are highly correlated but occur at different relative rates with different sensitizers. The sensitizer-specific capacities of dyes to induce inactivation changes could reflect ‘different abilities to penetrate to the region where inactivation occurs. Such a difference between Eosin Y and Rose Bengal was also found on squid axons, but was even more pronounced than on lobster such that external Rose Benegal led to no perceptible change in inactivation at all (Oxford et al., 1977). When applied internally Rose Bengal led to disruption of inactivation. Dye-specific interactions between sensitizer molecules and channel components could also account for non equality between dyes, but does not explain why Rose Bengal only modified inactivation from one side. It is not known what molecular moieties correspond to the three behavioral components of channel function (selectivity, activation, inactivation). Further revelations of dye-specific photochemical modification with as yet untried sensitizers could well provide new insights into the molecular basis for these aspects of excitable membrane function. In all of the previous work and in that reported here the relation between unblocked functional channels and photon dose (illumination time) shows an exponential decline with no lag (see, e.g. Fig. 2 in Pooler, 1972). From a target theory viewpoint this implies that channel block is a one photon-one channel interaction, with all channels belonging to a single population. The survival curve for the inactivation gating component cannot easily be defined in target theory terms. Increases in time-to-peak current occur because of a disruption of inactivation, but relating this to the survival of a specific inactivation entity is not possible. However, absence of a lag in the increase of time-to-peak over a wide variety of dose rates suggests that inactivation is modified as a one hit event also.
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The physical and chemical events leading to channel modification are still not clear. On the basis of data presently available and on analogy with simpler chemical systems we can speculate as follows. An individual sensitizer molecule diffuses into the lipid region of the membrane near the middle or inside surface and then binds to or lodges near one of several key sites on one channel. Absorption of a photon excites the sensitizer which then collides with an oxygen molecule, leaving it in its singlet state. The singlet oxygen molecule then oxidizes a susceptible amino acid residue at the key site. If the key site
is of one kind the channel becomes blocked. If it is of the other kind then a conformational change required for inactivation is interfered with. We hope that future experiments will help clarify whether this speculation or other possible mechanisms are an accurate account of the steps involved. AcknowledgementsThe authors gratefully acknowledge the technical assistance of L. Nielsen, C. Bane, and M. Neeley; chamber construction by M. Roberts; secretarial assistance by C. Guest and J. McLeod and generous use of the spectrophotometer in the laboratory of Dr. S. Hersey. Supported by NIH Grant NS09040.
REFERENCES
Allison, A. C. and M. R. Young (1964) Life Sci. 3, 147-1414. Auger, D. and A. Fessard (1933) Ann. Physiol. Physicochim. Biol. 9, 873-879. Chalazonitis, N. (1964) Photochem. Photobiol. 3, 539-559. Colquhoun, D. and J. M. Ritchie (1972) Mol. Pharmacol. 8, 285-292. DeGoeij, A. F. P. M., R. J. C. Van Straalen and J. Van Steveninck (1976) Clin. Chim. Acta 71. 485494.
Glad, B. W. and J. D. Spikes (1966) Radiat. Res. 27, 237-249. Govind, C. K. and F. Lang (1976) J. Comp. Neurol. 170.421433. Hille, B. (1975) J . Gen. Physiol. 66, 535-560. Hille, B. (1976) Ann. Rev. Physiol. 38, 139-152. Hille, B., A. M. Woodhull and B. I. Shapiro (1975) Phil. Trans. R. SOC. B 270, 301-318. Holtzman, E., A. R. Freeman and L. A. Kashmer (1971) J . Cell. Biol. 44, 438-445. Jori, G. (1975) Photochem. Photobiol. 21, 463-467. Julian, F. J., J. W. Moore and D. E. Goldman (1962a) J. Gen. Physiol. 45, 1195-1216. Julian, F. J., J. W. Moore and D. E. Goldman (1962b) J. Gen. Physiol. 45, 1217-1238. Kohli, R. P. and S. H. Bryant (1964) Experientia 20, 368-369. Kondo, M. and M. Kasai (1974) Photochem. Photobiol. 19, 3 5 4 1 . Lillie, R. S., M. A. Hinrichs and A. J. Kosman (1935) J . Cell. Comp. Physiol. 6, 487-503. Lippay, F. (1929) Pflugers Arch. ges. Physiol. 222, 616639. Lippay, F. (1930) Pflugers Arch. ges. Physiol. 224, 587-599. Lyudkovskaya, R. G. and L. P. Kayushin (1960) Biophysics. 5, 754-762. Narahashi, T., N. C. Anderson and J. W. Moore (1966) Science 153, 765-767. Oxford, G.S. and J. P. Pooler (1975) J . Gen. Physiol. 66, 765-779. Oxford, G. S.. J. Pooler and T. Narahashi (1977) J . Membr. Biol. 36, 159-174. Pooler, J. (1968) Biophys. J . 8, 1009-1026. Pooler, J. (1972) J . Gen. Physiol. 60, 367-387. Pooler, J. and G. S. Oxford (1973) J. Membr. Biol. 12, 339-348. Ross, W. N., B. M. Salzberg, L. B. Cohen, A. Grinvald, H. V. Davilla, A. S. Waggoner and C. H. Wang (1977) J . Membr. Biol. 33, 141-183. Simon, M. I. and H. Van Vunakis (1964) Arch. Biochem. Biophys. 105. 197-206. Supniewski, J. V. (1927) J . Physiol. 64, 30-38. Welsh, J. N. and M. H. Adams (1954) J. Bacteriol. 68, 122-127. Weil, L. and A. R. Buchert (1951) Arch. Biochem. Biophys. 34, 1-15. Weil, L. and J. Mayer (1950) Arch. Biochem. 29, 241-259. Williamson, C. E. and A. H. Corwin (1972) J. Colloid Interface Sci. 38, 567-576. Youtsey, K. J. and L. 1. Grossweiner (1967) Photochem. Photobiol. 6. 721-731.
003 1-8655/78/0801-0219SO2.00/0
KINETIC FACTORS GOVERNING SENSITIZED PHOTOOXIDATION OF EXCITABLE CELL MEMBRANES J. P. POOLER and DENNISP. VALENZENO Department of Physiology, Emory University, Atlanta, GA 30322, U.S.A. (Received 28 October 1977; accepted 14 February 1978)
Abstract-The kinetic factors which determine the rate at which Na+ channels in nerve membranes become photochemically modified were studied on giant axons from lobsters using the double sucrose gap voltage clamp technique. Axons were bathed in artificial sea water containing sensitizing dyes and illuminated from a Xe arc source with light in the visible region while being repetitively step depolarized. Successive values of peak Na+ current and time-to-peak were monitored and rate constants for their change served as the assay for magnitude of modification. Action spectra for four sensitizers in the fluorescein series exhibited red shifts of roughly 17 nm demonstrating that sensitizing species are not simply free in solution. Eosin Y diffuses to its sensitization sites with a half time of 70s indicating the existence of a major diffusion barrier which may mean that dye must penetrate to the interior of the membrane to be effective. Eosin Y is removed from sensitization sites by rinse with the same half time but shows two fractions: a faster fraction comprising 80%of sensitizing effectiveness and a slower fraction comprising 20%. The concentration dependence for Eosin Y is linear below 10 pA4 and shows a progressive saturation at higher values, where the relationship is difficult to determine because of shielding. Different sensitizers vary in their ability to sensitize block of channels vs disruption of inactivation, demonstrating separate processes for the two modifications. It is suggested that both modifications proceed from single photon absorption events by individual sensitizer molecules bound or located close to the modification sites on the channels.
INTRODUCI’ION
Photochemical modification is a useful tool to study structure-function relationships of macromolecules in solution and is also a process which can alter the functional behavior of living cells (see, e.g. Jori, 1975). Since the functional behavior of cells resides in the collective properties of their macromolecules it seems feasible to study structure-function relationships in living cells using the photo-chemical modification process. This approach has been initiated in several cell types, including muscle (Kondo and Kasai, 1974), erythrocytes (DeGoeij et al., 1976), and nerve cells (Pooler, 1968, 1972). Nerve cells seem particularly amenable to this approach because their functional behavior can be monitored precisely during the modification process without the possible complication of secondary effects which can develop in post-irradiation assays. Excitable cell behavior
The functional behavior of nerve axons consists of the propagation of nerve impulses, or action potentials. The generation of propagating action potentials resides in the opening and closing of surface membrane permeability pathways through which inorganic ions such as Na+ and K + enter and leave the cell. These membrane permeability pathways are often called pores, or more commonly, channels. For some nerve cells the behavioral properties of channels have been described with considerable precision, yet their molecular nature remains essentially unknown. General reasoning suggests that channels are pro219
teinaceous but there is little solid evidence to support even this mild conjecture. A common feature of mature neuronal membranes is the existence of “fast” sodium channels, so named because the kinetics of permeability change due to channel opening and closing is faster than for other channel types, and because these channels are more permeable to sodium than to any other normally present ion species. Opening and closing of N a + channels, or gating, is controlled by the membrane potential. From a state of rest, when almost all sodium channels are closed, a large depolarization triggers the opening of many channels (activation). A return of the potential to a polarized resting value closes them again (removal of activation), while maintaining the depolarization leads to a second kind of closing with somewhat slower kinetics called inactivation. This behavior is illustrated in Fig. 1. The behavior of Na’ channels is usually studied by means of the voltage clamp method whereby the level of membrane potential is controlled by electronic feedback. By controlling the potential the electrochemical gradient for ion flux is fixed. Changes in ion flux through the membrane (conveniently measured as an electric current) then reflect changes in the cumulative permeability of all N a + channels in the region of membrane under study. Previous investigations
Many classical studies on excitable tissues from a variety of species have demonstrated the susceptibility of excitable cells to sensitized photochemical modification (Lippay, 1929; Supniewski, 1927; Auger and
220 membrane potential
J. P. POOLER and DENNIS P. VALENZENO
-
tlnw
c
Figure I. Sodium channel behavior in voltage clamp. At A a large depolarization opens many sodium channels (activation). At B the membrane potential is returned to its original value causing the opened channels to close (removal of activation). At C a longer lasting depolarization leads to activation and then a spontaneous closure of
opened channels (inactivation). Fessard, 1933; Lyudkovskaya and Kayushin, 1960; Chalazanitis, 1964). Non-pigmented nerve cells, such as the lobster axons studied in the present experiments, are totally unresponsive to light in the absence of sensitizers. Most of the classical studies were carried out prior to, or without reference to, the channel hypothesis of excitability. A frequent finding has been light-induced depolarization, firing, and prolongation of electrically stimulated action potentials. The lightinduced firing rarely occurs unless the preparation is previously made hyperexcitable by bathing it in low Ca2+ solutions (see Pooler and Oxford, 1973). Voltage clamp studies have revealed certain characteristic modifications in channel behavior which are compatible with the earlier literature. These modifications include block of Na+ and K' channels and a complex disruption of sodium inactivation (Pooler, 1968, 1972; Oxford et al., 1977). The block of Na' channels and the modification of inactivation in unblocked channels occur in parallel during photooxidation (Pooler, 1972) while the activation component of gating is not changed (Pooler, 1972; and unpublished experiments). Shifts in the m and h conductance parameters of the Hodgkin-Huxley model do not occur (Oxford et a/., 1977; and unpublished experiments) suggesting that alterations in membrane surface charges are not involved. Block of sodium channels could occur by locking the gating components in the closed position, changing the ionic selectivity filter so that it is no longer permeable, or altering some other component so that ions cannot reach the gates or filter. Selectivity has not been studied carefully, but appears not to be changed (Pooler, 1972; Oxford et al., 1977). The time courses for the development of block and change in gating both proceed exponentially and all Na' channels, whether with modified gating or not, become blocked at long illumination times. This suggests that block and modification of the gating component are separate processes. The physical location of the separate functional components which can be modified is not known, however. General models of the Na' channel place the selectivity filter external to the gates
(Hille, 1975, 1976). Useful structure-function information could be obtained by establishing the location of sensitization sites within the three-dimensional architecture of the membrane. Knowing where sensitizing molecules must be in relation to components of the channel might reveal structural information on the functional components. Little is known of the chemical steps underlying excitable cell modification or of the physical factors governing its rate. Many workers have demonstrated an oxygen dependence (e.g. Lippay, 1930; Lillie et al., 1935; Kohli and Bryant, 1964; Ross et al., 1977), but the specific involvement of singlet oxygen has not been assessed. The possible need for binding of the sensitizer is not known. In order to get at these many unanswered questions it is necessary to quantify the factors which govern the magnitude of modification. These factors include dependence on sensitizer concentration, durations of exposure and rinse of the sensitizer, and intensity and wavelength of the illumination source. This information is particularly important for future work aimed at revealing the chemical events subsequent to photon absorption, and should be of utility to biologists using dyes as optical probes of membrane potential (see, e.g. Ross et al., 1977). An attempt was made previously to determine some of these factors (Pooler, 1972). Unfortunately the results were flawed because shielding by dye in the external bath distorted the results. Therefore an action spectrum for Eosin Y and an exposure time relation reported in the previous publication have been re-assessed here using a new chamber system and dye concentrations which obviate shielding artifacts. The new results show that our previous measure of action spectrum red shift was overestimated and that the time required for dye to penetrate into effective sites was underestimated. With the above background in mind the present experiments were initiated with the purpose of learning more about the basic kinetic factors which control photochemical modification of Na' channels, particularly with respect to the location and environment of the sensitizing molecules. MATERIALS AND METHODS
Experiments were performed on giant axons isolated from the circumesophageal connective nerve in lobster (Hornarus americanus) using a double sucrose gap and voltage clamp system for measurement and control of membrane current and voltage (Julian et al., 1962a, b). Axons were normally bathed in an artificial sea water (ASW)containing ions in the following millimolar concentrations: Na+ 428, K + 10, Caz+ 50, Mg2+ 8, C1- 546, SO:- 4, HEPES buffer 4. The pH was adjusted to 7.8 at the normal temperature of 2-3°C. Dyes for photosensitizing the axons (Chem Service Inc., West Chester, PA; Fisher Scientific Co., Pittsburg, PA) were used as supplied and dissolved in ASW. The illumination system is the same as the one described previously (Pooler, 1972). Briefly, the output of a lo00 W Xe arc was passed through glass filters to remove UV and IR and further filtered and attenuated with neutral density or bandpass interference filters. The beam was focused on a region of the nerve chamber covering the
Photooxidation of excitable membranes dye or rinse
i\-F
221
expressed as average values of these relative rate constants. Absorption spectra of sensitizers were measured with a double beam spectrophotometer (Varian Associates, model 635). RESULTS
Concentration dependence
Figure 2. Schematic view of middle portion of sucrose gap chamber. Flowing isosmotic sucrose (hatched areas) separates the Row of dye or ASW rinse solutions (dotted area) in the narrow central pool (diameter = 0.64mm) from wide side pools containing ASW and isosmotic KCI respectively. The distance between side pools is 2.75 mm. Drains (not shown) maintain constant volumes in each pool. The area of intense illumination completely covers the portion of axon in the central pool and part of that under sucrose. Only that portion of membrane between the sucrose flows in the central pool contributes to the measured current.
area of axon exposed to dye in the central pool. Figure 2 shows a schematic view of the chamber system in which a short segment of axon in the area between the sucrose streams is exposed to dye. Many experiments were performed on each axon by sequentially translating the axon from left to right in short steps, thereby bringing new nondye exposed regions of membrane into the central pool each time. For these experiments a chamber of new design with a short fixed-length light path through the dye solution was used and low concentrations (except where noted in the concentration dependence study) were employed to obviate shielding artifacts. Thirty-five mm film images of oscilloscope traces of membrane current and voltage were projected onto the platen of a Hewlett-Packard digitizer, digitized and analyzed using a calculator-plotter system (models 9864A. 9810. and 9862A. Hewlett-Packard Co., Palo Alto, CA). To separate sodium current from total membrane current each depolarizing clamp test pulse was preceded by a 30 mV depolarizing prepulse. Currents during the prepulse were linearly extrapolated to the potential of the test pulse and subtracted from total current to yield Na+ current. The holding potential was - 100 mV and test pulses were to a region of the positive limb of the Na+ current-voltage relation, normally about - 5 mV. A t this potential very little K' current flows until well past the peak in Na+ current so that variations in K' current have negligible effect on measured peak Na' currents. To assay the modification in Na' channel behavior. rate constants for changes in values of peak current and timeto-peak current were measured during illumination by applying six identical depolarizations during Illumination periods of 2.5-7.5 s. The successive decreases in peak current and increases in time-to-peak were subjected to a log regression analysis. The method is illustrated in Fig. 3. lllumination intensities were chosen so that normally at least 757; of the original current remained following illumination. There is considcrable variation in photochemical sensitivity between separate regions of an axon and between different axons. Axonal sensitivity as a function of rostra]caudal position was examined, but no consistent correlation could be discerned. To pool data from several axons with different sensitivities each rate constant from a given axon was scaled relative to the mean value obtained under a standard condition on the same axon. Normally every third trial was a standard condition. Most of the data is
The magnitude of sensitized photochemical modification as a function of sensitizer concentration was measured for Eosin Y (EY).Determining this relationship is somewhat difficult because of shielding by dye in the external bath at the highest concentration. A rough estimate of shielding based on the source intensity, transmission through a solution path length of 0.32 mm (one half the central pool diameter), and an action spectrum red shifted 17 nm relative to the free solution absorption spectrum (see section on action spectra) yields a value of no more than a 3% reduction in effective absorption at concentrations of 10pM and below, but a 24% reduction at l 0 0 p M , the highest used in this study. To reduce shielding a brief pre-illumination rinse can be used but this adds uncertainty to the value for concentration. Therefore, several experimental protocols were followed, some with and some without a pre-illumination rinse period. The results are presented in Fig. 4. Because the estimates of shielding are only approximate we did not attempt to correct the results for this factor. For concentrations up to 10 p M the magnitude of photochemical modification is linear with concentration. Above 10pM a progressive saturation occurs with a half maximum level in the vicinity of 50 p M . The complications mentioned above plus scatter in the data make it difficult to define precisely the relationship at the higher levels but linearity at
vqxL-'J-'J-
Illumlnmtbn tlnu
illumln8tlon time
Figure 3. Assays for magnitude of photochemical modification. During illumination the membrane is repeatedly step depolarized for durations of several milliseconds each time as shown in the top traces. Each depolarization gives rise to a characteristic activation and inactivation of sodium current as shown in the second row of traces. This characteristic pattern is progressively altered during illumination. Successive values of peak current (A,B,C) and times-to-peak current (a, b, c) are then plotted as functions of illumination time as shown in the bottom of the figure. Rate constants are calculated from'each plot and serve as the assay.
J. P. POOLER and DENNIS P. VALENZENO
222
I
3.5 min dye
block
0
20
40
60
dye concentration
80
100
(UM)
Figure 4. Concentration dependence for Eosin Y as sensitizer for block of sodium channels. Four separate experiments are shown with dye exposure and rinse protocols indicated in the figure. Points are means f S.E.M. of 8 to 15 values each. the lower levels, where shielding is not a problem, is clear. The linearity implies that excited sensitizer molecules lead to membrane alteration independent of each other rather than in a cooperative interaction in which the quantum yield is dependent on concentration.
wise at the switching valve the concentration in the central pool changed exponentially (time constant = 17 s), preceded by a 20 s absolute delay. A step change at the switching valve is therefore equivalent to a step change in the central pool delayed by 37 s.* In these
I
Ill '
Dye exposure time and rinse time dependence When axons are exposed to a given concentration of sensitizer the sensitizing effectiveness does not develop immediately, but instead rises along a quasiexponential time course. Similarly, when dye-exposed axons are rinsed prior to illumination the effectiveness is removed asymptotically. In dye exposure time experiments axons were subjected to a step rise in sensitizer concentration by quick-pulling new areas of membrane into the central pool which contained 5 pM EY. Illumination then followed after various time periods in the dark. The results are shown in Fig. 5 (upper). The sensitizing effectiveness rose to a plateau level, with a half time of about 70s. This time course presumably reflects the penetration of sensitizer through a diffusion barrier between the external bath and sensitization sites on or in the axon membrane. In rinse. time experiments a step fall in sensitizer concentration could not be attained because of the time required to rinse dye from the central pool. When sensitizer concentrations were changed step*At exposure times of a minute or less and concentrations of 10pM or less the sensitizing effect is proportional to the concentration-time product (Le., the time integral of concentration). Exposure to an exponentially falling concentration for a period of several time constants (theoretically infinity) is equivalent to exposure to the original concentration held constant for a period of one time constant. [: Ce-'"')dt = Cr = fi Cdt. Therefore the sensitizing effect of a 20 s exposure at constant concentration followed by exposure to an exponentially falling concentration with a 17 s time constant is the same as exposure to a constant concentration for 37 s.
rate of channel block
'
EXPOSURE TIME
0
2
8
4 6 time (min)
A
0
2
4 6 time (rnin)
8
Figure 5. Exposure time and rinse time dependence for sensitization of sodium channel block by 5pcM Eosin Y. In exposure time measurements untreated regions of axon were pulled into the central pool containing dye and illuminated for 2.5s after exposure for the times shown on the abscissa. In rinse time measurements regions of axon were exposed to dye in the central pool for 2min. The input to the central pool was then switched to a dyefree rinse solution and the axons were illuminated for 2.5 s after times shown on the abscissa. Dead space delay is equivalent to 37 s additional dye exposure and then a step drop to a zero concentration. Points are means f S.E.M. of 9 to 56 values each.
Photooxidation of excitable membranes
I
DBFL
4 50
223
ERB
500
wavelength inmi
-
s5c
---
500
1 %"
SSO
RB
Figure 6. Action spectra for sodium channel block and absorption spectra in free solution. Regions of axons were bathed in 5 ptM dye for 2 min and illuminated without rinse using bandpass filters with peak wavelengths shown on the abscissa. Points are means f S.E.M.of 6 to 63 values each. Absorption spectra were measured on the same solutions used to bathe the axons. All curves are scaled to the same height. Dyes used are Eosin Y (EY), Rose Bengal (RB), Erythrosin B (ERB) and Dibromofluorescein (DBFL). experiments axons were exposed to S p l 4 EY for 2min. The switching valve was then changed to a sensitizer-free solution and the axons were illuminated after various rinse times. The results are shown in Fig. 5 (lower). After correcting for dead space delay the time to remove half the sensitizing effectiveness was about 70s, the same as the half time for rise , effectiveness. At longer rinse times not all of the sensitizing effectiveness was removed, however, indicating that some of the dye was prevented from leaving the sensitization sites as fast as it diffused in. This suggests that sensitizing dye exists in two fractions. A larger fraction comprising about 80% diffuses out with the same ease with which it diffuses in while the remaining 20% is possibly bound after diffusing in, thus preventing its free diffusion back out. A smaller number of experiments using 4min dye exposures rather than 2 min dye exposures gave effectively the same results.
.
Action spectra
Action spectra for four sensitizers of the fluorescein series were determined using bandpass interference filters (Bausch and Lomb, Rochester, NY,series 78-44) to isolate wavelengths spaced every IOnm. The half width of the filters is approximately 8nm. The data was corrected for intensity variations arising from the arc lamp spectrum and filter characteristics by using a spectroradiometer (Instrumentation Specialities Co., Lincoln, NE, Model SR) to measure the total energy passing through the optical system when each filter was in place. Rate constants were divided by the appropriate intensity factor. The action spectra are shown in Fig. 6, along with the absorption spectra for the dyes in the solutions which bathed the axons. For all four dyes shown the action spectra are redshifted relative to the absorption spectra by roughly 17 nm. The shifts are not simple translations, however, since the long wavelength portions of the curves
Table 1. Differentialdye effectson channel block and disruption of inactivation. Sensitizer
k (time-to-peak)* k (channel block)
Correlation coefficient
Number of points
Dibromo-fluorescein Eosin Y Erythrosin B Rose Bengal
0.36 0.38 0.28 0.16
0.92 0.92 0.91 0.91
132 250 209 286
*Rate constants for increase in time-to-peak sodium current were plotted against rate constants for channel block and subjected to a linear regression analysis. The table lists the ratio of rate constants (slope of regression line), correlation coefficient, and number of points for each sensitizer.
J. P. POOLER and DENNIS P. VALENZENO
224
are shifted more than the short wavelength portions in some cases. These results indicate that the sensitizing molecules are probably not free in the bathing solution but exist in an altered environment within the matrix of the membrane and/or bound to a membrane site. Two modification processej
For all sensitizers used in these experiments the rate at which channels were blocked was highly correlated with the rate at which inactivation in unblocked channels was disrupted (assessed as an increase in time-to-peak current). However, the amount of increase in time-to-peak associated with a given fall in peak current varied from one sensitizer to another. To quantify this, rate constants for increase in timeto-peak were plotted against rate constants for fall in peak current for each sensitizer and subjected to a linear regression analysis. The results are presented in Table 1. Correlation coefficients between the two rate constants were greater than 0.9 for all sensitizers but the slope of the relationship varied from 0.38 to 0.12. With Eosin Y a photooxidation of a duration sufficient to block 50% of the channels led to an increase in time-to-peak of 30%, but for Rose Bengal the same 50% block led to only a 12% increase in time-to-peak. The differential ability of sensitizers to modify these two aspects of channel function, plus the fact that modified inactivation can only be detected in channels which are not blocked, means that separate physical events lead to the two alterations. DISCUSSION
tion peak is red shifted 9 nm when dissolved in decane containing monoolein. On the other hand the red shift may be a result of binding. The binding of Eosin Y to serum albumin red shifts its absorption by 15 nm (Youtsey and Grossweiner, 1967). In the present work the sensitizing species may become bound to an external component of the membrane. A third possibility is binding, but to a site within the membrane matrix away from the aqueous medium such that the environment of the bound dye is .largely non-polar. Sensitizing effectiveness increased linearly with dye concentration. Evidence of saturation at the highest levels appears, although uncertainties in shielding obscure this point. Saturation, if present, could mean that only dye molecules attached to saturable binding sites lead to sensitization or that aggregation or some other cooperative interaction reduces the quantum yield at high concentrations. A peak and then fall in quantum yield as a function of Eosin Y concentration was observed in the photoinactivation of tryp sin (Glad and Spikes, 1966), but shielding by peripheral dye at the higher concentrations was a problem in that and several other studies which showed a similar effect (e.g. Weil and Mayer, 1950; Weil and Buchert, 1951; Welsh and Adams, 1954; Simon and Van Vunakis, 1964). The EY washout experiments indicate the existence of two fractions of sensitizing molecules. The slow washout of the smaller (=20%) fraction suggests that this fraction of molecules is bound, while the larger and faster fraction either does not bind or is at least not rate limited by unbinding. If there are two fractions then the action spectrum must represent a composite of these two fractions. Attempts to numerically synthesize a composite action spectrum assuming that 20% of the effect is contributed by a red-shifted fraction and 80% by a non-shifted fraction do not yield a shape similar to the action spectrum measured here. The large fraction must be shifted to get a reasonable match. The apparent bound state of the smaller fraction and the requirement for a red shift of the larger fraction shows, as stated previously, that the molecules which sensitize are not simply free in aqueous solution.
The key findings in these experiments are the following: (1) The sensitizing molecules are probably bound and/or removed from water in a non-polar environment. (2) Sensitizers must cross a major diffusion barrier to reach the sites of sensitization. (3) The two aspects of Na' channel function which are modified probably involve independent physical processes. (4) The linearity of sensitizing effectiveness with concentration at low values implies that modification proceeds without interactions between sensitizer molLocation of the sensitization sites ecules. The dye exposure time experiments show that a The state oj the sensitizer significant diffusion barrier exists between the bathing The red shift of the action spectra relative to the medium and the regions which dye molecules must free solution absorption spectra for all four sensitizers reach in order to sensitize. The half time for maxiindicates clearly that most of the sensitizing species mum effectiveness is greater than I min. An imporare in a state other than free in aqueous solution. tant question to ask is whether this diffusion barrier There are several possible reasons for the shift. The is a component of the axon membrane or merely the dye fraction which actually sensitizes may be dis- Schwann cell layer outside. Lobster axons (diasolved in the hydrophobic lipid interior of the mem- meter = 100pm) are surrounded by a Schwann cell brane leading to a shift in the absorption spectrum layer (thickness = 5pm) and dye peripheral to the of this dissolved fraction to longer wavelengths. Dye Schwann cells in unlikely to sensitize. If the diffusion absorption spectra in general are sensitive to solvent barrier is the membrane itself than the sensitization polarity (see, e.g. Williamson and Corwin, 1972). Our site must be within the bulk of the membrane or at own measurement with EY indicates that its absorp- the inner surface. Numerous tubular pathways of
Photooxidation of excitable membranes 15-70nm diameter cross the Schwann cell layer between the external medium and the Schwann cellaxon space (Govind and Lang, 1976; Holtzman et al., 1971) which provide a direct aqueous path for molecules as large as the marker protein peroxidase. Sodium ions can diffuse across this barrier with a half time no greater than 1 s (unpublished experiments) and the pharmacologic agent 2,4,6-trinitrophenol (mol wt = 228) can diffuse to its site of action within this time span (Oxford and Pooler, 1975). From this information one would predict that dyes should be able to reach the external membrane surface within a few seconds, and certainly far faster than the measured speed for the development of sensitizing effectiveness. The slow time course suggests the presence of an additional diffusion barrier inside the Schwann cell space. This additional barrier could be part of the membrane. This interpretation is clouded by data on the kinetics of the action of tetrodotoxin, a neurotoxin (mol wt = 319) known to act at the external membrane surface (Narahashi et al., 1966). On lobster axons tetrodotoxin exerts its effect with a half time of several minutes (unpublished experiments). This slow speed has been interpreted on other preparations as uptake of toxin by non-specific binding sites which slows the rise in concentration at active sites (Colquhoun and Ritchie, 1972). With sensitizers significant binding to sites ineffective in sensitizing the membrane could similarly slow the rise in concentration at effective locations. Experiments with squid giant axons in which dyes were applied either externally, as in the present work, or intracellularly showed that sensitizing effectiveness was much greater with intracellular application (Oxford et al., 1977). These results on squid axons imply that the sensitization sites are nearer the internal membrane surface. If the sites are nearer the internal surface than externally applied dye of low permeability would not reach the same concentration as internally applied dye, since the cytoplasm would represent a large sink tending to remove dye from the sensitization sites as soon as it arrived. Permeability values for Eosin Y are not available but a study of dye uptake by several types of cells in culture led Allison and Young (1964) to conclude that Eosin Y is “excluded from living cells altogether.” Even though too little dye reaches the cytoplasm to color intracellular organelles it is still quite possible that Eosin Y can penetrate to the interior of excitable membranes sufficientlywell to sensitize channel modification. From all of the above considerations of dye penetration it is not possible to conclude with confidence just where the sensitization sites for channel block are located, but it seems likely that they are nearer the inside of the membrane than the outside. Do these sites correspond to a functional component of the sodium channel? Present day models of sodium channel structure place the selectivity filter near the outside and the gating components nearer the inside sur-
225
face (Hille, 1975, 1976). While gating is affected in both lobster and squid axons the reversal potential for sodium ions is not changed by photooxidation, suggesting no change in selectivity properties (Pooler, 1972; Oxford et al., 1977). Possibly the peripherally located selectivity component is not involved while the internally located gating components are the target. Excitable membranes in general possess surface charges on both the inside and outside which contribute to the electric field within the membrane (e.g. see Hille et al., 1975). Alteration of these surface charges changes the relation between conductance parameters and membrane potential. Since photooxidation does not appear to cause a shift of conductance parameters along the voltage axis (Oxford et al., 1977; and unpublished experiments) it does not Seem likely that‘ channel modification is brought about simply by altering surface charges. Modification of two components of channel function
Channel block and disruption of inactivation are highly correlated but occur at different relative rates with different sensitizers. The sensitizer-specific capacities of dyes to induce inactivation changes could reflect ‘different abilities to penetrate to the region where inactivation occurs. Such a difference between Eosin Y and Rose Bengal was also found on squid axons, but was even more pronounced than on lobster such that external Rose Benegal led to no perceptible change in inactivation at all (Oxford et al., 1977). When applied internally Rose Bengal led to disruption of inactivation. Dye-specific interactions between sensitizer molecules and channel components could also account for non equality between dyes, but does not explain why Rose Bengal only modified inactivation from one side. It is not known what molecular moieties correspond to the three behavioral components of channel function (selectivity, activation, inactivation). Further revelations of dye-specific photochemical modification with as yet untried sensitizers could well provide new insights into the molecular basis for these aspects of excitable membrane function. In all of the previous work and in that reported here the relation between unblocked functional channels and photon dose (illumination time) shows an exponential decline with no lag (see, e.g. Fig. 2 in Pooler, 1972). From a target theory viewpoint this implies that channel block is a one photon-one channel interaction, with all channels belonging to a single population. The survival curve for the inactivation gating component cannot easily be defined in target theory terms. Increases in time-to-peak current occur because of a disruption of inactivation, but relating this to the survival of a specific inactivation entity is not possible. However, absence of a lag in the increase of time-to-peak over a wide variety of dose rates suggests that inactivation is modified as a one hit event also.
J. P. POOLER and DENNISP. VALENZENO
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The physical and chemical events leading to channel modification are still not clear. On the basis of data presently available and on analogy with simpler chemical systems we can speculate as follows. An individual sensitizer molecule diffuses into the lipid region of the membrane near the middle or inside surface and then binds to or lodges near one of several key sites on one channel. Absorption of a photon excites the sensitizer which then collides with an oxygen molecule, leaving it in its singlet state. The singlet oxygen molecule then oxidizes a susceptible amino acid residue at the key site. If the key site
is of one kind the channel becomes blocked. If it is of the other kind then a conformational change required for inactivation is interfered with. We hope that future experiments will help clarify whether this speculation or other possible mechanisms are an accurate account of the steps involved. AcknowledgementsThe authors gratefully acknowledge the technical assistance of L. Nielsen, C. Bane, and M. Neeley; chamber construction by M. Roberts; secretarial assistance by C. Guest and J. McLeod and generous use of the spectrophotometer in the laboratory of Dr. S. Hersey. Supported by NIH Grant NS09040.
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