Neuron, Vol. 5, 675-684, November, 1990, Copyright @ 1990 by Cell Press

Neuraminidase Treatment Modifies the Function of Electroplax Sodium Channels in Planar iipid Bilayers Esperanza Recio-Pinto: William 6. Thornhill,+* Daniel S. Duch: Simon R. levinson,+ and Bernd W. Urban* *Departments of Anesthesiology and Physiology Cornell University Medical College New York, New York 10021 +Department of Physiology University of Colorado Medical School Denver, Colorado 80262.

Summary Sodium channels from several sources are covalently modified by unusually large numbers of negatively charged sialic acid residues. In the present studies, purified electroplax sodium channels were treated with neuraminidase to remove sialic acid residues and then examined for functional changes in planar lipid bilayers. Neuraminidase treatment resulted in a large depolarizing shift in the average potential required for channel activation. Additionally, desialidated channels showed a striking increase in the frequency of reversible transitions to subconductance states. Thus it appears that sialic acid residues play a significant role in the function of sodium channels, possibly through their influence on the local electric field and/or conformational stability of the channel molecule. Introduction The voltage-activated sodium channel is responsible for the initiation and propagation of the fast action potential in most excitable cells. As with all previously examined integral membrane proteins of the plasma membrane (Lennarz, 1983), the sodium channel is glycosylated and the sugar domain is most likely located at the extracellular side of the channel (Levinson et al., 1986). In the eel electroplax (Miller et al., 1983), rat brain Katterall 1986), and mammalian muscle (Barchi, 1983; Roberts and Barchi, 1987) the sugar domain is extensive, corresponding to 20%-30% of the molecular mass of the protein, of which 40%-46% is sialic acid residues. The importance of these sugar domains to sodium channel function is uncertain. In general, glycosylation of membrane proteins is thought to be involved in the biosynthetic processes of folding or targeting (Bradshaw et al., 1988; West, 1986). However, the large number of sialic acid residues (about 120 per channel) and their probable location on the external surface of the channel suggest that these moieties might also play a role in channel function through electrostatic *Present address: Department of Physiology and Biophysics, Mount Sinai School of Medicine, I-Gustave Levy Place, New York, New York 10029.

effects. In particular, past studies (Frankenhaeuser and Hodgkin, 1957; Neumcke and Stampfli, 1984; see Hille, 1984) have implicated the existence of an unknown number of fixed charges on the external surface of the membrane that influence the electric field sensed by the channel-gating mechanism. Furthermore, it has been suggested (Levine et al., 1983) that heavily sialidated membrane proteins may significantly affect the membrane surface electric potential, since at least some of these charged residues are likely to be close enough to the membrane to influence its local electric field. Alternatively, other investigations have shown that negative charges near the mouth of a cation-conducting pore may raise the conductance of such a channel (Apell et al., 1977; Green et al., 1987). Thus, if any of the sialic acid residues were close enough to the channel vestibule, it might be expected that the single-channel conductance would be raised above that of an unsialidated channel. The purified and reconstituted eel electroplax sodium channel is a convenient preparation to study the role of sialidation in channel function. It has been biochemically characterized and shown to be composed of a single 260 kd glycopeptide (Miller et al., 1983; Rosenberg et al., 1984; Duch and Levinson, 1987). It has been functionally characterized in the planar lipid bilayer system, where it was shown that its single-channel characteristics were similar to those of sodium channels from other sources (Recio-Pinto et al., 1987; Duch et al., 1989). In the present study, sialic acid residues were removed with neuraminidase, an exo-enzyme that serially cleaves glycosiditally bound sialic acid residues. Here we report that this treatment had significant effects on both steadystate activation and single-channel conductance. Some of the results of these experiments have previously appeared in preliminary form (Levinson et al., 1990). Results The Time Course of Neuraminidase Action In the present studies, the effects of enzyme treatment on the physicochemical (SDS-PAGE analysis) and functional (planar lipid bilayer) properties were correlated using the same channel preparation. Neuraminidase treatment altered the electrophoretic behavior of the eel sodium channel peptide in a time-dependent manner, decreasing the apparent molecular mass of the channel glycopeptide from 290 kd to about 220 kd (Figure 1; Figure 2A). Based on the observed mobility shifts, maximal desialidation appeared to be complete by 2-3 hr (Figure I), and treatment of the channel preparation for 24 hr at room temperature or 37°C did not result in any further mobility changes on SDS gels (data not shown). Thus, prepara-

NellrOn 676

c

5min

15min 30min lhr

2hr

3hr

4hr

5hr

290

1

A

234567

200 -

220

123456789 Figure 1. Time-Dependent Effects of Neuraminidase Seen with SDS-PAGE Analysis

Treatment

Lane 1, untreated sodium channel protein. Lanes 2-9, sodium channel protein treated with neuraminidase (Boehringer Mannheim, 0.30 U/ml) for the durations indicated. Treatment of the channel with neuraminidase (0.03 U/ml) from Sigma gave similar results (data not shown).

tions treated with neuraminidase for up to 5 hr were chosen for later functional studies. Exposing the preparation without enzyme to room temperature for 5 hr (Figure 2A, lane Z), or to neuraminidase that had been boiled for 10 min (Figure 2A, lanes 4 and 7) had no effect on the apparent molecular mass of the channel glycopeptide on SDS-PAGE. In addition, neuraminidase treatment seems to have affected most of the protein in the vesicles, as judged by the lack of a residual band at the position of the control. This observation is consistent with the measurements of Duch and Levinson (1987), who reported that channels reconstituted using these procedures were oriented in the vesicles in a highly asymmetric, right-side-out manner. On the other hand, thawing of vesicles that were frozen for long-term bilayer studies (see Experimental Procedures) probably resulted in membrane reorientation; thus some channels will also be in the inside-out orientation before bilayer reconstitution (Experimental Procedures). The neuraminidase preparations used for these studies were of the highest obtainable purity and were certified as protease-free by their suppliers (see Experimental Procedures; Figure 26). However, in light of the long incubation times, we thought it prudent to determine whether undetectable quantities of residual proteases could have acted on the channel preparation, thus possibly accounting for the observed effects. As described in Experimental Procedures, no evidence of proteolysis was found during neuraminidase treatment. The degree of desialidation achieved by long exposures to neuraminidase cannot be accurately determined from SDS-PAGE analysis. It is possible that channels retained a few sialic acid residues, especially since the channel band on SDS-PAGE was still rather broad, indicating that substantial glycosylation remained after extended enzyme exposure. Thus interpretation of the results of these studies is best viewed in the light of substantial, but not necessarily complete, removal of sialic acid residues (see Discussion).

116926645312114-

1

23456

230-

dfFigure 2. SDS-PAGE Analysis of Biochemically Purified Sodium Channels and Sodium Channel Core Poiypeptides Synthesized via In Vitro Translation before and after Treatment with Neuraminidase Treatment was for 5 hr at room temperature at the concentrations given in Experimental Procedures. (A) Treatment of biochemically purified sodium channels wrth neuraminidase. Lane 1 contains molecular weights standards; lane 2, control (neuraminidase absent); lane 3, treatment with neuraminidase from Boehringer Mannheim; lane 4, treatment with heat-denatured Boehringer Mannheim neuraminidase; lane 5, Boehringer Mannheim neuraminidase alone; lane 6, treatment with neuraminidase from Sigma; lane 7, treatment with heat-denatured Sigma neuraminidase. Samples were run on a 5%-30% acrylamide gradient gel and visualized by silver staining. (B) Treatment of in vitro synthesized sodium channel core polypeptide with neuraminidase. Lane 1, untreated sodium channel core polypeptide; lane 2, treatment with neuraminidase from Boehringer Mannheim; lane 3, treatment with neuraminidase from Sigma; lane 4, treatment with neuraminidase from V. cholerae in the presence of 1 mM calcium ions; lanes 5 and h treatment with trypsinkhymotrypsin, 5 &ml and 50 @ml each respectively.

Neuraminidase Effects on Purified Eel Na+ Channels 677

Steady-State Activation As in previous studies, channels were functionally characterized in the presence of batrachotoxin (see Experimental Procedures). The fractional open time (fJ of neuraminidase-treated channels showed voltage dependence, as previously reported for untreated channels (Recio-Pinto et al., 1987). However, the steady-state activation behavior of channels treated for 5 hr with neuraminidase was significantly more variable than that of control channels (see below), and a clear pattern emerged only after a large number of channels were studied. For example, separate channels could display very different midpoint potential values (V,), and a given channel could often change its V, value in a manner that showed no apparent predictable trend with time. Such a channel might also undergo frequent gating-state changes (e.g., changes in the average fractional open time while being held at a constant potential, or anomalous changes in f, in a direction opposite to that expected for their voltage dependence activation; see below; Recio-Pinto et al., 1987). Therefore, the average steady-state activation curves of all the observed channels will be presented first to show the overall effect of neuraminidase. Subsequently, data describing the variance in behavior of both individual and populations of treated channels will be presented to illustrate the wide range of steady-state activation behaviors. Average Steady-State Activation Curves The activation data for untreated channels were well fitted by a single Boltzmann distribution with a V, of -71 mV (Figure 3; Table I), in good agreement with previous results obtained under similar conditions

._.

-100

-50

so

l-h

IO0

Figure 3. Steady-State Activation Curves for Untreated and Channels Treated for 5 hr with Neuraminidase

Channels

The data were fitted with Equation 1 (dotted line) and Equation 2 (solid line). Open circles: untreated channels, n = 37 membranes, 136 channels (membrane mean * SEM). Filled squares: neuraminidasetreated channels, n = 24 membranes, 109 channels (membrane mean * SEM). Four batches of purified channels were used: three were treated with Boehringer Mannheim and one with Sigma neuraminidase.

(Recio-Pinto et al., 1987). Neither the fit of the data nor the derived activation parameters were substantially different when a two-component Boltzmann distribution was used, suggesting, on average, that control channels activated in a fairly consistent manner. Similar results were seen with channels treated with neuraminidase for 38 min CTable1). Other control conditions, such as exposing the channels to either heatdenatured neur-

Table 1. Effects of Neuraminidase V, Aggregate Control 5 hr Neu

Treatment

on Boltzmann

Activation

Parameters

z,

V?

z1

fml

V2

z2

# Membranes -

# Channels

-74 -65

1.39 1.16

0.930 0.733

+20 +36

1.35 0.81

37 24

136 109

Data -7l* -40

1 * 2a

1.02 * 0.03 0.66 f 0.02

Enzyme Controls Control 5 hr Neu Boiled Filtrate

-78 -38 -71 -68

f * * *

1.72 1.31 2.26 1.42

14 12 12 10

27 42 36 32

Time Dependence Control 30 min Neu 5 hr Neu

-69 -80 -47

& 4 f 3b f 20a

1.53 1.71 0.99

8 IO 5

20 35 10

7 11 6b 3b

Aggregate Data: Data are from Figure 3; four batches of purified channels were used: three were treated with Boehringer Mannheim and one with Sigma neuraminidase. The values were obtained with nonlinear regression curve-fitting methods (Experimental Procedures) using either a single (V, and z,) or a double (Vt, zt, fm,, Va, and z,) component Boltzmann distribution. Enzyme Control: One batch of purified channels was treated with neuraminidase from Sigma with the controls as given. Here, activation data from all channels within an individual membrane were averaged and then subjected to unweighted fits to a single Boltzmann activation component to obtain the membrane V, and z, value (RS/l; Bolt, Beranak, and Newman Software Products Corp., 1983). The ensembles of all membrane values were then averaged, and the V, means were analyzed for significant increases with the various manipulations using a one-tailed t-test. Time Dependence: One batch of purified channels was used. Neuraminidase was from Boehringer Mannheim, and the filtrate was from the Boehringer Mannheim neuraminidase. Statistical analysis was done as described above. a Significantly more positive than control (P < 0.05). b Not significantly more positive than control (P > 0.05). -

NelJVXl 678

aminidase or neuraminidase filtrate (Experimental Procedures) did not significantly affect the steadystate activation properties of the entire population (Table 1). After a 5 hr neuraminidase treatment, the average steady-state activation curves shifted significantly toward depolarizing potentials (V, = -40 mV) and became shallower (z, = 0.66 versus 1.02 for untreated) when fitted with a single Boltzmann distribution. Moreover, the trend of these plots was more complex than that of the control, as can be seen by the systematic deviations from the data of the single Boltzmann fit, and a much better fit was obtained using the twocomponent Boltzmann distribution (Figure 3; Table 1). The parameters derived from such fits suggest that enzyme treatments grossly resulted in two channel behaviors, namely those that were little affected (V, = -65 mV) and those whose activation voltages were shifted very markedly to positive potentials (V, = +35 mV). However, the improved fit using the two-component model does not necessarily imply the existence of two distinct channel populations with different activation characteristics. Rather, we suspect that the channels treated for 5 hr were a highly heterogeneous population with a broad range of gating behavior (e.g., see Figure 5), and given the scatter of the data, it did not appear useful to attempt to fit more than two activation components. Finally, neuraminidase treatment also increased the variation of observed f, values at most membrane potentials, as can be inferred by comparing the width of the error bars in controls and treated channels (Figure 3). Variability of Population Gating Behavior We have previously reported that untreated eel channels exhibit significant time- and sample-dependent variability in their activation behavior (Recio-Pinto et al., 1987). However, the gating behavior of the majority of the untreated channels was qualitatively similar. At positive and slightly negative potentials, channels remained mainly open, undergoing only occasional closures. At greater negative potentials, channel transitions between the open and closed levels monotonically increased until, at sufficiently high negative potentials, channels remained mostly closed (Figure 4A). By comparison, neuraminidase-treated channels showed a substantially larger variability in the voltage dependence of their fractional open-times and their gating occurred over a wider voltage range (Figures 4B, 4C, and 4D). In addition, neuraminidasetreated channels often displayed gating-state changes (discontinuous or nonmonotonic changes in the fractional open time), sometimes resulting in irregular activation curves in which f, tended to decrease as membrane potential became more positive (Figure 40). Neuraminidase treatment for short periods (5-30 min) apparently induced dramatic functional changes in a few channels, but significant functional changes in the channel population occurred only with longer treatment (5 hr).

A 40 -

-60 M

30 -

-70 *

1clr

“00

_i_l._ -150

B

,100

-50

1 0

50

_J 100

mV 95 -

-40 -nL?rr

90 WUl-Wl

-50 w

80 u&%%+

-60 w

m

,o 0.8

.B

Figure 4. Representative Examples of Steady-State Activation of Control and Neuraminidasexreated Channels Each part (A, 8, C, and D) shows current traces (left, 50 Hz) and the corresponding fractional open times (right). All membranes contained two channels, and f, = 1 corresponded to both channels being fully open. A dashed line indicates the current level at 0 mV, and channel openings are always upward. The time and current scales for all records are shown in (D). (A) Control: the voltage range at which the channels gated showed a large overlap. The combined V, values for the two channels was -69 mV (single Boltzmann fit). (B) 5 min neuraminidase treatment: these channels gated at very different potentials, showing V, values of -73 and +80 mV, with a combined activation midpoint of +8 mV. (C) 5 hr neuraminidase treatment: one of the channels underwent gating-state changes during activation measurement, resulting in scattered activation data; it had a conductance of 20 pS and a V, of -51 mV. The other channel had a lower conductance (IO pS) and a V, of -89 mV. The combined fit gave a V, of -63 mV. (D) 5 hr neuraminidase treatment: at least one of the channels started to undergo long and frequent closures during the observation. One of the channels (probably the one undergoing long closures) had a V, of -35 mV; the other channel had a V, of -67 mV. The combined fit gave a V, of -58 mV.

Neuraminidase Effects on Purified Eel Na+ Channels 679

60 50 40 30 20 IO 0

b.

-30

0

30

60

WW

Figure 5. Distribution of Membrane Average Midpoint Potentials for Untreated Channels and Channels Treated for 5 hr with Neuraminidase In both cases the membrane V, was obtained from a single Boltzmann component fit (Equation 1). Each bar segment represents a membrane, and its size indicates the number of channels present. Membranes were exposed to neuraminidase from Boehringer Mannheim or from Sigma. The mean and SEM of membrane V, values were, for control (A), -73 * 3 mV (n = 37 membranes, 136 channels) and, for 5 hr of neuraminidase treatment (B), -39 f 8 mV (n = 24 membranes, 107 channels). The difference of these V, values was highly significant (P < 0.001, nonpaired t-test).

As noted above, the increased variability in gating behavior for individual enzyme-treated channels was reflected in the increased variance of the points plotted in the averaged activation curves (Figure 3). The variability in gating was also analyzed by constructing histograms of the V, values for various membranes (Figure 5). Thus neuraminidase treatment for 5 hr increased the variability of V, values among membranes, as indicated bythe widening of the histogram, and shifted the average midpoint to depolarized potentials, as discussed above. Variability of Gating Behavior of Individual Channels For individual channels, neuraminidase treatment also increased the variability of the steady-state activation properties recorded at different times. A given channel could show large, but apparently random variations in its activation properties. Figure 6 illustrates different gating behaviors for the same single channel. They included shifts in individual V, values (V,), gating-state changes, and appearance of subconductance levels. To quantify this variability in gating behavior, the range of V, values in repeated measurements in each membrane was determined. Membranes with low

120

~90

-60

~30

0

30

60

Figure 6. Example of Variability of Midpoint Potential Values for a Single Channel Treated with Neuraminidase for 5 hr (Top) Each column shows current traces at various potentials for steady-state activation determinations recorded at different times. The individual V, value for each activation determination (V,) were obtained by fitting the data to Equation 1. Column 1 (leftmost): V, = -40 mV. Column 2: V, = -116 mV, 12 min later. Column 3: V, = -90 mV, 30 min. Column 4: V, = -77 mV, 85 min. Column 5: V, = -104 mV, 103 min. Column 6: V, = -106 mV, 110 min. The average V, value for this channel was -81 f 22 mV (mean f SD, n = 15 determinations). (Bottom) The channel fractional open times obtainedat time 0 (circles) and at 12 min (squares) shown as a function of the holding potential.

and constant numbers of channels W-10) were used. The range of the V, value significantly increased from 15 f 10 mV (mean f SD; n = 20 membranes, 55 channels) in control experiments to 41 It 28 mV (n = 13 membranes, 49 channels) after neuraminidase treatment for 5 hr (P < 0.01). Thus neuraminidase treatment for 5 hr increased the variability in V, seen in repetitive measurements on individual sodium channels themselves. Effects of Neuraminidase Treatment on Channel Conductance Transitions Untreated channels were not only relatively uniform in their activation behavior, but were also rather consistent in the conductance transitions exhibited (Fig-

Figure 7. Representative Current Traces of Untreated Channels and Channels Treated for 5 hr with Neuraminidase

0

5

IO

15

20

25

30

0

5

10

(Top trace) A membrane containing two untreated channels undergoing infrequent channel transitions of about the same magnitude. (Middle trace) A membrane containing more than one channel treated for 5 hr with neuraminidase. Channels underwent many and variable channel transitions. The number of channels in this membrane could not be determined, since it is not possible to distinguish between maximal channel conductances and subconductance states (see Experimental Procedures). (Bottom trace) A membrane containing one channel treated for 5 hr with neuraminidase undergoing channel closures (arrow) and subconductance states (asterisks). The direction of channel opening(O), the direction of channel closing (C), and the current level when all the channels are closed (dashed line) are indicated. (A-D) Frequency histograms of sodium channel transitions for untreated and neuraminidase-treated channels. Neuraminidase was from Boehringer Mannheim (0.30 U/ml). Only membranes with lifetimes X5 min were used. The total number of transitions for a given membrane was normalized to 1. For each conductance size the average and SEM (n = number of membranes) was calculated. (A) Transition histogram for untreated channels (16 membranes, 74 channels, 1282 transitions). (B) Channels treated for 5 min with neuraminidase (4 membranes, 11 channels, 1283 transitions). (C) Channels treated for 30 min with neuraminidase (12 membranes, 41 channels, 1623 transitions). (D) Channels treated for 5 hr with neuraminidase channels (13 membranes, 38 channels, 3105 transitions).

li

Conductance (pS)

ure 7, top trace), yielding transition histograms with major peaks around 18 pS (Figure 7A). After neuraminidase treatment, there was a marked increase in the number of smaller conductance transitions (Figure 7, middle and bottom traces; Figures 78, 7C, and 7D), particularly those in the range of 2-10 pS. However, in contrast to the shifts in activation midpoint (see above), these changes in transition level occurred even with short neuraminidase exposures (e.g., see Figures 7B and 7C). Prolonged exposure to the enzyme sometimes further broadened the transition histogram (Figure 7D), whereas in other experiments a clearly bimodal distribution resulted with peaks at approximately 5 and 18 pS (data not shown). Control conditions, such as exposing the channels to either neuraminidase filtrate or boiled neuraminidase, did not produce a significant increase in the number of small channel transitions (data not shown), in concert with the lack of effect on the V, value of the entire population (Table 1) and on the channel’s apparent molecular mass seen in SDS-PAGE (Figure

2A). In addition, single-channel transition histograms for untreated channels were not detectably affected by exposure to room temperature for 5 hr prior to their incorporation into bilayers. Discussion Neuraminidase treatment had three main effects on the function of batrachotoxin-modified electroplax sodium channels in planar bilayers. First, prolonged treatment with the enzyme caused dramatic changes in channel activation behavior, particularly the mean V,. Second, treated channels exhibited much increased dispersion in the distribution of V, values, both as a population and for repeated measurements of the same channel. Finally, the mainly unimodal conductance transitions observed in untreated channels became increasingly disperse as channels were exposed to enzyme action. One can consider several possible mechanisms behind these desialidation-induced changes, including

Neuraminidase Effects on Purified Eel Nat Channels 681

the electrostatic influences of either fixed or partially movable charges residing on sugar chains as well as the destabilization of protein conformation. Below we briefly consider these possibilities as causes of the functional changes that were observed. Sialic Acid Residues May Influence the Electric Field Near the Sodium Channel It has long been considered that fixed negative charges near the membrane surface might greatly influence the voltage-dependent gating of the sodium channel (see Introduction). Specifically, removal or screening of fixed negative charges on the outside membrane surface might be expected to increase the magnitude of the electric field within the membrane itself (assuming a negative membrane resting potential). For fixed charges near the channel molecule (such as sialic acid residues), such removal would likely exert a hyperpolarizing influence on putative channel-gating elements within the membrane. Thus an expected functional effect of decreasing the number of these fixed charges would be an increase in the depolarizing stimulus required to achieve a certain level of channel activation (see Hille, 1984, for a detailed discussion). Therefore, if sialic acid residues were to play such an electrostatic role in the steadystate activation behavior studied here, more bilayer depolarization should have been required to achieve the same fractional open time in desialidated channels when compared with untreated controls. In the present experiments, substantial depolarized shifts in the steady-state activation curves were indeed observed with channels subjected to prolonged neuraminidase exposure. In particular, a 31 mV change occurred in the mean V, (Table I), consistent with a significant role for sialic acid residues in determining the magnitude of the electric field affecting sodium channel activation. Since major changes in activation behavior were observed only after extensive desialidation (Figure 1; Table I), it would seem that only a fraction of sialic acid residues contributed to the effects seen. This observation would be consistent with an electrostatic mechanism if one considers both the mode of action of the enzyme and the likely arrangement of sialic acid residues on the protein. In this regard it has been shown that sialic acid residues on electroplax channels exist as long polymeric chains on the terminal ends of the core glycosyl groups (James and Agnew, 1987). Thus desialidation might be a lengthy process in which the sequential action of the exo-neuraminidase is progressively slowed as it attempts to remove sialic residues that are increasingly buried in the extensive glycocalyx of the channel as digestion proceeds. Changes in activation would only be observed upon prolonged digestion, since the sialic acid residues that had the most influence on the intramembrane electric field (i.e., those nearest the membrane surface) would be cleaved last and with the most diffic:uIty. Also consistent with this hypothesis is the ob-

servation that for untreated electroplax sodium channels, the V, value of the steady-state activation curve was the same in symmetrical 0.1 and 0.5 M NaCl (Recio-Pinto et al., 1987); such a result also suggests that negative charges influencing activation voltage sensitivity of eel electroplax sodium channels are sequestered from screening ions located in the bulk external aqueous phase. Such partial desialidation might also be expected to create a disperse population of channels with variable levels of associated sialic acid, accounting for the increased variation in activation curve midpoints (e.g., Figures 3 and 5) seen with treatment with neuraminidase for 5 hr. Possible Roles of Conformational Changes in Activation Variability and Conductance Behavior in Desialidated Channels In contrast to the striking effects on mean activation behavior, the increased variability of the activation bewith repeatedmeahavior seen in individualchannels surement (Figure 6; Results) would seem to be at variance with the expected electrostatic influences of fixed sialyl residues. Here, removal of putative fixed charges should have only depolarized activation curves and not necessarily increased their dispersion with time. In addition, the effect of desialidation on conductance levels was also unexpected from purely electrostatic considerations. Thus it is important to note that neuraminidase-treated single channels displayed reversible conductance transitions; if the subconductance states were the result of removal of fixed charges near the mouth of the pore, such transitions should have been irreversible. Rather, the conductance behavior observed in treated channels suggests that underlying reversible and discrete transitions in conformation of the channel molecule were responsible. In light of the above observations, it would seem possible that desialidation also resulted in enhanced movement of certain channel domains. In this regard it has already been proposed that stepwise changes in the position of the polysialyl moieties might lead to the abrupt changes in V, seen when untreated individual channels are observed in lipid bilayers (James and Agnew, 1987). Since sialic acid accounts for 12% of the mass of the electroplax sodium channel (Miller et al., 1983), it seems possible that ,partial desialidation might indeed lead to a changed position or flexible motion of the chains containing the remaining sialic acid residues, with a resultant increase in the time-dependent variability of channel activation and conductance behavior. Thus this mechanism is entirely consistent with an electrostatic effect of sugarassociated negative charge on channel properties. An alternative possibility is that removal of charge may allow flexibility in the protein core of the channel itself, leading to discrete conformational changes that might affect gating or conductance. Although our data do not allow a rigorous evaluation of any of the above mechanisms, the highly dispersed distribu-

Neuron 682

tions of both activation and conductance properties of desialidated channels (e.g., Figures 5 and 7) would require a large variety of stable positional changes in the related channel domains. Such a broad range of conformations would seem more consistent with the random movement of nonordered sugar groups than with a limited number of discrete transitions, which usually characterize conformational changes in highly aligned protein structures. Effects of Desialidation on Effective Cating Valence Compared with control channels, the V, values of neuraminidase-treated channels covered a wider voltage range, the frequency of anomalous gating-state changes was higher, and the channels could undergo long-lived channel closures even at depolarized potentials. These effects would be expected to contribute heavily to the shallower population steady-state activation curves of the neuraminidase-treated channels (Figure 3). As a consequence, it is not possible to say whether any of the decrease in the effective gating valence, z,, upon desialidation reflects a true change in the charge of the channel-gating mechanism as opposed to population heterogeneity or the increased gating variability of individual channels noted above. Other Considerations It should be noted that channels in the present study were of necessity modified by batrachotoxin to allow their activity to be observed in planar bilayers. Thus presently it cannot be ruled out that some or all of the effects of desialidation observed are related to batrachotoxin modification only and would not be seen in unmodified channels. In this regard, others have applied neuraminidase to intact tissues and have reported this treatment to be without discernible effect on sodium current properties (Frankenhaeuser et al., 1976). However, such exposures would have been much too brief (i.e., only up to 30 min at 23%) to have an observable effect on channel gating, especially considering that diffusion barriers in intact tissue would have further slowed access of the enzyme to the cell surface. Purified-reconstituted sodium channels, by contrast, can be treated for the long periods of time required to obtain substantial channel desialidation. We hope that the present studies will stimulate further investigations in preparations capable of withstanding the prolonged enzyme incubations required for functional modification. Possible Physiological Roles of Clycosylation The results of this study indicate that through posttranslational modifications, cells may be capable of modifying sodium channel activation and conductance characteristics. For example, by changing the number or pattern of attached sialic acid residues, and possibly other sugar residues, a cell could provide a channel with functional specificity (e.g., by determining the threshold for channel activation), or modulate its channel activity during development or

disease. In addition, our studies may provide a partial explanation for the frequently observed variability in single-channel gating and conductance reported in other studies of channels in bilayers (French et al., 1986; Green et al., 1987; Moczydlowski et al., 1984; Recio-Pinto et al., 1987) or intact tissues (Matteson and Armstrong 1982; Nagy et al., 1983; Gilly and Armstrong, 1984; Weiss and Horn, 1986; Stanley and Fozzard, 1987; Patlak, 1988). Overall, the present results suggest that posttranslational modifications as well as amino acid compositions should be considered when attempting to explain functional differences between sodium channels. Experimental Procedures Reconstitution of Purified Electroplax Sodium Channels into lipid Vesicles Eel electroplax sodium channels were purified and reconstituted as previously described (Duch and Levinson, 1987). Forthe studies here, channels were reconstituted in vesicles composed of a I:1 mixture of asolectin:egg phosphatidylcholine. The specific activity of the channel preparations after vesiculation ranged from 1400 to 2900 pmol of bound tetrodotoxin per mg of protein. Reconstituted preparations were either kept at 4OC for use within several days or stored at -80°C for later study. Neuraminidase Treatment and Biochemical Characterization Channels in lipid vesicles were treated with neuraminidase (an exo-glycosidase) before incorporation in lipid bilayers. Highly purified neuraminidase from Clostridium perfringens was obtained from Boehringer Mannheim GmbH, FRG, and Sigma Chemical Co., MO. All neuraminidase batches were the purest obtainable from these sources and were certified as being protease-free within detectable limits. As a further precaution against undetected proteolytic activity, EDTA (0.5 mM) was added during neuraminidase treatment and to the bilayer buffers (also see below). Neuraminidase hydrolysis was carried out for various exposure times of up to 5 hr at room temperature (22O-24V) using enzyme from Boehringer Mannheim (used at 0.30 U/ml, approximately 75 U per mg of protein at 37OC, NAN-lactose as substrate; applied to three separate channel preparations), or Sigma (type X, 0.03 U/ml, 160 U per mg of protein; applied to one channel preparation). Control channels were also exposed to room temperature for 5 hr in the absence of enzyme before their incorporation into planar lipid bilayers. Neuraminidase-treated samples and controls were analyzed on 5%-30% acrylamide gradient gels as previously described (Duch and Levinson, 1987) according to the method of taemmli (1970). Protein patterns were visualized via silver staining using a commercially obtained kit (New England Nuclear-DuPont). Specificity of Neuraminidase Action Protease activity was assessed using SDS-PAGE analysis to determine whether various conditions resulted in the disappearance of high molecular mass channel bands commensurate with the appearance of lower molecular mass species. First, protease activity that was dependent on divalent cations was not detected with the Clostridium enzymes used in this study, since neuraminidase treatment in the presence or absence of EDTA and magnesium did not affect the gel pattern (i.e., band number or density) of either the sodium channel peptide or the nonsialidated standard proteins (data not shown). Nevertheless, EDTA was always added during neuraminidase treatment and to the bilayer electrolyte solutions as a precautionary measure. As a more rigorous test for the presence of protease activity, we exposed the eel electroplax sodium channel core polypep tide (Thornhill and Levinson, 1987) to three different preparations of neuraminidase. The core polypeptide, synthesized via in vitro translation techniques (Thornhill and Levinson, 1987), is

Neuraminidase 683

Effects on Purified Eel Na+ Channels

a nonglycosylated precursor form of the channel protein. Thus neuraminidase activity itself would have no effect on the electrophoretic mobility of this moiety, and any observed mobility changes would have to be due to proteases or other compounds contaminating the enzyme preparation. Treatment of the core polypeptide for 5 hr at room temperature under the same buffer conditions used above with either Boehringer Mannheim or Sigma neuraminidase had nodetectableeffect on the molecular mass of the core polypeptide (Figure 28, lanes 1, 2, and 3). Treatment of myosin under these conditions also did not result in any detectable protease activity (data not shown). These methods are capable of detecting protease activity, since neuraminidase from Vibrio cholerae, which requires divalent ions for activation, did show protease activity in the presence of 1 mM calcium (Figure 2B, lane 4). This enzyme was therefore not used in these studies. Also, the addition of trypsin and chymotrypsin at two different concentrations cleaved the core polypeptide into peptides with molecular masses of less than about 25 kd (Figure 28, lanes 5 and 6), further demonstrating the proteolytic susceptibility of this moiety. We were also concerned that other compounds might have contaminated the neuraminidases and might separately affect the functional behavior of sodium channels in the bilayer. In particular, attempts to study the effects of enzyme treatment on channels after incorporation into planar bilayers were compromised by the increased instability of the bilayer membrane. Thus when bilayer membranes were exposed to Boehringer Mannheim neuraminidase alone (at concentrations at least one order of magnitude higher than those normally resulting from the addition of pretreated channel preparations), bilayer conductances became noisy, the membrane lifetime decreased significantly, and infrequently, irregular current transitions larger than 40 pS were detected (data not shown). Similar behavior was seen when bilayer membranes were exposed to an ultrafiltrate of the Boehringer Mannheim enzyme (Amicon Centricon, cutoff approximately 10,000 daltons), suggesting that a low molecular weight contaminant such as a detergent was responsible. Sigma neuraminidase did not cause any current transitions, but did tend to decrease membrane lifetime somewhat. To determine the effects of these contaminants on channel behavior, purified channels were exposed to neuraminidase filtrate or boiled neuraminidase before incorporation into bilayers. Neither condition had any functional effect on channels in bilayers when compared with untreated controls (see Table 1; Results). In sum, we conclude that the functional effects seen with neuraminidase treatment were due to removal of sialic acid residues that were associated with the channel protein. Single-Channel Measurements Single-channel electrophysiological characteristics were studied in a planar lipid bilayer system in the presence of batrachotoxin as previously described (Recio-Pinto et al., 1987). The symmetrical 1 ml Teflon chambers contained 0.1 M NaCl and 0.5 mM EDTA, buffered to pH 74 with 10 mM HEPES. Experiments were conducted at room temperature (22”-25’X). Planar bilayers were formed from neutral phospholipid solutions containing (4-l) phosphatidylethanolamine and phosphatidylcholine (Avanti Polar Lipids, Inc. Birmingham, AL) in decane (5% [w/v], 99.9% pure; Wiley Organics, Columbus, OH). After forming a stable lipid bilayer, 0.1-2.0 PI of vesiculated sodium channels was added to the cis bilayer chamber and currents were recorded under voltage-clamp conditions. A standard current-to-voltage amplifier was used with a lOlo n feedback resistor and a 150 Hz corner frequency, and output on a strip chart recorder was filtered at 10 and 50 Hz @-pole Bessel filter). The electrophysiological sign convention was used. All membrane potentials are quoted as intracellular minus extracellular potentials (Recio-Pinto et al., 1987). Steady-State Activation To analyze the voltage-dependent gating properties of channels in the bilayer, steady-state activation curves were constructed by measuring with a computer or manually (from strip chart

records) the channel fractional open time as a function of the applied membrane potential as previously described (RecioPinto et al., 1987; Duch et al., 1988). Membrane potentials were applied for 5-10 s and changed monotonically (from +I20 to -140 mV) in 5 mV steps. This voltage sequence was repeated as long as the channel number (maximal conductance) in a given membrane remained constant. At each holding potential, the time-averaged conductance of all the channels within each membrane was determined and normalized to yield fractional open times (by dividing by the number channels in the membrane and by the single-channel conductance). These fractional open times were then combined with thoseof other membranes to obtain the overall mean f,(V). Data points missing for a given membrane potential were obtained by extrapolation between values at higher and lower potentials (f5 mV). When channels were incorporated into the bilayers in both directions, the data were used only if the maximal and background conductance in both directions could be determined. Channels in each direction were treated as a separate membrane. In this study, 63 membranes had channels in only one direction and 16 membranes had channels in both directions given a total of 95 membranes (63 + [I6 x 21). Membranes contributed equally to the average steady-state activation analysis regardless of the number of channels each contained. The activation data were fitted using a two-level (open and closed) Boltzmann distribution for one or two components (Equations 1 and 2, respectively): f, I l/(1 + exp[-z,F&’ - VJIIKT} f,, = fm#

(1)

+ exp[-z,F(V - V#RT) + (1 - fml)i{l + expl-zzF(V - V>)]/RT)(2)

where z,, z,, and z2 are the effective valences of the apparent gating charges of each component and V,, V,, and V2 are the potentials at which the channels in each component were open half of the time. fm, is the maximal contribution of the first component to the total fractional open time. V is the membrane potential at which f,, was measured, F is the Faraday constant, R is the gas constant, and T is the absolute temperature. Best fits (defined as minimization of the sum of the variances of the experimental data from the fitted line) were obtained using nonlinear least squares methods implemented on a computer, and data were weighted inversely according to their experimentally determined variances as described by Bevington (1969). Single-Channel Conductance Analysis The term maximal single-channel conductance refers to the maximal conductance level that a given channel displayed during the recording period. The various lower conductance levels that the same channel could close or open to are termed subconductances. This term is only descriptive, as it does not imply any mechanism by which the channel conductance is lowered. Channels could display maximal single-channel conductances that varied greatly (~4-21 pS). Consequently, it was impossible to distinguish between low maximal conductances (