Dec 27, 1983 - Reconstitution of highly purified saxitoxin-sensitive Na+ -channels ... able channels were detected in the absence of any neuro- toxins.
The EMBO Journal vol.3 no.3 pp.509-515, 1984
Reconstitution of highly purified saxitoxin-sensitive Na + -channels into planar lipid bilayers
Wolfgang Hanke, Gunther Boheim, Jacques Barhanint, David Pauront and Michel Lazdunskil* Department of Cell Physiology, Ruhr-Universitat Bochum, Postfach 102148, D-4630 Bochum, FRG, and 'Centre de Biochimie du CNRS, Faculte des Sciences, Parc Valrose, F-06034 Nice Cedex, France *To whom reprint requests should be sent Communicated by Michel Lazdunski
Highly purified Na+-channels isolated from rat brain have been reconstituted into virtually solvent-free planar lipid bilayer membranes. Two different types of electrically excitable channels were detected in the absence of any neurotoxins. The activity of both channels was blocked by saxitoxin. The first channel type is highly selective for Na + over K + (- 10:1), it shows a bursting behavior, a conductance of 25 pS in Na +-Ringer and undergoes continuous opening and closing events for periods of minutes within a defined range of negative membranes voltages. The second channel type has a conductance of 150 pS and a lower selectivity for Na + and K + (2.2:1); only a few opening and closing events are observed with this channel after one voltage jump. The latter type of channel is also found with highly purified Na +-channel from Electrophorus electricus electroplax. A qualitative analysis of the physicochemical and pharmacological properties of the high conductance channel has been carried out. Channel properties are affected not only by saxitoxin but also by a scorpion (Centruroides suffusus suffusus) toxin and a sea anemone (Anemonia sulcata) toxin both known to be selective for the Na+-channel. The spontaneous transformation of the large conductance channel type into the small one has been considered; the two channel types may represent the expression of activity of different conformational states of the same protein. Key words: lipid bilayers/Na+-channels/reconstitution/saxitoxin
Introduction The rising phase of action potentials in most excitable systems is due to voltage-dependent Na +-channels. These channels have a rich pharmacology and they are blocked by toxins like tetrodotoxin or saxitoxin. These channels have been extensively studied using voltage-clamp techniques and, more recently, using patch-clamp techniques (Sigworth and Neher, 1980; Horn et al., 1981; Fenwick et al., 1982; Cachelin et al., 1983). Important progress has been made recently in the purification and biochemical characterization of the voltagedependent Na +-channel from the electric organ of Electrophorus electroplax (Moore et al., 1982; Miller et al., 1983; Norman et al., 1983), from rat brain (Hartshorne and Catterall, 1981; Barhanin et al., 1983a) and from skeletal muscle (Barchi et al., 1980). There is now general agreement that a large glycoprotein, mol. wt. 200 000 -270 000, is implicated in the structure of Na +-channels from all three sources. This (© IRL Press Limited, Oxford, England.
large glycoprotein not only contains the site which recognizes both tetrodotoxin and saxitoxin (Moore et al., 1982; Hartshorne and Catterall, 1981), but also contains the distinct site that recognizes scorpion neurotoxins (Norman et al., 1983; Barhanin et al., 1983a, 1983b). A number of investigators have studied the reconstitution of Na +-channel-containing membranes or of unpurified tetrodotoxin-sensitive Na +-channels into liposomes (Goldin et al., 1980; Villegas et al., 1980). More recently, two reports have described the functional reconstitution of more purified Na +-channels from rat brain (Talvenheimo et al., 1982) and rat sarcolemma (Tanaka et al., 1983). Reconstitution in these cases was measured by 22Na + flux techniques which can only be used after a chemical activation of Na +-channels by alkaloid neurotoxins like batrachotoxin and veratridine. Channel reconstitution in artificial planar bilayers has been successfully tried with different techniques and systems (Miller, 1978; Schindler and Quast, 1980; Boheim et al., 1981, 1982; Wilmsen et al., 1983). Very recently the incorporation of unpurified Na +-channels from rat brain synaptosomes into decane-containing bilayers was reported (Krueger et al., 1983). These authors observed single channel current fluctuations but again only in the presence of batrachotoxin. Blockage of these Na +-channels was observed with saxitoxin. The purpose of this paper is to report the first functional reconstitution (including pharmacological properties) of extensively purified Na +-channels protein into solvent-free planar bilayers. Results After formation of a stable lipid bilayer membrane on the hole in a teflon sandwich septum separating two aqueous compartments the vesicle preparation was added to the cis side. The concentration of added vesicles was adjusted so that, within 5-15 min, single channel current fluctuations appeared following adequate voltage-jumps. The single channel situation is generally stable for at least 30 min, i.e. no further fusion events were observed in these cases for this period of time. Figure la shows the SDS-gel electrophoresis pattern of the Na+-channel preparations at different levels of purification. The crude membrane preparation (P) from rat brain contains a large variety of proteins; an extensive purification of the Na+-channel protein is obtained after wheat germ agglutinin (WGA) chromatography, and the sucrose gradient step leads to one main broad band of mol. wt. 270 000 which represents the Na +-channel protein. Figure lb shows typical recordings indicating that the same type of channel is found after reconstitution of brain Na +-channel preparations of different levels of purification. In addition, Figure lb shows that the reconstitution of the Na +-channel extensively purified from E. electricus electroplax also gives a Na +channel of large conductance which is similar to the one observed with brain Na +-channel preparations. 509
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Fig. 1. (a) SDS-polyacrylamide gel electrophoresis of rat brain Na+ -channel preparations prior to reconstitution. Lane 1, Coomassie blue staining of the membrane preparation (P) (running gel 4- 12"7o polyacrylamide). Lanes 2 and 3, silver staining (running gel 4.5 15 % polyacrylamide) of WGA (lane 2) and sucrose gradient (lane 3) steps in the purification of the Na + -channel. Specific activities of Na + -channel preparations are 2, 320 and 2000 pmol of Titx-y binding sites per mg of protein under conditions corresponding to lanes 1, 2 and 3, respectively. (b) Single channel current fluctuations after incorporation of the various Na+ -channel preparations by fusion with planar lipid bilayer membranes. 1, crude synaptosomal fraction (P3) (3-6 x 10-5 g of protein/ml); 3, sucrose gradient purified preparation of rat brain (7 - 14 x 10- 9 g of protein/ml); 4, extensively purified Na + -channel preparation from Electrophorus electroplax (3-5 x 10-9 g of protein/ml). 'o' and 'c' correspond to open and closed states of the channel respectively. The left side calibration bar indicates 100 pS. Experimental conditions: virtuaily solvent-free planar bilayers formed from a mixture of 1,2-SOPC/S-PE/cholesterol (20:75:5, molar ratio). Salt solution on both membrane sides (in mM): 140 NaCI, 3 KCI, 0.5 MgSO4, 0.5 CaCI2, 25 Hepes-Tris at pH 7.4. Temperature: 22 -23°C. Voltage-jump from 0 mV. Applied voltages - 50 mV (1), 65 mV (3), 75 mV (4). -
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Current fluctuations presented in Figure 2, and obtained with a Na +-channel preparation from rat brain, clearly show that two different types of stepwise current events can be distinguished. This is attributed to the presence of a channel of large conductance and of a channel of small conductance. Under conditions in which a Na+-Ringer solution (140 mM NaCl, 3 mM KCl, 0.5 mM MgSO4, 0.5 mM CaCl2, 25 mM Hepes-Tris at pH 7.4) is present on both membrane sides as in Figure 2, the conductance of the 'large' channel is L = 150 pS. This channel type appears immediately after the voltage jump and it disappears rapidly. During a continuous pulse series the 'large' channel fails to show up at random after -250/o of the voltage jumps. The 'small' channel has a conductance of J\ SI = 25 pS and its properties will be analyzed in more detail below. Once observed this channel type may reappear for minutes at negative voltages. Both channel types adopt non-conducting states after jumps from negative to positive voltages as shown in the upper and middle traces of Figure 2. Although in most cases the small channel appears immediately after closure of the large one (see for example the control in Figure 5a), other situations occur which correspond to the spontaneous transformation of the channel type of large conductance into that of small conductance (Figure 2 middle and lower traces). In 1 -20o of the closure events of the large channel, the conductance does not 510 -A
return to the conductance level of the unmodified bilayer. Instead different states of conductance at XA S = 25 pS (middle trace) or at -A-S2 = 40 pS (lower trace) are adopted. In the lower trace the 25 pS level is reached only after an intermediate transformation step to 40 pS. Conversely, a spontaneous re-transformation corresponding to a conductance change from .ASI = 25 pS tOJ-.L = 150 pS was never
observed. The 'small' channel type has not been clearly identified in reconstitution experiments using the electric eel preparation because of increased current noise after reconstitution, probably due to the presence of small amounts of detergent (Lubrol PX for the electroplax preparation instead of Triton X-100 for the rat brain preparation). Consequently rat brain Na +-channel preparations have been used to characterize the two channel populations in more detail.
The small conductance channel The Na+-channel with a small conductance shows a pronounced voltage dependence of channel state distributions. Figure 3a shows that the channel exhibits bursts of activity. Within a burst period the channel is open most of the time at 40 mV and closes at more negative voltages. The open state probability, PO, changes from PO >0.95 at -40 mV to PO
