Reconstitution of highly purified saxitoxin-sensitive Na+-channels into planar lipid bilayers.

144

The ion conduction and voltage dependence of sodium channels purified from rat brain were investigated in planar lipid bilayers in the presence of batrachotoxin. Single channel currents are clearly resolved. Channel opening is voltage dependent and favored by depolarization. The voltage at which the channel is open 50% of the time is -91 ± 17 mV (SD, n = 22) and the apparent gating charge is -4. Tetrodotoxin reversibly blocks the ionic current through the sodium channels. The K; for the tetrodotoxin block is 8.3 nM at -50 mV and is voltage dependent with the K; increasing e-fold for depolarizations of 43 mV. The single channel conductance, y, is ohmic. At 0.5 M salt concentrations y = 25 pS for Na ', 3.5 pS for K+, and 1.2 pS for Rb+. This study demonstrates that the purified brain sodium channel-which consists of three polypeptide subunits: a (Mr 260,000), j31 (Mr 39,000), and (32 (Mr 37,000)-exhibits the same voltage dependence, neurotoxin sensitivity, and ionic selectivity associated with native sodium channels.The sodium channel controls the voltage-dependent changes in sodium conductance that occur during an action potential in electrically excitable tissues. Over the past three decades, sodium channel function was extensively characterized through the use of sophisticated electrophysiological techniques (reviewed in ref. 1). More recently, biochemical techniques were applied to identify the sodium channel protein and to purify it in a detergent-soluble form from eel electroplax (2, 3) rat brain (4,5), and rat muscle (6,7). The purified sodium channel protein from all three sources was reconstituted into phospholipid vesicles and shown to retain functional activity by measurements of neurotoxin-mediated 22Na' flux (8-11). The neurotoxins veratridine and batrachotoxin (BTX) are specific sodium channel activators that shift the voltage dependence of activation in the hyperpolarized direction and eliminate inactivation (12)(13)(14). Saxitoxin (STX) and tetrodotoxin (TTX) are specific sodium channel blockers that bind to a receptor site on the extracellular-facing side of the sodium channel and inhibit ion translocation (14,15 1To whom reprint requests should be addressed. 240The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Section: Discussioncontrasting

confidence: 98%

Functional reconstitution of the purified brain sodium channel in planar lipid bilayers.

Hartshorne¹,

Keller²,

Talvenheimo³

et al. 1985

144

“…The data in Fig. 1B (8,28). When the integrity of the Na' channel is preserved, such as in a node of Ranvier of rat sciatic nerve or in DRG cells, the antibody clearly labels the channel (Fig.…”

Section: Discussionmentioning

confidence: 91%

A monoclonal immunotoxin acting on the Na+ channel, with properties similar to those of a scorpion toxin.

Barhanin¹,

Meiri²,

Romey³

et al. 1985

We describe the properties of a monoclonal antibody against the Na' channel. The antibody, 72.38, competitively inhibited (Ki = 1.5 x 10-9 M) the binding of an '2SI4abeled toxin from the Brazilian scorpion Tityus serrulatus (12'I-TiTXy) to Na' channels of rat brain membranes. No signifcant inhibition of binding of a number of other Na' channel toxins was observed. The inhibition of 12'I-TiTXy binding also was observed with the solubilized Na' channel from rat brain membranes (K, = 2 x 10-9 M). Antibody 72.38antagonized 12'I-TiTXy binding to Na' channels from different animal species (fish, avian, and mammalian) and from different tissues (electroplax, brain, heart, and muscle). Moreover, 72.38 (17) antibodies directed against the Na+ channel have already been described; some of them have been reported to block nerve impulse (18).In this report, we describe the properties of a mAb that was generated against a membrane fraction from Electrophorus electricus electroplax and that has properties like those of a scorpion neurotoxin. The electrophysiological properties of its action on the Na+ channel are analyzed by using the voltage-clamp technique. Chicheportiche et al. (20). MATERIALS AND METHODSImmunization, Hybridization, and Cell Processing. Mice were immunized with an eel electroplax membrane preparation (4.3 pmol of TTX binding sites/mg of protein) and their spleen cells were fused with the nonproducing myeloma line NSO/1 for mAb production as detailed by Meiri et al. (18). Of 520 hybridomas obtained, 7 different clones producing relevant mAb, as determined by solid phase radioimmunoassay in the presence or absence of specific neurotoxins for the Na+ channel (18), were tested for their ability to affect the binding of some of these neurotoxins to rat brain membranes. The neurotoxins used were the iodinated derivatives of toxin II from the North African scorpion Androctonus australis (125I-AaH11), toxin V from the sea anemone Anemonia sulcata (125I-ASv), and toxin y from the Brazilian scorpion T. serrulatus (125I-TiTXy) and the tritiated derivative of tetrodotoxin ([3H] 1842The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

“…Native sodium channels from rat brain synaptosomes incorporated into planar bilayers had a 30-pS conductance in 500 mM NaCl (15), and batrachotoxin-activated sodium channels purified from rat brain exhibited a conductance of 25 pS with sodium concentrations identical to those used here (18). In another report, rat brain sodium channels said to contain only the 260-kDa protein were studied in planar bilayers in the absence of batrachotoxin, and multiple conductance states of 25 pS, 40 pS, and 150 pS were observed (24). These channel properties vary significantly from those reported in other preparations; the variations may be the result of different lipids used in the bilayers, differences in the purified channel constituents, or other undefined factors.…”

Section: Discussionmentioning

confidence: 99%

Voltage-dependent activation in purified reconstituted sodium channels from rabbit T-tubular membranes.

Furman¹,

Tanaka²,

Müeller³

et al. 1986

We have examined the voltage-dependent gating of batrachotoxin-modiflied sodium channels purified from rabbit T-tubular membranes in two ways. First, purified channels were reconstituted into planar bilayers and singlechannel properties were measured. Batrachotoxin-activated channels showed steep voltage-dependent activation with halfmaximal opening probabilities at potentials between -95 and -116 mV. The single-channel conductance (500 mM Na' cis, 200 mM Na' trans) averaged 20 pS and was independent of membrane potential. Channels usually inserted with their extracellular faces on the trans side of the bilayer; addition of tetrodotoxin to the cis side had no effect, whereas addition to the trans side blocked >95% of channel openings at -77 mV.A second approach was used to establish that this voltage dependence was a characteristic of the entire population of purified channels and not just those few channels observed in planar bilayers. Channels reconstituted into egg phosphatidylcholine vesicles were functionally oriented by inclusion of internal saxitoxin; vesicle membrane potentials were then generated by K+ gradients in the presence of valinomycin.After batrachotoxin activation, Vm was altered by shifts of KO'All of the specific 22Na+ influx activated by batrachotoxin and blocked by saxitoxin was found to be voltage sensitive, activating between predicted membrane potentials of -100 and -50 mV. The single-channel properties of the purified Ttubular sodium channel correspond closely to those seen with native sodium channels from rat sarcolemma. The voltagedependent activation of the batrachotoxin-modified reconstituted channel is the same as that seen with native channels in situ or in bilayers after exposure to this toxin. Most importantly, this voltage-dependent gating is a property of all of the purified channels capable of specific pharmacological activation.Voltage-sensitive sodium ion channels play a key role in the generation of action potentials in nerve and skeletal muscle. This ion channel has now been isolated from eel electroplax (1), rat skeletal muscle (2) and brain (3), rabbit skeletal muscle (4), and rat heart (5). In each case the isolated protein contained one large glycoprotein subunit of -260 kDa apparent molecular mass on NaDodSO4/PAGE; the mammalian channels may also contain one or more smaller subunits of -38 kDa (4-9). These proteins have been reconstituted into lipid vesicles and each is able to gate cation fluxes in response to pharmacological activation and block (10-13).A characteristic feature of the sodium channel is its capacity for activation in response to changes in membrane potential. The distinctive voltage-dependent currents recorded from large populations of sodium channels with conventional techniques are reflected at the single-channel level in the voltage-sensitive probability of channel opening events (14). Recently, methods have been developed to incorporate native sodium channels into artificial planar bilayers by vesicle fusion (15); these channels can be a...