Purification from rat sarcolemma of the saxitoxin-binding component of the excitable membrane sodium channel.

Abstract

The saxitoxin-binding component (SBC) of the excitable membrane sodium channel has been solubilized and purified from rat skeletal muscle sarcolemma. Phospholipid was required in mixed micelles with detergent for stability of the mammalian SBC. Even at optimal detergent-to-phospholipid ratio, the solubilized SBC showed significant temperaturedependent loss of specific toxin binding with time, necessitating maintenance of low temperatures during purification. Characteristics of saxitoxin binding to the solubilized material closely resembled those seen in intact membranes. A weak anion-exchange column was synthesized; it provided rapid 10-to 20-fold purification of the solubilized SBC. Additional necessary purification was obtained by chromatography on immobilized wheat germ agglutinin. Specific saxitoxin-binding activity of the purified material averaged t1500 pmol of saxitoxin bound per mg of protein. Three bands were present in this material on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified material sedimented on a sucrose gradient with an apparent s20,w of 9.9 S.Action potentials recorded in a wide variety of excitable membranes share a mechanism based on transient sequential increases in membrane conductance to Na+ and K+ (1). These voltage-and time-dependent conductance changes are mediated by separate membrane pores or channels presumably formed by intrinsic proteins spanning the bilayer (2). A large body of information has been amassed concerning the current gating properties of these channels during the 30 years since the development of the voltage clamp technique (1, 3). Remarkably little is known, however, about their biochemical features or molecular structure.Although successful solubilization of sodium channel components was reported as early as 1972 (4), further biochemical analysis has been hindered by difficulties encountered in stabilizing the solubilized channel and in identifying the channel or its components during subsequent purification. The availability of radiolabeled neurotoxins such as tetrodotoxin (TTX) and saxitoxin (STX), which bind with high affinity and specificity within the ion pathway of the channel, has provided a fruitful approach to one of these problems (5). Recent recognition of the role of phospholipids in stabilizing the solubilized channel protein in a conformation capable of binding toxin represents a major advance in biochemical studies of the channel protein (6). On the basis of their findings in this regard, Agnew et al. have accomplished the solubilization and purification of a sodium channel TTX-binding component from eel electroplax (7).Sodium channel density in mammalian tissue is generally much lower than that in eel electroplax, introducing an additional complication to the characterization of the channel in a mammalian system. We report here the successful solubilization and purification of the sodium channel STX-binding component (SBC) from a mammalian source, rat sarcolemma. The techniques used in this purification take advant...

Purification of the saxitoxin receptor of the sodium channel from rat brain.

“…However, the recovery ofbound STX receptor from the concanavalin A column was only 7-12%, whereas recovery from the WGA column was 65-85%. Similar results with WGA columns were obtained by Barchi et al (11) using STX receptor solubilized from rat skeletal muscle sarcolemma. After loading of a 10 ml WGA-Sepharose column with the eluate from a DEAE-Sephadex column, the column was washed with 50 ml of a high ionic strength buffer to minimize nonspecific ionic interactions with.the column.…”

“…More recently, stabilization of the solubilized receptor by phosphatidylcholine and TTX (8,9) or by phosphatidylcholine and Ca2" (10) has allowed substantial purification of the STX/TIX receptor from eel electroplax (8), rat skeletal muscle sarcolemma (11), and rat brain (12).…”

New studies of the excitatory sodium currents in heart muscle.

“…The functional state of these isolated channels can be studied by their incorporation into artificial bilayers. Good progress has been made in the chemical isolation of Na + channels (Catterall and Nirenberg, 1973;Goldin et al, 1980;Barchi et al, 1980;Agnew, 1984). At least two subunits have been identified, one of 270,000 and the other of 39,000 daltons (Beneski and Catterall, 1980).…”

Removal of sodium channel inactivation in squid axon by the oxidant chloramine-T.

“…Biochemical characterization of these proteins has recently begun (Agnew et al, 1980 ;Barchi et al, 1980 ;Costa et al, 1982). Nonetheless, the "functional" amino acid residues of the sodium channel that mediate the permeability changes during depolarization remain obscure.…”