The sodium channel from rat brain

A procedure is described for purification of the so- dium channel 1380-fold from rat brain to essential homogeneity. The channel is solubilized in Triton X-100 and stabilized by addition of phosphatidylcholine and 10 mM CaClz. It is purified by sequential chromatography on DEAE-Sephadex, hydroxylapatite, and wheat germ agglutinin/Sepharose followed by sedimentation through sucrose gradients. The final prep- aration binds 2910 pmol of saxitoxin (STX)/mg of protein or 0.9 mol of STX/mol of sodium channel of M, -316,000. Three polypeptide subunits comprise 90% of the silver stain intensity on sodium dodecyl sulfate-polyacrylamide gels of the pure protein and migrate as a stoichiometric complex coincident with STX-binding activity in sucrose gradient sedimentation: a with M, - 260,000, 81 with M, - 39,000, and 82 with M, -37,000. The a subunit, both purified and in intact synaptosomes, is shown to behave anomalously during sodium dodecyl sulfate-polyacrylamide gel electropho- resis exhibiting an unusually high extrapolated electrophoretic free mobility. A subunit stoichiometry of ~r ~( @l) ~ (B2)~ is proposed. the inhibitors A, and phenylmethylsulfonyl were all obtained from Sigma. Measurement of PHISTX Binding-13H]STX bound to purified sodium channels in column fractions was measured by rapid gel filtration through 2-ml columns of Sephadex G-50 as described pre- viously (14, 20) after a 5-min incubation at 0 "C with sufficient 13H] STX to saturate >95% of the binding sites. This technique underes- timates [3H]STX binding by 15% relative to equilibrium dialysis measurements. This underestimation was corrected in the calcula- tions of STX bound. Nonspecific binding was determined in the presence of 1 p~ TTX and subtracted from the total binding in all data shown. Equilibrium dialysis as described in Ref. 20 was used to measure 13H]STX binding in experiments to determine the concen- tration dependence of toxin binding and to determine the amount of active STX receptor in the most highly purified preparations. Assay-Peterson's modification the Lowry protein assay (21) for membrane proteins was used to determine protein concentra- tion using bovine serum albumin as a standard. proteolytic degradation STX protease inhibitors active each of the four classes of protease were added to every solution used during the purification. Phenylmethylsulfonyl fluoride (0.1 mM) was used to inhibit serine proteases. used to inhibit thiol proteases. transition for activity of metalloproteases without the Ca2+ in most solutions. A inhibit proteases with activated carboxylic acid groups.