The sodium channel from rat brain. Reconstitution of neurotoxin-activated ion flux and scorpion toxin binding from purified components

  • Tamkun M
  • Talvenheimo J
  • Catterall W
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Abstract

The rat brain Na+ channel was purified to homogeneity and reconstituted into pure egg phosphatidylcholine vesicles or vesicles composed of a mixture of egg phosphatidylcholine and rat brain lipid. In each case, the binding affinities at 37 degrees C for saxitoxin (STX) and tetrodotoxin (TTX) are nearly identical with those measured for intact Na+ channels. Approximately 50% of the reconstituted channels are oriented right-side-out. Veratridine stimulates the initial rate of 22Na+ uptake 8- to 15-fold with a K0.5 of 28 microM. External TTX blocks the fraction of Na+ channels oriented right-side-out with a Ki of 14 nM. All of the veratridine-stimulated 22Na+ uptake is blocked when TTX is present on both sides of the vesicle membrane, or when tetracaine is added to the external medium. The veratridine-activated reconstituted Na+ channel transports cations with a permeability ratio of PNa+/PRb+/PCa+ = 1.0:0.25:0.12. We estimate that at least 30% and perhaps as many as 70% of the reconstituted channels are active. Purified sodium channels reconstituted in egg phosphatidylcholine vesicles do not bind 125I-scorpion toxin (125I-LqTx). In contrast, when incorporated into vesicles containing rat brain lipids, 76% of the Na+ channels bound 125I-LqTx with an average KD of 80 nM. Thermal denaturation of purified Na+ channels at 36 degrees C prior to reconstitution causes a parallel loss of both the [3H]STX- and 125I-LqTx-binding activity measured after reconstitution. Sea anemone toxin II displaces bound 125I-LqTx with a KD 60-fold greater than that of unlabeled LqTx. These data indicate that the alpha, beta 1, and beta 2 subunits of the sodium channel are sufficient for reconstitution of both selective, veratridine-stimulated ion transport and 125I-LqTx binding.

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Authors

  • Gina LuuDepartment of Biochemistry, University of Wisconsin at Madison

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  • M. M. Tamkun

  • J. A. Talvenheimo

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