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The functional reconstitution of the voltageregulated Na channel purified from the electroplax of Electrophorus electricus is described. Reconstitution was achieved by removing detergent with Bio-Beads SM-2 followed by freeze-thaw-sonication in the presence of added liposomes. This preparation displayed heat-stable binding of 3H-labeled tetrftlotoxin (TTX) (Kd = 33 nM). 22Na' influx was stimulated 2-to 5-fold by alkaloid neurotoxins and blocked by TTX Among a variety of pharmacological agents that are functional probes of the Na channel, four classes of compounds have been especially useful: (i) tetrodotoxin (TTX) and saxitoxin (STX) bind with high affinity to an external site to block ion conductance; (ii) lipid-soluble alkaloid toxins, including veratridine and batrachotoxin (BTX), alter gating mechanisms to cause persistent channel activation; (iii) local anesthetics, including derivatives of procaine and lidocaine, interact with an internal site to block conductance; and (iv) peptide toxins isolated from species of scorpion and sea anemone perturb gating so as to enhance the effects of the alkaloid toxins (for reviews see refs. 1-3). Tritiated TTX and STX have been used to monitor the purification of the TTX/STX-binding components from eel electroplax (4, 5), rat muscle sarcolemma (6), and rat brain synaptosomes (7). The reported peptide compositions of the proteins from these tissues are somewhat different. Whereas the TTXbinding activity of the eel electroplax copurifies to homogeneity with a single, heavily glycosylated peptide of Mr 260,000-300,000 (5), additional peptides of Mr 37,000 and 39,000 are present in preparations from brain (8), and peptides of Mr 45,000, 38,000, and 39,000 appear in preparations from muscle (9). Because TTX and STX binding identifies only a part of the Na channel, these differences may reflect losses of important peptide subunits during isolation, incomplete purification, proteolytic degradation, or actual differences in the proteins from different tissues or species.To determine whether the isolated proteins are functionally intact, the TTX/STX-binding components of muscle (10) and brain (11,12) were previously reconstituted into phospholipid vesicles and alkaloid-stimulated, TTX-blocked 22Na+ influx was demonstrated. In this communication we report the incorporation of the Na channel purified from electroplax into lipid vesicles. Alkaloid toxins, local anesthetics, and TTX all modified Na+ influx in a manner consistent with their known pharmacology. The large glycopeptide accounted for -80% of the protein present in the preparations tested. Furthermore, evidence is presented that the smaller peptides present probably are not required for functional reconstitution. MATERIALS AND METHODSMaterials. Citrate-free TTX was the kind gift of Y. Kishi, Harvard University, and it was tritiated by the Wilzbach procedure and purified as described (4). The toxin was of specific activity 59.1 Ci/mol (1 Ci = 37 GBq) and =65% radiochemical purity as determined by frog sciatic nerve ...

The tetrodotoxin-binding protein purified from electroplax of Electrophorus electricus has been reincorporated into multilamellar vesicles that were used for patch recording. When excised patches of these reconstituted membranes were voltage clamped in the absence of neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties qualitatively and quantitatively similar to those reported for Na channels from nerve and muscle cells, including uniform single-channel conductances of the appropriate magnitude (-11 pS in 95 mM Na+), mean open times of =1.9 msec, and 7-fold selectivity for Na+ over K+. Currents averaged from many depolarizations showed initial voltage-dependent activation and subsequent inactivation. In the presence of batrachotoxin, channels were observed with markedly different properties, including conductances of 20-25 pS (95 mM Na+), mean open times of -28 msec, and no indication of inactivation. Collectively, these findings indicate that the tetrodotoxin-binding protein of electroplax is a voltage-regulated sodium channel.

Recent immunohistochemical evidence from the rat, indicating that gamma-aminobutyric acid (GABA)-containing fibres of central nervous origin project to the pars intermedia of the pituitary1,2, prompts inquiry into the function of this innervation. There is electrophysiological evidence that GABA acts directly on melanotrophs isolated from rat, through bicuculline-blockable receptors, to increase Cl- conductance and thereby drive the membrane potential towards the Cl- equilibrium potential in these cells, resulting in depolarization at rest or reduction of the depolarization caused by excess K+ (ref. 3). As voltage-dependent Ca channels can participate in the regulation of secretion in these cells4, we have now examined the effect of GABA on hormone output and find that it first stimulates and then inhibits spontaneous secretion of melanocyte-stimulating hormone (MSH) and inhibits K+-evoked secretion. Moreover, our pharmacological evidence suggests that similar receptors are involved in the secretory and the electrophysiological responses. A function of the GABAergic innervation may therefore be to regulate hormone output by acting directly on the melanotrophs, and this regulation may be affected by the changes in electrical properties induced by GABA.

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