Purified and unpurified sodium channels from eel electroplax in planar lipid bilayers.

Abstract

Highly purified sodium channel protein from the electric eel, Electrophorus electricus, was reconstituted into liposomes and incorporated into planar bilayers made from neutral phospholipids dissolved in decane. The purest sodium channel preparations consisted of only the large, 260-kD tetrodotoxin (TTX)-binding polypeptide. For all preparations, batrachotoxin (BTX) induced long-lived single-channel currents (25 pS at 500 mM NaCl) that showed voltage-dependent activation and were blocked by TTX. This block was also voltage dependent, with negative potentials increasing block. The permeability ratios were 4.7 for Na+:K + and 1.6 for Na+:Li +. The midpoint for steady state activation occurred around -70 mV and did not shift significantly when the NaCI concentration was increased from 50 to 1,000 mM. Veratridine-induced single-channel currents were about half the size of those activated by BTX. Unpurified, nonsolubilized sodium channels from E. electricus membrane fragments were also incorporated into planar bilayers. There were no detectable differences in the characteristics of unpurified and purified sodium channels, although membrane stability was considerably higher when purified material was used. Thus, in the eel, the large, 260-kD polypeptide alone is sufficient to demonstrate single-channel activity like that observed for mammalian sodium channel preparations in which smaller subunits have been found.

Effects of veratridine on sodium currents and fluxes

“…The reason is that the latter alkaloid keeps the channels open almost continuously on depolarization whereas in veratridine, even at maximal concentrations, the channels close part of the time. Also, ymax, measured at saturating Na + concentrations, is about twice as high in the presence of batrachotoxin than of veratridine, independent of the channel origin as in rat skeletal muscle: 21 vs 10 pS (Garber and Miller 1987), rat brain: 30 vs 9 pS (Krueger et al 1983;Corbett and Krueger 1989), and in another study 24 vs 10 pS (Cukierman 1991), eel electroplax: 25 vs 13 pS (Recio-Pinto et al 1987) and lobster nerve: 16 vs 10 pS (Castillo et al 1992). Veratridinemodified channels in natural membranes show lower conductances (see previous section), most probably because they have been determined at lower (natural) cation concentrations.…”

Sodium Channel Inactivation: Molecular Determinants and Modulation

“…GTX-modified channels also have a reduced conductance (508). Likewise sodium channels from eel electroplax inserted in planar bilayers have reduced conductances in the sequence unmodified Ͼ BTX Ͼ GTX Ͼ VT (115,364).…”

Energy Conversion in Protocells with Natural Nanoconductors

“…The challenges to this technology lay in creating stable and selective channels and ion pumps-natural membrane proteins are currently purified and inserted into lipid bilayers [91,92]. Synthetic counterparts of ion channels can be synthesized by protein engineering, such as α-hemolysin and its mutants [63,71].…”