Na+ channels of lobster nerve membrane were incorporated into soybean liposomes by the freeze-thaw-sonication procedure. Electron microscopy showed that the vesicular pattern of membranes and liposomes was broken and reassembled during the process. The amount of membrane protein incorporated depended linearly on the amount of membrane protein added to the liposomes for reconstitution. The 22Na flux into the reconstituted vesicles was increased by veratridine (0.5 mM) or grayanotoxin I (150 microM) and the increment was abolished by tetrodotoxin (Ki = 4 to 5 nM). The drug-sensitive 22Na influx depended linearly on the amount of membrane protein incorporated. No response to the drugs was found in protein-free vesicles or in vesicles reconstituted with membrane heated at 50 degrees C for 15 min. A fraction of digitonin-treated membranes had a higher Na+ channel activity. The Na+ channel was able to discriminate between Na+ and K+ or Rb+.
Primary cultures of rat embryonic central nervous system were grown for 3 to 4 weeks in vitro. Impalement of neurons by means of high-resistance glass pipettes revealed that firing of action potentials could either be elicited by application of short square pulses or occurred spontaneously. The presence of the neurotoxin apamin caused a hyperpolarization of the membrane potential accompanied by an increase of the spike amplitude as well as an increase of membrane resistance. The significance of this effect is discussed in terms of a postsynaptic effect of this toxin.
