Reconstitution of neurotoxin-modulated ion transport by the voltage-regulated sodium channel isolated from the electroplax of Electrophorus electricus.
The functional reconstitution of the voltage-regulated 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 tetrodotoxin (TTX) (Kd = 33 nM). 22Na+ influx was stimulated 2- to 5-fold by alkaloid neurotoxins and blocked by TTX. Veratridine activated Na+ influx with a K1/2 of 18 microM, and this activation was blocked by TTX precisely in parallel with specific [3H]TTX binding. Batrachotoxin stimulated 22Na+ flux more effectively than did veratridine. No effect of the peptide anemone toxin II was found. Insertion of the Na channel into membranes resulted in 60-70% of the TTX-binding sites facing the vesicle exterior. Thus, external TTX partially inhibited flux, whereas blockade was complete when TTX was also equilibrated with the vesicle interior. The lipid-soluble local anesthetics tetracaine and dibucaine inhibited flux completely. QX-222, a charged derivative of lidocaine, blocked only a fraction of the channels, apparently those oriented inside-out. Purified samples were predominantly composed of the Mr 260,000-300,000 glycopeptide but contained variable quantities of smaller peptides. Veratridine-dependent flux and peptide compositions were determined on fractions across a gel filtration column profile. Stimulated flux codistributed only with the large peptide.
D000779:Anesthetics, Local / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D004557:Electric Organ / Q000502:physiology*; D004593:Electrophorus; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D009498:Neurotoxins / Q000633:toxicity*; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels; D013779:Tetrodotoxin / Q000378:metabolism / Q000633:toxicity*; D014701:Veratridine / Q000494:pharmacology
D000779:Anesthetics, Local; D002352:Carrier Proteins; D007473:Ion Channels; D009498:Neurotoxins; D015222:Sodium Channels; C023790:tetrodotoxin-binding protein; D013779:Tetrodotoxin; D014701:Veratridine; D012964:Sodium