Pore formation by tetanus toxin, its chain and fragments in neuronal membranes and evaluation of the underlying motifs in the structure of the toxin molecule.
The pore-forming activity of tetanus toxin, its chains and fragments was studied on membrane patches from spinal cord neurons of fetal mice using the outside-out patch-clamp configuration. 1. The dichain tetanus toxin forms pores at pH 5, but not at pH 7.4. The elementary pore conductance is 38.4 +/- 1.1 pS and nonselective for small cations. The open probability of the pores is voltage-dependent and increases with membrane depolarisation. The pores activate at +80 mV with a time constant of about 20 ms and deactivate at -80 mV with two time constants of about 2 ms and 10 ms. Besides the elementary pore conductance, larger pore conductances which are multiples of the elementary conductance were observed. With increasing conductances, their frequency of occurrence decreases exponentially. 2. The light chain of tetanus toxin alone does not form pores in neuronal membranes at pH 5 or at pH 7.4. 3. The heavy chain of tetanus toxin forms pores at pH 5 as well as at pH 7.4. The single pore conductance increases from 35.0 +/- 1.2 pS at pH 5 to 43.2 +/- 1.8 pS at pH 7.4. The pores allow mono- and divalent cations and chloride ions to pass. Only at pH 5 do they have a voltage dependence with time constants identical to those obtained with tetanus toxin. 4. Secondary structure predictions show a high density of presumably helically organized elements in fragment beta 2 (45 kDa) of the heavy chain between residues 700-850.(ABSTRACT TRUNCATED AT 250 WORDS)
Beise
J
J
Rudolf-Buchheim-Institut für Pharmakologie der Justus-Liebig-Universität, Giessen, Germany.
Hahnen
J
J
Andersen-Beckh
B
B
Dreyer
F
F
D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005069:Evaluation Studies as Topic; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008970:Molecular Weight; D009474:Neurons / Q000187:drug effects* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D013116:Spinal Cord / Q000166:cytology / Q000187:drug effects; D013329:Structure-Activity Relationship; D013744:Tetanus Toxin / Q000737:chemistry / Q000494:pharmacology*
D010446:Peptide Fragments; D013744:Tetanus Toxin