Gating and permeability of ion channels produced by botulinum toxin types A and E in PC12 cell membranes.
Botulinum neurotoxin (BoNT) is known to produce cationic channels in artificial bilayers. This study examined ion channels formed by BoNT in native membranes from cultured PC12 cells under conditions approximating those thought to occur during toxin internalization. Membrane patches were excised from PC12 cells using patch electrodes and exposed to symmetrical solutions containing either 200 mM CsCl, RbCl or KCl. The patch pipettes also contained 1-5 microg/ml BoNT buffered to pH 5.3 while the bath solutions were buffered to pH 7.0. In the presence of toxin, bursts of ion channel openings were observed. These toxin-induced channels were most active with a negative voltage applied to the same side as the toxin (cis). The increased activity at negative voltages was due to an increase in mean open time of e-fold per 120 mV and a decrease in mean closed time between bursts of e-fold per 110 mV. The shorter mean closed time within a burst was independent of membrane voltage. While BoNT-induced ion channels started as a single conductance level of 27 pS (KCl), 34 pS (RbCl) or 46 pS (CsCl) they typically increased in roughly equal steps to five or more times the original channel conductance. These higher conductance BoNT 'channels' opened and closed synchronously and could be distinguished from superposition of multiple independent channels. Despite differences in putative transmembrane sequences between BoNT/A and BoNT/E, both serotypes evidenced the same channel conductance and mean open time.
Sheridan
R E
RE
Neurotoxicology Branch, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010-5425, USA.
D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000737:chemistry / Q000633:toxicity*; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000633:toxicity*; D002462:Cell Membrane / Q000187:drug effects*; D002586:Cesium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D007473:Ion Channels / Q000187:drug effects*; D008969:Molecular Sequence Data; D009465:Neuromuscular Agents / Q000633:toxicity*; D016716:PC12 Cells; D018408:Patch-Clamp Techniques; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D012413:Rubidium / Q000494:pharmacology
D002712:Chlorides; D007473:Ion Channels; D009465:Neuromuscular Agents; D002586:Cesium; D011189:Potassium Chloride; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C028019:cesium chloride; D012413:Rubidium; C032710:rubidium chloride; C096326:botulinum toxin type E