title: Large diameter of palytoxin-induced Na/K pump channels and modulation of palytoxin interaction by Na/K pump ligands.
abstract: Palytoxin binds to Na/K pumps to generate nonselective cation channels whose pore likely comprises at least part of the pump's ion translocation pathway. We systematically analyzed palytoxin's interactions with native human Na/K pumps in outside-out patches from HEK293 cells over a broad range of ionic and nucleotide conditions, and with or without cardiotonic steroids. With 5 mM internal (pipette) [MgATP], palytoxin activated the conductance with an apparent affinity that was highest for Na(+)-containing (K(+)-free) external and internal solutions, lowest for K(+)-containing (Na(+)-free) external and internal solutions, and intermediate for the mixed external Na(+)/internal K(+), and external K(+)/internal Na(+) conditions; with Na(+) solutions and MgATP, the mean dwell time of palytoxin on the Na/K pump was about one day. With Na(+) solutions, the apparent affinity for palytoxin action was low after equilibration of patches with nucleotide-free pipette solution. That apparent affinity was increased in two phases as the equilibrating [MgATP] was raised over the submicromolar, and submillimolar, ranges, but was increased by pipette MgAMPPNP in a single phase, over the submillimolar range; the apparent affinity at saturating [MgAMPPNP] remained approximately 30-fold lower than at saturating [MgATP]. After palytoxin washout, the conductance decay that reflects palytoxin unbinding was accelerated by cardiotonic steroid. When Na/K pumps were preincubated with cardiotonic steroid, subsequent activation of palytoxin-induced conductance was greatly slowed, even after washout of the cardiotonic steroid, but activation could still be accelerated by increasing palytoxin concentration. These results indicate that palytoxin and a cardiotonic steroid can simultaneously occupy the same Na/K pump, each destabilizing the other. The palytoxin-induced channels were permeable to several large organic cations, including N-methyl-d-glucamine(+), suggesting that the narrowest section of the pore must be approximately 7.5 A wide. Enhanced understanding of palytoxin action now allows its use for examining the structures and mechanisms of the gates that occlude/deocclude transported ions during the normal Na/K pump cycle.
authors.affiliation: Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, NY 10021-6399, USA.
authors.forename: Pablo
authors.initials: P
authors.lastname: Artigas
authors.forename: David C
authors.initials: DC
authors.lastname: Gadsby
mesh_terms: D000178:Acrylamides / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002316:Cardiotonic Agents / Q000494:pharmacology; D002478:Cells, Cultured; D003064:Cnidarian Venoms; D004347:Drug Interactions; D004553:Electric Conductivity; D006168:Guinea Pigs; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D007668:Kidney / Q000166:cytology; D008024:Ligands; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011188:Potassium / Q000493:pharmacokinetics; D012964:Sodium / Q000493:pharmacokinetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D000095702:Polyether Toxins
chemical_list: D000178:Acrylamides; D000255:Adenosine Triphosphate; D002316:Cardiotonic Agents; D003064:Cnidarian Venoms; D008024:Ligands; D010042:Ouabain; D011188:Potassium; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; C010272:palytoxin; D000095702:Polyether Toxins