title: The polyamine binding site in inward rectifier K+ channels.
abstract: Strongly inwardly rectifying potassium channels exhibit potent and steeply voltage-dependent block by intracellular polyamines. To locate the polyamine binding site, we have examined the effects of polyamine blockade on the rate of MTSEA modification of cysteine residues strategically substituted in the pore of a strongly rectifying Kir channel (Kir6.2[N160D]). Spermine only protected cysteines substituted at a deep location in the pore, between the "rectification controller" residue (N160D in Kir6.2, D172 in Kir2.1) and the selectivity filter, against MTSEA modification. In contrast, blockade with a longer synthetic polyamine (CGC-11179) also protected cysteines substituted at sites closer to the cytoplasmic entrance of the channel. Modification of a cysteine at the entrance to the inner cavity (169C) was unaffected by either spermine or CGC-11179, and spermine was clearly "locked" into the inner cavity (i.e., exhibited a dramatically slower exit rate) following modification of this residue. These data provide physical constraints on the spermine binding site, demonstrating that spermine stably binds at a deep site beyond the "rectification controller" residue, near the extracellular entrance to the channel.
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA. cnichols@cellbio.wustl.edu
authors.forename: Harley T
authors.initials: HT
authors.lastname: Kurata
authors.forename: Laurence J
authors.initials: LJ
authors.lastname: Marton
authors.forename: Colin G
authors.initials: CG
authors.lastname: Nichols
mesh_terms: D000818:Animals; D001665:Binding Sites; D015317:Biogenic Polyamines / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D003545:Cysteine / Q000032:analysis; D004594:Electrophysiology; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D008564:Membrane Potentials / Q000502:physiology; D008954:Models, Biological; D018408:Patch-Clamp Techniques; D011073:Polyamines / Q000378:metabolism* / Q000494:pharmacology; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding; D011700:Putrescine / Q000494:pharmacology; D013096:Spermine / Q000378:metabolism / Q000494:pharmacology; D000099161:Kcnj11 Channel
chemical_list: D015317:Biogenic Polyamines; D003545:Cysteine; D005020:Ethyl Methanesulfonate; D011073:Polyamines; D024661:Potassium Channels, Inwardly Rectifying; D011700:Putrescine; D013096:Spermine; D000099161:Kcnj11 Channel; C512143:CGC 11179; C489461:Kir2.1 channel; C093171:methanethiosulfonate ethylammonium