title: Locale and chemistry of spermine binding in the archetypal inward rectifier Kir2.1.
abstract: Polyamine block of inwardly rectifying potassium (Kir) channels underlies their steep voltage dependence observed in vivo. We have examined the potency, voltage dependence, and kinetics of spermine block in dimeric Kir2.1 constructs containing one nonreactive subunit and one cysteine-substituted subunit before and after modification by methanethiosulfonate (MTS) reagents. At position 169C (between the D172 "rectification controller" and the selectivity filter), modification by either 2-aminoethyl MTS (MTSEA) or 2-(trimethylammonium)ethyl MTS (MTSET) reduced the potency and voltage dependence of spermine block, consistent with this position overlapping the spermine binding site. At position 176C (between D172 and the M2 helix bundle crossing), modification by MTSEA also weakened spermine block. In contrast, MTSET modification of 176C dramatically slowed the kinetics of spermine unblock, with almost no effect on potency or voltage dependence. The data are consistent with MTSET modification of 176C introducing a localized barrier in the inner cavity, resulting in slower spermine entry into and exit from a "deep" binding site (likely between the D172 rectification controller and the selectivity filter), but leaving the spermine binding site mostly unaffected. These findings constrain the location of deep spermine binding that underlies steeply voltage-dependent block, and further suggest important chemical details of high affinity binding of spermine in Kir2.1 channels-the archetypal model of strong inward rectification.
authors.affiliation: Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. harley.kurata@ubc.ca
authors.forename: Harley T
authors.initials: HT
authors.lastname: Kurata
authors.forename: Emily A
authors.initials: EA
authors.lastname: Zhu
authors.forename: Colin G
authors.initials: CG
authors.lastname: Nichols
mesh_terms: D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D008253:Macaca mulatta; D008698:Mesylates / Q000494:pharmacology; D008958:Models, Molecular; D018408:Patch-Clamp Techniques; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding; D013096:Spermine / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
chemical_list: C489461:Kir2.1 channel; D008698:Mesylates; D024661:Potassium Channels, Inwardly Rectifying; C093171:methanethiosulfonate ethylammonium; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D013096:Spermine; D005020:Ethyl Methanesulfonate; D003545:Cysteine