Molecular basis of inward rectification: polyamine interaction sites located by combined channel and ligand mutagenesis.
Polyamines cause inward rectification of (Kir) K+ channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSEA+ modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter.
Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
Harley T
HT
Kurata
L Revell
LR
Phillips
Thierry
T
Rose
Gildas
G
Loussouarn
Stefan
S
Herlitze
Hariolf
H
Fritzenschaft
Decha
D
Enkvetchakul
Colin G
CG
Nichols
Thomas
T
Baukrowitz
D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D011073:Polyamines / Q000494:pharmacology*; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014982:Xenopus laevis; D000099161:Kcnj11 Channel
D011073:Polyamines; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; D000099161:Kcnj11 Channel