Spatial localization of the K+ channel selectivity filter by mutant cycle-based structure analysis.
The structurally well-characterized scorpion toxin Agitoxin2 inhibits ion permeation through Shaker K+ channels by binding to the external pore entryway. Scanning mutagenesis identified a set of inhibitor residues critical for making energetic contacts with the channel. Using thermodynamic mutant cycle analysis, we have mapped channel residues relative to the known inhibitor structure. This study constrains the position of multiple channel residues within the pore-forming loops; in one stretch, we have been able to map five out of seven contiguous residues to the inhibitor interaction surface, including those involved in ion selectivity. One interaction in particular, that of K27M on the inhibitor with Y445F on the channel, is unique in that it depends on the K+ ion concentration. These results reveal a shallow vestibule formed by the pore loops at the K+ channel entryway. The selectivity filter is located at the center of the vestibule close to (approximately 5 A) the extracellular solution.
Ranganathan
R
R
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Lewis
J H
JH
MacKinnon
R
R
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics; D015640:Ion Channel Gating / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012604:Scorpion Venoms / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012605:Scorpions / Q000235:genetics; D051637:Shaker Superfamily of Potassium Channels; D013816:Thermodynamics
D000596:Amino Acids; D029721:Drosophila Proteins; D015221:Potassium Channels; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; C496929:Sh protein, Drosophila; D051637:Shaker Superfamily of Potassium Channels; C087603:agitoxin 2; D011188:Potassium