Flexibility of the Kir6.2 inward rectifier K(+) channel pore.
Interactions of sulfhydryl reagents with introduced cysteines in the pore-forming (Kir6.2) subunits of the K(ATP) channel were examined. 2-Aminoethyl methanethiosulfonate (MTSEA(+)) failed to modify Cd(2+)-insensitive control-Kir6.2 channels, but rapidly and irreversibly modified Kir6.2[L164C] (L164C) channels. Although a single Cd(2+) ion is coordinated by L164C, four MTSEA(+) "hits" can occur, each sequentially reducing the single-channel current. A dimeric fusion of control-Kir6.2 and L164C subunits generates Cd(2+)-insensitive channels, confirming that at least three cysteines are required for coordination, but MTSEA(+) modification of the dimer occurs in two hits. L164C channels were not modified by bromotrimethyl ammoniumbimane (qBBr(+)), even though qBBr(+) caused voltage-dependent block (as opposed to modification) that was comparable to that of MTSEA(+) or 3-(triethylammonium)propyl methanethiosulfonate (MTSPTrEA(+)), implying that qBBr(+) can also enter the inner cavity but does not modify L164C residues. The Kir channel pore structure was modeled by homology with the KcsA crystal structure. A stable conformation optimally places the four L164C side chains for coordination of a single Cd(2+) ion. Modification of these cysteines by up to four MTSEA(+) (or three MTSPTrEA(+), or two qBBr(+)) does not require widening of the cavity to accommodate the derivatives within it. However, like the KcsA crystal structure, the energy-minimized model shows a narrowing at the inner entrance, and in the Kir6.2 model this narrowing excludes all ions. To allow entry of ions as large as MTSPTrEA(+) or qBBr(+), the entrance must widen to >8 A, but this widening is readily accomplished by minimal M2 helix motion and side-chain rearrangement.
Loussouarn
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G
Departments of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
Phillips
L R
LR
Masia
R
R
Rose
T
T
Nichols
C G
CG
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002104:Cadmium / Q000494:pharmacology*; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D007202:Indicators and Reagents / Q000494:pharmacology; D008698:Mesylates / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018583:Pliability; D017354:Point Mutation; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics; D024661:Potassium Channels, Inwardly Rectifying; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid
D007202:Indicators and Reagents; D008698:Mesylates; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D000644:Quaternary Ammonium Compounds; C093171:methanethiosulfonate ethylammonium; D002104:Cadmium; C014674:methyl methanethiosulfonate; C074502:methanethiosulfonate; C027344:bromotrimethylammoniobimane; D005020:Ethyl Methanesulfonate; D008741:Methyl Methanesulfonate; D003545:Cysteine