期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 98, 期 7, 页码 4227-4232出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.061452698
关键词
inner pore; Cd2+; MTS reagents; subconductance
资金
- NHLBI NIH HHS [HL54171, R01 HL054171] Funding Source: Medline
Interactions of sulfhydryl reagents with introduced cysteines in the pore-terming (Kir6.2) subunits of the K-ATP channel were examined. 2-Aminoethyl methanethiosulfonate (MTSEA(+)) failed to modify Cd2+-insensitive control-Kir6.2 channels, but rapidly and irreversibly modified Kir6.2[L164C] (L164C) channels. Although a single Cd2+ 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 Cd2+-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 las 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 Cd2+ 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 Angstrom, but this widening is readily accomplished by minimal M2 helix motion and side-chain rearrangement.
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