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 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 (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 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 Å, but this widening is readily accomplished by minimal M2 helix motion and side-chain rearrangement.