Cytoplasmic vestibule of the weak inward rectifier Kir6.2 potassium channel

Abstract

Intracellular application of certain charged methanethiosulfonate (MTS) reagents modified and irreversibly inhibited Kir6.2 channels when cysteine substitutions were introduced at positions Ile-210, Ile-211, or Ser-212 within the putative cytoplasmic region. Inhibition depends on the spatial dimensions of the MTS reagents. Reaction of MTS reagents, having head diameters of 7.6-8.2 A, with cysteines introduced at position Ser-212 must occur in more than two subunits of the tetrameric Kir6.2 complex to inhibit channel activity. MTS reagents with head diameters less than 6.6 A modified cysteines without causing channel inhibition. An MTS reagent with a head diameter of approximately 10 A could neither modify nor inhibit the channels. Channel inhibition is interpreted as blockage of the intracellular vestibule by MTS reagents that enter the channel vestibule and react with the cysteine residues at vestibule-lining positions. Data are consistent with the hypothesis that residues Ile-210-Ser-212 line a funnel-shaped vestibule of 20-25 A in diameter, which remains unchanged during channel gating.