title: Interactions of external K+ and internal blockers in a weak inward-rectifier K+ channel.
abstract: We investigated the effects of changing extracellular K(+) concentrations on block of the weak inward-rectifier K(+) channel Kir1.1b (ROMK2) by the three intracellular cations Mg(2+), Na(+), and TEA(+). Single-channel currents were monitored in inside-out patches made from Xenopus laevis oocytes expressing the channels. With 110 mM K(+) in the inside (cytoplasmic) solution and 11 mM K(+) in the outside (extracellular) solution, these three cations blocked K(+) currents with a range of apparent affinities (K(i) (0) = 1.6 mM for Mg(2+), 160 mM for Na(+), and 1.8 mM for TEA(+)) but with similar voltage dependence (zδ = 0.58 for Mg(2+), 0.71 for Na(+), and 0.61 for TEA(+)) despite having different valences. When external K(+) was increased to 110 mM, the apparent affinity of all three blockers was decreased approximately threefold with no significant change in the voltage dependence of block. The possibility that the transmembrane cavity is the site of block was explored by making mutations at the N152 residue, a position previously shown to affect rectification in Kir channels. N152D increased the affinity for block by Mg(2+) but not for Na(+) or TEA(+). In contrast, the N152Y mutation increased the affinity for block by TEA(+) but not for Na(+) or Mg(2+). Replacing the C terminus of the channel with that of the strong inward-rectifier Kir2.1 increased the affinity of block by Mg(2+) but had a small effect on that by Na(+). TEA(+) block was enhanced and had a larger voltage dependence. We used an eight-state kinetic model to simulate these results. The effects of voltage and external K(+) could be explained by a model in which the blockers occupy a site, presumably in the transmembrane cavity, at a position that is largely unaffected by changes in the electric field. The effects of voltage and extracellular K(+) are explained by shifts in the occupancy of sites within the selectivity filter by K(+) ions.
authors.affiliation: Department of Physiology and Biophysics, Weill-Cornell Medical College, New York, NY 10065, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Yang
authors.forename: Johan
authors.initials: J
authors.lastname: Edvinsson
authors.forename: Lawrence G
authors.initials: LG
authors.lastname: Palmer
mesh_terms: D000818:Animals; D005110:Extracellular Space / Q000737:chemistry; D042541:Intracellular Space / Q000737:chemistry; D015640:Ion Channel Gating; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials; D020125:Mutation, Missense; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D019789:Tetraethylammonium / Q000494:pharmacology; D014981:Xenopus
chemical_list: D026902:Potassium Channel Blockers; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; D019789:Tetraethylammonium; D012964:Sodium; D008274:Magnesium; D011188:Potassium