Hydrophobic mutations alter the movement of Mg2+ in the pore of voltage-gated potassium channels.
The permeation pathways of the voltage-gated K+ channels Kv3.1 and ShakerB delta 6-46 (ShB delta) were studied using Mg2+ block. Internal Mg2+ blocked both channels in a voltage-dependent manner, and block was partially relieved by external K+, consistent with Mg2+ binding within the pore. The kinetics of Mg2+ block was much faster for Kv3.1 than for ShB delta. Fast block of Kv3.1 was transferred to ShB delta with transplantation of the P-region, but not of S6. The difference in the P-region, causing the change in Mg2+ binding kinetics, was attributed to ShB delta (V443) and its analog Kv3.1(L401), because in both channels leucine at this position gave fast block, whereas valine gave slow block. For Kv3.1 the major determinant of the voltage dependence of Mg2+ binding resided primarily in the off rate, whereas for Kv3.1(L401V) the voltage dependence resided primarily in the on rate, consistent with a change in the rate-limiting barrier for Mg2+ binding. Our data suggest that hydrophobic residues at positions 401 of Kv3.1 and 443 of ShB delta act as barriers to the movement of Mg2+ in the pore.
Harris
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Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Isacoff
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D055592:Biophysical Phenomena; D001703:Biophysics; D005260:Female; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D008564:Membrane Potentials; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D017354:Point Mutation; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051637:Shaker Superfamily of Potassium Channels; D051670:Shaw Potassium Channels; D014982:Xenopus laevis
D009479:Neuropeptides; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011993:Recombinant Fusion Proteins; D051637:Shaker Superfamily of Potassium Channels; D051670:Shaw Potassium Channels; D008274:Magnesium