title: Modulation of Kir1.1 inactivation by extracellular Ca and Mg.
abstract: Kir1.1 inactivation, associated with transient internal acidification, is strongly dependent on external K, Ca, and Mg. Here, we show that in 1 mM K, a 15 min internal acidification (pH 6.3) followed by a 30 min recovery (pH 8.0) produced 84 ± 3% inactivation in 2 mM Ca but only 18 ± 4% inactivation in the absence of external Ca and Mg. In 100 mM external K, the same acidification protocol produced 29 ± 4% inactivation in 10 mM external Ca but no inactivation when extracellular Ca was reduced below 2 mM (with 0 Mg). However, chelation of external K with 15 mM of 18-Crown-6 (a crown ether) restored inactivation even in the absence of external divalents. External Ca was more effective than external Mg at producing inactivation, but Mg caused a greater degree of open channel block than Ca, making it unlikely that Kir1.1 inactivation arises from divalent block per se. Because the Ca sensitivity of inactivation persisted in 100 mM external K, it is also unlikely that Ca enhanced Kir1.1 inactivation by reducing the local K concentration at the outer mouth of the channel. The removal of four surface, negative side chains at E92, D97, E104, and E132 (Kir1.1b) increased the sensitivity of inactivation to external Ca (and Mg), whereas addition of a negative surface charge (N105E-Kir1.1b) decreased the sensitivity of inactivation to Ca and Mg. This result is consistent with the notion that negative surface charges stabilize external K in the selectivity filter or at the S(0)-K binding site just outside the filter. Extracellular Ca and Mg probably potentiate the slow, K-dependent inactivation of Kir1.1 by decreasing the affinity of the channel for external K independently of divalent block. The removal of external Ca and Mg largely eliminated both Kir1.1 inactivation and the K-dependence of pH gating, thereby uncoupling the selectivity filter gate from the cytoplasmic-side bundle-crossing gate.
authors.affiliation: Department of Physiology and Biophysics, Chicago Medical School, Rosalind Franklin University, North Chicago, Illinois, USA. henry.sackin@rosalindfranklin.edu
authors.forename: Henry
authors.initials: H
authors.lastname: Sackin
authors.forename: Mikheil
authors.initials: M
authors.lastname: Nanazashvili
authors.forename: Hui
authors.initials: H
authors.lastname: Li
authors.forename: Lawrence G
authors.initials: LG
authors.lastname: Palmer
authors.forename: Lei
authors.initials: L
authors.lastname: Yang
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D005110:Extracellular Space / Q000378:metabolism*; D005260:Female; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000187:drug effects*; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D008958:Models, Molecular; D017354:Point Mutation; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D055672:Static Electricity; D013499:Surface Properties
chemical_list: D024661:Potassium Channels, Inwardly Rectifying; D008274:Magnesium; D011188:Potassium; D002118:Calcium