title: Identification of the Intracellular Na+ Sensor in Slo2.1 Potassium Channels.
abstract: Slo2 potassium channels have a very low open probability under normal physiological conditions, but are readily activated in response to an elevated [Na(+)]i (e.g. during ischemia). An intracellular Na(+) coordination motif (DX(R/K)XXH) was previously identified in Kir3.2, Kir3.4, Kir5.1, and Slo2.2 channel subunits. Based loosely on this sequence, we identified five potential Na(+) coordination motifs in the C terminus of the Slo2.1 subunit. The Asp residue in each sequence was substituted with Arg, and single mutant channels were heterologously expressed in Xenopus oocytes. The Na(+) sensitivity of each of the mutant channels was assessed by voltage clamp of oocytes using micropipettes filled with 2 M NaCl. Wild-type channels and four of the mutant Slo2.1 channels were rapidly activated by leakage of NaCl solution into the cytoplasm. D757R Slo2.1 channels were not activated by NaCl, but were activated by the fenamate niflumic acid, confirming their functional expression. In whole cell voltage clamp recordings of HEK293 cells, wild-type but not D757R Slo2.1 channels were activated by a [NaCl]i of 70 mM. Thus, a single Asp residue can account for the sensitivity of Slo2.1 channels to intracellular Na(+). In excised inside-out macropatches of HEK293 cells, activation of wild-type Slo2.1 currents by 3 mM niflumic acid was 14-fold greater than activation achieved by increasing [NaCl]i from 3 to 100 mM. Thus, relative to fenamates, intracellular Na(+) is a poor activator of Slo2.1.
authors.affiliation: From the Nora Eccles Harrison Cardiovascular Research and Training Institute and.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Thomson
authors.affiliation: From the Nora Eccles Harrison Cardiovascular Research and Training Institute and.
authors.forename: Angela
authors.initials: A
authors.lastname: Hansen
authors.affiliation: From the Nora Eccles Harrison Cardiovascular Research and Training Institute and Department of Internal Medicine, Division of Cardiovascular Medicine, University of Utah, Salt Lake City, Utah 84112 sanguinetti@cvrti.utah.edu.
authors.forename: Michael C
authors.identifier: http://orcid.org/0000-0001-8974-0969
authors.initials: MC
authors.lastname: Sanguinetti
mesh_terms: D000818:Animals; D001665:Binding Sites; D057809:HEK293 Cells; D006801:Humans; D009544:Niflumic Acid / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000081033:Potassium Channels, Sodium-Activated; D012965:Sodium Chloride / Q000378:metabolism*; D014981:Xenopus
chemical_list: C480494:KCNT2 protein, human; D015221:Potassium Channels; D000081033:Potassium Channels, Sodium-Activated; D012965:Sodium Chloride; D009544:Niflumic Acid
keywords: Slick; Slo2.1; Xenopus; biophysics; gating; patch clamp; potassium channel