Stabilization of the activity of ATP-sensitive potassium channels by ion pairs formed between adjacent Kir6.2 subunits.
ATP-sensitive potassium (KATP) channels are formed by the coassembly of four Kir6.2 subunits and four sulfonylurea receptor subunits (SUR). The cytoplasmic domains of Kir6.2 mediate channel gating by ATP, which closes the channel, and membrane phosphoinositides, which stabilize the open channel. Little is known, however, about the tertiary or quaternary structures of the domains that are responsible for these interactions. Here, we report that an ion pair between glutamate 229 and arginine 314 in the intracellular COOH terminus of Kir6.2 is critical for maintaining channel activity. Mutation of either residue to alanine induces inactivation, whereas charge reversal at positions 229 and 314 (E229R/R314E) abolishes inactivation and restores the wild-type channel phenotype. The close proximity of these two residues is demonstrated by disulfide bond formation between cysteine residues introduced at the two positions (E229C/R314C); disulfide bond formation abolishes inactivation and stabilizes the current. Using Kir6.2 tandem dimer constructs, we provide evidence that the ion pair likely forms by residues from two adjacent Kir6.2 subunits. We propose that the E229/R314 intersubunit ion pairs may contribute to a structural framework that facilitates the ability of other positively charged residues to interact with membrane phosphoinositides. Glutamate and arginine residues are found at homologous positions in many inward rectifier subunits, including the G-protein-activated inwardly rectifying potassium channel (GIRK), whose cytoplasmic domain structure has recently been solved. In the GIRK structure, the E229- and R314-corresponding residues are oriented in opposite directions in a single subunit such that in the tetramer model, the E229 equivalent residue from one subunit is in close proximity of the R314 equivalent residue from the adjacent subunit. The structure lends support to our findings in Kir6.2, and raises the possibility that a homologous ion pair may be involved in the gating of GIRKs.
Lin
Yu-Wen
YW
Center for Research on Occupational and Environmental Toxicology, Oregon Health & Science University, Portland, OR 97239, USA.
Jia
Taiping
T
Weinsoft
Anne M
AM
Shyng
Show-Ling
SL
D000255:Adenosine Triphosphate / Q000502:physiology; D000409:Alanine / Q000737:chemistry; D019943:Amino Acid Substitution; D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D019281:Dimerization; D018698:Glutamic Acid / Q000737:chemistry; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D051379:Mice; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D017354:Point Mutation; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D018129:Phosphatidylinositol Phosphates; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits; C060974:phosphatidylinositol 3,4,5-triphosphate; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D000409:Alanine