Critical protein domains and amino acid residues for gating the KIR6.2 channel by intracellular ATP.
K(ATP) channels couple intermediary metabolism to cellular excitability. Such a property relies on the inherent ATP-sensing mechanism known to be located in the Kir6 subunit. However, the molecular basis for the ATP sensitivity remains unclear. Here we showed evidence for protein domains and amino acid residues essential for the channel gating by intracellular ATP. Chimerical channels were constructed using protein domains of Kir6.2 and Kir1.1, expressed in HEK293 cells, and studied in inside-out patches. The N and C termini, although important, were inadequate for channel gating by intracellular ATP. Full ATP sensitivity also required M1 and M2 helices. Cytosolic portions of the M1 and M2 sequences were crucial, in which six amino acid residues were identified, i.e., Thr76, Met77, Ala161, Iso162, Leu164, and Cys166. Site-specific mutation of any of them reduced the ATP sensitivity. Construction of these residues together with the N/C termini produced ATP sensitivity identical to the wild-type channels. The requirement for specific membrane helices suggests that the Kir6.2 gating by ATP is not shared by even two closest relatives in the K(+) channel family, although the general gating mechanisms involving membrane helices appear to be conserved in all K(+) channels.
Department of Biology, Georgia State University, 24 Peachtree Center Avenue, Atlanta, Georgia 30302, USA.
Jianping
J
Wu
Hailan
H
Piao
Asheebo
A
Rojas
Runping
R
Wang
Ying
Y
Wang
Ningren
N
Cui
Yun
Y
Shi
Fuxue
F
Chen
Chun
C
Jiang
D000255:Adenosine Triphosphate / Q000378:metabolism*; D020816:Amino Acid Motifs; D002460:Cell Line; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
C086989:KCNJ1 protein, human; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate