TY - JOUR
T1 - Molecular determinants of cardiac KATP channel activation by epoxyeicosatrienoic acids
AU - Lu, Tong
AU - Hong, Min Pyo
AU - Lee, Hon Chi
PY - 2005/5/13
Y1 - 2005/5/13
N2 - We have previously reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolites of arachidonic acid, are potent stereospecific activators of the cardiac KATP channel. The epoxide group in EET is critical for reducing channel sensitivity to ATP, thereby activating the channel. This study is to identify the molecular sites on the KATP channels for EET-mediated activation. We investigated the effects of EETs on Kir6.2ΔC26 with or without the coexpression of SUR2A and on Kir6.2 mutants of positively charged residues known to affect channel activity coexpressed with SUR2A in HEK293 cells. The ATP IC50 values were significantly increased in Kir6.2 R27A, R50A, K185A, and R201A but not in R16A, K47A, R54A, E67A, R192A, R195A, K207A, K222A, and R314A mutants. Similar to native cardiac KATP channel, 5 μM 11,12-EET increased the ATP IC 50 by 9.6-fold in Kir6.2/SUR2A wild type and 8.4-fold in Kir6.2ΔC26. 8,9- and 14,15-EET regioisomers activated the Kir6.2 channel as potently as 11,12-EET. 8,9- and 11,12-EET failed to change the ATP sensitivity of Kir6.2 K185A, R195A, and R201A, whereas their effects were intact in the other mutants. 14,15-EET had a similar effect with K185A and R201A mutants, but instead of R195A, it failed to activate Kir6.2R192A. These results indicate that activation of Kir6.2 by EETs does not require the SUR2A subunit, and the region in the Kir6.2 C terminus from Lys-185 to Arg-201 plays a critical role in EET-mediated Kir6.2 channel activation. Based on computer modeling of the Kir6.2 structure, we infer that the EET-Kir6.2 interaction may allosterically change the ATP binding site on Kir6.2, reducing the channel sensitivity to ATP.
AB - We have previously reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolites of arachidonic acid, are potent stereospecific activators of the cardiac KATP channel. The epoxide group in EET is critical for reducing channel sensitivity to ATP, thereby activating the channel. This study is to identify the molecular sites on the KATP channels for EET-mediated activation. We investigated the effects of EETs on Kir6.2ΔC26 with or without the coexpression of SUR2A and on Kir6.2 mutants of positively charged residues known to affect channel activity coexpressed with SUR2A in HEK293 cells. The ATP IC50 values were significantly increased in Kir6.2 R27A, R50A, K185A, and R201A but not in R16A, K47A, R54A, E67A, R192A, R195A, K207A, K222A, and R314A mutants. Similar to native cardiac KATP channel, 5 μM 11,12-EET increased the ATP IC 50 by 9.6-fold in Kir6.2/SUR2A wild type and 8.4-fold in Kir6.2ΔC26. 8,9- and 14,15-EET regioisomers activated the Kir6.2 channel as potently as 11,12-EET. 8,9- and 11,12-EET failed to change the ATP sensitivity of Kir6.2 K185A, R195A, and R201A, whereas their effects were intact in the other mutants. 14,15-EET had a similar effect with K185A and R201A mutants, but instead of R195A, it failed to activate Kir6.2R192A. These results indicate that activation of Kir6.2 by EETs does not require the SUR2A subunit, and the region in the Kir6.2 C terminus from Lys-185 to Arg-201 plays a critical role in EET-mediated Kir6.2 channel activation. Based on computer modeling of the Kir6.2 structure, we infer that the EET-Kir6.2 interaction may allosterically change the ATP binding site on Kir6.2, reducing the channel sensitivity to ATP.
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U2 - 10.1074/jbc.M414065200
DO - 10.1074/jbc.M414065200
M3 - Article
C2 - 15760904
AN - SCOPUS:21444460965
SN - 0021-9258
VL - 280
SP - 19097
EP - 19104
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 19
ER -