科研之友
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摘要
The atrial G protein (heterotrimeric guanine nucleotide-binding protein)-regulated inwardly rectifying K+ (GIRK1 and GIRK4) heterotetrameric channels underlie the acetylcholine-induced K+ current responsible for vagal inhibition of heart rate and are activated by the G protein beta gamma subunits (G beta gamma). We used a multistage protein-protein docking approach with data from published structures of GIRK1 and G beta gamma to generate an experimentally testable interaction model of G beta gamma docked onto the cytosolic domains of the GIRK1 homotetramer. The model suggested a mechanism by which G beta gamma promotes the open state of a specific cytosolic gate in the channel, the G loop gate. The predicted structure showed that the G beta subunit interacts with the channel near the site of action for ethanol and stabilizes an intersubunit cleft formed by two loops (LM and DE) of adjacent channel subunits. Using a heterologous expression system, we disrupted the predicted GIRK1- and G beta gamma-interacting residues by mutation of one protein and then rescued the regulatory activity by mutating reciprocal residues in the other protein. Disulfide cross-linking of channels and G beta gamma with cysteine mutations at the predicted interacting residues yielded activated channels. The mechanism of G beta gamma-induced activation of GIRK4 was distinct from GIRK1 homotetramers. However, GIRK1-GIRK4 heterotetrameric channels activated by G beta gamma displayed responses indicating that the GIRK1 subunit dominated the response pattern. This work demonstrated that combining computational with experimental approaches is an effective method for elucidating interactions within protein complexes that otherwise might be challenging to decipher.
- 出版日期2013-8-13
