Abstract
Activation of substance P receptors, which are coupled to Gαq, inhibits the Kir3.1/3.2 channels, resulting in neuronal excitation. We have shown previously that this channel inactivation is not caused by reduction of the phosphatidylinositol 4,5-bisphosphate level in membrane. Moreover, Gαq immunoprecipitates with Kir3.2 (J Physiol564:489–500, 2005), suggesting that Gαq interacts with Kir3.2. Positive immunoprecipitation, however, does not necessarily indicate direct interaction between the two proteins. Here, the glutathione transferase pull-down assay was used to investigate interaction between Gαq and the K+ channels. We found that Gαq interacted with N termini of Kir3.1, Kir3.2, and Kir3.4. However, Gαq did not interact with the C terminus of any Kir3 or with the C or N terminus of Kir2.1. TRPC6 is regulated by the signal initiated by Gαq. Immunoprecipitation, however, showed that Gαq did not interact with TRPC6. Thus, the interaction between Gαq and the Kir3 N terminus is quite specific. This interaction occurred in the presence of GDP or GDP-AlF–4. The Gαq binding could take place somewhere between residues 51 to 90 of Kir3.2; perhaps the segment between 81 to 90 residues is crucial. Gβγ, which is known to bind to N terminus of Kir3, did not compete with Gαq for the binding, suggesting that these two binding regions are different. These findings agree with the hypothesis (J Physiol564:489–500, 2005) that the signal to inactivate the Kir3 channel could be mainly transmitted directly from Gαq to Kir3.
Footnotes
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This work was supported by National Institutes of Health grants AG06093, GM59427, NS41441, GM61454, and T32-HL07692 T.Ko. and S.N. equally contributed to this article.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.032508.
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ABBREVIATIONS: SP, substance P; GIRK, G protein-coupled inwardly rectifying K+ channels; GST, glutathione transferase; HEK, human embryonic kidney; PIP2, phosphatidylinositol 4,5-bisphosphate; RGS, regulator of G protein signaling; GRK2-RH, RGS homology domain of G protein-coupled receptor kinase 2.
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↵1 Current affiliation: Department of Rehabilitation Medicine, Montefiore Medical Center, The University Hospital for the Albert Einstein College of Medicine, Bronx, New York.
- Received November 8, 2006.
- Accepted February 12, 2007.
- The American Society for Pharmacology and Experimental Therapeutics