Abstract
Inward rectifier K+ (Kir) channels are activated by phosphatidylinositol-(4,5)-bisphosphate (PIP2), but G protein-gated Kir (KG) channels further require either G protein βγ subunits (Gβγ) or intracellular Na+ for their activation. To reveal the mechanism(s) underlying this regulation, we compared the crystal structures of the cytoplasmic domain of KG channel subunit Kir3.2 obtained in the presence and the absence of Na+. The Na+-free Kir3.2, but not the Na+-plus Kir3.2, possessed an ionic bond connecting the N terminus and the CD loop of the C terminus. Functional analyses revealed that the ionic bond between His-69 on the N terminus and Asp-228 on the CD loop, which are known to be critically involved in Gβγ- and Na+-dependent activation, lowered PIP2 sensitivity. The conservation of these residues within the KG channel family indicates that the ionic bond is a character that maintains the channels in a closed state by controlling the PIP2 sensitivity.
Highlights
Role of Ionic Bond between N Terminus and CD Loop in Conformational Changes in Cytoplasmic Domain of Kir3.2—In crystal structures of full-length Kir channels (4 – 6, 17), the channels are presumed to be in the closed state, with the physical constraints for ion permeation in the transmembrane the Naϩ-plus Kir3.2 was found at the position equivalent to domain
The conformation of the cytoplasmic domain in these His-68 in the Naϩ-free structure (Fig. 3B). It could complete structures is comparable with those of isolated cytobe His-69 that geometrically made an ionic bond with Asp-228. plasmic domains lacking the transmembrane domain (18 –20)
The basal current amplitude associated with PIP2 of the mutant it could be speculated that, in the closed state, the attachKir3.2 which substituted a glutamine for His-69 (H69Q) was ment of the transmembrane domain had little effect upon the high (30.4 Ϯ 4.8%, n ϭ 9)
Summary
Crystallization, Data Collection, and Model Building of Cytoplasmic Domain of Kir3.2—The longest isoform of mouse Kir3.2 (Kir3.2c) is made up of 425 amino acids with two more amino acids on the N terminus compared with human Kir3.2. His-69 and Asp-228 in mouse Kir3.2 correspond to His-67 and Asp-226 in human Kir3.2, respectively (27). The concatemer of cytoplasmic N and C termini of Kir3.2 (amino residues 53–74 and 200 –381, respectively) was expressed in bacteria and purified without the addition of Naϩ as described previously (20). Crystals were grown by vapor diffusion at 4 °C by mixing equal volumes of protein solution (5 mg/ml) and reservoir solution of 7.5–12.5% (v/v) ethanol, 0.1 M imidazole
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