Diverse Kir modulators act in close proximity to residues implicated in phosphoinositide binding

Abstract

Inwardly rectifying potassium (Kir) channels were the first shown to be directly activated by phosphoinositides in general and phosphatidylinositol bisphosphate (PIP(2)) in particular. Atomic resolution structures have been determined for several mammalian and bacterial Kir channels. Basic residues, identified through mutagenesis studies to contribute to the sensitivity of the channel to PIP(2), have been mapped onto the three dimensional channel structure and their localization has given rise to a plausible model that can explain channel activation by PIP(2). Moreover, mapping onto the three-dimensional channel structure sites involved in the modulation of Kir channel activity by a diverse group of regulatory molecules, revealed a striking proximity to residues implicated in phosphoinositide binding. These observations support the hypothesis that the observed dependence of diverse modulators on channel-PIP(2) interactions stems from their localization within distances that can affect PIP(2)-interacting residues.

Figure 1. Residues affecting PIP₂ sensitivity form a pocket that involves both the N- and C-termini of Kir channels

A, the known cytosolic structure of Kir3.1 with a modelled transmembrane domain based on KirBac3.1. The cyan-highlighted residues are conserved basic amino acids sensitive to PIP₂ as suggested by functional evidence tested on Kir2.1. B, surface Kir3.1 model with PIP₂ sites shown in cyan. Top view of the four (1–4) subunits of the Kir3.1 channel that face the membrane, without the transmembrane domain. The channel is represented as a transparent surface model to emphasize the important surface-accessible residues.

Figure 2. Gβγ induces stabilization of Kir3 channel-PIP₂ interactions (A) and the localization of Gβγ-interacting sites relative to PIP₂-sensitive residues (B, C)

A, PIP₂ antibody rapidly blocked Kir3.4* currents. Perfusion with DTT reversed this inhibition, presumably by reducing the disulphide bond of the antibody. Inside-out macropatches were bathed in a solution containing flouride; vanadate, (pyrophospate). The summary data (n = 3) depict T₅₀ (Time required for half maximal current inhibition by the PIP₂ antibody) values for the PIP₂ antibody-mediated block of Kir3.4* in the presence and absence of Gβγ. Following activation by Gβγ of the channels in the same patch, PIP₂ antibody blocked the K⁺ current with slower kinetics than before Gβγ treatment (P < 0.01). B, the known cytosolic structure of Kir3.1 with a modelled transmembrane domain based on KirBac3.1 showing putative Gβγ-interacting residues highlighted in red, in addition to the cyan-highlighted residues that affect channel–PIP₂ interactions. C, surface Kir3.1 model with PIP₂ and Gβγ sites shown from the same orientation as described in Fig. 1C. The colours match the colours used in Fig. 2B.

Figure 3. Na⁺-induced stabilization of Kir3 channel-PIP3 interactions (A) and the localization of a Na⁺-interacting Asp residue relative to PIP₂–sensitive residues (B, C)

A, Inside-out macropatches excised in a Na⁺-free Mg²⁺-containing solution were subjected to multiple, brief exposures to Na⁺ ions, which stimulated channel activity, the amplitude of which declined as a function of the time following excision. The dashed lines represent fits to the peak currents elicited by the brief applications of Na⁺ (denoted by asterisks). Kir3.4*(D223N) on the other hand failed to produce Na⁺-mediated stimulation of K⁺ currents. Following patch excision, Na⁺ application during the slow course of rundown not only failed to stimulate activity, but it also caused a small current inhibition (denoted by asterisks). The current rundown kinetics of the Kir3.4*(D223N) mutant were much slower than those of Kir3.4*. Moreover, no significant difference in rundown kinetics was seen in the presence (dashed line is the fit to the current troughs elicited by the brief exposures to Na⁺) or absence of Na⁺ ions. The summary data (n = 4) show the effect of Na⁺ ions on the current rundown kinetics for Kir3.4* (P < 0.005) and Kir3.4*(D223N). B, the known cytosolic structure of Kir3.1 with a modelled transmembrane domain based on KirBac3.1 showing the putative Na⁺ interacting residue, N217, highlighted in blue, in addition to the cyan-highlighted residues that affect channel–PIP₂ interactions. N217 is located at the equivalent position to the position of D223 in Kir3.4*. C, surface Kir3.1 model with PIP₂ and N217, the Na⁺-interacting residue, shown from the same orientation as described in Fig. 1C. The colours match the colours used in Fig. 3B.

Figure 4. Localization of phosphorylation sites of the Kir 3.1 channel relative to tis PIP₂–sensitive residues

A, the known cytosolic structure of Kir3.1 with a modelled transmembrane domain showing the phosphorylation sites suggested by functional evidence highlighted in orange, in addition to the cyan-highlighted residues that affect channel–PIP₂ interactions. B, surface Kir3.1 model with PIP₂ and the putative phosphorylation sites, shown from the same orientation as described in Fig. 1C. The colours match the colours used in Fig. 4A.

Figure 5. Localization of pH–sensitive residues relative to Kir1.1 PIP₂–sensitive residues

A, an homology model of Kir1.1 based on the cytosolic structure of Kir2.1 and the transmembrane domain of KirBac3.1. Sites highlighted in green show residues, mutation of which affects pH sensitivity, in addition to the cyan-highlighted residues that affect channel–PIP₂ interactions as determined experimentally (Lopes et al. 2002). B, surface Kir1.1 model with PIP₂ and the sites that affect pH sensitivity, shown from the same orientation as described in Fig. 1C. The colours match the colours used in Fig. 5A.