Xie Lai-Hua, John Scott A, Ribalet Bernard, Weiss James N
Cardiovascular Research Laboratory, Rm 3645 MRL Building, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
J Physiol. 2008 Apr 1;586(7):1833-48. doi: 10.1113/jphysiol.2007.147868. Epub 2008 Feb 14.
Inwardly rectifying potassium (Kir) channels are gated by the interaction of their cytoplasmic regions with membrane-bound phosphatidylinositol-4,5-bisphosphate (PIP(2)). In the present study, we examined how PIP(2) interaction regulates channel availability and channel openings to various subconductance levels (sublevels) as well as the fully open state in the strong inward rectifier Kir2.1 channel. Various Kir2.1 channel constructs were expressed in Xenopus oocytes and single channel or macroscopic currents were recorded from inside-out patches. The wild-type (WT) channel rarely visited the subconductance levels under control conditions. However, upon reducing Kir2.1 channel interaction with PIP(2) by a variety of interventions, including PIP(2) antibodies, screening PIP(2) with neomycin, or mutating PIP(2) binding sites (e.g. K188Q), visitation to the sublevels was markedly increased before channels were converted to an unavailable mode in which they did not open. No channel activity was detected in channels with the double mutation K188A/R189A, a mutant which exhibits extremely weak interaction with PIP(2). By linking subunits together in tandem dimers or tetramers containing mixtures of WT and K188A/R189A subunits, we demonstrate that one functional PIP(2)-interacting WT subunit is sufficient to convert channels from the unavailable to the available mode with a high open probability dominated by the fully open state, with similar kinetics as tetrameric WT channels. Occasional openings to sublevels become progressively less frequent as the number of WT subunits increases. Quantitative analysis reveals that the interaction of PIP(2) with WT subunits exerts strong positive cooperativity in both converting the channels from the unavailable to the available mode, and in promoting the fully open state over sublevels. We conclude that the interaction of PIP(2) with only one Kir2.1 subunit is sufficient for the channel to become available and to open to its full conductance state. Interaction with additional subunits exerts positive cooperativity at multiple levels to further enhance channel availability and promote the fully open state.
内向整流钾（Kir）通道通过其胞质区域与膜结合的磷脂酰肌醇-4,5-二磷酸（PIP₂）的相互作用而门控。在本研究中，我们研究了PIP₂相互作用如何调节通道的可用性、通道向各种亚电导水平（亚水平）以及强内向整流Kir2.1通道的完全开放状态的开放情况。各种Kir2.1通道构建体在非洲爪蟾卵母细胞中表达，并从内向外膜片记录单通道或宏观电流。野生型（WT）通道在对照条件下很少达到亚电导水平。然而，通过多种干预措施减少Kir2.1通道与PIP₂的相互作用，包括使用PIP₂抗体、用新霉素筛选PIP₂或突变PIP₂结合位点（如K188Q），在通道转变为不可用模式（即不开放）之前，对亚水平的访问显著增加。在具有双突变K188A/R189A的通道中未检测到通道活性，该突变体与PIP₂的相互作用极其微弱。通过将亚基以串联二聚体或包含WT和K188A/R189A亚基混合物的四聚体形式连接在一起，我们证明一个功能性的与PIP₂相互作用的WT亚基足以将通道从不可用模式转变为可用模式，并以完全开放状态为主导的高开放概率打开，其动力学与四聚体WT通道相似。随着WT亚基数量的增加，偶尔向亚水平的开放逐渐变得不那么频繁。定量分析表明，PIP₂与WT亚基的相互作用在将通道从不可用模式转变为可用模式以及促进完全开放状态超过亚水平方面都发挥了强烈的正协同作用。我们得出结论，PIP₂仅与一个Kir2.1亚基的相互作用就足以使通道变得可用并开放至其完全电导状态。与其他亚基的相互作用在多个水平上发挥正协同作用，以进一步提高通道可用性并促进完全开放状态。
