Epshtein Yulia, Chopra Arun P, Rosenhouse-Dantsker Avia, Kowalsky Gregory B, Logothetis Diomedes E, Levitan Irena
Department of Medicine, Pulmonary Section, University of Illinois, Chicago, IL 60612, USA.
Proc Natl Acad Sci U S A. 2009 May 12;106(19):8055-60. doi: 10.1073/pnas.0809847106. Epub 2009 Apr 29.
A variety of ion channels are regulated by cholesterol, a major lipid component of the plasma membrane whose excess is associated with multiple pathological conditions. However, the mechanism underlying cholesterol sensitivity of ion channels is unknown. We have recently shown that an increase in membrane cholesterol suppresses inwardly rectifying K(+) (Kir2) channels that are responsible for maintaining membrane potential in a variety of cell types. Here we show that cholesterol sensitivity of Kir2 channels depends on a specific region of the C terminus of the cytosolic domain of the channel, the CD loop. Within this loop, the L222I mutation in Kir2.1 abrogates the sensitivity of the channel to cholesterol whereas a reverse mutation in the corresponding position in Kir2.3, I214L, has the opposite effect, increasing cholesterol sensitivity. Furthermore, the L222I mutation has a dominant negative effect on cholesterol sensitivity of Kir2.1 WT. Mutations of 2 additional residues in the CD loop in Kir2.1, N216D and K219Q, partially affect the sensitivity of the channel to cholesterol. Yet, whereas these mutations have been shown to affect activation of the channel by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], other mutations outside the CD loop that have been previously shown to affect activation of the channel by PI(4,5)P(2) had no effect on cholesterol sensitivity. Mutations of the lipid-facing residues of the outer transmembrane helix also had no effect. These findings provide insights into the structural determinants of the sensitivity of Kir2 channels to cholesterol, and introduce the critical role of the cytosolic domain in cholesterol dependent channel regulation.
多种离子通道受胆固醇调节，胆固醇是质膜的主要脂质成分，其过量与多种病理状况相关。然而，离子通道胆固醇敏感性的潜在机制尚不清楚。我们最近发现，膜胆固醇增加会抑制内向整流钾通道（Kir2），该通道负责维持多种细胞类型的膜电位。在此我们表明，Kir2通道的胆固醇敏感性取决于通道胞质结构域C末端的一个特定区域，即CD环。在这个环内，Kir2.1中的L222I突变消除了通道对胆固醇的敏感性，而Kir2.3中相应位置的反向突变I214L则有相反的效果，增加了胆固醇敏感性。此外，L222I突变对Kir2.1野生型的胆固醇敏感性具有显性负效应。Kir2.1的CD环中另外两个残基N216D和K219Q的突变部分影响通道对胆固醇的敏感性。然而，尽管这些突变已被证明会影响通道被膜磷脂磷脂酰肌醇4,5 - 二磷酸[PI(4,5)P(2)]激活，但先前已证明影响通道被PI(4,5)P(2)激活的CD环外的其他突变对胆固醇敏感性没有影响。外跨膜螺旋面向脂质的残基的突变也没有影响。这些发现为Kir2通道对胆固醇敏感性的结构决定因素提供了见解，并揭示了胞质结构域在胆固醇依赖性通道调节中的关键作用。
