Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.
Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA; Center for Public Health Genomics, Department of Biomedical Engineering, School of Engineering &Applied Science, University of Virginia, Charlottesville, VA 22904, USA.
J Struct Biol. 2024 Jun;216(2):108091. doi: 10.1016/j.jsb.2024.108091. Epub 2024 Apr 17.
Cholesterol is a negative regulator of a variety of ion channels. We have previously shown that cholesterol suppresses Kir2.2 channels via residue-residue uncoupling on the inter-subunit interfaces within the close state of the channels (3JYC). In this study, we extend this analysis to the other known structure of Kir2.2 that is closer to the open state of Kir2.2 channels (3SPI) and provide additional analysis of the residue distances between the uncoupled residues and cholesterol binding domains in the two conformation states of the channels. We found that the general phenomenon of cholesterol binding leading to uncoupling between specific residues is conserved in both channel states but the specific pattern of the uncoupling residues is distinct between the two states and implies different mechanisms. Specifically, we found that cholesterol binding in the 3SPI state results in an uncoupling of residues in three distinct regions; the transmembrane domain, membrane-cytosolic interface, and the cytosolic domain, with the first two regions forming an envelope around PI(4,5)P2 and cholesterol binding sites and the distal region overlapping with the subunit-subunit interface characterized in our previous study of the disengaged state. We also found that this uncoupling is dependent upon the number of cholesterol molecules bound to the channel. We further generated a mutant channel Kir2.2 with a single point mutation in a residue proximal to the PI(4,5)P2 binding site, which is predicted to be uncoupled from other residues in its vicinity upon cholesterol binding and found that this mutation abrogates the sensitivity of Kir2.2 to cholesterol changes in the membrane. These findings suggest that cholesterol binding to this conformation state of Kir2.2 channels may destabilize the PI(4,5)P2 interactions with the channels while in the disengaged state the destabilization occurs where the subunits interact. These findings give insight into the structural mechanistic basis for the functional effects of cholesterol binding to the Kir2.2 channel.
胆固醇是多种离子通道的负调节剂。我们之前已经表明，胆固醇通过通道紧密状态下的亚基间界面上的残基解偶联来抑制 Kir2.2 通道（3JYC）。在这项研究中，我们将这种分析扩展到 Kir2.2 的另一个已知结构，该结构更接近 Kir2.2 通道的开放状态（3SPI），并对通道两种构象状态下解偶联残基与胆固醇结合域之间的残基距离进行了额外分析。我们发现，胆固醇结合导致特定残基之间解偶联的一般现象在两种通道状态下都是保守的，但在两种状态下，解偶联残基的特定模式是不同的，这意味着不同的机制。具体来说，我们发现 3SPI 状态下的胆固醇结合导致三个不同区域的残基解偶联；跨膜域、膜胞质界面和胞质域，前两个区域形成一个围绕 PI(4,5)P2 和胆固醇结合位点的包膜，而远端区域与我们之前研究的脱离状态下的亚基间界面重叠。我们还发现，这种解偶联依赖于结合到通道上的胆固醇分子的数量。我们进一步生成了一个突变通道 Kir2.2，其在靠近 PI(4,5)P2 结合位点的残基上有一个单点突变，该突变预测在胆固醇结合时与其附近的其他残基解偶联，并且发现该突变消除了 Kir2.2 对膜中胆固醇变化的敏感性。这些发现表明，胆固醇结合到 Kir2.2 通道的这种构象状态可能会使 PI(4,5)P2 与通道的相互作用不稳定，而在脱离状态下，亚基相互作用会导致不稳定。这些发现为胆固醇结合到 Kir2.2 通道的功能效应的结构机制基础提供了深入了解。
