The conformational changes required for activation and K+ conduction in inward-rectifier K+ (Kir) channels are still debated. These structural changes are brought about by lipid binding. It is unclear how this process relates to fast gating or if the intracellular and extracellular regions of the protein are coupled. Here, we examine the structural details of KirBac1.1 reconstituted into both POPC and an activating lipid mixture of 3:2 POPC:POPG (wt/wt). KirBac1.1 is a prokaryotic Kir channel that shares homology with human Kir channels. We establish that KirBac1.1 is in a constitutively active state in POPC:POPG bilayers through the use of real-time fluorescence quenching assays and Förster resonance energy transfer (FRET) distance measurements. Multidimensional solid-state NMR (SSNMR) spectroscopy experiments reveal two different conformers within the transmembrane regions of the protein in this activating lipid environment, which are distinct from the conformation of the channel in POPC bilayers. The differences between these three distinct channel states highlight conformational changes associated with an open activation gate and suggest a unique allosteric pathway that ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, selectivity filter loop, and the pore helix. We also identify specific residues involved in this conformational exchange that are highly conserved among human Kir channels.

中文翻译：

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固态NMR和功能分析显示，将KirBac1.1门控到开放激活状态后，构象变化。

内向整流器K +（Kir）通道中激活和K +传导所需的构象变化仍然存在争议。这些结构变化是由脂质结合引起的。尚不清楚该过程与快速门控如何相关，或者蛋白质的细胞内和细胞外区域是否偶联。在这里，我们检查了重构为POPC和3：2 POPC：POPG（wt / wt）的活化脂质混合物的KirBac1.1的结构细节。KirBac1.1是原核Kir通道，与人类Kir通道具有同源性。我们通过使用实时荧光猝灭测定和福斯特共振能量转移（FRET）距离测量，确定了POPC：POPG双层中KirBac1.1处于组成型活性状态。多维固态NMR（SSNMR）光谱实验显示，在这种活化脂质环境中，蛋白的跨膜区域内有两个不同的构象异构体，与POPC双层通道的构象不同。这三个不同通道状态之间的差异突出显示了与打开的激活门相关的构象变化，并提出了一种独特的变构途径，该路径通过两个跨膜螺旋，转塔，选择性过滤器环和孔螺旋之间的相互作用将选择性过滤器与激活门联系起来。我们还确定了参与此构象交换的特定残基，这些残基在人类Kir通道之间高度保守。这与POPC双层中通道的构型不同。这三个不同通道状态之间的差异突出显示了与打开的激活门相关的构象变化，并提出了一种独特的变构途径，该路径通过两个跨膜螺旋，转塔，选择性过滤器环和孔螺旋之间的相互作用将选择性过滤器与激活门联系起来。我们还确定了参与此构象交换的特定残基，这些残基在人类Kir通道之间高度保守。这与POPC双层中通道的构型不同。这三个不同通道状态之间的差异突出显示了与打开的激活门相关的构象变化，并提出了一种独特的变构途径，该路径通过两个跨膜螺旋，转塔，选择性过滤器环和孔螺旋之间的相互作用将选择性过滤器与激活门联系起来。我们还确定了参与此构象交换的特定残基，这些残基在人类Kir通道之间高度保守。炮塔，选择性过滤器回路和孔隙螺旋。我们还确定了参与此构象交换的特定残基，这些残基在人类Kir通道之间高度保守。炮塔，选择性过滤器回路和孔隙螺旋。我们还确定了参与此构象交换的特定残基，这些残基在人类Kir通道之间高度保守。