Henze Erik, Ehrlich Jacqueline J, Robertson Janice L, Gelsleichter Eric, Kawate Toshimitsu
Department of Molecular Medicine, Cornell University, Ithaca, NY 14853.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.
Proc Natl Acad Sci U S A. 2024 Dec 17;121(51):e2411898121. doi: 10.1073/pnas.2411898121. Epub 2024 Dec 13.
Pannexin 1 (Panx1) constitutes a large pore channel responsible for the release of adenosine triphosphate (ATP) from apoptotic cells. Strong evidence indicates that caspase-mediated cleavage of the C-terminus promotes the opening of the Panx1 channel by unplugging the pore. However, this simple pore-plugging mechanism alone cannot account for the observation that a Panx1 construct ending before the caspase cleavage site remains closed. Here, we show that a helical region located immediately before the caspase cleavage site, referred to as the "C-terminal activating domain (CAD)", plays a pivotal role in facilitating Panx1 activation. Electrophysiology and mutagenesis studies uncovered that two conserved leucine residues within the CAD play a pivotal role. Cryoelectron microscopy (Cryo-EM) analysis of the construct ending before reaching the CAD demonstrated that the N terminus extends into an intracellular pocket. In contrast, the construct including the CAD revealed that this domain occupies the intracellular pocket, causing the N terminus to flip upward within the pore. Analysis of electrostatic free energy landscape in the closed conformation indicated that the intracellular side of the ion permeation pore may be occupied by anions like ATP, creating an electrostatic barrier for anions attempting to permeate the pore. When the N terminus flips up, it diminishes the positively charged surface, thereby reducing the drive to accumulate anions inside the pore. This dynamic change in the electrostatic landscape likely contributes to the selection of permeant ions. Collectively, these experiments put forth a mechanism in which C-terminal cleavage liberates the CAD, causing the repositioning of the N terminus to promote Panx1 channel opening.
泛连接蛋白1（Panx1）构成一个大孔通道，负责将三磷酸腺苷（ATP）从凋亡细胞中释放出来。有力证据表明，半胱天冬酶介导的C末端切割通过疏通孔道来促进Panx1通道的开放。然而，仅这种简单的孔道堵塞机制无法解释以下观察结果：在半胱天冬酶切割位点之前结束的Panx1构建体仍然关闭。在这里，我们表明，位于半胱天冬酶切割位点之前紧邻的一个螺旋区域，称为“C末端激活域（CAD）”，在促进Panx1激活中起关键作用。电生理学和诱变研究发现，CAD内的两个保守亮氨酸残基起关键作用。对在到达CAD之前结束的构建体进行的冷冻电子显微镜（Cryo-EM）分析表明，N末端延伸到一个细胞内口袋中。相比之下，包含CAD的构建体显示该结构域占据了细胞内口袋，导致N末端在孔道内向上翻转。对封闭构象中的静电自由能景观分析表明，离子渗透孔的细胞内侧可能被ATP等阴离子占据，为试图渗透孔道的阴离子形成一个静电屏障。当N末端向上翻转时，它减少了带正电的表面，从而降低了在孔道内积累阴离子的驱动力。这种静电景观的动态变化可能有助于选择渗透离子。总的来说，这些实验提出了一种机制，即C末端切割释放CAD，导致N末端重新定位以促进Panx1通道开放。
