Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca²⁺/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation—measured by changes in DAPI uptake rate—was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (∼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca²⁺ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.

Pannexin1 (Panx1) 通道在脊椎动物细胞中普遍表达，并被广泛接受为三磷酸腺苷 (ATP) 释放膜通道。Panx1 的激活与特定酪氨酸残基的磷酸化或其 C 末端结构域的切割有关。在目前的工作中，我们确定了一个残基 (S394) 作为 Ca ²⁺的推定磷酸化位点/钙调蛋白依赖性激酶 II (CaMKII)。在用大鼠 Panx1 (rPanx1) 转染的 HeLa 细胞中，膜拉伸 (MS) 诱导的激活（通过 DAPI 摄取率的变化来衡量）通过 Piezo1 的敲低或钙调蛋白或 CaMKII 的药理学抑制显着降低。通过定点诱变，我们生成了对 MS 失去敏感性的 rPanx1S394A-EGFP（增强型绿色荧光蛋白）和模拟磷酸化的 rPanx1S394D-EGFP，在没有 MS 刺激或 C 末端切割的情况下显示出高 DAPI 摄取率。使用全细胞膜片钳和外切贴片配置，我们发现 rPanx1-EGFP 和 rPanx1S394D-EGFP 通道在 ±100 mV 之间的所有电压下均显示电流，具有外向整流的类似单通道电流和单一电导（~30至 70 pS）。然而，使用细胞连接配置，我们发现 rPanx1S394D-EGFP 通道显示增加的自发单一事件，独立于 MS 刺激。计算机研究表明，S394 的磷酸化导致选择性过滤器的构象变化并增加横向隧道的平均体积，从而使 ATP 通过这些管道释放，DAPI 直接从通道口吸收到细胞质空间。这些结果可以解释在细胞质 Ca 增加时激活 rPanx1 的一种可能机制 允许 ATP 通过这些管道释放，DAPI 直接从通道口吸收到细胞质空间。这些结果可以解释在细胞质 Ca 增加时激活 rPanx1 的一种可能机制 允许 ATP 通过这些管道释放，DAPI 直接从通道口吸收到细胞质空间。这些结果可以解释在细胞质 Ca 增加时激活 rPanx1 的一种可能机制²⁺信号由 C 末端结构域未切割的各种生理条件引发。