Suganthan Haushe, Le Han, Elbassiouny Ahmed, Rajkumar Anthony, Raveendran Vineeth A, Pressey Jessica C, Woodin Melanie A, Chang Belinda S W, Buck Leslie T
Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.
Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.
Neuroscience. 2025 Jul 23;579:10-23. doi: 10.1016/j.neuroscience.2025.05.032. Epub 2025 May 22.
The western painted turtle is the most anoxia-tolerant tetrapod known, surviving ∼ 4 months at 3 °C without oxygen. In the mammalian brain, absence of oxygen leads to hyper-excitability and cell death within minutes. A major mechanism by which painted turtles survive anoxia is a large increase of γ-aminobutyric acid (GABA) in the brain leading to a dominating Cl conductance that clamps membrane potential near the reversal potential of the GABA receptor. Whole-cell GABA receptor currents are known to increase with the onset of anoxia because of increased presynaptic GABA release, we hypothesized that GABA receptor currents may also exhibit a large increase due to increased channel open time. To investigate this, we used cell-attached single-channel patch-clamp electrophysiological techniques to measure GABA receptor open times (P) during a normoxic to anoxic transition in pyramidal neurons in turtle brain cortical sheets. GABA receptor P significantly increased 13-fold with the onset of anoxia and was blocked by the inclusion of the protein kinase C (PKC) activator PMA phorbol-12-myristate-13-acetate. Indicating the receptor was regulated by covalent modification. To investigate the molecular evolutionary mechanisms underlying these adaptations, we used codon-based likelihood models to detect changes in selective pressure amongst the GABA receptor subunit genes. We found positive selection in GABRB2 and GABRB3 at sites near their ligand binding interface, likely impacting channel kinetics associated with hypoxia-tolerance. The elucidation of the adaptations associated with increased hypoxia tolerance furthers our understanding of physiological adaptations to extreme low-oxygen environments.
西部锦龟是已知最耐缺氧的四足动物，在3摄氏度无氧条件下可存活约4个月。在哺乳动物大脑中，缺氧会在几分钟内导致过度兴奋和细胞死亡。锦龟在缺氧状态下存活的一个主要机制是大脑中γ-氨基丁酸（GABA）大量增加，导致Cl电导占主导地位，从而将膜电位钳制在GABA受体的反转电位附近。已知全细胞GABA受体电流会随着缺氧的开始而增加，这是由于突触前GABA释放增加，我们推测GABA受体电流也可能因通道开放时间增加而大幅增加。为了研究这一点，我们使用细胞贴附式单通道膜片钳电生理技术来测量龟脑皮层切片中锥体神经元在常氧到缺氧转变过程中GABA受体的开放时间（P）。随着缺氧的开始，GABA受体P显著增加了13倍，并被蛋白激酶C（PKC）激活剂佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）所阻断。这表明该受体受共价修饰调节。为了研究这些适应性背后的分子进化机制，我们使用基于密码子的似然模型来检测GABA受体亚基基因之间选择性压力的变化。我们发现在GABRB2和GABRB3靠近其配体结合界面的位点存在正选择，这可能影响与耐缺氧相关的通道动力学。对与耐缺氧能力增强相关的适应性的阐明进一步加深了我们对极端低氧环境生理适应性的理解。
