Increased GABAA receptor open probability: Adaptive mechanisms to cope with anoxia in the painted turtle.

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 GABAA receptor. Whole-cell GABAA receptor currents are known to increase with the onset of anoxia because of increased presynaptic GABA release, we hypothesized that GABAA 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 GABAA receptor open times (Popen) during a normoxic to anoxic transition in pyramidal neurons in turtle brain cortical sheets. GABAA receptor Popen 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 GABAA 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.