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