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In response to low ambient oxygen levels the western painted turtle brain undergoes a large depression in metabolic rate which includes a decrease in neuronal action potential frequency. This involves the arrest of N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) currents and paradoxically an increase in γ-aminobutyric acid receptor (GABAR) currents in turtle cortical neurons. In a search for other oxygen-sensitive channels we discovered a Ca(2+)-activated K(+) channel (K(Ca)) that exhibited a decrease in open time in response to anoxia. Single-channel recordings of K(Ca) activity were obtained in cell-attached and excised inside-out patch configurations from neurons in cortical brain sheets bathed in either normoxic or anoxic artificial cerebrospinal fluid (aCSF). The channel has a slope conductance of 223pS, is activated in response to membrane depolarization, and is controlled in a reversible manner by free [Ca(2+)] at the intracellular membrane surface. In the excised patch configuration anoxia had no effect on K(Ca) channel open probability (P(open)); however, in cell-attached mode, there was a reversible fivefold reduction in P(open) (from 0.5 ± 0.05 to 0.1 ± 0.03) in response to 30-min anoxia. The inclusion of the potent protein kinase C (PKC) inhibitor chelerythrine prevented the anoxia-mediated decrease in P(open) while drip application of a phorbol ester PKC activator decreased P(open) during normoxia (from normoxic 0.4 ± 0.05 to phorbol-12-myristate-13-acetate (PMA) 0.1 ± 0.02). Anoxia results in a slight depolarization of turtle pyramidal neurons (∼8 mV) and an increase in cytosolic [Ca(2+)]; therefore, K(Ca) arrest is likely important to prevent Ca(2+) activation during anoxia and to reduce the energetic cost of maintaining ion gradients. We conclude that turtle pyramidal cell Ca(2+)-activated K(+) channels are oxygen-sensitive channels regulated by cytosolic factors and are likely the reptilian analog of the mammalian large conductance Ca(2+)-activated K(+) channels (BK channels)., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Oxygen depleted environments are relatively common on earth and represent both a challenge and an opportunity to organisms that survive there. A commonly observed survival strategy to this kind of stress is a lowering of metabolic rate or metabolic depression. Whether metabolic rate is at a normal or a depressed level the supply of ATP (glycolysis and oxidative phosphorylation) must match the cellular demand for ATP (protein synthesis and ion pumping), a condition that must of course be met for long-term survival in hypoxic and anoxic environments. Underlying a decrease in metabolic rate is a corresponding decrease in both ATP supply and ATP demand pathways setting a new lower level for ATP turnover. Both sides of this equation can be actively regulated by second messenger pathways but it is less clear if they are regulated differentially or even sequentially with the onset of anoxia. The vertebrate brain is extremely sensitive to low oxygen levels yet some species can survive in oxygen depleted environments for extended periods and offer a working model of brain survival without oxygen. Hypoxia tolerant vertebrate brain will be the primary focus of this review; however, we will draw upon research involving hypoxia/ischemia tolerance mechanisms in liver and heart to offer clues to how brain can tolerate anoxia. The issue of regulating ATP supply or demand pathways will also be addressed with a focus on ion channel arrest being a significant mechanism to reduce ATP demand and therefore metabolic rate. Furthermore, mitochondria are ideally situated to serve as cellular oxygen sensors and mediator of protective mechanisms such as ion channel arrest. Therefore, we will also describe a mitochondria based mechanism of ion channel arrest involving ATP-sensitive mitochondrial K(+) channels, cytosolic calcium and reaction oxygen species concentrations.

Earlier studies identified two main defense strategies against hypoxia in hypoxia tolerant animals: (1) reduction in energy turnover, and (2) improved energetic efficiency of those metabolic processes that remain. We used two model systems from the highly anoxia-tolerant aquatic turtle: (1) tissue slices of brain cortex (to probe cell level electrophysiological responses to oxygen limitation), and (2) isolated liver hepatocytes (to probe signalling and defense). In the latter, a cascade of processes underpinning hypoxia defense begins with an oxygen sensor that is probably a heme protein and a signal transduction pathway that leads to the specific activation of some genes (increased expression of several proteins) and to specific down-regulation of other genes (decreased expression of several other proteins). The pathway seems to have characteristics in common with oxygen-regulated control elements in other cells. The probable roles of the oxygen sensing and signal transduction system include coordinate down-regulation of energy demand and energy supply pathways in metabolism. Because of this coordination, hypoxia tolerant cells stay in energy balance even as they down-regulate to extremely low levels of ATP turnover. The main ATP-demanding processes in normoxia (protein synthesis, protein degradation, glucose synthesis, urea synthesis and maintenance of electrochemical gradients) are all turned down to variable degrees during anoxia or extreme hypoxia. Most striking is the observation that ion pumping is the main energy sink in anoxia-despite reductions in cell membrane permeability ("channel arrest"). Neurons also show a much lower permeability than do homologous mammalian cells but, in this case under acute anoxia, there is no further change in cell membrane conductivity. We consider that, through this recent work, it is becoming evident how normoxic maintenance ATP turnover rates can be down-regulated by an order of magnitude or more-to a new hypometabolic steady state that is prerequisite for surviving prolonged hypoxia or anoxia. The implications of these developments extend to many facets of biology and medicine.