Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

Central neurons undergo spreading depolarization (SD) which shuts down brain function during acute metabolic demand. SD migrates across gray matter at 1-5 mm/min and has evolved in insects (and perhaps in the vertebrates) to induce brain silence under the stress of anoxia, head injury or sudden temperature change. SD is behaviorally protective by reducing movement but can kill neurons if it lasts many minutes. SD immediately follows failure of the Na/K ATPase but the molecular mechanism linking it the massive inward current driving SD is unknown and elusive. Blockade of voltage- or ligand-gated channels does not prevent the SD-like anoxic depolarization (AD). The molecular action the marine poison (palytoxin, Ptox) is known to specifically bind the Na/K pump at picomolar amounts, converting it from an ATP-requiring transporter to an open cationic channel. The sudden Na+ influx and K+ efflux we show emulates SD at a mere 10-100 nM in live brain slices of mouse or rat. Our hypothesis is that severe metabolic stress likewise converts the Na/K ATPase into a channel that drives SD, evoking the neuronal shutdown that is prevalent across animal classes. Thus the molecular action of Ptox can provide insight to CNS shutdown.
The proposal has 3 Objectives: 1) Show with membrane patch recording by our HQP Peter G. that the Na/K ATPase opens to drive SD in amphibian and mammalian neurons of the higher brain. Direct demonstration of pump conversion to a channel requires recording across membrane patches from neurons. A Ptox-evoked 12 pS single channel conductance represents opening of a single pump transporter. 2) Demonstrate that conversion from pump to channel also drives SD in locust and Drosophilia where the molecular details of SD can be assessed in a large cohort using genetically variable strains. With the expertise of HQP Dr. Kristin S. we will extend patch experiments to insect brain. 3) Our pilot data from rat show SD is evoked by rapid temperature shifts to 40 or to 13 o C, similar to heat- and chill-coma in insects. We will study brain slices from homeotherms under variable temperature, simulating poikilothermy to directly compare with naturally cold-blooded animals such as frog and insect.
Shutdown elicited by SD reduces oxidative injury and bypasses partial depolarization that drives epileptiform/spastic activity. As well SD inactivates both Na + channels...and the animal itself. But in homeotherms this immediate behavioral protection lasts only minutes and quickly leads to neuronal death, a process we do not understand. My Canadian lab together with my 3 HQPs represent the first neuroscience facility to directly study the CNS Na/K pump and the channels that open as it fails. We will identify the SD channel by showing a) it activates under metabolic stress; b) it is not blocked by inhibitors of standard channels across animal species; and c) it may share properties similar to the pump channel induced by Ptox.