As expected for a KATP-mediated current, current “run-up” was absent in Kir6.2−/− neurons or in wild-type neurons that were incubated in 200 μM tolbutamide prior to recording ( Figure 5C). To test for a whole-cell correlate of the high resting single channel Popen observed in cell-attached

patches, we performed experiments with high ATP (4 mM) in the pipette, with the idea that as the ATP washes into the cell it might inhibit any initially active KATP channels. Indeed, we found that conductance in Bad−/− neurons decreased INCB28060 in vivo within the first minute after break-in with high ATP and then remained constant ( Figure 5D). This “washdown” was not seen in wild-type cells ( Figure 5D). It was also eliminated in Bad−/− neurons if they were preincubated with tolbutamide ( Figure 5E) or in neurons from animals that lacked both BAD and Kir6.2 ( Figure 5D), confirming that this initial high conductance was due to KATP channels. Current washdown also occurred in neurons from BadS155A mice ( Figure 5D),

showing that it is BAD’s metabolic, rather than apoptotic, function that is responsible for increasing KATP conductance. The marked increase in KATP channel open probability in Bad−/− DGNs suggests a link between KATP channel conductance and seizure protection in these mice and predicts that BAD’s effect on seizure sensitivity may be mediated by the KATP channel. To test this prediction, we generated Bad−/−; Kir6.2−/− double mutant mice and tested their sensitivity to KA in parallel with single Bad−/− or Kir6.2−/− mutants. Ablation of the selleck inhibitor Kir6.2 subunit in the Bad null

genetic background substantially diminished the seizure resistance phenotype of Bad−/− mice ( Figures 6A and 6B). Single deletion of the Kir6.2 subunit did not sensitize Thymidine kinase mice to acute seizures, arguing against an orthogonal or additive effect on seizures. These findings provide genetic evidence that the KATP channel is required for mediating BAD’s effect on neuronal excitability. Using a combination of genetic models and multiple experimental approaches ranging from mitochondrial respirometry in primary neural cultures and slice electrophysiology to behavioral and electrographic seizure monitoring in vivo, we provide a vertical analysis of BAD’s effect on neural carbon substrate utilization, neuronal excitability, and seizure susceptibility. Our observations suggest that BAD imparts reciprocal effects on glucose and ketone body consumption through a phosphoregulatory mechanism that modifies S155 within its BH3 domain. Specifically, BAD deficiency or interference with its phosphorylation is associated with diminished mitochondrial metabolism of glucose and a concomitant metabolic preference for ketone bodies. An electrophysiologic consequence of this metabolic shift is a marked increase in the open probability of the metabolically sensitive KATP channel.