KATP channel activity and slow oscillations in pancreatic beta cells are regulated by mitochondrial ATP production

Pancreatic beta cells secrete Insulin in response to plasma glucose. The ATP-sensitive Potassium Channel (K_ATP ) links glucose metabolism to islet electrical activity in these cells by responding to increased cytosolic [ATP]/[ADP]. It was recently proposed that Pyruvate Kinase (PK) in close proximity to beta cell K_ATP locally produces the ATP that inhibits K_ATP activity. This proposal was largely based on the observation that applying phosphoenolpyruvate (PEP) and ADP to the cytoplasmic side of excised inside-out patches inhibited K_ATP . To test the relative contributions of local vs. mitochondrial ATP production, we recorded K_ATP activity using mouse beta cells and INS-1 832/13 cells. In contrast to prior reports, we could not replicate inhibition of K_ATP activity by PEP + ADP. However, when the pH of the PEP solutions was not corrected for the addition of PEP, strong channel inhibition was observed as a result of the well-known action of protons to inhibit K_ATP . In cell-attached recordings, perifusing either a PK activator or an inhibitor had little or no effect on K_ATP channel closure by glucose, further suggesting that PK is not an important regulator of K_ATP . In contrast, addition of mitochondrial inhibitors robustly increased K_ATP activity. Finally, by measuring the [ATP]/[ADP] responses to imposed calcium oscillations in mouse beta cells, we found that Oxidative Phosphorylation could raise [ATP]/[ADP] even when ADP was at its nadir during the burst silent phase, in agreement with our mathematical model. These results indicate that ATP produced by mitochondrial Oxidative Phosphorylation is the primary controller of K_ATP in pancreatic beta cells. KEY POINTS: Phosphoenolpyruvate (PEP) plus adenosine diphosphate does not inhibit K_ATP activity in excised patches. PEP solutions only inhibit K_ATP activity if the pH is unbalanced. Modulating Pyruvate Kinase has minimal effects on K_ATP activity. Mitochondrial inhibition, in contrast, robustly potentiates K_ATP activity in cell-attached patches. Although the ADP level falls during the silent phase of calcium oscillations, mitochondria can still produce enough ATP via Oxidative Phosphorylation to close K_ATP . Mitochondrial Oxidative Phosphorylation is therefore the main source of the ATP that inhibits the K_ATP activity of pancreatic beta cells.

KATP channels; [ATP]/[ADP] ratio; beta cells; glucose response; insulin secretion; oxidative phosphorylation; pH modulation; pyruvate kinase.