Glucose Generates Sub-plasma Membrane ATP Microdomains in Single Islet β-Cells

Increases in the concentration of free ATP within the islet beta-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+ (KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free ATP in subdomains of single living MIN6 and primary beta-cells. Resting ATP in the cytosol (ATPc), in the mitochondrial matrix (ATPm), and beneath the plasma membrane (ATPpm) were similar ( approximately 1 mM). Elevations in extracellular glucose concentration (3-30 mM) increased free ATP in each domain with distinct kinetics. Thus, sustained increases in ATPm and ATPpm were observed, but only a transient increase in ATPc. However, detectable increases in ATPc and ATPpm, but not ATPm, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high K+ had little effect on the apparent ATPc and ATPm increases but augmented the ATPpm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high K+. These data support a model in which glucose increases ATPm both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of ATPpm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration.