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

Increases in the concentration of free ATP within the islet β-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K⁺(K_ATP) 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 β-cells. Resting [ATP] in the cytosol ([ATP]_c), in the mitochondrial matrix ([ATP]_m), and beneath the plasma membrane ([ATP]_pm) were similar (∼1 mm). Elevations in extracellular glucose concentration (3–30 mm) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]_m and [ATP]_pm were observed, but only a transient increase in [ATP]_c. However, detectable increases in [ATP]_c and [ATP]_pm, but not [ATP]_m, required extracellular Ca²⁺. Enhancement of glucose-induced Ca²⁺ influx with high [K⁺] had little effect on the apparent [ATP]_c and [ATP]_m increases but augmented the [ATP]_pm 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 [ATP]_m both through enhanced substrate supply and by progressive Ca²⁺-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]_pm, which may be essential for the sustained closure of K_ATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration.