Visualization of Spatially Controlled Glucokinase Activation in Living Pancreatic Beta Cells using an Optimized FRET-Based Biosensor

Abstract

Glucokinase (GCK) activity in pancreatic beta cells limits the rate of glucose metabolism and controls the glucose threshold for insulin secretion. Regulation of GCK activity by cell surface receptors can occur through activation of nitric oxide synthase (NOS) on insulin secretory granules, and subsequent S-nitrosation of GCK. It is thought that interaction between NOS and GCK is essential to the S-nitrosation reaction. Nonetheless, activation of an existing Förster resonance energy transfer (FRET)-based sensor suggests that the activation occurs diffusively. To examine whether GCK activation has a spatial regulatory component, we developed an improved GCK biosensor using cyan fluorescent protein derived from mCerulean3. Circularly permuted mCerulean3 proteins were constructed with the intent of optimizing rotational positioning of the FRET pair. Several cpmCer3 variants were constructed. The brightest variant, cpmCer174, retains a very high quantum yield (> 0.8) similar to mCerulean3, and produced the highest contrast sensor when incorporated into the FRET-GCK biosensor. Expression in pancreatic beta cells revealed evidence supporting tight spatial control over GCK activation at the insulin secretory granules. These results have numerous implications for the nature of post-translational regulation of glucose sensing in pancreatic beta cells.