BPS2026 – KATP gating conformational changes measured using FRET

ATP-sensitive potassium (K ATP ) channels play a critical role in human metabolic regulation. Structural studies using cryo-electron microscopy have predicted a large conformational rearrangement within these channels during transitions between inhibitory and active states. However, it remains unclear whether such conformational changes occur under physiological conditions. In this study, we employ Förster resonance energy transfer (FRET) to investigate the spatial dynamics between the Kir6.2 and SUR1 subunits of the K ATP channel in living cells. We tagged Kir6.2 with cyan fluorescent protein (CFP) and SUR1 with yellow fluorescent protein (YFP) and co-expressed the tagged subunits in HEK293T cells. Using fluorescence spectroscopy, we established a baseline FRET signal indicating subunit proximity. To test the hypothesis that activation is accompanied by a large conformational change, we measured FRET efficiency in the presence of pharmacological inhibitors (glibenclamide) and activators (diazoxide). Our results reveal a significant alteration in FRET signal between these states, suggesting that a substantial conformational shift indeed accompanies channel modulation. The FRET signal from intact HEK293T cells arose from mature channel proteins at the plasma membrane, but also from trafficking/assembly intermediates in the ER/Golgi. Therefore, we repeated our experiments in plasma membrane fragments isolating by unroofing. In unroofed membranes, we observed a similar conformational change in the presence of diazoxide. MgADP was required to support this conformational change. These findings provide evidence for existing structural models in the context of living cells and offer insight into the dynamic regulation of K ATP channels.