Analysis of Large Conductance Ca2+-Activated K+ (BK) Channels Dynamics in Living Cells

The title of my project is : "Analysis of Large Conductance Ca2+-Activated K+ (BK) Channels Dynamics in Living Cells", and the abstract:

"Large conductance Ca2+-activated K+ (BK) channels are expressed in human β-cells regulating insulin secretion. BK channel activation is driven by both intracellular Ca2+ and voltage, and they are essential for rapid membrane repolarisation.
We developed novel methods to analyse the dynamics and distribution of BK channels at the level of single molecules in cell membranes using super-resolution microscopy. We expressed a cDNA construct that encodes the BK-channel α-subunit (BKα) fused to a photoactivatable fluorescent protein mutant of mCherry (PAmCherry) and localized single channel molecules using photoactivatable localization microscopy (PALM). Additionally, particle tracking of BK channels was done with live cell PALM imaging. We also analysed BK channel localization with gated stimulated emission depletion (g-STED).
In order to study the proximity of BK channels with the secretory machinery, cells were transfected with our BKα-EGFP construct and they were immunolabeled for Syntaxin-1, a plasma membrane protein involved in vesicle fusion. The co-localization analysis suggested that both BKα and Syntaxin-1 may not co-localize.
On the other hand, electrophysiological analysis with patch-clamp was done to study the conductance properties of BK channels. Further experiments will consist of integrating both TIRF imaging and patch-clamp for the study of BK channels activity and dynamics. Since previous electrophysiological studies suggested that N-type calcium- and BK-channel molecules must be in close proximity in the plasma membrane, we will run both imaging and electrophysiology experiments to determine whether they co-localize."