Submitted on January 5, 2005
Accepted on March 3, 2005
FRET-Based Voltage Probes For Confocal Imaging: Membrane Potential Oscillations Throughout Pancreatic Islets
Andrey Kuznetsov1*, Vytautas P Bindokas2, Jeremy D Marks3, and Louis H Philipson1
1 Medicine, The University of Chicago, Chicago, IL, USA
2 Neurobiology, Pharmacology and Physiology, The University of Chicago, Chicago, IL, USA
3 Pediatrics, The University of Chicago, Chicago, IL, USA
* To whom correspondence should be addressed. E-mail: akuznets{at}uchicago.edu.
Insulin secretion is dependent on coordinated pancreatic islet physiology. Here we overcame limitations of cellular electrophysiology to optically determine cell membrane potential (Vm) throughout an islet, employing a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence resonance energy transfer (FRET) with the fluorescent donor CC2-DMPE and acceptor DiSBAC2(3) in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from Vm -70 to +50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium (TEA). Essentially every responding cell in a mouse islet displayed similar time-dependent changes in Vm. When Vm was measured simultaneously with intracellular calcium, all active cells showed tight coupling of Vm to islet cell calcium changes. Our findings indicate that FRET-based voltage-sensitive dyes with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans.
