FRET-based voltage probes for confocal imaging: membrane potential oscillations throughout pancreatic islets.
Insulin secretion is dependent on coordinated pancreatic islet physiology. In the present study, we found a way to overcome the limitations of cellular electrophysiology to optically determine cell membrane potential (V(m)) throughout an islet by using a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence (Förster) resonance energy transfer (FRET) with the fluorescent donor N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine and the acceptor bis-(1,3-diethylthiobarbiturate) trimethine oxonol in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from V(m) -70 to +50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium. Essentially, every responding cell in a mouse islet displayed similar time-dependent changes in V(m). When V(m) was measured simultaneously with intracellular Ca2+, all active cells showed tight coupling of V(m) to islet cell Ca2+ changes. Our findings indicate that FRET-based, voltage-sensitive dyes used in conjunction with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans.
Kuznetsov
Andrey
A
Department of Medicine, MC1027, Division of Biological Sciences, Pritzker School of Medicine, University of Chicago, 5841 S. Maryland Ave., Chicago, Illinois 60637, USA.
Bindokas
Vytautas P
VP
Marks
Jeremy D
JD
Philipson
Louis H
LH
D000818:Animals; D001463:Barbiturates / Q000737:chemistry* / Q000494:pharmacology*; D002462:Cell Membrane / Q000502:physiology; D004396:Coloring Agents; D003374:Coumarins / Q000737:chemistry* / Q000494:pharmacology*; D004983:Ethanolamines / Q000737:chemistry* / Q000494:pharmacology*; D031541:Fluorescence Resonance Energy Transfer; D066298:In Vitro Techniques; D007515:Islets of Langerhans / Q000502:physiology*; D007555:Isoxazoles / Q000737:chemistry* / Q000494:pharmacology*; D008564:Membrane Potentials; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009991:Oscillometry; D013858:Thiobarbiturates
D001463:Barbiturates; D004396:Coloring Agents; D003374:Coumarins; D004983:Ethanolamines; D007555:Isoxazoles; C502247:N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine; D013858:Thiobarbiturates; C025284:bis(1,3-diethylthiobarbiturate)trimethineoxonol