Intracellular communication through gap junctions is critical in many biological processes including the modulation of neural activity, coordination of cardiac cells, and biogenesis of cancer cells. The classic method for visualizing gap junction connectivity involves charging specific cells with a fluorophore or biotin derivative that is permeable to gap junctions. Current techniques to introduce small molecules into cells are invasive, inefficient, and limited in the number of cells that can be studied in an experiment. We aim to develop small molecule approaches that address the limitations of current methods and are pursuing two strategies. The first involves fixable fluorogenic substrates for E.coli nitroreductase (NTR). We synthesized a fluorogenic Oregon Green that is masked with nitroimidizole groups (OG-Im2 , Figure 1A). The NTR-catalyzed reduction of the nitro group causes the expulsion of imidazole moiety and releases fluorescent Oregon Green tracer dye. OG-Im2  is stable to endogenous reductases in several cell lines including neurons, but is activated rapidly in cells expressing exogenous NTR. Appending this substrate with a fixable group (amine, hydrazide, etc.) could allow post hoc visualization of gap junction connectivity in tissue. The second strategy involves placing the nitroimidizole mask on O6-benzylguanine (BG) to form BG-Im  (Figure 1B), thereby blocking reactivity with the cognate SNAP-tag protein. In this strategy, we express NTR in one cell type and SNAP-tag in another cell-type. Visualization of labeled SNAP-tag protein indicates gap junction communication between these two cellular populations.

Novel small molecule approaches for studying gap junction connectivity of neuronal cells

248th National Meeting (2014)

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