No abstract

The cyclic nucleotide-binding domain (CNBD) functions as a regulatory domain of many proteins involved in cyclic nucleotide signalling. We developed a straightforward and reliable binding assay based on intermolecular fluorescence resonance energy transfer (FRET) between an adenosine-3', 5'-cyclic monophosphate analogue labelled with fluorescein and a recombinant CNBD of human EPAC1 tagged with a cyan fluorescence protein (CFP). The high FRET efficiency of this method (~ 80%) allowed us to perform several types of binding experiments with nanomolar range of sample using conventional equipment. In addition, the CFP tag on the CNBD enabled us to perform a specific binding experiment using an unpurified protein. Considering these advantages, this technique is useful to study poorly characterized CNBDs.

The mitochondrial BK Ca channel (mitoBK Ca ) is a splice variant of plasma membrane BK Ca (Maxi-K, BK Ca , Slo1, K Ca 1.1). While a high-resolution structure of mitoBK Ca is not available yet, functional and structural studies of the plasma membrane BK Ca have provided important clues on the gating of the channel by voltage and Ca 2+ , as well as the interaction with auxiliary subunits. To date, we know that the control of expression of mitoBK Ca , targeting and voltage-sensitivity strongly depends on its association with its regulatory β1-subunit, which overall participate in the control of mitochondrial Ca 2+ -overload in cardiac myocytes. Moreover, novel regulatory mechanisms of mitoBK Ca such as β-subunits and amyloid-β have recently been proposed. However, major basic questions including how the regulatory BK Ca -β1-subunit reaches mitochondria and the mechanism through which amyloid-β impairs mitoBK Ca channel function remain to be addressed.