Abstract
Large-conductance calcium activated K+ channels (BK) play a crucial role in the control of neuronal excitability. At the presynaptic terminals, BK channels interact with voltage-gated Ca2+ channels (VGCCs), in spatially restricted domains called “Ca2+ nanodomains”, providing a negative feedback mechanism for Ca2+ influx and regulating neurotransmitter release. These properties make BK channels ideal candidates to develop new genetically encoded calcium indicators to report changes in [Ca2+] restricted to nanodomains. Based on FRETable BK channels previously developed in our laboratory, we have generated a potential Ca2+ sensor (BK-860cpGFP) by inserting a circularly permutated green fluorescent protein (cpGFP) into the BK amino acid position 860. Here we show that BK function is not altered by the cpGFP insertion. Addition of ionomycin to BK-860cpGFP-transfected HEK293 cells induced an increase in cpGFP fluorescence, which was associated to Ca2+ binding to specific sites in the BK channel. Simultaneous imaging and electrophysiology experiments in excised membrane patches allowed us to characterize the Ca2+ and voltage-sensitivity of the fluorescent signal. By using the superresolution microscopy technique STORM, we have also demonstrated that BK-860cpGFP forms complexes with VGCCs similar to those formed with wild-type BK channels. To further increase signal-to-noise ratio of BK-860cpGFP, we have introduced a modification that has showed to improve the dynamic range of sensors signals, consisting on the alteration/shortening of the amino acid linkers between the host protein and the cpGFP. Optimized BK-860cpGFP linker-versions were selected after screening using a fluorescence assay with a plate reader. Tests of BK sensors function in a neuronal preparation are currently underway.
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