Development of low-affinity, membrane-targeted Ca²⁺ sensors suitable for measuring presynaptic Ca²⁺

1. Our aim is to measure near-membrane Ca²⁺ flux within the presynaptic terminals of central neurons by modifying new genetically encoded Ca²⁺ sensors to develop tools capable of measuring localized Ca ²⁺ signals. 2. We used standard recombinant DNA technologies to generate the DNA coding for a fusion construct of a modified fluorescent 'pericam' Ca²⁺ biosensor with a presynaptic P2X7 receptor (P2X7R). The Ca²⁺ sensitivity of the biosensor was modified by rational site-directed mutagenesis of the calmodulin portion of the pericam. 3. Biosensor-receptor fusions were transfected into expression systems for evaluation. Expression studies in HEK-293 cells showed that biosensor-receptor fusion construct-delivered protein was localized exclusively to the plasma membrane, confirming that fusion did not affect the ability of the receptor to undergo normal protein synthesis and trafficking. 4. The Ca²⁺- dependent fluorescence of the pericam portion of the fusion protein was also retained. Site-direct mutagenesis within the calmodulin moiety of the pericam significantly reduced the Ca²⁺ affinity of the complex. The dynamic range of the sensor following this modification is better matched to the higher Ca²⁺ levels expected within presynaptic Ca²⁺ microdomains.