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

Abstract

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 co..