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  • Title: Development of low-affinity, membrane-targeted Ca2+ sensors suitable for measuring presynaptic Ca2+.
    Author: Monif M, Smart ML, Reid CA, Williams DA.
    Journal: Clin Exp Pharmacol Physiol; 2004 Dec; 31(12):885-9. PubMed ID: 15659054.
    Abstract:
    1. Our aim is to measure near-membrane Ca(2+) flux within the presynaptic terminals of central neurons by modifying new genetically encoded Ca(2+) 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' Ca(2+) biosensor with a presynaptic P2X7 receptor (P2X7R). The Ca(2+) 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 Ca(2+)-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 Ca(2+) affinity of the complex. The dynamic range of the sensor following this modification is better matched to the higher Ca(2+) levels expected within presynaptic Ca(2+) microdomains.
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