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.
|Number of pages||5|
|Journal||Clinical and Experimental Pharmacology and Physiology|
|Publication status||Published - 1 Dec 2004|
- P2X7 receptors
- Presynaptic terminals