Development of low-affinity, membrane-targeted Ca2+ sensors suitable for measuring presynaptic Ca2+.
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.
Monif
M
M
Department of Physiology, The University of Melbourne, Parkville, Victoria 3010, Australia.
Smart
M L
ML
Reid
C A
CA
Williams
D A
DA
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D017729:Presynaptic Terminals / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology
D002118:Calcium