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

M. Monif, Megan L Smart, C. A. Reid, D. A. Williams

Research output: Contribution to journalArticleResearchpeer-review

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 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.

Original languageEnglish
Pages (from-to)885-889
Number of pages5
JournalClinical and Experimental Pharmacology and Physiology
Volume31
Issue number12
DOIs
Publication statusPublished - 1 Dec 2004
Externally publishedYes

Keywords

  • Biosensors
  • Calcium
  • P2X7 receptors
  • Pericams
  • Presynaptic terminals

Cite this

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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 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.",
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Development of low-affinity, membrane-targeted Ca2+ sensors suitable for measuring presynaptic Ca2+. / Monif, M.; Smart, Megan L; Reid, C. A.; Williams, D. A.

In: Clinical and Experimental Pharmacology and Physiology, Vol. 31, No. 12, 01.12.2004, p. 885-889.

Research output: Contribution to journalArticleResearchpeer-review

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