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1. Our aim us 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 Ca2+ 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 constructs 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+ micro-domains. [ABSTRACT FROM AUTHOR]

1. Our aim us 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 Ca2+ 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 constructs 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+ micro-domains. [ABSTRACT FROM AUTHOR]

Mal de Meleda is a rare autosomal recessive genodermatosis caused by mutations in the ARS B (SLURP1) gene, with possible founder effects in the Mediterranean and Adriatic regions. We report an affected individual from Indonesia without known consanguinity in the family, suggesting that SLURP1 gene mutations are ubiquitous. Recognition of the phenotype can be confirmed by genetic testing, thus facilitating genetic counselling., (© 2014 The Australasian College of Dermatologists.)