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Ca2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo. © 2008 Elsevier Inc. All rights reserved.

The FASEB Journal www.fasebj.org March 6, 2006 The FASEB Journal vol. 20 no. 4 A59 The heterogeneity of NaPi protein dynamics and NaPi cotransport activity in renal brush border membranes Judith Tamara Blaine1, Hideaki Takahashi1, Nicholas Barry1, Michelle Digman2, Claudia Lee2, Sophia Breusegem1, William Mantulin2, Enrico Gratton2 and Moshe Levi1 Author Affiliations Abstract Alterations in renal proximal tubule brush border membrane (BBM) cholesterol, sphingomyelin, and glycosphingolipid content play an important role in regulating the activity of the sodium-phosphate cotransporter (NaPi). The molecular mechanisms of how alterations in lipid composition may modulate NaPi activity are not known. We have fractionated BBM prepared from rat kidney using detergent- free density gradient ultracentrifugation (OptiPrep). We have found that the NaPi protein preferentially partitions into lipid rafts. To determine the potential consequences of the partitioning of the NaPi transporter in these lipid domains we have i) measured NaPi transport activity and ii) used fluctuation correlation spectroscopy (FCS) methods to determine NaPi diffusion. Partitioning of NaPi protein into lipid rafts results in i) decreased NaPi cotransport activity and ii) decreased diffusion of NaPi protein. Similar results were obtained in BBM isolated from the superficial cortex (SC) versus the juxtamedullary cortex (JMC). The three- fold decrease in NaPi cotransport activity in JMC-BBM is associated with a 3-fold decrease in NaPi protein diffusion. Our results therefore indicate that partitioning of NaPi protein into lipid rafts results in impairment of its activity and diffusion.