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Nicotinamide adenine dinucleotide-dependent binding of the neuronal Ca2+ sensor protein GCAP2 to photoreceptor synaptic ribbons.

ABSTRACT: Guanylate cyclase activating protein 2 (GCAP2) is a recoverin-like Ca2+-sensor protein known to modulate guanylate cyclase activity in photoreceptor outer segments. GCAP2 is also present in photoreceptor ribbon synapses where its function is unknown. Synaptic ribbons are active zone-associated presynaptic structures in the tonically active photoreceptor ribbon synapses and contain RIBEYE as a unique and major protein component. In the present study, we demonstrate by various independent approaches that GCAP2 specifically interacts with RIBEYE in photoreceptor synapses. We show that the flexible hinge 2 linker region of RIBEYE(B) domain that connects the nicotinamide adenine dinucleotide (NADH)-binding subdomain with the substrate-binding subdomain (SBD) binds to the C terminus of GCAP2. We demonstrate that the RIBEYE-GCAP2 interaction is induced by the binding of NADH to RIBEYE. RIBEYE-GCAP2 interaction is modulated by the SBD. GCAP2 is strongly expressed in synaptic terminals of light-adapted photoreceptors where GCAP2 is found close to synaptic ribbons as judged by confocal microscopy and proximity ligation assays. Virus-mediated overexpression of GCAP2 in photoreceptor synaptic terminals leads to a reduction in the number of synaptic ribbons. Therefore, GCAP2 is a prime candidate for mediating Ca2+-dependent dynamic changes of synaptic ribbons in photoreceptor synapses.

SUBMITTER: Venkatesan JK

PROVIDER: S-EPMC3900572 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Nicotinamide adenine dinucleotide-dependent binding of the neuronal Ca2+ sensor protein GCAP2 to photoreceptor synaptic ribbons.

Venkatesan Jagadeesh Kumar JK Natarajan Sivaraman S Schwarz Karin K Mayer Sabine I SI Alpadi Kannan K Magupalli Venkat Giri VG Sung Ching-Hwa CH Schmitz Frank F

The Journal of neuroscience : the official journal of the Society for Neuroscience 20100501 19

Guanylate cyclase activating protein 2 (GCAP2) is a recoverin-like Ca2+-sensor protein known to modulate guanylate cyclase activity in photoreceptor outer segments. GCAP2 is also present in photoreceptor ribbon synapses where its function is unknown. Synaptic ribbons are active zone-associated presynaptic structures in the tonically active photoreceptor ribbon synapses and contain RIBEYE as a unique and major protein component. In the present study, we demonstrate by various independent approach ...[more]

PMID: 20463219

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Project description:AimsAccurate analysis of dinucleotide redox cofactors nicotinamide adenine dinucleotide phosphate reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide reduced (NADH), and nicotinamide adenine dinucleotide (NAD+) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms.ResultsWe compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues: a cold aqueous buffer commonly used in enzyme assays with and without detergent, hot aqueous buffer, and cold organic mixtures (80% methanol, buffered 75% acetonitrile, and acidic 40:40:20 acetonitrile:methanol:water with either 0.02 M or 0.1 M formic acid). Extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). To monitor the metabolite interconversion, cells were grown in 13C6-glucose medium, and unlabeled standards were spiked into the extraction solvents. Interconversion between the oxidized and reduced forms was substantial except for the enzyme assay buffer with detergent, 80% methanol and 40:40:20 acetonitrile:methanol:water, with the 0.1 M formic acid mix giving the least interconversion and best recoveries. Absolute NAD+, NADH, NADP+, and NADPH concentrations in cells and mouse tissues were measured with this approach.InnovationWe found that the interconversion between the reduced and oxidized forms during extraction is a major barrier to accurately measuring NADPH/NADP+ and NADH/NAD+ ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards.ConclusionExtraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.

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Nicotinamide adenine dinucleotide-dependent binding of the neuronal Ca2+ sensor protein GCAP2 to photoreceptor synaptic ribbons.

Publications

Nicotinamide adenine dinucleotide-dependent binding of the neuronal Ca2+ sensor protein GCAP2 to photoreceptor synaptic ribbons.

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