View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1742 (2004) 151–160 http://www.elsevier.com/locate/bba Review Annexin 7 mobilizes calcium from endoplasmic reticulum stores in brainB W.D. Watsona,1, M. Srivastavab,1, X. Leightonb, M. Glasmanb, M. Faradayc, L.H. Fossamd, H.B. Pollardb,*, A. Vermae aNeurology Department, National Naval Medical Center, Bethesda, MD 20814, United States bDepartment of Anatomy, Physiology, and Genetics, 4301 Jones Bridge Road, Bethesda, MD 20814, United States cDepartment of Medical Psychology, Bethesda, MD 20814, United States dDivision of Neuropharmacological Drug Products, CDER, FDA, Rockville, MD 20852, United States eDepartment of Neurology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, United States Received 18 September 2004; received in revised form 5 October 2004; accepted 12 October 2004 Available online 29 October 2004 Abstract Mobilization of intracellular calcium from inositol-1,4,5-triphosphate (IP3)-sensitive endoplasmic reticulum (ER) stores plays a prominent role in brain function. Mice heterozygous for the annexin A7 (Anx7) gene have a profound reduction in IP3 receptor function in pancreatic islets along with defective insulin secretion. We examined IP3-sensitive calcium pools in the brains of Anx7 (+/À) mice by utilizing ATP/Mg2+-dependent 45Ca2+ uptake into brain membrane preparations and tissue sections. Although the Anx7 (+/À) mouse brain displayed similar levels of IP3 binding sites and thapsigargin-sensitive 45Ca2+ uptake as that seen in wild-type mouse brain, the Anx7 (+/À) mouse brain Ca2+ pools showed markedly reduced sensitivity to IP3. A potent and saturable Ca2+-releasing effect of recombinant ANX7 protein was demonstrated in mouse and rat brain membrane preparations, which was additive with that of IP3. We propose that ANX7 mobilizes Ca2+ from an endoplasmic reticulum-like pool, which can be recruited to enhance IP3-mediated Ca2+ release. D 2004 Elsevier B.V. All rights reserved. Keywords: Annexin 7; Knockout mouse; Endoplasmic reticulum; Calcium; IP3 receptor; Signaling; Brain 2+ 1. Introduction second messenger-induced [Ca ]cyt changes. Inositol-1,4,5- triphosphate (IP3), an intracellular second messenger gen- Precise regulation of cytoplasmic calcium levels erated in response to neurotransmitter receptor activation, 2+ ([Ca ]cyt) is required to control the electrical, secretory, mediates the release of calcium from a subset of intracellular and metabolic activities of brain cells. Cytoplasmic calcium ER stores by binding to its receptors (IP3R; 2). The IP3R1 is levels are controlled by a diverse set of mechanisms, the predominant subtype of IP3 receptor in the central including calcium binding proteins; plasma membrane nervous system and allows variable control of local intra- channels, pumps, and exchangers; mitochondria; and endo- cellular calcium concentrations, which can modulate the plasmic reticulum (ER) uptake and release mechanisms [1]. efficacy of both excitatory and inhibitory inputs [2]. The ER calcium machinery has a major impact on neuronal Homozygote mutants for IP3R1 die in utero while hetero- zygotes manifest epilepsy with polyspike electroencephalo- grams, opisthotonus, motor discoordination, ataxia, and B The opinions and assertions contained herein are the private views of altered synaptic function and plasticity [3]. These observa- the authors and are not to be construed as official or reflecting the views of tions indicate the importance of IP3-regulated Ca2+ release the Department of Defense. * Corresponding author. Tel.: +1 301 295 3661; fax: +1 301 295 2822. mechanisms in normal brain function. Proteins that interact E-mail address: [email protected] (H.B. Pollard). with the ER and with the IP3R directly can also influence 1 Dr. Watson and Dr. Srivastava are to be considered as co-first authors. IP3-mediated Ca2+ release. For example, the homer family of 0167-4889/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2004.10.008 152 W.D. Watson et al. / Biochimica et Biophysica Acta 1742 (2004) 151–160 proteins enhances IP3-mediated calcium release by bringing was maintained at 23 8C at 50% relative humidity on a 12-h plasma membrane proteins such as metabotropic receptors reverse light/dark cycle (lights on at 1900 h). Behaviors into close proximity with IP3R [4]. Coupling of IP3-sensitive were measured during the dark portion of the circadian and IP3-insensitive compartments by putative GTP-binding cycle for response stability and for face validity (i.e., proteins has also been proposed as a mechanism for animals were in the active portion of their daily cycle). increasing agonist mobilization of calcium pools [5]. Calm- Open-field apparatus: Open-field activity was measured odulin and Transient Receptor Potential channels (TRPs) also using an Omnitech Electronics Digiscan infrared photocell appear to regulate the function of IP3 receptors [6]. system [Test box model RXYZCM (16 TAO); Omnitech Members of the annexin gene family are a diverse group Electronics, Columbus, OH], located in a dedicated room of highly conserved Ca2+-dependent phospholipid binding constructed so that sound is kept to a minimum. Animals proteins found ubiquitously in higher organisms [7]. were placed singly in a 40Â40Â30-cm clear Plexiglas Although most annexins have poorly understood functions, arena. A Plexiglas lid with multiple 3.5-cm diameter they contain a conserved C-terminal region, which in many ventilation holes was placed on top of the arena. A photocell members of the annexin gene family possesses calcium array measured horizontal locomotor activity using 16 pairs channel activity [8]. Annexin A7 (ANXA7; ANX7; of infrared photocells located every 2.5 cm from side-to-side synexin) is a Ca2+-dependent membrane fusion protein with and 16 pairs of infrared photocells located front-to-back in a intrinsic GTPase activity. Ca2+, GTP, and PKC are proposed plane 2 cm above the floor of the arena. Data were to converge on ANXA7 to drive membrane fusion activity automatically gathered and transmitted to a computer via required during exocytosis [9]. Besides this activity, an Omnitech Model DCM-I-BBU analyzer. The apparatus ANXA7 has increasingly been implicated in the regulation monitored animal activity continuously with data recorded of intracellular calcium homeostasis in several cell types and as cumulative activity every 5 min for a total testing period species [11–13]. We have shown that Anx7 heterozygous of 2 h. Once animals were placed in the test arenas, the knockout Anx7 (+/À) mice display an insulin secretion experimenter turned off the lights and left animals undis- deficit from pancreatic islets of Langerhans due to defective turbed during the testing period. For data analysis purposes, intracellular pancreatic Ca2+ signaling processes with a activity was summed over the total 2-h testing period. Total marked reduction in IP3-sensitive Ca2+ [13]. Given the activity over the 2-h testing period was used to determine importance of ER Ca2+ in brain function and the reduced whether or not Anx7 (+/À) knockout male and female mice expression of Anx7 in all tissues of Anx7 (+/À) mice, we and wild-type littermates exhibit differences in activity. explored the possible impact of this deficiency on brain ER During the Baseline Phase (10 days), animals were calcium pools. acclimated to the facility and were handled every day to minimize any stress that might occur as a result of routine handling for body weight measurement, open-field testing, 2. Methods and startle testing. Animals underwent two acclimation exposures to the open-field apparatus to minimize effects of 2.1. Animals stress or novelty. Acclimation sessions consisted of placing the animal in the apparatus for 1 h on two separate days. Adult male Sprague–Dawley rats (300–350 g) were Baseline open-field measurements were then obtained. All obtained from Charles River. The Anx7 targeting vector, the statistical analysis tests were two-tailed. Results are sig- transfection and selection of embryonic stem cells, the nificant at Pb0.05, unless otherwise noted. preparation of chimeras, and the breeding protocols were as described by Srivastava et al. [13]. All animals described in 2.3. Tissue preparation this article were the progeny of at least six successive backcrosses into the wild-type C57BL6/J strain. Adult wild-type mice, Anx7 (+/À) mice or Sprague– Dawley rats were sacrificed using CO2 narcosis followed by 2.2. Behavioral studies decapitation. Brains were rapidly removed, minced with a razor, and placed in ice-cold homogenization buffer (10 ml/ To determine whether knockout of the Anx7 gene has any g) containing 25 mM N-2hydroxyethylpiperazine-NV-2 general effects on behavior, we tested open-field behavior in ethanesulfonic acid (HEPES, pH 7.3 with potassium 28 7-month-old mice: 7 male Anx7 (+/À) mice with 7 wild- hydroxide), 0.25 M sucrose, 100 mM EDTA, and protease type male littermates and 7 female Anx7 (+/À) mice with 7 inhibitors (100 mg/ml phenylmethanesulfonylfluoride, 10 wild-type female littermates. Open-field testing was per- Ag/ml leupeptin, and 10 Ag/ml aprotinin). A glass-teflon formed essentially as described previously [14]. Animals dounce homogenizer was used to homogenize tissue using were individually housed in standard polycarbonate shoe- five up-and-down strokes of a T-Line laboratory stirrer box cages (42Â20.5Â20 cm) on hardwood chip bedding (Talboys Engineering, Montrose, PA) at 70% RPM, (Pine-Dri). Throughout the study, the animals had contin- followed by ultracentrifugation at 100,000Âg with a Sorvall uous access to rodent chow and water. The housing room TH-641 swinging bucket rotor in a Sorvall Ultra Pro 80 at 4 W.D. Watson et al. / Biochimica et Biophysica Acta 1742 (2004) 151–160 153 8C for 60 min. The pellet, containing whole cell membranes, digitonin at 25 8C for 15 min. Slides were incubated in assay was rinsed and resuspended using ice-cold homogenization buffer (same as used for membranes) at 37 8C for 60 min.