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	Session 1: Pumps

TOP Session 1: Pumps Session 2: Cardiac and... Session 3: Neurodegeneration Session 4: Central Nervous... Session 5: TRP Channels

One of the distinctive properties of the calcium signal is ambivalence. When the control of the cellular homeostasis of calcium becomes defective, calcium becomes a conveyor of doom. Dysfunctions of the calcium signal may involve proteins that process it and/or proteins that transport calcium across membranes to buffer its concentration in cells (e.g., pumps). Genetic defects of the plasma membrane calcium pump (PMCA) produce hereditary deafness. Calcium plays an essential role in the hearing process. It enters the stereocilia of hair cells of the Corti organ through mechanoelectrical transduction (MET) channels opened by the deflection of the hair bundles and is exported back to the endolymph that bathes them by an unusual splicing isoform (w/a) of the PMCA2 pump. Precise control of the homeostasis of endolymph calcium is essential to operation of the MET channels. Because the PMCA2 pump is the only system that ejects calcium to the endolymph, it has a vital role in the control of its homeostasis. The w/a isoform carries inserts at site A in the N-terminal half of the pump structure and at site C in its C-terminal tail. The C insert induces premature truncation of the pump. Ablation or missense mutations of the pump cause deafness, as described for the first time in G283S mutation of the deafwaddler (dfw) mouse. In organotypic cultures, Ca²⁺ imaging of vestibular hair cells has shown that the dissipation of stereociliary Ca²⁺ transients induced by Ca²⁺ uncaging was compromised in the dfw (and PMCA2 KO) mice. Novel deafness-inducing mutations have been identified in two human families. That identified in our laboratory (G293) was close to the site of the dfw mutation. The wild-type PMCA2 w/a isoform and its G283S and G293S mutants were overexpressed in CHO cells. The other splice variants of PMCA2 (w/b, z/a, and z/b) were also expressed as controls. Recombinant aequorin was used to monitor Ca²⁺. At variance with the other PMCA2 isoforms, the w/a variant reacted poorly to the arrival of a Ca²⁺ pulse induced in CHO cells by InsP₃. The G293S and G283S mutations did not further reduce the poor ability of the w/a variant to become rapidly activated but delayed the longer term dissipation of the Ca²⁺ transients, compromising the long-term nonactivated export of calcium from the stereocilia and thus its homeostasis in the endolymph.

A digenic mechanism was operational in the G293S human family case. The family was screened for mutations in cadherin 23, which had been shown to accentuate hearing loss in the other human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who instead presented the PMCA2 mutation.

2. Unique Gating Properties of CLC Anion Channel Splice Variants are Determined by Altered CBS Domain Secondary Structure. SONYA DAVE,^1,2 JONATHAN SHEEHAN,³ and KEVIN STRANGE,^{1,2 1}Department of Anesthesiology, ²Department of Molecular Physiology and Biophysics, and ³Center for Structural Biology, Vanderbilt University Medical Center, Nashville, TN 37232

Eukaryotic CLCs are homodimeric proteins with large cytoplasmic C termini containing two cystathionine-β-synthase (CBS) domains. The role played by CBS domains in CLC channel structure and gating is unclear. clh-3 encodes two splice variants, CLH-3a and CLH-3b, of a Caenorhabditis elegans CLC channel that exhibit striking differences in gating kinetics and sensitivity to depolarizing voltages, pH and Cl^–. Mutagenesis studies have shown that the C terminus of CLH-3b gives rise to its unique gating properties. Splice variation of the C terminus includes the last six amino acids in the second CBS domain (CBS2). Mutating these amino acids in CLH-3b to those present in CLH-3a gives rise to CLH-3a gating properties. Similarly, CLH-3b gating properties are induced in CLH-3a by mutating the last six amino acids of CBS2 to those present in CLH-3b. To define how these amino acids alter CBS2 structure, we developed homology models based on crystal structures of C-terminal cytoplasmic domains in vertebrate CLCs using Modeller and Rosetta software. CBS domains have a highly conserved secondary structure consisting of an N-terminal β sheet (β1) followed by an helix (1), two β sheets (β2 and β3), and an helix (2). Our models show that the six–amino acid splice variation is part of 2. Measuring the length of this helix in 10,000 candidate structures generated by Rosetta demonstrated that the splice variation has a higher probability of forming a helical turn in CLH-3b. Thus, 2 of CBS2 in CLH-3a is predicted to be shorter compared with that of CLH-3b. The model also suggests that the length of 2 could alter an interface with either a transmembrane helix that comprises part of the channel pore and/or the interface between cytoplasmic C-terminal monomers. We are currently carrying out modeling and mutagenesis studies to determine how CBS2 splice variation alters these interfaces and channel gating.

3. The Dopamine Transporter as a Possible Mediator of Extrasynaptic Transmitter Release. MARIA DEL PILAR GOMEZ,^1,2 JUAN MANUEL ANGUEYRA,^1,2 ISABELLE MINTZ,³ and ENRICO NASI,^{1,2 1}Universidad Nacional de Colombia, Bogota, Colombia; ²Marine Biological Laboratory, Woods Hole, MA 02543; ³Northwestern University, Evanston, IL 60208

The canonical mode of chemical communication among nerve cells is vesicular release of the neurotransmitter (NT); however, scattered reports suggest alternative mechanisms operating in a Ca-independent manner in regions that are devoid of NT-filled vesicles. This study intended to develop an experimental model to test the proposition that the release of dopamine reported in the pars compacta of the Substantia nigra (SN) of rat brain, which is comprised only of somatas and proximal dendrites, may be mediated by reverse-mode operation of the dopamine transporter (DAT). The task requires the use of isolated cells in which localization of the required machinery can be ascertained, and the release of NT can be monitored under conditions that exclude conventional exocytosis. We established by Western blot analysis that DAT expresses massively in the SN; this is essential if DAT functions effectively as a release mechanism, compensating for the sluggishness of its transport cycle. Second, we confirmed by immunocytochemistry that the DAT is present in the membrane of the soma and proximal dendrites of SN neurons. Next, we turned to the conditions that would promote reversal of the transport cycle: localized increase in [Na⁺]_i and sustained depolarization. We used confocal microscopy on dual-labeled cells with anti-DAT Abs as well as monoclonal Abs against voltage-dependent sodium channels. We observed that DAT and Nav 1.2 colocalize. Moreover, current-clamp recordings indicate the presence of plateau potentials providing the sustained depolarization that may be needed, together with [Na⁺]_i, to drive a reversal of DAT transport. Finally, amperometric measurements on voltage-clamped SN cells show oxidation currents that are graded with depolarization and attenuated by GBR-12395, a specific antagonist of DAT. These preliminary observations lend credence to the conjecture that this transporter may indeed contribute to the release of dopamine in the pars compacta of the Substantia nigra. (Supported by National Institutes of Health [National Institute on Drug Abuse] grant RO1 DA016298.)

4. Examining the Ancient Phototransduction Mechanisms in a Primitive Chordate. MARÍA DEL PILAR GOMEZ,^1,2 JUAN MANUEL ANGUEYRA,^1,2 and ENRICO NASI,^{1,2 1}Universidad Nacional de Colombia, Bogota, Colombia; ²Marine Biological Laboratory, Woods Hole, MA 02543

Two lineages of photoreceptors underlie spatial vision in different organisms: microvillar, which is typical of invertebrates, and ciliary, which is like rods and cones. Additional photosensitive cells have recently been identified among the ganglion cells of mammalian retina; these mediate nonvisual light sensitivity, which is crucial for the regulation of circadian rhythms, the pupillary reflex, and the modulation of melatonin secretion. A novel photopigment, melanopsin, underlies these photoresponses, and current evidence points to the possibility that its transduction mechanisms may be akin to the lipid signaling scheme of microvillar receptors rather than the cyclic-nucleotide cascade of rods and cones. However, rapid progress toward rigorously testing this conjecture has been hindered by the difficulty of subjecting melanopsin-expressing cells to physiological analysis because of their extreme scarcity (<1% of retinal ganglion cells). Recently, it has been discovered that melanopsin has an ancient origin in the vertebrates and expresses in two morphologically distinct classes of cells (pigmented ocelli and Joseph cells) scattered along the neural tube of a primitive prechordate, the Amphioxus. Should these be bona fide photoreceptors, this organism could constitute an appealing model to investigate melanopsin-mediated light transduction. By microdissection and enzymatic dissociation of the neural tube of Amphioxus, we obtained morphologically intact isolated cells representative of both cell types and used patch-electrode recoding to determine that they are indeed primary photoreceptors. The spectral sensitivity is reminiscent of that of melanopsin-expressing mammalian ganglion cells. Both cell types produce a transient depolarizing receptor potential; under voltage clamp, the photocurrent strongly rectifies in the inward direction and does not revert at potentials up to 100 mV. Manipulations of extracellular calcium and of cytosolic Ca-buffering capacity alter the photocurrent in a manner that parallels the effect of calcium on the light response of invertebrate microvillar receptors, providing preliminary supporting evidence for a kinship between the two phototransduction cascades.

5. Smelling Light: a Novel Olfactory-like Phototransduction Cascade. MARÍA DEL PILAR GOMEZ,^1,2,3 FRANCISCA SILVA,² JUAN FELIPE DIAZ,^1,3 and ENRICO NASI,^{1,3, 1}Universidad Nacional de Colombia, Bogota, Colombia; ²Boston University School of Medicine, Boston, MA 02118; ³Marine Biological Laboratory, Woods Hole, MA 02543

The distal photoreceptors of mollusk Pecten irradians functionally resemble those of vertebrate rods and cones in that (1) the light-sensing structures are modified cilia, (2) the receptor potential is hyperpolarizing, and (3) cGMP operates as an internal messenger. However, key properties of these cells diverge from those of their vertebrate counterparts: (1) they do not express transducin, (2) the light-induced changes in cGMP reflect regulation of its synthesis rather than hydrolysis, and (3) the receptor potential is caused by an increase in a K-selective conductance. We obtained a full-length clone of the subunit of a G_o from Pecten retina cDNA and localized it to the distal retina by in situ hybridization; functional data support its involvement in the photoresponse. A β subunit was also identified. A novel guanylate cyclase (GC) mediates downstream effects of light: the photoresponse is insensitive to manipulations of NO (ruling out a soluble GC) and of [Ca²⁺]_i (unlike rod/cone GCs); moreover, regulation by a G-protein is not contemplated by either canonical GC class. Western blots with antibodies raised against the nonconventional 12-TMD GC of Paramecium detected a band of the appropriate molecular mass (242 kD); such GCs have been reported to be regulated by G_o in Dictyostelium. PCR amplifications yielded the sequence of two catalytic domains. The last element of the phototransduction cascade of Pecten, the light-dependent ion channel, represents the first documented functional link between cyclic-nucleotide–dependent channels and K-selective channels, two ancestrally related protein families. Antibodies raised against the olfactory cyclic nucleotide–gated channel CNG2 but not anti-CNG1 or anti-CNG3 recognize a protein of z73 kD in Western blots and selectively stain the distal retina, localizing primarily in the ciliary appendages of the hyperpolarizing photoreceptors, as revealed by confocal fluorescence microscopy. A novel cascade is proposed to underlie light transduction, which parallels that of olfactory receptor cells.

6. Identification of Ste20 Kinase Regulatory Phosphorylation Sites in a Cell Cycle– and Cell Volume– sensitive Clc Anion Channel. REBECCA A. FALIN,^1,2,3 REBECCA MORRISON,^1,2,3 AMY-JOAN L. HAM,⁴ and KEVIN STRANGE,^{1,2,3 1}Department of Anesthesiology, ²Department of Pharmacology, ³Department of Molecular Physiology and Biophysics, and ⁴Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University Medical Center, Nashville, TN 37232

Members of the ClC superfamily of transport proteins function as anion channels and Cl^–/H⁺ exchangers in plasma and intracellular organelle membranes and play key roles in diverse and fundamental physiological processes. Despite intensive study, little is known about how ClCs are regulated. clh-3 encodes two splice variants, CLH-3a and CLH-3b, of a Caenorhabditis elegans voltage-gated ClC Cl^– channel. CLH-3b is expressed in the worm oocyte and is activated during meiotic cell cycle progression or in response to cell swelling. Channel activation is brought about by serine/threonine dephosphorylation mediated by the type 1 phosphatases GLC-7 and GLC-7β. GCK-3 is a Ste20 kinase and homologue of mammalian PASK/SPAK, which regulates cell volume–sensitive cation-coupled Cl^– cotransporters. GCK-3 binds to a 101–amino acid C-terminal splice insert unique to CLH-3b. Knockdown of GCK-3 in worm oocytes constitutively activates CLH-3b. When coexpressed in HEK293 cells with CLH-3b, GCK-3 dramatically inhibits channel activity and alters gating kinetics and voltage sensitivity. Cell swelling reverses the effects of kinase inhibition. Using mass spectrometry, we identified two serine residues, S742 and S747, which are phosphorylated when CLH-3b is coexpressed with GCK-3. These residues conform to a recently identified Ste20 kinase phosphorylation motif. GCK-3–mediated channel inhibition is prevented when either residue is mutated to alanine. When S742 and S747 are mutated individually to the phosphomimetic amino acids aspartate or glutamate, channel activity is normal and fully inhibited by GCK-3. Unlike the wild-type channel, cell swelling did not reverse this inhibition. Mutation of both S742 and S747 to aspartate or glutamate constitutively inhibits CLH-3b and renders it insensitive to GCK-3 activity. We conclude that both S742 and S747 must be phosphorylated for inhibition of CLH-3b and that both residues must be dephosphorylated for channel activation in response to cell swelling.

7. Rostafuroxin Antagonizes Adducin-mediated Activation of Na-KATPase–Src Signaling Pathway in Congenic Hypertensive Rats. M. FERRANDI,¹ I. MOLINARI,¹ M.P. RASTALDI,² P. FERRARI,¹ and G. BIANCHI,^{3 1}Prassis Research Institute, Sigma-Tau, 20019 Settimo Milanese, Milan, Italy; ²Fondazione D'Amico per la Ricerca sulle Malattie Renali, San Carlo Hospital, 20122 Milan, Italy; ³San Raffaele Hospital, Vita-Salute San Raffaele University, 20132 Milan, Italy

Adducin mutations are associated with genetic hypertension in humans and Milan hypertensive rats (MHS). Mutated Adducin affects actin cytoskeleton organization and increases renal Na-K pump activity in MHS rats and in transfected cells. In congenic rats (NA) having the mutated -adducin locus introgressed into the normotensive MNS background, hypertension was associated with activation of the Na-KATPase–Src-EGFr– and v-integrin–dependent signaling pathways within renal caveolae. Treatment of NA rats with the antihypertensive compound Rostafuroxin (100 µg/kg os) normalized blood pressure and the enhanced expression of Na-KATPase subunits, Src-Tyr⁴¹⁸, EGFr, and integrins, as demonstrated by immunofluorescence analysis on renal tissues and Western blotting on renal caveolae. In a cell-free system, human recombinant adducin dose-dependently activated Src kinase Tyr⁴¹⁸ phosphorylation, with the mutated variant displaying a higher apparent affinity than the wild type (46 ± 3.8 vs. 86 ± 5.7 nM; P < 0.01). In turn, Src-dependent Tyr phosphorylation of the mutated adducin in the absence or presence of Na-KATPase was higher than the wild-type adducin (35%; P < 0.05). Src-SH₂ and SH₃ domain–GST pull-down and competition experiments with Src-SH₂ peptides showed that mutated adducin preferentially associated to Src-SH₂ rather than Src-SH₃ domain (85 vs. 35%), whereas the wild type equally bound to Src domains (50%). Both adducins did not interact with the Src-kinase domain. 10^–11 M Rostafuroxin selectively reduced the Src-Tyr⁴¹⁸, adducin-, and Na-KATPase–Tyr phosphorylation (–30%; P < 0.05) induced by the mutated, but not wild-type, adducin. This effect occurred at a site within the phospho-Tyr–binding pocket of Src-SH₂ domain, where the mutated, but not the wild-type, adducin binds.

These findings provide a molecular explanation for the adducin-mediated activation of signaling pathways associated with hypertension and related organ complications and shed light on the molecular mechanism of Rostafuroxin antihypertensive effects.

8. Disruption of Plasma Membrane Calcium ATPase Isoform 2 (PMCA2) Alters Glutamate-evoked Calcium Transients in Enriched Cultures of Purkinje Cells. AMANDA K. LEE,¹ LARRY D. GASPERS,² ANDREW P. THOMAS,² and STELLA ELKABES,^{1 1}Department of Neurology and Neuroscience and ²Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ 07101

The cerebellum plays critical roles in motor coordination, learning, and certain cognitive functions. Cerebellar deficits are observed in several neurological disorders, including ataxia, multiple sclerosis, and autism spectrum disorders. Purkinje cells (PC), the output neurons of the cerebellar cortex, receive glutamatergic inputs that signal through metabotropic glutamate receptor 1 (mGluR1) and AMPA receptors (AMPA-R). Both receptors are important in cerebellar development, function, and plasticity. PCs also highly express a neuronal Ca²⁺ pump, PMCA2. Morphological, cellular, and molecular alterations in the cerebellum of PMCA2^–/– mice have been reported, emphasizing the importance of PMCA2 in the development of this region.

We have previously found that PMCA2 forms a complex with mGluR1, its downstream effectors, and AMPA-R subunits, GluR2/3 (Kurnellas, M.P., A.K. Lee, H. Li, L. Deng, D.J. Ehrlich, and S. Elkabes. 2007. Mol. Cell Neurosci. 34:178–188). The current studies investigated the role of PMCAs and especially PMCA2 in the clearance of AMPA-R and mGluR1-mediated Ca²⁺ transients. Using enriched PC cultures, we found that carboxy eosin (CE), a PMCA inhibitor, increases the amplitude of AMPA-R–mediated Ca²⁺ responses and slows the decay of Ca²⁺ transients. In contrast, CE decreases the amplitude of mGluR1-mediated Ca²⁺ transients and accelerates the clearance rate. Interestingly, we observed a reduction in the amplitude of AMPA-R–mediated Ca²⁺ transients in PCs of PMCA2^–/– mouse cerebellum. This may be caused by developmental changes, including alterations in PC dendrites (Empson, R.M., M.L. Garside, and T. Knöpfel. 2007. J. Neurosci. 27:3753–3758), synapses, expression of AMPA-R, and endoplasmic reticulum Ca²⁺ homeostasis. In contrast, an increase in AMPA-R–mediated Ca²⁺ transients occurred in PCs of PMCA2^+/– mouse cerebellum, reminiscent of the aforementioned effects observed after inhibition of PMCAs by CE. These data indicate that disruption of PMCA activity or expression differentially alters glutamate receptor–mediated Ca²⁺ signaling in cerebellar PCs. Future studies will determine how reductions in PMCA2 affect mGluR1 and AMPA-R–mediated responses in PCs and whether PMCA2 plays an essential role in PC pathology.

9. Development of an Intracellular cAMP "Sponge". KONSTANTINOS LEFKIMMIATIS,^1,2 JESSICA ROY,^1,2 MEERA SRIKANTHAN,^1,2 MARY PAT MOYER,³ SILVANA CURCI,^1,2 and ALDEBARAN M. HOFER,^{1,2 1}VA Boston Healthcare System and ²Department of Surgery, Brigham and Women's Hospital, Harvard Medical School, West Roxbury, MA 02132; ³NCELL Corporation LLC, San Antonio, TX 78249

Cyclic AMP is a fundamental second messenger known for its ability to modulate a wide variety of cellular responses. How one simple signaling molecule is able to simultaneously control so many (sometimes contradictory) cellular functions is an extremely active area of investigation. The existence of spatially distinct cAMP microdomains has been invoked to explain this paradox, but do these signaling microdomains really exist? To determine the importance of these putative cAMP domains, it would be extremely useful to selectively perturb the cAMP concentration in situ, limiting its ability to activate localized effectors. This strategy has been successfully used for other soluble second messengers ("InsP3 sponge" for InsP3 and BAPTA for Ca⁺²). Here, we present the generation and validation of a novel buffer for cAMP based on the cAMP-binding domains of PKA regulatory subunit type Iβ (RIβ). To quantitatively assess the expression of our "cAMP sponge" construct at the single-cell level, we labeled it with the fluorescent protein mCherry. As measured by FRET-based sensors for cAMP, expression of sponge constructs targeted to cytoplasm, plasma membrane, and nucleus in NCM460 cells significantly reduced cAMP responses to 10 nM PGE2, 10 nM VIP, and 5 µM forskolin. As expected, attenuation of agonist-induced cAMP signals also resulted in reduction in PKA activation as measured by the FRET-based sensor AKAR2. Interestingly, we found that the extra cAMP buffering power provided by the cAMP sponge can be compensated by second messenger derived from connected neighboring cells that do not express the exogenous buffer, revealing new aspects of gap junction–mediated communication. This new tool should also prove valuable for assessing the importance of cAMP microdomains in secretion, migration, and transcriptional regulation.

10. How the Sarcoplasmic Reticulum Ca²⁺-ATPase Pumps Ca²⁺ and is Inhibited by Thapsigargin. JESPER V. MØLLER,¹ CLAUS OLESEN,¹ MARTIN PICARD,² PREBEN MORTH,² ANNE-MARIE L. WINTER,² POUL NISSEN,² SØREN B. CHRISTENSEN,³ and HELMER SØHOEL,^{3 1}Institute of Physiology and Biophysics and ²Department of Molecular Biology, PUMPKIN Research Center, Aarhus University, DK-8000 Aarhus C, Denmark; ³Department of Medicinal Chemistry, University of Copenhagen, DK-1165 Copenhagen K, Denmark

The recent explosion of structures of sarcoplasmic reticulum (SR) Ca²⁺-ATPase in various functional states, solved at atomic resolution by x-ray diffraction analysis of protein crystals, enables a description of the whole enzymatic transport cycle of this actively Ca²⁺/H⁺-exchanging protein. We have recently analyzed three new structures (one E1P and two E2P) in detail (Olesen, C., M. Picard, A.M. Winther, C. Gyrup, J.P. Morth, C. Oxvig, J.V. Møller, and P. Nissen. 2007. Nature. 450: 1036–1042). They reveal how cytoplasmic Ca²⁺ after phosphorylation of Ca²⁺-ATPase by ATP is occluded inside the membranous domain and released by opening of the compact 10-transmembrane domain into three lobes to expose the intramembranously bound Ca²⁺ toward the lumenal space. This is consistent with a transport mechanism in which the Ca²⁺-binding sites communicate either with the cytoplasm or with the SR lumen. The new E2P structures were obtained without stabilization by thapsigargin (TG). They show how TG, when bound to the Ca²⁺-ATPase, is situated at the protein–lipid interphase in a preformed notch between transmembrane segments M3, M5, and M7, with minor adjustments of some of the amino aid side chains. They also provide hints to why TG inhibits the binding of Ca²⁺. These structural data are useful in the attempt to synthetize new sesquiterpene prodrugs (Søhoel, H., A.M. Jensen, J.V. Møller, P. Nissen, S.R. Denmeade, J.T. Isaacs, C.E. Olsen, and S.B. Christensen. 2006. Bioorg. Med. Chem. 14:2810–2815) that can be targeted to produce apoptosis in prostatic cancer cells (Denmeade, S.R., C.M. Jakobsen, S. Janssen, S.R. Khan, E.S. Garrett, H. Lilja, S.B. Christensen, and J.T. Isaacs. 2003. J. Natl. Cancer Inst. 95:990–1000).

11. Secretory Pathway Stress as a Common Disease Mechanism in SERCA2 or SPCA1 Ca²⁺ Pump Deficiency. GARY E. SHULL, GBOLAHAN OKUNADE, MARIAN L. MILLER, and VIKRAM PRASAD, Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45231

Gene-targeting and human genetics studies show that heterozygous-null mutations in the genes encoding the SERCA2 (Atp2a2) or SPCA1 (Atp2c1) Ca²⁺ pumps, which are expressed in endoplasmic reticulum (ER) or Golgi apparatus, respectively, cause squamous cell tumors in mice and acantholytic skin disease (Darier Disease and Hailey-Hailey Disease) in humans. Despite species differences in disease manifestations, the similarities between the target cell (keratinocytes in both) and phenotypic effects of the two Ca²⁺ pump deficiencies suggest a common disease mechanism. In the case of SERCA2 deficiency, the involvement of ER stress responses is a likely possibility. Loss of both copies of the SPCA1 gene in mice leads to embryonic death at gestation day 10.5; however, cardiovascular development and hematopoiesis, which are often associated with death at this stage, were normal. Apoptosis was increased in null mutant embryos, suggesting a general loss of cell viability. Membrane structures such as caveolae, junctional complexes, and desmosomes were normal, which is consistent with a functional secretory pathway; however, Golgi membranes were dilated, had fewer stacked leaflets, and were greatly expanded in amount. These studies of SPCA1-null embryos reveal clear evidence of Golgi stress, with both proapoptotic and prosurvival stress responses that are analogous to the well-known ER stress responses observed when SERCA2 activity is deficient. The available data support a model in which prosurvival responses to ER or Golgi stress predominate in mouse keratinocytes and lead to cancer, whereas proapoptotic responses predominate in humans and lead to the acantholytic skin diseases Darier Disease or Hailey-Hailey Disease.

	Session 2: Cardiac and Skeletal Muscle

TOP Session 1: Pumps Session 2: Cardiac and... Session 3: Neurodegeneration Session 4: Central Nervous... Session 5: TRP Channels

 
12. The _1S III-IV Loop Influences DHPR Gating but is Not Directly Involved in Excitation–Contraction Coupling Interactions with RyR1. ROGER A. BANNISTER and KURT G. BEAM, Department of Physiology and Biophysics, University of Colorado-Denver, Aurora, CO 80045

In skeletal muscle, coupling between the dihydropyridine receptor (DHPR) and type 1 ryanodine receptor (RyR1) underlies excitation–contraction (EC) coupling. The III-IV loop of the DHPR _1S subunit binds to a segment of RyR1 in vitro (Leong, P., and D.H. MacLennan. 1998. J. Biol. Chem. 273:29958–29964), and mutations in the III-IV loop have been linked to malignant hyperthermia (Monnier, N., V. Procaccio, P. Stieglitz, and J. Lunardi. 1997. Am. J. Hum. Genet. 60:1316–1325), raising the possibility that this loop is directly involved in signal transmission from the DHPR to RyR1. To clarify the role of the _1S III-IV loop in EC coupling, we examined the functional properties of a chimera (GFP-_1S[III-IVa]) in which the III-IV loop of the divergent _1A isoform replaced that of _1S. Dysgenic myotubes expressing GFP- _1S[III-IVa] yielded myoplasmic Ca²⁺ transients that were z65% smaller than those of GFP-_1S and that displayed an z10-mV hyperpolarizing shift in voltage dependence of activation. Relative to GFP-_1S, L-type Ca²⁺ currents mediated by GFP-_1S[III-IVa] were z40% smaller and activated at z5 mV less depolarized potentials. The altered gating of GFP-_1S[III-IVa] was accentuated by exposure to ±Bay K 8644, which caused a much larger hyperpolarizing shift in activation compared with its effect on GFP-_1S. GFP-_1S[III-IVa] channels appeared to traffic less efficiently to the membrane, as indicated by an z65% reduction in voltage-dependent charge movements, which was similar in magnitude to the reduction of maximal Ca²⁺ transients. Collectively, our observations indicate that the _1S III-IV loop is not directly involved in EC coupling but indirectly influences EC coupling by regulating DHPR gating.

13. Interplay of Calcium and Calmodulin Binding Motif in Regulation of Cardiac Sodium Channel Gating. SUBRATA BISWAS, DEBORAH DISILVESTRE, YANLI TIAN, VICTORIA L. HALPERIN, and GORDON F. TOMASELLI, Department of Medicine, Division of Cardiology, Johns Hopkins University, Baltimore, MD 21287

Intracellular Ca²⁺ can mediate bimodal regulation of the voltage-gated cardiac sodium channel (Na_V1.5) through its C terminus (CT) via the calmodulin (CaM)-binding IQ motif and the direct Ca²⁺-binding EF hand–like (EFL) motif of the channel. Although the EFL and IQ motifs are at opposite ends of the proximal structured portion of the CT-Na_V1.5, mutations in either motif have been associated with arrhythmogenic changes in expressed Na_V1.5 currents. Ca²⁺ binding to Na_V1.5 shifts steady-state inactivation in the depolarizing direction and delays entry into inactivated states. Mutation of the four EFL residues E1788A, D1790A, D1792A, and E1799A (Na_V1.5_4X) stabilizes inactivation compared with the wild-type channel and abolishes the Ca²⁺ sensitivity of inactivation gating. Modulation of the steady-state availability of Na_V1.5 by intracellular Ca²⁺ is more pronounced after the truncation of CT proximal to the IQ motif (Na_V1.5₁₈₈₅), which retains the EFL. In contrast, CaM has no apparent effect on Na_V1.5 channel function at physiological [Ca²⁺], yet FRET between fluorophore-conjugated CaM and CT of intact Na_V1.5 channels demonstrate CaM tethering to channel, indicating a latent CaM regulation of the wild-type channel. Mutating the EFL unmasks CaM-mediated regulation of the kinetics and voltage dependence of inactivation. The latent CaM modulation of inactivation is eliminated by mutation of the IQ motif (Na_V1.5_4X-IQ/AA). Thus, Ca²⁺ binding to the EFL motif plays a critical role in controlling Na_V1.5 availability in the heart through direct binding of Ca²⁺ independent of any CaM–IQ interaction. Unlike other isoforms of the Na channel, the IQ–CaM interaction in the CT-Na_V1.5 is latent under physiological conditions but may become manifest in the presence of disease-causing mutations in the CT of Na_V1.5 (particularly in EFL), contributing to the production of potentially lethal ventricular arrhythmias. However, the concerted action of the two motifs finely tunes normal Na channel inactivation.

14. Calcium Transients Evoked by Electrical Stimulation are In Part Dependent on ATP Release and P2X/P2Y Receptor Activation in Skeletal Muscle Cells. SONJA BUVINIC,¹ JAVIERA LÓPEZ,² JUAN PABLO HUIDOBRO-TORO,² JORDI MOLGÓ,³ and ENRIQUE JAIMOVICH,^{1 1}Centro de Estudios Moleculares de la Célula, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Casilla 70005, Santiago, Chile; ²Centro de Regulación Celular y Patología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile; ³Laboratoire de Neurobiologie Cellulaire et Moléculaire, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France

ATP released to the extracellular medium by all cell types fulfill several physiological roles through activation of plasma membrane P2X (ion channels) or P2Y (metabotropic) receptors. Several subtypes of these receptors are differentially activated by ATP, UTP, or their metabolites (ADP and UDP) generated by ectonucleotidase activities. Skeletal muscle cells express several P2X and P2Y receptor subtypes, and ATP is profusely released during muscle activity. We previously demonstrated that in skeletal myotubes, depolarizing stimuli induces two calcium signals: a fast signal associated with excitation–contraction coupling and a slow signal that has an important component in the nucleus and regulates gene expression. Here, we propose that extracellular nucleotides released by electrical stimulation are in part responsible for intracellular calcium homeostasis. In rat skeletal myotubes, we demonstrated that a tetanic stimulus (45 Hz, 400 pulses, 1 mseg each) rapidly increased extracellular levels of ATP, ADP, and AMP from 15 s to 3 min, with different half-life times. Exogenous ATP applications induced a dose-dependent increase in intracellular calcium with an EC₅₀ value of 7.8 ± 3.1 µM. Exogenous ADP, UTP, and UDP also promote calcium transients. By RT-PCR, we detected mRNA expression for P2X_1-7 and P2Y_1,2,4,6,11 in these cells. Both fast- and slow-calcium signals evoked by tetanic stimulation were partially inhibited by either 10–100 µM suramin (nonselective P2X/P2Y blocker) or 2 U/ml apyrase (nucleotidase that metabolizes ATP and ADP to AMP). In hemidiaphragm preparations, we demonstrated that apyrase reduces both twitch- and tetanus-evoked increase in tension. Our results suggest that nucleotides endogenously released during skeletal muscle activity act through P2X and P2Y receptors to modulate both calcium homeostasis and muscle physiology. (Supported by FONDAP 15010006, FONDAP 13980001, and Fondecyt 3080016.)

15. Maintenance of Muscle Cell Membrane Integrity and the Pathogenesis of Muscular Dystrophy. KEVIN P. CAMPBELL, Department of Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, Howard Hughes Medical Institute, University of Iowa, Iowa City, IA 52242

Muscular dystrophy is a group of inherited myogenetic disorders characterized by progressive muscle weakness and wasting caused by muscle fiber necrosis. A large number of genes associated with various types of muscular dystrophies have been identified. Interestingly, 12 of these muscular dystrophic genes are related to the dystrophin–glycoprotein complex (DGC). This multisubunit complex is present at the muscle sarcolemma and connects the basement membrane surrounding the muscle fiber to the intracellular cytoskeleton. The genes affected encode for integral proteins of the DGC, or extracellular matrix proteins that bind the DGC, or enzymes necessary for glycosylation of the central DGC component, dystroglycan. Emerging cell and molecular data on mouse models and patient biopsies strongly support the hypothesis that defects in dystroglycan are central to the pathogenesis of structural and functional brain abnormalities seen in congenital MD. Another class of muscular dystrophies in which repair, not structure, of the plasma membrane is abnormal is linked to mutations in dysferlin. The elucidation of the molecular pathogenesis of various forms of muscular dystrophy is providing new therapeutic strategies for the treatment of these diseases.

16. Effect of Adrenergic Stimulation on Resting [Ca²⁺] in the Sarcoplasmic Reticulum (SR) of Frog Skeletal Muscle Assessed with the Indicator Tetramethylmurexide (TMX). GABOR GYURKOVICS and PAUL C. PAPE, Université de Sherbrooke Faculté de Médecine et des Sciences de la Santé, Département de Physiologie et Biophysique, Sherbrooke, Québec J1H5N4, Canada (Sponsor: Paul C. Pape)

Several past studies indicated that adrenergic stimulation enhances the directly stimulated twitch response of fast-twitch skeletal muscle. Arreola et al. (Arreola, J., J. Calvo, M.C. García, and J.A. Sánchez. 1987. J. Physiol. 393:307–330) reported that the potentiation required a train of closely spaced action potentials (aps) and attributed this requirement to the build up of voltage activation of L-type Ca channels during the train of aps. In contrast to this idea, other researchers report that adrenaline greatly enhances the first twitch response after adrenergic stimulation, which suggests that the enhanced Ca entry occurred in the resting state (Gonzalez-Serratos, H., L. Hill, and R. Valle-Aguilera. 1981. J. Physiol. 315:267–282). To resolve this issue, we evaluated the effect of adrenergic stimulation (10 µM isoproterenol) on the fraction of the Ca indicator tetramethylmurexide (TMX) complexed with Ca (denoted f_Ca), an indication of [Ca²⁺] in the SR and, less directly, the total Ca content in the fiber (Pape, P.C., K. Fénelon, C.R. Lamboley, and D. Stachura. 2007. J. Physiol. 581.1:319–367). These experiments were done on cut fibers from frog mounted in a double-Vaseline gap chamber and held at the normal resting potential of –90 mV. For the main set of results, the internal solution was nominally physiological (high K-glutamate) with 0.1 mM EGTA and no added Ca. The external solution was normal Ringers. Before the addition of isoproterenol to the external solution, f_Ca had a maintained, steady value of 0.08 (SEM = 0.02; n = 8). With an exponential delay () after the start of isoproterenol exposure ( = 7.07 min; SEM = 0.55 min), f_Ca progressively increased, reaching a sustained rate of increase of 0.0039/min (SEM = 8). Isoproterenol had no effect on f_Ca with no Ca in the external solution. These results indicate that adrenergic stimulation causes a significant net entry of Ca in resting muscle.

17. Mitochondrial Calcium Signaling in Skeletal Muscle Disease. GYÖRGY HAJNÓCZKY, CHRISTOPHER J. BUZAS, MUQING YI, PAL PACHER, and HENRY ROSENBERG, Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107

Calcium oscillations exert physiological control on mitochondrial energy metabolism but also induce mitochondrial membrane permeabilization and ensuing cell death. The specificity of the mitochondrial calcium signaling is dependent on the coincident presence of stress factors like reactive oxygen species (ROS), ceramide, and on the amount and spatiotemporal pattern of the Ca²⁺ transfer to the mitochondria. The latter factor is coming into focus based on recent evidence revealing that many human cardiac/skeletal muscle and neurodegenerative diseases are associated with mutations of the intracellular Ca²⁺ release channels and their regulatory proteins and present early mitochondrial impairments. Sensitization of IP3R/RyR-mediated Ca²⁺ release is caused by a variety of mutations associated with malignant hyperthermia (MH)/central core disease, idiopathic paroxysmal ventricular tachycardia, and Huntington disease. However, dependence of the mitochondrial injury on the altered Ca²⁺ mobilization remains elusive. To test the hypothesis that mutations promoting Ca²⁺ release through RyR1 may elicit mitochondrial Ca²⁺ overload and make mitochondria vulnerable to membrane permeabilization, we obtained primary skeletal myoblasts from MH-negative (MHN) and malignant hyperthermia susceptible (MHS; e.g., RyR1 A2347V) individuals and investigated the effect of halothane, a toxic anesthetic for MH patients, on mitochondrial morphology and function. Imaging of cytosolic [Ca²⁺] ([Ca²⁺]_c) and mitochondrial matrix [Ca²⁺] ([Ca²⁺]_m) showed that the halothane-induced [Ca²⁺]_c signal is propagated to the mitochondria, giving rise to a large [Ca²⁺]_m elevation. The halothane-induced [Ca²⁺]_c signal was also associated with a decrease in mitochondrial membrane potential and in NAD(P)H fluorescence. In a side-by-side comparison of MHN and MHS myotubes, accentuated halothane-induced depolarization was observed in the MHS cells. After permeabilization of the plasma membrane, both halothane and Ca²⁺ pulses evoked a relatively large mitochondrial depolarization in MHS cells. This was associated with release of cytochrome c from mitochondria to the cytosol, activating a cell killing mechanism. Thus, the halothane-induced sarcoplasmic reticulum Ca²⁺ mobilization is effectively propagated to the mitochondria. In MHS cells, the enhanced Ca²⁺ mobilization may provide a mechanism to damage the mitochondria. Furthermore, halothane may also directly target a mitochondrial factor that synergizes with [Ca²⁺]_m to cause impairment of the mitochondrial membrane barrier and metabolic function.

18. The Total Ca Contents of Skeletal Muscle from Frogs and Rats – Implications for the Ca-binding Capacity of Calsequestrin. CÉDRIC R.H. LAMBOLEY and PAUL C. PAPE, Université de Sherbrooke Faculté de Médecine et des Sciences de la Santé, Département de Physiologie et Biophysique, Sherbrooke, Québec J1H5N4, Canada (Sponsor: Cedric Lamboley)

Results in Table III of Pape et al. (Pape, P.C., K. Fénelon, C.R. Lamboley, and D. Stachura. 2007. J. Physiol. 581.1:319–367) indicate that >90% of the total Ca in the sarcoplasmic reticulum (SR) is complexed to the high capacity Ca-binding protein calsequestrin in resting fast-twitch muscle of frog. In stark contrast to this result, Royer et al. (Royer, L., S. Pouvreau, Y. Wang, G. Meissner, J. Zhou, A. Nori, P. Volpe, J.W. Bain, D.A. Riley, R. Fitts, and E. Rios. 2008. Biophys. J. 94:2685) recently reported no significant changes in total releasable Ca when calsequestrin (CSQ1) is knocked down in fast-twitch skeletal muscle of mice. To resolve this issue, we developed a quantitative method for measuring the total amount of Ca in a muscle (or any other tissue, for that matter) using the Ca-dependent UV absorbance properties of the high-affinity Ca buffer BAPTA. In brief, whole fast-twitch muscle (ileo-fibularis from frog and EDL from rat) were placed in a Ringers (frog) or Krebs solution (rat) with 0 Ca and 0.1 mM EGTA for z1.5 h to remove external Ca. The muscle was then added to a solution containing 0.1 mM BAPTA and homogenized with a blender. After removal and weighing of small pieces of tendon, an additional 0.1-mM BAPTA solution was added to give a total volume of 50 ml. 1% Triton X-100 was added to permeabilize membranes. Total muscle Ca ([Ca_T]; given in units millimoles per 1,000 grams of wet weight of muscle, less the tendon weight) was determined from absorbances at 306 nm of the supernatant and the supernatant with three levels of Ca: 0Ca, Ca, and with addition of a known Ca standard. In frog, the average value for [Ca_T] of 1.89 (SEM = 0.1; n = 5) is very close (within 1%) to that expected from previous measurements in cut fibers (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259–336). In rat, the average value of 3.3 (SEM = 0.2; n = 3) indicates that Ca binding to calsequestrin must be even more important in rat than in frog.

19. Heart Failure and Sudden Cardiac Death: Causes and Cures. ANDREW R. MARKS, Department of Physiology and Cellular Biophysics, Clyde and Helen WU Center for Molecular Cardiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032

Heart failure and sudden cardiac death are leading causes of mortality. Developing effective therapy has been hampered because of a lack of understanding of the mechanisms that cause these disorders. We have shown that "leaky" ryanodine receptor/calcium release channels (RyR2) contribute to heart failure and sudden cardiac death in humans and relevant animal models. RyR2 is the major calcium release channel on the sarcoplasmic reticulum and is required for excitation-contraction coupling. RyR2 becomes leaky in failing hearts as a result of PKA hyperphosphorylation of the channel that results from chronic activation of the "fight or flight" stress response. PKA hyperphosphorylation decreases the binding affinity of calstabin2, a subunit of the channel, rendering the channels leaky. Treatment with a novel 1,4 benzothiazepine derivative known as a rycal that enhances the affinity of calstabin2 for PKA-phosphorylated RyR2 improves cardiac function and prevents sudden cardiac death in animal models.

20. Calcium Dysfunction in Ankyrin-based Human Cardiac Arrhythmias. PETER J. MOHLER, Department of Internal Medicine and Department of Physiology, University of Iowa Carver College of Medicine, Iowa City, IA 52242

A number of serious human genetic diseases are caused by abnormal targeting and expression of ion channel and transporters at specialized membrane domains. Our research focuses on the molecular mechanisms underlying ion channel and transporter targeting in cardiac and other excitable cells. In particular, we are interested in the role of the membrane-associated ankyrin family of polypeptides in the targeting and function of ion channels and transporters. Our recent work establishes that loss-of-function mutation in ankyrin-B is the basis for a new human cardiac arrhythmia syndrome associated with bradycardia, abnormal heart rate variability, repolarization defects, and polymorphic tachyarrhythmia in response to stress and/or exercise. Additionally, our work revealed that reduction of ankyrin-B in mice results in reduced levels and abnormal localization of Na/Ca exchanger, Na/K ATPase, and InsP3 receptor at transverse-tubule/sarcoplasmic reticulum sites in cardiomyocytes and leads to altered Ca²⁺ signaling and extrasystoles that provide a rationale for the arrhythmia. Current work is focused on two ankyrin-based areas of research directly related to human disease. Specifically, we are interested in identifying the molecular determinants of ankyrin-B–dependent targeting of ion channels and transporters to specialized T tubule/SR membranes required for normal calcium homeostasis and cardiac function. Additionally, we are actively characterizing the role of ankyrin-G in targeting of voltage-gated sodium channel Nav1.5 (SCN5A) to excitable membranes of ventricular cardiomyocytes.

21. Cross Talk between Myostatin and IGF-1 Transduction Pathways Mediated by Calcium in Skeletal Muscle Cells. JUAN ANTONIO VALDÉS and ALFREDO MOLINA, Departamento de Ciencias Biológicas, Facultad de Ciencias de la Salud, Universidad Andrés Bello, Santiago, Chile

Skeletal muscle growth and development are very complex and regulated processes that begin during the early embryonic muscle myogenesis and continue throughout life during muscle regeneration and repair. The myogenic program involves several coordinated steps regulated by extracellular cues, like myostatin and insulin-like growth factor-1 (IGF-1). IGF-1 is a positive regulator in proliferation and differentiation of skeletal muscle cells, whereas myostatin is a member of the transforming growth factor β superfamily that acts as a negative regulator of skeletal muscle mass. These growth factors exert their antagonist functions, activating different transduction pathways mediated by their receptors. Myostatin has been shown to bind to Activin type II receptors, activating a signal transduction pathway that leads to phosphorylation of the transcription factors Smad2/3, which ultimately leads to suppression of myogenesis. On the other hand, almost all biological actions are mediated by binding to the IGF-1 receptor, activating transduction pathways like phosphoinositide 3-kinase (PI3K)–phospholipase C (PLC) that will induce inositol triphosphate (IP3) production and subsequently calcium release from intracellular stores. However, experimental data obtained by our group strongly suggest a negative feedback through a cross talk between IGF-1 and myostatin receptors mediated by calcium: (1) myostatin inhibits calcium release induced by IGF-1 in C2C12 myoblast; (2) myostatin inhibits the activation of NFAT, a calcium-dependent transcription factor; (3) myostatin inhibits NFAT-dependent transcription during myoblast differentiation and fusion. Considering the fact that IGF-1 can induce calcium release from IP3R intracellular stores mediated by the activation of PI3K-PLC, we propose that myostatin diminishes the PI3K or PLC activity, thereby inhibiting the IP3-induced calcium release and subsequent activation of calcium-dependent transduction pathways. Actually, we are performing experiments to analyze the contribution of IP3 pathways in the cross talk between myostatin and IGF-1.

	Session 3: Neurodegeneration

TOP Session 1: Pumps Session 2: Cardiac and... Session 3: Neurodegeneration Session 4: Central Nervous... Session 5: TRP Channels

 
22. REST-dependent Epigenetic Remodeling of AMPA Receptor Ca²⁺ Permeability is Critical to Ischemia-induced Neuronal Death. M.V.L. BENNETT, K.M. NOH, A. FOLLENZI, T. MIYAWAKI, J.M. GREALLY, and R.S. ZUKIN, Albert Einstein College of Medicine, Bronx, NY 10461

AMPA receptors (AMPARs) mediate fast synaptic transmission at excitatory synapses of the CNS and are crucial to neuronal development, synaptic plasticity, and structural remodeling. The subunit composition and Ca²⁺ permeability of AMPA receptors are not static but are dynamically remodeled in a cell- and synapse-specific manner. The precise mechanisms underlying the switch in AMPAR phenotype remain controversial. Here, we show that after a 10-min period of global ischemia, the transcriptional repressor REST acts via epigenetic modifications to repress GluR2 expression, leading to formation of GluR2-lacking Ca²⁺-permeable AMPARs by neurons in the adult hippocampal CA1 (but not CA3). Increased Ca²⁺ influx in response to glutamate released at excitatory synapses or by activated astrocytes leads to neuronal death specific to CA1. Global ischemia promotes assembly of the transcriptional repressor REST, CoREST, mSin3A, and HDACs (the REST corepressor complex) over the GluR2 promoter specifically in CA1 pyramidal neurons. The REST–corepressor complex promotes deacetylation of the core histone protein H3, dimethylation of H3 at lysine 9 (H3K9me2) but not lysine 4 (H3K4), and association with MeCP2, an epigenetic signature of gene silencing. Inhibition of REST by lentiviral delivery of a recombinant mutant REST (REST-VP16) or of a REST RNA interference (RNAi) sequence into the hippocampus of rats before the ischemic insult rescues GluR2 expression and ameliorates hippocampal injury. These findings document a causal role for REST-dependent epigenetic remodeling of target genes that regulate AMPAR Ca²⁺ permeability in delayed neurodegeneration after ischemia and identify REST as a novel therapeutic target for ischemic stroke.

23. Src Family Kinases Link Death Receptors to the Apoptotic Calcium Machinery. DARREN BOEHNING, ASKAR AKIMZHANOV, and XINMIN WANG, Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX 77555

Inositol 1,4,5-trisphosphate receptor (IP₃R) channels mediate apoptotic calcium release in response to both intrinsic and extrinsic cell death signals. We recently demonstrated that apoptotic calcium release mediated by the death receptor Fas is dependent upon phospholipase C (PLC)– activity and subsequent IP₃R-mediated calcium release. However, the mechanism by which Fas receptor is coupled to PLC- activation is unknown. Here, we show that Fas receptor ligation in T cells is associated with the recruitment and activation of src family kinases canonically associated with T cell receptor engagement. Genetic inactivation of Lck and Zap70 eliminated PLC- phosphorylation, activation, and subsequent calcium release. These effects could be rescued by stably overexpressing the respective kinases. Recruitment and activation of Lck and Zap70 after Fas receptor clustering was dependent on segregation of these signaling components to lipid rafts, where they combined with the canonical components of the death-induced signaling complex (DISC). These results reveal the surprising finding that Fas receptor signaling mimics T cell receptor signaling and uncovers a novel pathway that is absolutely required for apoptotic calcium release and cell death after death receptor activation.

24. Absence of InsP₃R Ca²⁺ Signals Induce AMPK-dependent Prosurvival Autophagy. CESAR CARDENAS, KING-HO CHEUNG, JUN YANG, HORIA VAIS, and KEVIN FOSKETT, Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

Inositol trisphosphate receptor (InsP₃R) Ca²⁺ release channels are ubiquitously expressed as three different isoforms. Knockdown of InsP₃R expression induced autophagy, a conserved cell survival mechanism that may also promote cell death (Criollo, A., M.C. Maiuri, E. Tasdemir, I. Vitale, A.A. Fiebig, D. Andrews, J. Molgó, J. Díaz, S. Lavandero, F. Harper, et al. 2007. Cell Death Differ. 14:1029–1039). Alternately, InsP₃R-mediated Ca²⁺ release was shown to induce autophagy (Høyer-Hansen, M., L. Bastholm, P. Szyniarowski, M. Campanella, G. Szabadkai, T. Farkas, K. Bianchi, N. Fehrenbacher, F. Elling, R. Rizzuto, et al. 2007. Mol. Cell. 25:193–205). In normal growth medium, a significantly higher percentage of chicken DT40 B cells genetically deficient in all InsP₃R isoforms (KO) were autophagic compared with InsP₃R-expressing WT cells as measured by electron microscopy, LC3-II formation, and LC3-GFP localization. Viability was not different between the two lines in normal growth medium, but KO cells were resistant to nutrient depletion–induced cell death. Pharmacologic inhibition of autophagy was without effect on WT cell survival in normal growth medium, whereas it caused the death of KO cells, and it eliminated enhanced resistance of KO cells to nutrient deprivation, indicating that autophagy becomes activated as a prosurvival mechanism in the absence of InsP₃R expression. Autophagy was induced specifically by loss of InsP₃R because expression of rat InsP₃R-3 in KO cells reduced autophagy to control levels. Ca²⁺ release through InsP₃R was required because blocking InsP₃R with Xestospongin B induced autophagy, and expression of permeation and gating-deficient InsP₃R failed to reduce autophagy in KO cells. Suppression of autophagy was specific for InsP₃R because expression of RyR failed to reduce autophagy in KO cells. The metabolic sensor kinase AMPK was activated in KO cells, although phosphorylation of mTOR was unchanged. Inhibition of AMPK with compound C or addition methyl-pyruvate reduced autophagy. We conclude that InsP₃R-mediated Ca²⁺ signaling is fundamentally required to preserve optimal bioenergetic status in cells. Its absence triggers a compensatory prosurvival autophagy mediated by AMPK and unidentified downstream effectors that do not include mTOR.

25. Deviant Ryanodine Receptor–mediated Calcium Release Alters Synaptic Activity in Presymptomatic Alzheimer's Disease Mice. SHREAYA CHAKROBORTY, IVAN GOUSSAKOV, MEGAN MILLER, and BETH STUTZMANN, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064

ER calcium signaling is critical for many neuronal signaling events, including modulation of synaptically evoked responses and long-term plasticity. In neurons expressing Alzheimer's disease (AD)–linked presenilin (PS) mutations, ER calcium dysregulation occurs early in the disease process, before the onset of histopathology and memory deficits. Neuronal compartments associated with dense synaptic contacts, such as dendritic spine heads and distal processes, show particularly high relative increases in RyR-sensitive calcium release compared with nontransgenic mice. However, it is largely unknown how synaptic transmission and plasticity mechanisms are affected by exaggerated ER calcium release.

In this study, ryanodine (RyR)-mediated alterations in calcium signaling and synaptic activity were examined in cortical and hippocampal neurons from young (6–8 wk old) triple transgenic (3xTg-AD) and nontransgenic (NonTg) control mice. Using two-photon calcium imaging and electrophysiological techniques, we demonstrate greatly exaggerated (>10-fold) calcium signals in dendritic spine heads and dendritic processes of pyramidal neurons from the AD mice relative to NonTgs. Under control conditions, membrane electrical properties, basal synaptic transmission, and plasticity mechanisms appear similar between the mouse strains. However, manipulating the RyR-calcium stores reveals remarkably different patterns of calcium signaling underlying these synaptic and membrane properties. Blocking the RyR with dantrolene has little effect on synaptic transmission and plasticity in the NonTg animals, yet it increases paired-pulse facilitation and synaptic strength and converts LTP expression to LTD in the 3xTg-AD mice. Activation of RyR by caffeine also alters synaptic transmission differently in AD mice compared with NonTgs. Synaptic stimulation concurrent with RyR activation reveals significantly increased calcium release in spine heads and dendrites in 3xTg-AD neurons. These findings demonstrate that 3xTg-AD pyramidal neurons operate under an aberrant yet seemingly functional calcium signaling and synaptic transmission system long before AD histopathology onset. These early signaling alterations may underlie the later synaptic breakdown and cognitive deficits characteristic of AD.

26. Mechanism of Calcium Disruption in Alzheimer's Disease by Presenilin Regulation of InsP₃ Receptor Channel Gating. KING-HO CHEUNG,¹ DIANA SHINEMAN,^2,4 MARIOLY MULLER,¹ CESAR CARDENAS,³ LIJUAN MEI,¹ JUN YANG,¹ TAISUKE TOMITA,⁵ TAKESHI IWASUBO,⁵ VIRGINIA M.-Y. LEE,^2,4 and J. KEVIN FOSKETT,^{1,3 1}Department of Physiology, ²Department of Pathology and Laboratory Medicine, ³Department of Cell and Developmental Biology, and ⁴Center for Neurodegenerative Disease Research, University of Pennsylvania, Philadelphia, PA 19104; ⁵Department of Neuropathology and Neuroscience, University of Tokyo, Tokyo 113-0033, Japan

Mutations in presenilins (PS) are the major cause of familial Alzheimer's disease (FAD) and have been associated with calcium (Ca²⁺) signaling abnormalities. Recently, it was proposed that PS may contain an intrinsic Ca²⁺ and monovalent ion permeability that is impaired in FAD-associated mutant PS (Tu, H., O. Nelson, A. Bezprozvanny, Z. Wang, S.F. Lee, Y.H. Hao, L. Serneels, B. De Strooper, G. Yu, and I. Bezprozvanny. 2006. Cell. 126:981–993). Here, using a different electrophysiological technique, nuclear patch clamping, we have been unable to verify that either wild-type (WT) or mutant PS form ion channels in native ER membranes. However, we have identified a molecular interaction of WT and FAD mutant PS with the inositol 1,4,5-trisphosphate receptor (InsP₃R) Ca²⁺ release channel. Both WT and FAD mutant PS1 (M146L) and PS2 (N141I) interact biochemically with the InsP₃R, as evidenced by coimmunoprecipitation using cell culture and brain lysates. In two model cell systems, nuclear patch clamp electrophysiology revealed that FAD mutant PS1 (M146L) and PS2 (N141I) exert profound stimulatory effects on InsP₃R channel gating activity in response to both saturating and suboptimal levels of InsP₃. The stimulatory activity results primarily from destabilization of the channel closed state. These functional interactions result in InsP₃R-dependent exaggerated cellular Ca²⁺ signaling in FAD PS-expressing cells in response to agonist stimulation as well as enhanced low level Ca²⁺ signaling in unstimulated cells. Parallel studies in InsP₃R-expressing and -deficient cells revealed that enhanced Ca²⁺ release from the endoplasmic reticulum as a result of the specific interaction of PS1-M146L with the InsP₃R stimulates amyloid β processing, an important feature of AD pathology. These observations provide molecular insights into the Ca²⁺ dysregulation hypothesis of AD pathogenesis and suggest novel targets for therapeutic intervention.

27. Bcl-2 Interacts with the InsP₃R to Increase Ca²⁺ Oscillations and Antioxidant Capacity. EMILY ECKENRODE,³ LIJUAN MEI,¹ J. KEVIN FOSKETT,^1,2 and CARL WHITE,^{3 1}Department of Physiology and ²Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104; ³Department of Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064

Expression of Bcl-2 inhibits apoptosis by a variety of mechanisms. Some of the physiological effects of Bcl-2 are mediated by modulation of the endoplasmic reticulum (ER)–localized inositol trisphosphate receptor Ca²⁺ release channel (InsP₃R). In addition, Bcl-2 expression repeatedly correlates with elevated levels of reduced glutathione (GSH), providing increased cellular antioxidant capacity and apoptotic resistance. This study examined the role of the Bcl-2–InsP₃R interaction in regulating Ca²⁺ homeostasis, apoptosis, and total cellular glutathione.

Using the DT40 cell line genetically deficient in all InsP₃Rs (DT40-TKO), stable lines were generated that overexpressed Bcl-2 in both the WT and TKO background. In permeabilized cells, experiments measuring ER Ca²⁺ flux revealed that Bcl-2 expression increased the apparent sensitivity of InsP₃R-dependent Ca²⁺ release to low levels of InsP₃. In intact cells, Ca²⁺ imaging experiments demonstrated that Bcl-2 expression in the WT increased both the frequency of spontaneous Ca²⁺ oscillations and the number of oscillating cells. Importantly, in WT and TKO expressing cells, Bcl-2 protected against apoptotic stimuli, although it provided significantly more protection in the InsP₃R-expressing WT cells. This was correlated with a significant increase in steady-state levels of GSH in WT but not TKO cells expressing Bcl-2.

Together, these data suggest that modulation of InsP₃R-dependent Ca²⁺ signaling by Bcl-2 is required to increase the cellular pool of reduced GSH. This supports a novel model in which Bcl-2 expression leads to InsP₃R-dependent redox adaptations that enable cells to better withstand an apoptotic stimulus.

28. Calcium Involvement in Toxic β Amyloid Peptide's Rapid Suppression of Kv1.1 Channel Activity. JOSEPH FARLEY, ALEC SEXTON, BRENT HALLAHAN, and JORDAN RAYNOR, Indiana University, Neuroscience Program, Bloomington, IN 47405

Substantial evidence implicates the amyloid β (Aβ1-42) peptide in the pathogenesis of Alzheimer's Disease (AD). Exactly how Aβ1-42 kills neurons remains unclear; most accounts view disruption of Ca²⁺ homeostasis as critical. Previous studies indicate that Aβ1-42 affects diverse neuronal K⁺ currents, but the molecular composition of the contributing channel subunits has generally been undefined. Kv1.1 is a voltage-dependent K⁺ channel responsible for repolarization of action potentials in many mammalian neurons, including those affected by AD. Suppression of Kv1.1 activity would be expected to increase Ca²⁺ influx. We report that Aβ1-42 produces a profound and rapid suppression of Kv1.1 currents, and this is partially dependent on [Ca²⁺]_i. Homomeric -subunit Kv1.1 channels were expressed in Xenopus oocytes, and macroscopic Kv1.1 currents were measured using standard voltage-clamp methods. Bath application of 1 µM Aβ1-42 produced an z50% decrease in Kv1.1 current (at 30 min), with no change in voltage dependency nor any indication of use-dependent pore block. Solvent and Aβ40-1 control peptide experiments produced little change (7%). Dose-response studies indicate a clear suppression of Kv1.1 current by Aβ1-42 concentrations as low as 1 nM. We have previously studied Ca²⁺-mediated suppression of Kv1.1 currents in oocytes by a pathway involving tyrosine phosphorylation, PKC, and calcineurin (PP2B) activation. We find that suppression of Kv1.1 by Aβ1-42 is partially mediated by this pathway. Although suppression was not dependent on Ca²⁺ influx, because Aβ1-42 addition to a Ca²⁺-free bath still produced strong suppression of K⁺ currents (z50%), partial abrogation (z50%) was produced by incubating oocytes in the Ca²⁺-chelator BAPTA-AM, indicating that [Ca²⁺]_i plays a role. Cyclosporine A (a PP2B inhibitor) also partially (z50%) blocked Kv1.1 suppression. Our results suggest that Aβ1-42 suppression of Kv1.1 (and related K⁺) channels may represent one of the earliest steps in AD neurotoxicity and that [Ca²⁺]_i is involved.

29. Heterogeneous Ligand Sensitivities of Single Insp₃ Receptor Ca²⁺-release Channels within Endoplasmic Reticulum Membrane Patches. MARISABEL FERNÁNDEZ-MONGIL, SUMAN DATTA, J. KEVIN FOSKETT, and DON-ON DANIEL MAK, Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104

Modulation of cytoplasmic-free Ca²⁺ concentration ([Ca²⁺]_i) by the ubiquitous inositol 1,4,5-trisphosphate (InsP₃) receptor (InsP₃R), an intracellular Ca²⁺-release channel localized to the endoplasmic reticulum (ER) membrane, regulates numerous physiological processes. A fundamental yet controversial aspect of the InsP₃-mediated Ca²⁺ signaling is the widely observed phenomenon of quantal Ca²⁺ release: the ability of cells to have graded release of Ca²⁺ from intracellular stores in response to incremental levels of extracellular agonist or cytoplasmic [InsP₃]. Many schemes have been proposed to account for this phenomenon, including Ca²⁺ stores containing InsP₃R with different ligand sensitivities, Ca²⁺ stores with different InsP₃R densities, and regulation of InsP₃R channel activities by [Ca²⁺] in the ER lumen. Here, we applied rapid solution exchange techniques in nuclear patch-clamp experiments with membrane patches obtained in the cytoplasmic–side out configuration to expose the cytoplasmic side of InsP₃R channels in the same membrane patches to alternating buffers containing different ligand (InsP₃ and Ca²⁺) concentrations under constant lumenal [Ca²⁺]. We observed that even in the same membrane patch, a larger number of InsP₃R channels was activated by more favorable ligand conditions in a graded manner up to the optimal ligand conditions (optimal [Ca²⁺]_i and saturating [InsP₃]). Such graded activation of InsP₃R channels in the same nuclear membrane patches was not only observed in endogenous InsP₃R channels in insect Sf9 cells with only one InsP₃R gene but also in recombinant rat type 3 InsP₃R channels stably transfected into DT40 cells with all three genes for the endogenous InsP₃R isoforms knocked out. Thus, a population of InsP₃R channels homogeneous in their primary sequences in the same Ca²⁺ store can nevertheless exhibit graded heterogeneous sensitivities to activation by [InsP₃] and [Ca²⁺]_i. (Supported by National Institutes of Health grant 5R01GM074999.)

30. Secretases, Oxidative Stress, and Perturbed Calcium Homeostasis in AD and Stroke. MARK P. MATTSON, Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, MD 21224

In this presentation, I will describe how cleavage of the β-amyloid precursor protein (APP) by , β and secretases influences neuronal calcium homeostasis in the contexts of synaptic plasticity and Alzheimer's disease. In brief, sequential cleavages of APP by β and secretases generate amyloid β peptide (Aβ). Aβ induces membrane-associated oxidative stress that impairs the function of ion-motive ATPases and glutamate and glucose transporters, thereby promoting elevations of intracellular calcium levels, which impairs synaptic function and renders neurons vulnerable to excitotoxicity. Cleavage of APP by secretase generates sAPP-, which activates a signaling pathway involving cGMP production and activation of potassium channels that hyperpolarizes neurons. Cleavage of Notch by secretase generates the Notch NICD, which translocates to the nucleus and modifies the expression of genes encoding proteins that may enhance responses of neurons to glutamate, thereby playing a role in synaptic plasticity. However, under conditions of energetic compromise, as in stroke, Notch activation renders neurons vulnerable to calcium overload and cell death. By modifying APP and Notch processing, secretase inhibitors are valuable research tools and also hold potential as neurotherapeutic agents.

31. Is the Basis of Some Neurodegenerative Diseases Aberrant Control of an Ancient Self-replicating Protein Template? JULIE E.M. MCGEOCH, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138

Water entrapped by a self-replicating membrane of polymer hydrophobic protein has been suggested as the first material-based topology of the universe (McGeoch, J. 2008. Harvard da Vinci Group, May 12). That a similar system existed when cells first arose on earth 3.8 million yr ago is supported by evidence that the nucleotide code of one hydrophobic polymer, proteolipid, is the most conserved throughout Archaea, Eubacteria, and Eukaria. Water-tight hydrogen-bonded β sheets of proteolipid can stack as 6-Å-deep layers around centrally entrapped ordered water, providing a topology that separates charge, and intermittent -helical configurations confer ion channel function for the transport of cations and water between the layers (McGeoch, J.E., and M.W. McGeoch. 2008. J.R. Soc. Interface. 5:311–318). Here, we hypothesize that this ancient system of separating charge between insulating layers of hydrophobic protein was incorporated into the first cells as the fundamental component for information storage, later becoming a nervous system. The advent of nucleotide code–based cell chemistry evolved proteins to interact with this system, some of which suppressed its inclination to self-replicate from a template. Today, certain diseases of the nervous system in Homo sapiens involving aggregated β-sheet protein might have their basis in age-related imperfect transcription/translation or code mutations, rendering the code-based system of control aberrant. We suggest that Batten's (NCL's) and Alzheimer's disease might be in this category. The ratio of cell proteolipid mass to its P1 and P2 code translation should be higher than that for another control polymer like a skin keratin if there is ancient template-based self-replication for proteolipid, and, in Batten's disease, it should be even higher as a result of aberrant control of the template system.

32. Mechanisms of Altered N-Methyl-D-Aspartate Receptor–mediated Calcium Signaling in the YAC Mouse Model of Huntington's Disease. A. MILNERWOOD,¹ C. GLADDING,¹ M.R. HAYDEN,^2,3 T.H. MURPHY,^1,2 and L.A. RAYMOND,^{1,2 1}Department of Psychiatry, ²Brain Research Centre, and ³Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, BC V6T 1Z4

Huntington's disease (HD) is an autosomal dominantly inherited neurodegenerative disease predominantly involving the striatal medium-sized spiny neurons (MSNs). HD is caused by expansion of a CAG repeat in the HD gene, resulting in a polyglutamine (polyQ) expansion >35 near the N terminus of the protein, huntingtin, and age of onset is inversely correlated with polyQ repeat length. Previous work in human HD brain tissue suggested that overactivation of N-methyl-D-aspartate (NMDA)–type glutamate receptors (NMDARs) contributes to the selective loss of striatal MSNs. We have tested this hypothesis using the YAC transgenic HD mouse model, expressing full-length human huntingtin with 18 polyQ (normal; YAC18) or 46, 72, or 120 polyQ (pathologically expanded; YAC46, YAC72, and YAC128, respectively). Striatal neurons from YAC mice expressing expanded polyQ huntingtin show enhanced sensitivity to NMDA-induced apoptotic cell death compared with MSNs from wild-type or YAC18 mice both in vivo and in cultures from postnatal mice. In MSN cultures, sensitization to NMDAR-mediated apoptosis results from increased activation of the mitochondrial permeability transition (mPT) pore and the intrinsic pathway, in part because of enhanced NMDAR activity and calcium mishandling. However, less is known about the mechanisms underlying increased striatal sensitivity to excitotoxicity in these HD mice in vivo. Previous studies demonstrate that apoptotic or survival pathways can be preferentially triggered by activation of extrasynaptic or synaptic NMDARs, respectively. In whole cell patch clamp recordings from cortico-striatal brain slices, we found evidence for increased extrasynaptic NMDAR activity in YAC128 MSNs compared with YAC18 MSNs. NMDAR synaptic current amplitude is increased in YAC128 mice under conditions of augmented glutamate release but not with low levels of synaptic activity. Moreover, treatment with a glutamate uptake inhibitor results in a significant increase in NMDAR-mediated holding current and action potential–evoked charge transfer in YAC128 but not YAC18 MSNs, suggesting activation of a population of extrasynaptic NMDARs in the YAC128 MSNs. Consistent with these electrophysiological data, subcellular fractionation shows enhanced expression of NR1 in the striatal nonpostsynaptic plasma membrane fraction from YAC128 compared with YAC18 mice, whereas no differences are detected in the striatal postsynaptic fractions or either fraction from cortical tissue. These data demonstrate a link between an inherited neurodegenerative disease and extrasynaptic NMDAR neurotransmission. (Supported by the Canadian Institutes of Health Research, the Michael Smith Foundation for Health Research, the Huntington Disease Society of American, the Huntington Society of Canada, and the HighQ Foundation.)

33. Enhanced CREB Phosphorylation by FAD Mutant Presenilin-1 (M146L)–associated Exaggerated Ca²⁺ Signaling. MARIOLYMÜLLER, KING-HOCHEUNG, CÉSARCÁRDENAS, LIJUANMEI, and J. KEVIN FOSKETT, Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104

Mutations in presenilin 1 (PS1) and presenilin 2 (PS2) genes account for the majority of early onset familial Alzheimer's Disease (FAD). Accumulating evidence suggests that disruption of intracellular Ca²⁺ signaling may play a proximal and perhaps central role in the pathogenesis of AD. Nevertheless, possible links between disrupted intracellular Ca²⁺ homeostasis and cell signaling are obscure. Here, we demonstrate that PS1-FAD mutant M146L constitutively enhances phosphorylation of the transcription factor cAMP-response element-binding protein (CREB) in a Ca²⁺-dependent way. Immunoblotting using p-CREB–specific antibody revealed that CREB is constitutively phosphorylated in M146L-expressing PC12 cells. This phosphorylation was completely abolished by depletion of endoplasmic reticulum Ca²⁺ stores with thapsigargin, suggesting an important role of Ca²⁺ release in this phenomenon. By use of specific pharmacological inhibitors, both CAMKIV and Ca²⁺-dependent protein kinase C but not MAPK were involved in CREB phosphorylation, reinforcing the idea that Ca²⁺ is a primary signal for CREB phosphorylation. The activity of the InsP₃ receptor Ca²⁺ release channel is potentiated by PS1-FAD mutant M146L. Inhibition of PLC, the enzyme responsible for the IP₃ generation, treatment of the cells with Xestospongin B, a specific InsP₃ receptor inhibitor, or RNAi against InsP₃ receptor type 1, the main neuronal isoform, each completely inhibited PS1-FAD mutant M146L-dependent constitutive CREB phosphorylation. Our results demonstrate that exaggerated Ca²⁺ signaling through activation of the InsP₃ receptor in FAD PS1 mutant-expressing PC12 cells affects CREB phosphorylation and may suggest a pathway involved in AD pathogenesis.

34. Calcium Signaling Defects in Familial Alzheimer's Disease Patient: Relevance for Alzheimer's Disease. OMAR NELSON, HUARUI LIU, and IIYA BEZPROZVANNY, Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390

Mutations in presenilin 1 (PS1) and presenilin 2 (PS2) are responsible for 40% of all early onset familial Alzheimer's disease (FAD) cases in which a genetic cause has been identified. In addition, a number of mutations in PS1 have been associated with the occurrence of frontal temporal dementia (FTD), although a formal proof of their causal involvement has not been provided. Presenilins (PSs) are highly conserved transmembrane proteins that support cleavage of the amyloid precursor protein by -secretase. Recently, we discovered that PSs also function as passive endoplasmic reticulum Ca²⁺ leak channels (Tu, H., O. Nelson, A. Bezprozvanny, Z. Wang, S.F. Lee, Y.H. Hao, L. Serneels, B. De Strooper, G. Yu, and I. Bezprozvanny. 2006. Cell. 126:981–993). We further found that PS1-M146V, E9, L166P, A246E, E273A, G384A, P436Q FAD, and PS2-N141I FAD mutations in PSs affected their ER Ca²⁺ leak function. In contrast, the ER Ca²⁺ leak function of PS1 appeared unaffected by A79V FAD mutation. Similar to FAD A79V, neither of the FTD-associated mutations in PS1 (L113P, G183V, and Rins352) affected ER Ca²⁺ leak function in our experiments (Tu, H., O. Nelson, A. Bezprozvanny, Z. Wang, S.F. Lee, Y.H. Hao, L. Serneels, B. De Strooper, G. Yu, and I. Bezprozvanny. 2006. Cell. 126:981–993; Nelson, O., H. Tu, T. Lei, M. Bentahir, B. de Strooper, and I. Bezprozvanny. 2007. J. Clin. Invest. 117:1230–1239). To further validate our findings, we use Ca²⁺ imaging experiments to evaluate ER Ca²⁺ leak in lymphoblast from FAD patients harboring mutations in PS1 (M139V, M146L, H163Y, K239E, V261F, A426P, A431E, P264L, R269G, and C410Y), PS2-N141I, -R406W, APP-V717L, and young and old sporadic AD cases. In addition, Ca²⁺ rescue experiments with the PS1 FAD mutations were performed in PS-null mouse embryonic fibroblasts (DKO) to validate ER Ca²⁺ leak function. We found that PS1 FAD mutations M139V, M146L, H163Y, K239E, V261F, A426P, A431E, and PS2-N141I abolished ER Ca²⁺ leak in lymphoblast and Ca²⁺ rescue experiments in DKO, whereas PS1 (P264L, R269G, E9, and C410Y), -R406W, APP-V717L, and young and old sporadic AD had no effect of ER Ca²⁺ leak in lymphoblast. Our observations are consistent with the potential role of disturbed Ca²⁺ homeostasis in AD pathogenesis. (Supported by Alzheimer's Association research grant IIRG-06-24703 and the McKnight Brain Disorders Award to I. Bezprozvanny and by the National Institutes of Health Predoctoral Fellowship Award for Minority Students [F31- AG031692-02] to O. Nelson.)

35. Role of Ca²⁺ Signaling During the Unfolded Protein Response (UPR). R. MADELAINE PAREDES,¹ PATRICIA CAMACHO,^1,2 and JAMES D. LECHLEITER,^{1,2 1}Department of Cellular and Structural Biology and ²Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229

Two of the main functions of the endoplasmic reticulum (ER) are Ca²⁺ storage and protein folding (Berridge, M.J. 2002. Cell Calcium. 32:235–249). Although it has been suggested that these two functions are associated (Li, Y., and P. Camacho. 2004. J. Cell Biol. 164:35–46), it is not clear how. It is known that disturbing the balance of any function of the ER generates ER stress (Rutkowski, D.T., and R.J. Kaufman. 2004. Trends Cell Biol. 14:20–28). Under ER stress, a process known as the unfolded protein response (UPR) is induced. The UPR process is an attempt by the cell to restore balance and maintain normal function in the ER, which otherwise results in cell death.

Previous work in our group has shown a dependent and direct relationship between Ca²⁺ and members of the protein-folding machinery such as Calreticulin (CRT), a luminal chaperone, and ERp57, an oxidoreductase that promotes disulfide bond formation during protein folding. In that work, it is demonstrated that under high Ca²⁺ concentrations in the lumen of the ER, ERp57 forms a complex with CRT, and that complex can bind the SERCA 2b pump, inhibiting its activity. On the other hand, when luminal Ca²⁺ concentrations are low, the CRT–ERp57 complex can no longer bind SERCA 2b, and the pump is able to function properly, bringing Ca²⁺ back into the lumen of the ER (Li, Y., and P. Camacho. 2004. J. Cell Biol. 164:35–46).

Additional data from our group indicate that treatment of Xenopus oocytes with an inhibitor of protein glycosylation (Tunicamycin) that mimics ER stress increases cytosolic Ca²⁺ (in conditions of zero extracellular Ca²⁺), suggesting a partial depletion of luminal Ca²⁺ subsequent to ER stress (unpublished data).

To monitor the effect of luminal Ca²⁺ on protein folding, we have developed an imaging assay to monitor accumulation of protein in the ER. In brief, we label a luminal protein, CPY (carboxypeptidase Y), that is normally secreted outside the cell with strawberry fluorescent protein (SFP). CPY is known to misfold and accumulate in the ER when a single point mutation is introduced into the protein (Mancini, R., M. Aebi, and A. Helenius. 2003. J. Biol. Chem. 278:46895–46905). We labeled the single point mutant of CPY with cyan fluorescent protein (CFP) to image its ER accumulation. Our preliminary data indicate that thapsigargin-depleted ER Ca²⁺ stores increases accumulation of SFP-tagged wild-type CPY. Moreover, prolonged partial depletion of luminal Ca²⁺ by low concentrations of thapsigargin also induces protein misfolding accumulation in the ER. Treatment of oocytes with both high and low concentrations of thapsigargin induced ER stress as monitored by phosphorylation of PERK and phosphorylation of eIF2.

Collectively, these data suggest that Ca²⁺ depletion from the ER can be both a causative effect as well as a consequence of protein misfolding in the UPR. (Supported by grants PO1 AG19316-06 and RO1 AG29461-01.)

36. Rosiglitazone Treatment Prevents Mitochondrial Dysfunction in Mutant Huntingtin–Expressing Cells. RODRIGO A. QUINTANILLA,¹ YOUNGMAN JIN,² and GAIL V.W. JOHNSON,^{1,2 1}Department of Anesthesiology and ²Department of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642

Defects in calcium regulation and mitochondrial function have been implicated in the pathogenesis of Huntington's disease (HD). To evaluate the potential role of the calcium deregulation and loss of mitochondrial function in the pathogenesis of HD, we used striatal cells that express wild-type (STHdh^Q7/Q7) or mutant (STHdh^Q111/Q111) huntingtin protein at physiological levels. In these studies, we examined cytosolic calcium levels, mitochondria calcium changes, and mitochondrial membrane potential using confocal microscopy. Treatment of mutant cells with thapsigargin resulted in a pronounced decrease in mitochondrial calcium uptake, an increase in reactive oxygen species (ROS) production, and a significant decrease in mitochondrial membrane potential. These events were partially prevented by pretreatment with cyclosporine A (CsA), indicating a potential role for permeability transition pore (PTP) opening in this mitochondrial dysfunction. In addition, we evaluated the potential neuroprotective role of PPAR activation in preventing the loss of mitochondrial function in HD using striatal cells. PPAR activation by rosiglitazone totally prevented the mitochondrial dysfunction and oxidative stress that occurred when striatal cells expressing mutant huntingtin were challenged with pathological increases in calcium. The beneficial effects of rosiglitazone were mediated by activation of PPAR, as all protective effects were prevented by the specific PPAR receptor antagonist GW9662. Additionally, we observed that mutant huntingtin–expressing cells presented with significant impairment of the PPAR signaling pathway, including decreases in PPAR expression and reduced PPAR transcriptional activity. In addition, we observed that treatment of striatal cells with rosiglitazone increased mitochondrial mass levels, suggesting a role for the PPAR pathway in mitochondrial function in striatal cells. All together, this evidence indicates that PPAR activation by rosiglitazone attenuates mitochondrial dysfunction in mutant huntingtin–expressing striatal cells, and this could be an important therapeutic avenue to ameliorate the mitochondrial dysfunction that occurs in HD.

37. Functional Studies of a Novel Gene Linked to Late Onset Alzheimer's Disease. ADAMP. SIEBERT,¹ HORAVAIS,¹ LIJUANMEI,¹ PHILIPPEMARAMBAUD,³ and J. KEVIN FOSKETT,^{1,2 1}Department of Physiology and ²Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104; ³Litwin-Zucker Research Center for the Study of Alzheimer's Disease and Memory Disorders, Feinstein Institute for Medical Research, Manhasset, NY 11030

Alzheimer's Disease is a common form of dementia involving slowly developing and ultimately fatal neurodegeneration. Mutations in presenilins and amyloid precursor protein cause the majority of early onset autosomal dominant familial cases of the disease. However, complex interactions between different genetic variants and environmental factors are believed to modulate the risk of the vast majority of late onset Alzheimer's disease. A prevailing idea, the calcium hypothesis, proposes that sustained changes in intracellular calcium homeostasis provide the common pathway for age-associated brain changes. Recently, we identified CALHM1, a gene of unknown function, located on chromosome 10 within 1.6 Mb of the late onset Alzheimer's Disease marker D10S1671. The frequency of a nonsynonymous single-nucleotide polymorphism (SNP) in CALHM1, which results in an amino acid substitution, is significantly increased in independent cohorts of AD cases in French and British populations. In preliminary studies, we have determined that CALHM1 expression induces novel plasma membrane ionic con