Gonul Velicelebi

STIM1, an essential and conserved component of store-operated Ca2+ channel function

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

Modulation of γ-Secretase Reduces β-Amyloid Deposition in a Transgenic Mouse Model of Alzheimer's Disease

Alzheimers disease (AD) is characterized pathologically by the abundance of senile plaques and neurofibrillary tangles in the brain. We synthesized over 1200 novel gamma-secretase modulator (GSM) compounds that reduced Abeta(42) levels without inhibiting epsilon-site cleavage of APP and Notch, the generation of the APP and Notch intracellular domains, respectively. These compounds also reduced Abeta(40) levels while concomitantly elevating levels of Abeta(38) and Abeta(37). Immobilization of a potent GSM onto an agarose matrix quantitatively recovered Pen-2 and to a lesser degree PS-1 NTFs from cellular extracts. Moreover, oral administration (once daily) of another potent GSM to Tg 2576 transgenic AD mice displayed dose-responsive lowering of plasma and brain Abeta(42); chronic daily administration led to significant reductions in both diffuse and neuritic plaques. These effects were observed in the absence of Notch-related changes (e.g., intestinal proliferation of goblet cells), which are commonly associated with repeated exposure to functional gamma-secretase inhibitors (GSIs).

Human neuronal voltage-dependent calcium channels: studies on subunit structure and role in channel assembly.

Voltage-dependent calcium (Ca2+) channels, expressed in the CNS, appear to be multimeric complexes comprised of at least alpha 1, alpha 2 and beta subunits. Previously, we cloned and expressed human neuronal alpha 1, alpha 2 and beta subunits to study recombinant channel complexes that display properties of those expressed in vivo. The alpha 1B-mediated channel subtype binds omega-conotoxin (CgTx) GVIA with high affinity and exhibits properties of N-type voltage-dependent Ca2+ channels. Here we describe several alpha 2 and beta splice variants and report results on the expression of omega-CgTx GVIA binding sites, assembly of the subunit complex and biophysical function of alpha 1B-mediated channel complexes containing some of these splice variants. We optimized recombinant expression in human embryonic kidney (HEK) 293 cells of alpha 1B alpha 2b beta 1 subunit complexes by controlling the expression levels of subunit mRNAs and monitored cell surface expression by binding of omega-CgTx GVIA to the alpha 1B subunit. Co-expression of either alpha 2b or beta 1 subunits with an alpha 1B subunit increased expression of binding sites while the most efficient expression was achieved when both alpha 2b and beta 1 subunits were co-expressed with an alpha 1B subunit. The presence of alpha 2b affects the affinity of omega-CgTx GVIA binding and barium (Ba2+) current magnitudes, although it does not appear to alter kinetic properties of the Ba2+ current. This is the first evidence of an alpha 2 subunit modulating the binding affinity of a cell-surface Ca2+ channel ligand. Our results demonstrate that alpha 1, alpha 2 and beta subunits together contribute to the efficient assembly and functional expression of voltage-dependent Ca2+ channel complexes.

Comparative structure of human neuronal α2–α7 and β2–β4 nicotinic acetylcholine receptor subunits and functional expression of the α2, α3, α4, α7, β2, and β4 subunits

AbstractcDNA clones encoding human neuronal nicotinic acetylcholine receptor α2, α3, α4, α5, α6, α7, β2, β3, and β4 subunits were isolated from brainstem, hippocampus, prefrontal cortex, substantia nigra, thalamus, and IMR32 libraries. Human α2 and α6 and full-length β3 and β4 clones have not been previously reported. Deduced amino acid sequences of the α2, α6, β3, and β4 predicted mature peptides are 503 residues (56.9 kDa), 464 residues (53.7 kDa), 440 residues (50.8 kDa), and 477 residues (54.1 kDa), respectively. These sequences show 84 (α2), 87 (α6), 89 (β3), and 84% (β4) identity to the corresponding rat sequences. The amino termini of the human α2 and β3 mature peptides contain 23 and six additional residues, respectively, compared to those of rat α2 and β3. Recombinant receptors were expressed inXenopus laevis oocytes injected with in vitro transcripts encoding either α7 alone or α2, α3, or α4 in pairwise combination with β2 or β4. Inward currents were elicited by the application of acetylcholine (1–100 µM) and other agonists; these responses were blocked 65–97% by application of 10 µM d-tubocurare, confirming functional expression of human nicotinic receptors.

Characterization of the recombinant human neuronal nicotinic acetylcholine receptors α3β2 and α4β2 stably expressed in HEK293 cells

Abstract HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations α3β2 or α4β2. [ 3 H]-(±)Epibatidine ([ 3 H]-(±)EPI) bound to membranes from A3B2 (α3β2) and A4B2.2 (α4β2) cells with K d values of 7.5 and 33.4 pM and B max values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca 2+ concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-β-erythroidine (DHβE) in A3B2 cells and MEC=DHβE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in α3β2 and α4β2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca 2+ , respectively. Our results indicate that stably expressed α3β2 and α4β2 human nAChRs are pharmacologically and functionally distinct.

Functional Characterization of Human N‐Methyl‐d‐Aspartate Subtype 1A/2D Receptors

Abstract: The human NMDAR2D subunit was cloned, and the pharmacological properties of receptors resulting from injection of transcripts encoding human NMDAR1A and NMDAR2D subunits in Xenopus oocytes were characterized by profiling NMDA receptor agonists and antagonists. We found that glutamate, NMDA, glycine, and d‐serine were significantly more potent on hNMDAR1A/2D than on hNMDAR1A/2A or hNMDAR1A/2B. Also, the potencies of NMDA and glycine were higher for hNMDAR1A/2D than for hNMDAR1A/2C. Ifenprodil was more potent at hNMDAR1A/2B than at hNMDAR1A/2D, whereas 5,7‐dichlorokynurenate was more potent at hNMDAR1A/2A than at hNMDAR1A/2D. As measured in transiently transfected human embryonic kidney 293 cells, the maximal inward current in the presence of external Mg2+ occurred at −40 mV, and full block was not observed at negative potentials. Kinetic measurements revealed that the higher affinity of hNMDAR1A/2D for both glutamate and glycine relative to hNMDAR1A/2A and hNMDA1A/2B can be explained by slower dissociation of each agonist from hNMDAR1A/2D. The hNMDAR1A/2D combination represents a pharmacologically and functionally distinct receptor subtype and may constitute a potentially important target for therapeutic agents active in the human CNS.

Fluorescence techniques for measuring ion channel activity.

Publisher Summary Changes in intracellular free calcium concentration ([Ca 2+ ] i ) play a crucial role in cellular physiology. A number of cell surface receptors and channels are known to regulate [Ca 2+ ] i through different molecular mechanisms. Therefore, the functional and pharmacologic properties of many of these cell surface receptors and ion channels can be studied effectively by measuring changes in [Ca 2+ ] i in intact cells. For drug discovery efforts, several ion channel and receptor systems have been targeted that play different roles in neuronal physiology and pathophysiology. These molecular targets include voltage- and ligand-gated ion channels: the human neuronal voltage-gated calcium channels (VGCCs), ligand-gated nicotinic acetylcholine receptor channels (NAChRs), ionotropic N -methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and kainate-type excitatory amino acid receptor (EAA) channels. All of these channels mediate elevation of [Ca 2+ ] i via Ca 2+ influx from the extracellular medium upon depolarization or activation by agonist. This chapter describes the experimental methods with particular emphasis on the validation of the assay for human VGCCs, NAChRs, and NMDA receptors.

Structural characterization of vasoactive intestinal peptide receptors from rat lung membranes

The presence of specific receptors for vasoactive intestinal peptide (VIP) in numerous cell and membrane preparations has been well documented.’ The binding of VIP to cell-surface receptors correlates with the stimulation of adenylate cyclase activity and elevation of intracellular concentration of CAMP, leading to the biological effects of the peptide.2 Hence, structural and functional characterization of VIP receptors will be integral to understanding the molecular mechanism of action of VIP. Our studies have focused on the characterization of VIP receptors from rat lung. There is evidence that the lung may be an important target organ for the actions of VIP based on the demonstration of VIP innervation associated with airways and pulmonary vessels,’ bronchodilatory effects of VIP in humans,’ and its relaxant effects on pulmonary vascular smooth muscle.’ In this study, we describe characterization of VIP receptors from rat lung membranes both in the membrane-bound and detergent-solubilized states. In the former case, rat lung membrane proteins were covalently labeled with [‘2’I]VIP using a bifunctional cross-linker and a 55-kDa species was identified as a ligand-binding unit of rat lung VIP receptors. Cross-linking studies have been reported in greater detail elsewhere.6 In the latter case, [ 12’I]VIP-labeled, membrane-bound receptors were solubilized in Triton X-100 and studied by gel filtration and sucrose density gradient sedimentation, yielding an apparent molecular weight of 270,000 for the [ 12’I]VIPreceptor complex in Triton X-100 solution.

Limited proteolysis of the vasoactive intestinal peptide receptor: comparison of its folded structure in the membrane-bound and detergent-solubilized states

Limited proteolysis was used to probe and compare the conformation of the rat lung vasoactive intestinal peptide (VIP) receptor in membrane-bound and detergent-solubilized states. It had been shown previously that the activity of the detergent-solubilized VIP receptor is sensitive to the nature of the detergent used for extraction (Patthi, S., Simerson S. and Velicelebi, G. (1988) J. Biol. Chem., 263, 19363-19369). Receptors that were extracted from the membrane using digitonin retained the ability to bind 125I-VIP, while those solubilized in Triton X-100 displayed little or no detectable activity. In order to correlate the differences observed in the activity of the receptor with its folded state, membrane-bound and detergent-solubilized receptors were covalently labeled with 125I-VIP and subjected to limited proteolysis using trypsin, chymotrypsin or carboxypeptidase Y. Digitonin-solubilized receptors most closely resembled the membrane-bound protein in terms of protease sensitivity and proteolytic cleavage products. By contrast, receptors solubilized in Triton X-100 displayed increased sensitivity to proteases and produced distinctly different proteolytic patterns. Thus, the differences observed in the activities of receptors solubilized in digitonin and those solubilized in Triton X-100 could be correlated with detectable differences in the conformation of the protein in each respective detergent solution. These results suggest that digitonin provides an environment that is more compatible with the native folded state of the receptor, similar to its conformation in the membrane.