Intraluminal Loop Research Articles

Cellugyrin (synaptogyrin-2) dependent pathways are used by bacterial cytolethal distending toxin and SARS-CoV-2 virus to gain cell entry.

Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is capable of intoxicating lymphocytes macrophages, mast cells and epithelial cells. Following Cdt binding to cholesterol, in the region of membrane lipid rafts, the CdtB and CdtC subunits are internalized and traffic to intracellular compartments. These events are dependent upon, cellugyrin, a critical component of synaptic like microvesicles (SLMVCg+). Target cells, such as Jurkat cells, rendered unable to express cellugyrin are resistant to Cdt-induced toxicity. Similar to Cdt, SARS-CoV-2 entry into host cells is initiated by binding to cell surface receptors, ACE-2, also associated with cholesterol-rich lipid rafts; this association leads to fusion and/or endocytosis of viral and host cell membranes and intracellular trafficking. The similarity in internalization pathways for both Cdt and SARS-CoV-2 led us to consider the possibility that cellugyrin was a critical component in both processes. Cellugyrin deficient Calu-3 cells (Calu-3Cg-) were prepared using Lentiviral particles containing shRNA; these cells were resistant to infection by VSV/SARS-CoV-2-spike pseudotype virus and partially resistant to VSV/VSV-G pseudotype virus. Synthetic peptides representing various regions of the cellugyrin protein were prepared and assessed for their ability to bind to Cdt subunits using surface plasmon resonance. Cdt was capable of binding to a region designated the middle outer loop (MOL) which corresponds to a region extending into the cytoplasmic surface of the SLMVCg+. SARS-CoV-2 spike proteins were assessed for their ability to bind to cellugyrin peptides; SARS-CoV-2 full length spike protein preferentially binds to a region within the SLMVCg+ lumen, designated intraluminal loop 1A. SARS-CoV-2-spike protein domain S1, which contains the receptor binding domains, binds to cellugyrin N-terminus which extends out from the cytoplasmic surface of SLMV. Binding specificity was further analyzed using cellugyrin scrambled peptide mutants. We propose that SLMVCg+ represent a component of a common pathway that facilitates pathogen and/or pathogen-derived toxins to gain host cell entry.

Functional and evolutionary analysis of host Synaptogyrin-2 in porcine circovirus type 2 susceptibility.

Mammalian evolution has been influenced by viruses for millions of years, leaving signatures of adaptive evolution within genes encoding for viral interacting proteins. Synaptogyrin-2 (SYNGR2) is a transmembrane protein implicated in promoting bacterial and viral infections. A genome-wide association study of pigs experimentally infected with porcine circovirus type 2b (PCV2b) uncovered a missense mutation (SYNGR2 p.Arg63Cys) associated with viral load. In this study, CRISPR/Cas9-mediated gene editing of the porcine kidney 15 (PK15, wtSYNGR2+p.63Arg) cell line generated clones homozygous for the favorable SYNGR2 p.63Cys allele (emSYNGR2+p.63Cys). Infection of edited clones resulted in decreased PCV2 replication compared to wildtype PK15 (P<0.05), with consistent effects across genetically distinct PCV2b and PCV2d isolates. Sequence analyses of wild and domestic pigs (n>700) revealed the favorable SYNGR2 p.63Cys allele is unique to domestic pigs and more predominant in European than Asian breeds. A haplotype defined by the SYNGR2 p.63Cys allele was likely derived from an ancestral haplotype nearly fixed within European (0.977) but absent from Asian wild boar. We hypothesize that the SYNGR2 p.63Cys allele arose post-domestication in ancestral European swine. Decreased genetic diversity in homozygotes for the SYNGR2 p.63Cys allele compared to SYNGR2 p.63Arg, corroborates a rapid increase in frequency of SYGNR2 p.63Cys via positive selection. Signatures of adaptive evolution across mammalian species were also identified within SYNGR2 intraluminal loop domains, coinciding with the location of SYNGR2 p.Arg63Cys. Therefore, SYNGR2 may reflect a novel component of the host-virus evolutionary arms race across mammals with SYNGR2 p.Arg63Cys representing a species-specific example of putative adaptive evolution.

Disruption of ER ion homeostasis maintained by an ER anion channel CLCC1 contributes to ALS-like pathologies

Although anion channel activities have been demonstrated in sarcoplasmic reticulum/endoplasmic reticulum (SR/ER), their molecular identities and functions remain unclear. Here, we link rare variants of Chloride Channel CLIC Like 1 (CLCC1) to amyotrophic lateral sclerosis (ALS)-like pathologies. We demonstrate that CLCC1 is a pore-forming component of an ER anion channel and that ALS-associated mutations impair channel conductance. CLCC1 forms homomultimers and its channel activity is inhibited by luminal Ca2+ but facilitated by phosphatidylinositol 4,5-bisphosphate (PIP2). We identified conserved residues D25 and D181 in CLCC1 N-terminus responsible for Ca2+ binding and luminal Ca2+-mediated inhibition on channel open probability and K298 in CLCC1 intraluminal loop as the critical PIP2-sensing residue. CLCC1 maintains steady-state [Cl–]ER and [K+]ER and ER morphology and regulates ER Ca2+ homeostasis, including internal Ca2+ release and steady-state [Ca2+]ER. ALS-associated mutant forms of CLCC1 increase steady-state [Cl–]ER and impair ER Ca2+ homeostasis, and animals with the ALS-associated mutations are sensitized to stress challenge-induced protein misfolding. Phenotypic comparisons of multiple Clcc1 loss-of-function alleles, including ALS-associated mutations, reveal a CLCC1 dosage dependence in the severity of disease phenotypes in vivo. Similar to CLCC1 rare variations dominant in ALS, 10% of K298A heterozygous mice developed ALS-like symptoms, pointing to a mechanism of channelopathy dominant-negatively induced by a loss-of-function mutation. Conditional knockout of Clcc1 cell-autonomously causes motor neuron loss and ER stress, misfolded protein accumulation, and characteristic ALS pathologies in the spinal cord. Thus, our findings support that disruption of ER ion homeostasis maintained by CLCC1 contributes to ALS-like pathologies.

The Sarcoplasmic Reticulum Oxidoreductase System Modulates Luminal Ca2+ Regulation of the Ryanodine Receptor in Cardiac Disease

The sarcoplasmic reticulum (SR) Ca2+ release ryanodine receptor channel (RyR2) is susceptible to posttranslational modifications by reactive oxygen species (ROS), impairing luminal Ca2+ regulation and increasing channel activity. Despite significant research efforts, exact mechanisms underlying redox-mediated increase of RyR2 function in cardiac disease remain elusive. We tested whether the oxidoreductase family of proteins that dynamically regulate the oxidative environment in the SR are involved in this process. In ventricular myocytes (VMs) from rats with hypertrophy induced by thoracic aortic banding (TAB), expression of SR oxidoreductase Ero1α was significantly increased compared to Sham VMs. Adenoviral expression of intra-SR ROS probe ERroGFP in cultured TAB rat VMs revealed enhanced oxidative stress in the vicinity of RyR2, in parallel with increased RyR2 oxidation, measured with DNP antibody. Ero1α overexpression in control VMs recapitulated a similar phenotype. Conversely, pharmacological inhibition of Ero1α with EN460 (20 μM) reduced arrhythmogenic potential in ex vivo optically mapped TAB rat hearts by reducing SR Ca2+ leak, and suppressed Ca2+ spark frequency in TAB VMs, indicating stabilization of RyR2 function. Utilizing a heterologous HEK293 cell system and intra-ER Ca2+ sensor R-CEPIAer to probe Ca2+ release dynamics of human RyR2, Ero1α overexpression significantly increased Ca2+ wave frequency, mimicking effects of strong oxidative agent DTDP. Knockdown of endogenous Ero1α had opposite effects. Furthermore, mutation of two intraluminal cysteines of RyR2 in the second intraluminal loop, potentially sensitive to modification by ROS, resulted in more frequent Ca2+ waves and reduced basal intra-ER [Ca2+]. These findings suggest upregulation of SR oxidoreductase Ero1α in cardiac disease is detrimental to luminal Ca2+ homeostasis by modulating RyR2 activity via ROS. Additionally, we have identified two potential intraluminal residues of the channel that may be sensitive to redox modification.

Chapter 3: Molecular basis for the therapeutic effectiveness of botulinum neurotoxin type A.

The utility of botulinum neurotoxin type A (BoNT/A) for treating overactive muscles and endocrine glands is attributable to a unique conflation of properties honed to exploit and inactivate synaptic transmission. Specific, high-affinity coincident binding to gangliosides plus an intraluminal loop of synaptic vesicle protein 2 (SV2) by the heavy chain (HC) of BoNT/A confers selectivity for presynaptic nerve terminals and subsequent uptake by endocytosis. Upon vesicle acidification, the HC forms a channel for transmembrane transfer of the light chain to the cytosol, as observed by single channel recordings. The light chain is a Zn(2+) -dependent endoprotease that cleaves and inactivates SNAP-25, thereby blocking exocytotic release of transmitters, a discovery that revealed the pivotal role of the latter in synaptic vesicle fusion. A di-leucine motif in BoNT/A light chain stabilizes this protease, contributing to its longevity inside nerves. The ubiquity of SV2 and SNAP-25 has prompted re-evaluation of the nerve types susceptible to BoNT/A. In urology, there is emerging evidence that BoNT/A blocks neuropeptide release from afferent nerves, exocytosis of acetylcholine and purines from efferent nerves, and possibly ATP release from the urothelium. Suppression by BoNT/A of the surface expression of nociceptor channels on bladder afferents might also contribute to its improvement of urological sensory symptoms.

C-terminal Domain of Chromogranin B Regulates Intracellular Calcium Signaling

The versatility of intracellular calcium as a second messenger is seen in its ability to mediate opposing events such as neuronal cell growth and apoptosis. A leading hypothesis used to explain how calcium regulates such divergent signaling pathways is that molecules responsible for maintaining calcium homeostasis have multiple roles. For example, chromogranin B (CGB), a calcium binding protein found in secretory granules and in the lumen of the endoplasmic reticulum, buffers calcium and also binds to and amplifies the activity of the inositol 1,4,5-trisphosphate receptor (InsP(3)R). Previous studies have identified two conserved domains of CGB, an N-terminal domain (N-CGB) and a C-terminal domain (C-CGB). N-CGB binds to the third intraluminal loop of the InsP(3)R and inhibits binding of full-length CGB. This displacement of CGB decreases InsP(3)R-dependent calcium release and alters normal signaling patterns. In the present study, we further characterized the role of N-CGB and identified roles for C-CGB. The effect of N-CGB on calcium release depended upon endogenous levels of cellular CGB, whereas the regulatory effect of C-CGB was apparent regardless of endogenous levels of CGB. When either full-length CGB or C-CGB was expressed in cells, calcium transients were increased. Additionally, the calcium signal initiation site was altered upon C-CGB expression in neuronally differentiated PC12 and SHSY5Y cells. These results show that CGB has numerous regulatory roles and that CGB is a critical component in modulating InsP(3)R-dependent calcium signaling.

Triadin Binding to the C-Terminal Luminal Loop of the Ryanodine Receptor is Important for Skeletal Muscle Excitation–Contraction Coupling

Ca2+ release from intracellular stores is controlled by complex interactions between multiple proteins. Triadin is a transmembrane glycoprotein of the junctional sarcoplasmic reticulum of striated muscle that interacts with both calsequestrin and the type 1 ryanodine receptor (RyR1) to communicate changes in luminal Ca2+ to the release machinery. However, the potential impact of the triadin association with RyR1 in skeletal muscle excitation–contraction coupling remains elusive. Here we show that triadin binding to RyR1 is critically important for rapid Ca2+ release during excitation–contraction coupling. To assess the functional impact of the triadin-RyR1 interaction, we expressed RyR1 mutants in which one or more of three negatively charged residues (D4878, D4907, and E4908) in the terminal RyR1 intraluminal loop were mutated to alanines in RyR1-null (dyspedic) myotubes. Coimmunoprecipitation revealed that triadin, but not junctin, binding to RyR1 was abolished in the triple (D4878A/D4907A/E4908A) mutant and one of the double (D4907A/E4908A) mutants, partially reduced in the D4878A/D4907A double mutant, but not affected by either individual (D4878A, D4907A, E4908A) mutations or the D4878A/E4908A double mutation. Functional studies revealed that the rate of voltage- and ligand-gated SR Ca2+ release were reduced in proportion to the degree of interruption in triadin binding. Ryanodine binding, single channel recording, and calcium release experiments conducted on WT and triple mutant channels in the absence of triadin demonstrated that the luminal loop mutations do not directly alter RyR1 function. These findings demonstrate that junctin and triadin bind to different sites on RyR1 and that triadin plays an important role in ensuring rapid Ca2+ release during excitation–contraction coupling in skeletal muscle.

Identification of residues participating in the interaction between an intraluminal loop of inositol 1,4,5-trisphosphate receptor and a conserved N-terminal region of chromogranin B

The inositol 1,4,5-trisphosphate receptor (IP3R) is a membrane channel that conducts calcium ions from the intracellular calcium stores. Despite a wealth of information on the cytoplasmic regulation of the IP3R, little is known about its regulation on the luminal side of the calcium stores. Here, we report studies on the IP3R intraluminal loop L3-2 and a conserved N-terminal region of chromogranin B. The IP3R loop is an important part of the channel's pore-forming region, and the chromogranin peptide has been shown to competitively inhibit calcium signaling by IP3R. Using the NMR titration approach, we showed that a part of the L3-2 is involved in a specific interaction with the chromogranin B peptide. Further NMR resonance assignments revealed that the 14th–20th residues of L3-2 are the keys to the binding to the chromogranin B peptide. Through detailed analysis of the data, we suggest a mechanism of IP3R regulation by chromogranin B involving conformational exchanges of the L3-2 region. Our report presents the findings of the first study on the interaction between the luminal loop of the IP3 receptor and its regulator at residue-resolution. The approaches described here should help to guide further studies on the interactions between the IP3R and other luminal side regulators.

Occurrence of atypical Ca 2+ transients in triadin-binding deficient-RYR1 mutants

Triadin in the junctional sarcoplasmic reticulum (SR) of skeletal muscle cells has been suggested to interact with ryanodine receptor 1 (RYR1) via its KEKE motifs. Recently, we showed that amino acid residues D4878, D4907, and E4908 in RYR1 are critical for triadin-binding in vitro [J.M. Lee, S.H. Rho, D.W. Shin, C. Cho, W.J. Park, S.H. Eom, J. Ma, D.H. Kim, Negatively charged amino acids within the intraluminal loop of ryanodine receptor are involved in the interaction with triadin, J. Biol. Chem. 279 (2004) 6994–7000]. In order to test whether a disruption of the triadin-binding site(s) in RYR1 affects SR Ca 2+ release, alanine-substituted single (D4878A, D4907A, and E4908A) and triple (RYR1-TM) mutants of D4878, D4907, and E4908 were expressed in RYR1-null myotubes. Co-immunoprecipitation experiments showed a 50–60% decrease of triadin brought down in the D4907A and RYR1-TM complexes compared to the triadin–wtRYR1 complex. Ca 2+ imaging experiments using Fluo-4-AM showed atypical caffeine responses in myotubes expressing D4907A and RYR1-TM characterized by either a lack of or slower activation and faster inactivation of Ca 2+ transients. The results suggest that disruption of interaction between triadin and RYR1 impairs RYR1 function and SR Ca 2+ release.

Negatively Charged Amino Acids within the Intraluminal Loop of Ryanodine Receptor Are Involved in the Interaction with Triadin

In mammalian striated muscles, ryanodine receptor (RyR), triadin, junctin, and calsequestrin form a quaternary complex in the lumen of sarcoplasmic reticulum. Such intermolecular interactions contribute not only to the passive buffering of sarcoplasmic reticulum luminal Ca2+, but also to the active Ca2+ release process during excitation-contraction coupling. Here we tested the hypothesis that specific charged amino acids within the luminal portion of RyR mediate its direct interaction with triadin. Using in vitro binding assay and site-directed mutagenesis, we found that the second intraluminal loop of the skeletal muscle RyR1 (amino acids 4860-4917), but not the first intraluminal loop of RyR1 (amino acids 4581-4640) could bind triadin. Specifically, three negatively charged residues Asp4878, Asp4907, and Glu4908 appear to be critical for the association with triadin. Using deletional approaches, we showed that a KEKE motif of triadin (amino acids 200-232) is essential for the binding to RyR1. Because the second intraluminal loop of RyR has been previously shown to contain the ion-conducting pore as well as the selectivity filter of the Ca2+ release channel, and Asp4878, Asp4907, and Glu4908 residues are predicted to locate at the periphery of the pore assembly of the channel, our data suggest that a physical interaction between RyR1 and triadin could play an active role in the overall Ca2+ release process of excitation-contraction coupling in muscle cells.

Proteolysis of type I inositol 1,4,5-trisphosphate receptor in WB rat liver cells.

A comparison of the basal degradation of type I Ins P3Rs [L- myo -inositol 1,4,5-trisphosphate receptor], measured by pulse-chase analysis or by analysis of immunoreactive Ins P3Rs after cycloheximide addition, indicated that the small pool of newly synthesized radioactive Ins P3Rs degraded relatively rapidly compared with the large pool of mature Ins P3Rs. An antibody (Ab) against a peptide sequence within the IL-3 (third intraluminal loop) of the receptor (IL-3 Ab) was used to identify protected proteolytic fragments that may accumulate in cells. The IL-3 Ab recognized a 56 kDa fragment in both WB rat liver cells and A7R5 smooth-muscle cells. Gel filtration experiments indicated that the 56 kDa fragment was monomeric and, based on reactivity to other Abs, was missing the cytosol-exposed N- and C-terminal segments of the receptor. The addition of the lysosomal protease inhibitor chloroquine resulted in the rapid disappearance of the 56 kDa band. This effect was mimicked by the cysteine protease inhibitors leupeptin, N -acetyl-L-leucyl-L-leucyl-L-methioninal and N -acetyl-leucyl-leucyl-norleucinal. Lactacystin and NH4Cl were less effective. A second fragment of 16 kDa containing the C-terminus accumulated only when the cells were treated with NH4Cl, and not with any of the other inhibitors tested. No N-terminal-reactive fragments were observed. We propose that mature Ins P3R tetramers dissociate into monomers and that the 56 kDa fragment is a cleavage intermediate of the monomer representing the six transmembrane domains. Angiotensin-II-stimulated down-regulation of Ins P3Rs in WB cells has been shown to involve the ubiquitin/proteasome pathway. Angiotensin-II treatment of WB cells neither resulted in the accumulation of any new fragments nor increased the levels of the 56 or 16 kDa fragments. We conclude that basal and agonist-stimulated degradations of Ins P3Rs occur by different pathways. The agonist-mediated pathway involves the concerted removal and proteolysis of the entire receptor molecule from the endoplasmic reticulum membrane without the appearance of intermediate intraluminal fragments.

Molecular Determinants of Ion Permeation and Selectivity in Inositol 1,4,5-Trisphosphate Receptor Ca2+ Channels

We tested the hypothesis that key residues in a putative intraluminal loop contribute to determination of ion permeation through the intracellular Ca(2+) release channel (inositol 1,4,5-trisphosphate receptors (IP(3)Rs)) that is gated by the second messenger inositol 1,4,5-trisphosphate (IP(3)). To accomplish this, we mutated residues within the putative pore forming region of the channel and analyzed the functional properties of mutant channels using a (45)Ca(2+) flux assay and single channel electrophysiological analyses. Two IP(3)R mutations, V2548I and D2550E, retained the ability to release (45)Ca(2+) in response to IP(3). When analyzed at the single channel level; both recombinant channels had IP(3)-dependent open probabilities similar to those observed in wild-type channels. The mutation V2548I resulted in channels that exhibited a larger K(+) conductance (489 +/- 13 picosiemens (pS) for V2548I versus 364 +/- 5 pS for wild-type), but retained a Ca(2+) selectivity similar to wild-type channels (P(Ca(2+)):P(K(+)) approximately 4:1). Conversely, D2550E channels were nonselective for Ca(2+) over K(+) (P(Ca(2+)):P(K(+)) approximately 0.6:1), while the K(+) conductance was effectively unchanged (391 +/- 4 pS). These results suggest that amino acid residues Val(2548) and Asp(2550) contribute to the ion conduction pathway. We propose that the pore of IP(3)R channels has two distinct sites that control monovalent cation permeation (Val(2548)) and Ca(2+) selectivity (Asp(2550)).

Interaction of Chromogranin B and the Near N-terminal Region of Chromogranin B with an Intraluminal Loop Peptide of the Inositol 1,4,5-Trisphosphate Receptor

Given the interaction of the inositol 1,4,5-trisphosphate receptor (IP(3)R) with chromogranins A (CGA) and B (CGB), two major Ca(2+) storage proteins of secretory granules that have been shown to be IP(3)-sensitive intracellular Ca(2+) store of neuroendocrine cells, we have investigated the potential interaction of the intraluminal loop regions of the IP(3)R with both intact CGB and the conserved near N-terminal region of CGB. The interaction studies carried out with CGB and glutathione S-transferase fusion proteins of intraluminal loop regions of bovine type 1 IP(3)R showed that CGB interacts with intraluminal loop 3-2 (the second loop formed between transmembrane regions 5 and 6) of the IP(3)R at both pH 5.5 and 7.5. Analytical ultracentrifugation studies also indicated that CGB interacts with the same intraluminal loop region of the IP(3)R and the interaction was much stronger than that between CGA and the loop. Moreover, the conserved near N-terminal region of CGB also interacted with the intraluminal loop region of the IP(3)R. The CGB interaction with the IP(3)R intraluminal loop peptide at pH 7.5 showed a DeltaG(0) value of -8.1 kcal/mol at 37 degrees C for a 1:1 stoichiometry, indicating a K(d) of approximately 1.9 micrometer. These results give insight into the molecular organization of the IP(3)-sensitive Ca(2+) store.

Thermodynamic molecular switch in macromolecular interactions.

It is known that most living systems can live and operate optimally only at a sharply defined temperature, or over a limited temperature range, at best, which implies that many basic biochemical interactions exhibit a well-defined Gibbs free energy minimum as a function of temperature. The Gibbs free energy change, deltaG(o) (T), for biological systems shows a complicated behavior, in which deltaG(o)(T) changes from positive to negative, then reaches a negative value of maximum magnitude (favorable), and finally becomes positive as temperature increases. The critical factor in this complicated thermodynamic behavior is a temperature-dependent heat capacity change (deltaCp(o)(T) of reaction, which is positive at low temperature, but switches to a negative value at a temperature well below the ambient range. Thus, the thermodynamic molecular switch determines the behavior patterns of the Gibbs free energy change, and hence a change in the equilibrium constant, Keq, and/or spontaneity. The subsequent, mathematically predictable changes in deltaH(o)(T), deltaS(o)(T), deltaW(o)(T), and deltaG(o)(T) give rise to the classically observed behavior patterns in biological reactivity, as demonstrated in three interacting protein systems: the acid dimerization reaction of alpha-chymotrypsin at low pH, interaction of chromogranin A with the intraluminal loop peptide of the inositol 1,4,5-triphosphate receptor at pH 5.5, and the binding of L-arabinose and D-galactose to the L-arabinose binding protein of Escherichia coli. In cases of protein unfolding of four mutants of phage T4 lysozyme, no thermodynamic molecular switch is observed.

Interaction between an intraluminal loop peptide of the inositol 1,4,5-trisphosphate receptor and the near N-terminal peptide of chromogranin A

The near N-terminal region of chromogranin A (CGA) has been shown to be the secretory vesicle membrane binding region, and tetrameric chromogranin A has been demonstrated to bind four molecules of an intraluminal loop peptide of the inositol 1,4,5-trisphosphate (IP 3) receptor. It was therefore necessary to determine whether the conserved near N-terminal region of CGA interacts with the intraluminal loop region of the IP 3 receptor. In the present study, we found that the proposed anchor region of CGA, the conserved near N-terminal region, does indeed interact with the intraluminal loop region of the IP 3 receptor at the intravesicular pH of 5.5, further strengthening the case for the potential interaction between tetrameric chromogranins and tetrameric IP 3 receptors in the cell.

Radioligand binding and immunoautoradiographic evidence for a lack of toxicity to dopaminergic nerve terminals in human cocaine overdose victims

Radioligand binding to and immunolabeling of transport sites associated with monoamine-containing synaptic vesicles affords a novel approach for mapping the integrity of dopaminergic (DAergic) nerve terminals. The present study used [ 125I]iodovinyltetrabenzine ([ 125I]TBZ) and a fusion protein antibody directed at the large intraluminal loop of the neuronal vesicular monoamine transporter (hVMAT2-loop) as probes to assess the effects of chronic cocaine use on the integrity of DAergic nerve terminals in the striatum of cocaine fatalities. Visualization of [ 125I]TBZ binding in human brain revealed a distinct pattern of labeling throughout the rostral-caudal extent of the striatum. Saturation binding of [ 125I]TBZ in striatal membranes demonstrated a single high affinity site ( K d = 2.3 ± 0.9 nM and B max = 55.5 ± 8.1 pmol/g tissue) with a pharmacological profile (tetrabenazine ≥ iodovinyltetrabenazine > ketanserin ≥ reserpine > haloperidol > GBR 12909) consistent with the specific labeling of hVMAT2. Quantitative in vitro autoradiography demonstrated no significant alteration in the density of [ 125I]TBZ binding sites in the anterior and posterior sectors of the striatum in cocaine fatalities with and without preterminal excited delirium as compared to drug-free and age-matched control subjects. Similarly, the levels of hVMAT2-loop immunoreactivity were not significantly different across control and cocaine fatality groups. The results demonstrate the lack of an alteration in [ 125I]TBZ binding sites and hVMAT2 protein in the striatum from a young cohort of cocaine fatalities. Since striatal VMAT2 is primarily associated with DAergic nerve terminals, these results suggest that chronic cocaine use failed to affect the integrity of striatal DAergic nerve terminals.

Thermodynamic study of the pH-dependent interaction of chromogranin A with an intraluminal loop peptide of the inositol 1,4,5-trisphosphate receptor.

The secretory vesicles of adrenal chromaffin cells have previously been identified as a major inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, and their Ca2+ store role has been attributed to the presence of chromogranin A, a high capacity, low affinity Ca2+ binding protein. Chromogranin A has since been shown to exist primarily in a dimeric state at pH 7.5 and primarily in a tetrameric state at the intravesicular pH of 5.5 and has also been shown to interact with the membrane proteins of secretory vesicles at pH 5.5, including a 260-kDa protein reactive to IP3 receptor antibody [Yoo, S. H. (1994) J. Biol. Chem. 269, 12001-12006]. In a recent study, chromogranin A was shown to interact with one of the intraluminal loop regions of the IP3 receptor at pH 5.5 but not at pH 7.5 [Yoo, S. H., & Lewis, M. S. (1994) FEBS Lett. 341, 28-32]. To gain further insight, we have studied the temperature dependence of the pH-dependent interaction of chromogranin A with the intraluminal peptide of the the IP3 receptor by analytical ultracentrifugation, using multiwavelength scan analysis, and found that four molecules of the intraluminal domain peptide of the IP3 receptor bound to each chromogranin A tetramer with delta Go values ranging from -23.6 to -27.6 kcal mol-1 in the absence and presence of 35 mN Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

PH-dependent interaction of an intraluminal loop of inositol 1,4,5-trisphosphate receptor with chromogranin A

The inositol 1,4,5-trisphosphate (IP 3)-sensitive Ca 2+ store role of the secretory vesicles of adrenal medullary chromaffin cells is attributed to the presence of high capacity, low affinity Ca 2+ binding protein chromogranin A. Chromogranin A has recently been shown to interact with the protein component(s) on the intraluminal side of the secretory vesicle membrane at the intravesicular pH of 5.5 but to dissociate from them at the near physiological pH of 7.5. Further, one of the chromogranin A-interacting membrane proteins was tentatively identified as the IP 3 receptor. Therefore, the pH-dependent potential interaction of the intraluminal loop domains of the IP 3 receptor with chromogranin A was studied by analytical ultracentrifugation utilizing synthetic intraluminal loop peptides of the IP 3 receptor labeled with 5-hydroxy-tryptophan at the N-terminus as a chromophore. One of the intraluminal loop domains was found to interact with chromogranin A at pH 5.5 but not at pH 7.5, suggesting the importance of the intraluminal loop domain in transmitting Ca 2+ mobilization signals to chromogranin A.

Oxygen-induced retinopathy: ultrastructure of vitreous new vessels in the kitten model

Oxygen-induced retinopathy was produced by exposing 3-day-old kittens to 80% oxygen between 60 and 105 hours. They were then allowed to survive in room air until day 15, 21 or 28 when they were sacrificed after perfusion with colloidal carbon. Specimens were prepared for transmission electron microscopy. Ninety separate vitreous capillaries from oxygen-treated animals were examined. A total of 235 intercellular junctions were examined, 116 of them from the 15-day old animals. In the 15-day old animals, five junctions of 116 were open and the remainder were tight. No open junctions were seen in 21- or 28-day-old animals. In one capillary from a 15-day animal, fenestrated endothelium was seen in an aberrant, intraluminal loop of endothelium which formed no part of the blood/tissue barrier. The wall thickness of the vitreous new vessels seemed to decrease and the number of vesicles and vacuoles appeared to increase with increasing age. The basement membrane of the vitreous new vessels was scanty. In some sections, cells, presumably macrophages, were seen outside the new vessels. It is concluded that the increased permeability of the vitreous new vessels in 15-day-old animals can be explained by, and is possibly totally due to, the presence of open endothelial junctions.