Program in Molecular Physiology
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The Program in Molecular Physiology (PMP) brings together a group of resident and visiting scientists who share common interests in the molecular bases of cellular physiology. Through diverse subjects the member laboratories focus on cellular plasticity and the properties of molecular transport mechanisms. A variety of experimental approaches are used ranging from molecular and biochemical methodologies, through biophysics, to advanced optical and electrochemical imaging techniques.
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ArticleImprovements in simultaneous sodium and calcium imaging( 2019-01-08)High speed imaging of ion concentration changes in neurons is an important and growing tool for neuroscientists. We previously developed a system for simultaneously measuring sodium and calcium changes in small compartments in neurons (Miyazaki and Ross, 2015). We used this technique to analyze the dynamics of these ions in individual pyramidal neuron dendritic spines (Miyazaki and Ross, 2017). This system is based on high speed multiplexing of light emitting diodes (LEDs) and classic organic indicators. To improve this system we made additional changes, primarily incorporating lasers in addition to the LEDs, more sophisticated imaging protocols, and the use of newer sodium and calcium indicators. This new system generates signals with higher signal to noise ratio (S/N), less background fluorescence, and less photodynamic damage. In addition, by using longer wavelength indicators instead of indicators sensitive in the UV range, it allows for the incorporation of focal uncaging along with simultaneous imaging, which should extend the range of experiments.
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ArticleNeurovascular interaction and the pathophysiology of diabetic retinopathy(Hindawi Publishing, 2011)Diabetic retinopathy (DR) is the most severe of the several ocular complications of diabetes, and in the United States it is the leading cause of blindness among adults 20 to 74 years of age. Despite recent advances in our understanding of the pathogenesis of DR, there is a pressing need to develop novel therapeutic treatments that are both safe and efficacious. In the present paper, we identify a key mechanism involved in the development of the disease, namely, the interaction between neuronal and vascular activities. Numerous pathological conditions in the CNS have been linked to abnormalities in the relationship between these systems. We suggest that a similar situation arises in the diabetic retina, and we propose a logical strategy aimed at therapeutic intervention.
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ArticleDistribution of postsynaptic density (PSD)-95 and Ca2+/calmodulin-dependent protein kinase II at the PSD(Society for Neuroscience, 2003-12-03)Postsynaptic densities (PSDs) contain proteins that regulate synaptic transmission. We determined the positions of calcium/calmodulin-dependent protein kinase II (CaMKII) and PSD-95 within the three-dimensional structure of isolated PSDs using immunogold labeling, rotary shadowing, and electron microscopic tomography. The results show that all PSDs contain a central mesh immediately underlying the postsynaptic membrane. Label for PSD-95 is found on both the cytoplasmic and cleft sides of this mesh, averaging 12 nm from the cleft side. All PSDs label for PSD-95. The properties of CaMKII labeling are quite different. Label is virtually absent on the cleft sides of PSDs, but can be heavy on the cytoplasmic side at a mean distance of 25 nm from the cleft. In tomograms, CaMKII holoenzymes can be visualized directly, appearing as labeled, tower-like structures reflecting the 20 nm diameter of the holoenzyme. These towers protrude from the cytoplasmic side of the central mesh. There appears to be a local organization of CaMKII, as judged by fact that the nearest-neighbor distances are nearly invariant over a wide range of labeling density for CaMKII. The average density of CaMKII holoenzymes is highly variable, ranging from zero to values approaching a tightly packed state. This variability is significantly higher than that for PSD-95 and is consistent with an information storage role for CaMKII.
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ArticlePersistent accumulation of calcium/calmodulin-dependent protein kinase II in dendritic spines after induction of NMDA receptor-dependent chemical long-term potentiation(Society for Neuroscience, 2004-10-20)Calcium/calmodulin-dependent protein kinase II (CaMKII) is a leading candidate for a synaptic memory molecule because it is persistently activated after long-term potentiation (LTP) induction and because mutations that block this persistent activity prevent LTP and learning. Previous work showed that synaptic stimulation causes a rapidly reversible translocation of CaMKII to the synaptic region. We have now measured green fluorescent protein (GFP)-CaMKIIα translocation into synaptic spines during NMDA receptor-dependent chemical LTP (cLTP) and find that under these conditions, translocation is persistent. Using red fluorescent protein as a cell morphology marker, we found that there are two components of the persistent accumulation. cLTP produces a persistent increase in spine volume, and some of the increase in GFP-CaMKIIα is secondary to this volume change. In addition, cLTP results in a dramatic increase in the bound fraction of GFP-CaMKIIα in spines. To further study the bound pool, immunogold electron microscopy was used to measure CaMKIIα in the postsynaptic density (PSD), an important regulator of synaptic function. cLTP produced a persistent increase in the PSD-associated pool of CaMKIIα. These results are consistent with the hypothesis that CaMKIIα accumulation at synapses is a memory trace of past synaptic activity.
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PreprintNovel γ-carboxyglutamic acid-containing peptides from the venom of Conus textile( 2006-04-25)The cone snail is the only invertebrate system in which the vitamin K dependent carboxylase (or γ-carboxylase) and its product γ-carboxyglutamic acid (Gla)1 have been identified. It remains the sole source of structural information of invertebrate γ-carboxylase subtrates. Four novel γ- carboxyglutamic acid (Gla)1 containing peptides were purified from the venom of Conus textile and characterized by biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have 6 Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-L-bromotryptophan. Gla(2)-TxVI/A and Gla(2)- TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids –46 to –27) were nearly identical and highly hydrophobic. The predicted propeptide region (–20 to –1) that contains the γ-carboxylation recognition site (γ-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conus γ-carboxylase and synthetic peptide substrates localized the γ-CRS of Gla(1)-TxVI to the region –14 to –1 of the polypeptide precursor: the Km was reduced from 1.8 mM for Gla (1)-TxVI lacking a propeptide to 24 μM when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km 10-fold.
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PreprintCa2+ signalling, voltage-gated Ca2+ channels and praziquantel in flatworm neuromusculature( 2005-10)Transient changes in calcium (Ca2+) levels regulate a wide variety of cellular processes, and cells employ both intracellular and extracellular sources of Ca2+ for signaling. Praziquantel, the drug of choice against schistosomiasis, disrupts Ca2+ homeostasis in adult worms. This review will focus on voltage-gated Ca2+ channels, which regulate levels of intracellular Ca2+ by coupling membrane depolarization to entry of extracellular Ca2+. Ca2+ channels are members of the ion channel superfamily and represent essential components of neurons, muscles, and other excitable cells. Ca2+ channels are membrane protein complexes in which the pore-forming α1 subunit is modulated by auxiliary subunits such as β and α2δ. Schistosomes express two Ca2+ channel β subunit subtypes: a conventional subtype similar to β subunits found in other vertebrates and invertebrates; and a novel variant subtype with unusual structural and functional properties. The variant schistosome β subunit confers praziquantel sensitivity to an otherwise praziquantel-insensitive mammalian Ca2+ channel, implicating it as a mediator of praziquantel action.
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PreprintVoltage-gated calcium channel subunits from platyhelminths : potential role in praziquantel action( 2006-02-07)Voltage-gated calcium (Ca2+) channels provide the pathway for Ca2+ influxes that underlie Ca2+-dependent responses in muscles, nerves, and other excitable cells. They are also targets of a wide variety of drugs and toxins. Ca2+ channels are multisubunit protein complexes consisting of a pore-forming α1 subunit and other modulatory subunits, including the β subunit. Here, we review the structure and function of schistosome Ca2+ channel subunits, with particular emphasis on variant Ca2+ channel β subunits (Cavβvar) found in these parasites. In particular, we examine the role these β subunits may play in the action of praziquantel, the current drug of choice against schistosomiasis. We also present evidence that Cavβvar homologs are found in other praziquantel-sensitive platyhelminths such as the pork tapeworm, Taenia solium, and that these variant β subunits may thus represent a platyhelminth-specific gene family.
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PreprintSchistosoma mansoni : use of a fluorescent indicator to detect nitric oxide and related species in living parasites( 2005-12-20)Nitric oxide (NO) is synthesized enzymatically by nitric oxide synthase (NOS). Several groups have previously presented evidence for NOS activity and immunoreactivity in several parasitic platyhelminths, including schistosomes. Here, we use 4,5-diaminofluorescein-2 diacetate (DAF-2 DA), a fluorescent indicator of NO, to detect NO in living schistosomes. In adult worms, DAF-2 fluorescence is found selectively in epithelial-like cells. Fluorescence increases when worms are incubated in L-arginine, the precursor of NO synthesis, and decreases dramatically in the presence of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), indicating that predicted NO release may be NOS-dependent, and that enzymatic NO signaling pathways may play an important role in schistosome physiology.
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PreprintInhibition of phosphatase activity facilitates the formation and maintenance of NMDA-induced calcium/calmodulin-dependent protein kinase ii clusters in hippocampal neurons( 2004-10-04)The majority of hippocampal neurons in dissociated cultures and in intact brain exhibit clustering of CaMKII into spherical structures with an average diameter of 110 nm when subjected to conditions that mimic ischemia and excitotoxicity (Tao-Cheng et al., 2001). Because clustering of CaMKII would reduce its effective concentration within the neuron, it may represent a cellular strategy to prevent excessive CaMKII-mediated phosphorylation during episodes of Ca2+ overload. Here we employ a relatively mild excitatory stimulus to promote sub-maximal clustering for the purpose of studying the conditions for the formation and disappearance of CaMKII clusters. Treatment with 30 µM NMDA for 2 min produced CaMKII clustering in ~15 percent of dissociated hippocampal neurons in culture, as observed by pre-embedding immunogold electron microscopy. These CaMKII clusters could be labeled with antibodies specific to the phospho form (Thr286) of CaMKII, suggesting that at least some of the CaMKII molecules in clusters are autophosphorylated. To test whether phosphorylation is involved in the formation and maintenance of CaMKII clusters, the phosphatase inhibitors calyculin A (5 nM) or okadaic acid (1 µM) were included in the incubation medium. With inhibitors more neurons exhibited CaMKII clusters in response to 2 min NMDA treatment. Furthermore, 5 min after the removal of NMDA and Ca2+, CaMKII clusters remained and could still be labeled with the phospho-specific antibody. In contrast, in the absence of phosphatase inhibitors, no clusters were detected 5 min after the removal of NMDA and Ca2+ from the medium. These results suggest that phosphatases type 1 and/or 2A regulate the formation and disappearance of CaMKII clusters.