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Biomedical subjects

G J Augustine

Publications and source records attributed to G J Augustine.

At least 19 recordsLinked to original sources
PubMed1998

Regulation of neurotransmitter release kinetics by NSF.

NSF (N-ethylmaleimide-sensitive factor) is an adenosine triphosphatase (ATPase) that contributes to a protein complex essential for membrane fusion. The synaptic function of this protein was investigated by injecting, into the giant presynaptic terminal of squid, peptides that inhibit the ATPase activity of NSF stimulated by the soluble NSF attachment protein (SNAP). These peptides reduced the amount and slowed the kinetics of neurotransmitter release as a result of actions that required vesicle turnover and occurred at a step subsequent to vesicle docking. These results define NSF as an essential participant in synaptic vesicle exocytosis that regulates the kinetics of neurotransmitter release and, thereby, the integrative properties of synapses.

Adenosine Triphosphatases↗
PubMed1998

Rabphilin-3A: a multifunctional regulator of synaptic vesicle traffic.

We have investigated the function of the synaptic vesicle protein Rabphilin-3A in neurotransmitter release at the squid giant synapse. Presynaptic microinjection of recombinant Rabphilin-3A reversibly inhibited the exocytotic release of neurotransmitter. Injection of fragments of Rabphilin-3A indicate that at least two distinct regions of the protein inhibit neurotransmitter release: the NH2-terminal region that binds Rab3A and is phosphorylated by protein kinases and the two C2 domains that interact with calcium, phospholipid, and beta-adducin. Each of the inhibitory fragments and the full-length protein had separate effects on presynaptic morphology, suggesting that individual domains were inhibiting a subset of the reactions in which the full-length protein participates. In addition to inhibiting exocytosis, constructs containing the NH2 terminus of Rabphilin-3A also perturbed the endocytotic pathway, as indicated by changes in the membrane areas of endosomes, coated vesicles, and the plasma membrane. These results indicate that Rabphilin-3A regulates synaptic vesicle traffic and appears to do so at distinct stages of both the exocytotic and endocytotic pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Role of intrinsic synaptic circuitry in collicular sensorimotor integration.

The superficial gray layer of the superior colliculus contains a map that represents the visual field, whereas the underlying intermediate gray layer contains a vector map of the saccades that shift the direction of gaze. These two maps are aligned so that a particular region of the visual field is represented directly above the neurons that orient the highest acuity area of the retina toward that region. Although it has been proposed that the transmission of information from the visuosensory to the motor map plays an important role in the generation of visually guided saccades, experiments have failed to demonstrate any functional linkage between the two layers. We examined synaptic transmission between these layers in vitro by stimulating the superficial layer while using whole-cell patch-clamp methods to measure the responses of intermediate layer neurons. Stimulation of superficial layer neurons evoked excitatory postsynaptic currents in premotor cells. This synaptic input was columnar in organization, indicating that the connections between the layers link corresponding regions of the visuosensory and motor maps. Excitatory postsynaptic currents were large enough to evoke action potentials and often occurred in clusters similar in duration to the bursts of action potentials that premotor cells use to command saccades. Our results indicate the presence of functional connections between the superficial and intermediate layers and show that such connections could play a significant role in the generation of visually guided saccades.

Action Potentials↗
PubMed1997

Disruption of syntaxin-mediated protein interactions blocks neurotransmitter secretion.

The membrane protein syntaxin participates in several protein-protein interactions that have been implicated in neurotransmitter release. To probe the physiological importance of these interactions, we microinjected into the squid giant presynaptic terminal botulinum toxin C1, which cleaves syntaxin, and the H3 domain of syntaxin, which mediates binding to other proteins. Both reagents inhibited synaptic transmission yet did not affect the number or distribution of synaptic vesicles at the presynaptic active zone. Recombinant H3 domain inhibited the interactions between syntaxin and SNAP-25 that underlie the formation of stable SNARE complexes in vitro. These data support the notion that syntaxin-mediated SNARE complexes are necessary for docked synaptic vesicles to fuse.

Amino Acid Sequence↗
PubMed1997

Pharmacologically distinct presynaptic calcium channels in cerebellar excitatory and inhibitory synapses.

We have used whole-cell patch clamp recordings and pharmacological blockers of Ca channels to compare the pharmacology of Ca channels that mediate synaptic transmission at the three types of synapses innervating Purkinje cells in rat cerebellar slices. Both parallel fiber and climbing fiber excitatory synapses were sensitive to the P-type Ca channel blocker, omega-AgaIVA and the P/Q/N-type channel blocker, omega-conotoxin MVIIC. Transmission at inhibitory interneuronal synapses was not suppressed by these toxins, or by the N-type (omega-conotoxins GVIA and MVIIA) or L-type (nimodipine) channel blockers. Inhibitory transmission could be inhibited by Ni2+ and amiloride, but only at concentrations (IC50 approximately 300 microM) that affect other types of Ca channels. These results indicate that excitatory and inhibitory presynaptic terminals of the cerebellar cortex possess different types of voltage-gated Ca channels. The excitatory terminals contain P-type, Q-type and N-type Ca channels, with P-type channels playing the most prominent role. The inhibitory terminals possess quite different type(s) of Ca channel. The heterogeneous distribution of Ca channel types should impart unique properties to transmitter release from the excitatory and inhibitory terminals.

Animals↗
PubMed1997

Chemical two-photon uncaging: a novel approach to mapping glutamate receptors.

Functional mapping of neurotransmitter receptors requires rapid and localized application of transmitter. The usefulness of caged glutamate for this purpose has been limited, because photolysis by unfocused light above and below the target cell limits depth resolution. This problem is eliminated by using a double-caged glutamate that requires absorption of two photons for conversion to active glutamate, resulting in a substantial improvement in spatial resolution over conventional caged glutamate. This method was used to map the distribution of glutamate receptors on hippocampal pyramidal neurons. A higher density of AMPA receptors was found on distal apical dendrites than on basal or primary apical dendrites, suggesting that synaptic efficacy is locally heterogeneous. Such "chemical two-photon uncaging" offers a simple, general, and economical strategy for spatially localized photolysis of caged compounds.

2-Amino-5-phosphonovalerate↗
PubMed1996

Alterations in calcium channel currents underlie defective insulin secretion in a transgenic mouse.

A transgenic mouse overexpressing a mutant form of calmodulin (CaM-8) that is selectively targeted to pancreatic beta-cells has an impaired ability to secrete insulin in response to elevated blood glucose. Fluorescence measurements of cytosolic Ca2+ concentration ([Ca2+]i) showed that intracellular Ca2+ rises produced by glucose were smaller than normal in beta-cells of CaM-8 mice. Glucose utilization rates were not different between the CaM-8 and control beta-cells, suggesting that glucose metabolism was unperturbed by CaM-8. Ion channel defects were implicated in the phenotype of CaM-8 beta-cells because treatment of these cells with tolbutamide, a blocker of ATP-sensitive K+ channels, produced smaller than normal amounts of insulin secretion and Ca2+ rises. Depolarization with elevated extracellular K+ also produced smaller Ca2+ rises in beta-cells from CaM-8 mice. Whole-cell patch-clamp recordings revealed that Ca2+ channel currents of beta-cells from CaM-8 mice were half as large as Ca2+ currents in control cells, while the currents carried by delayed rectifier and ATP-sensitive K+ channels were similar in magnitude in both cell types. We conclude that expression of the CaM-8 form of calmodulin causes a down-regulation of Ca2+ channel currents, which reduces Ca2+ entry and accumulation when glucose stimulates closure of the ATP-sensitive K+ channels. The reduction in intracellular Ca2+ accumulation then prevents an adequate amount of insulin from being secreted from beta-cells of CaM-8 mice.

Adenosine Triphosphate↗
PubMed1996

Adaptation of Ca(2+)-triggered exocytosis in presynaptic terminals.

Rapid increases in Ca2+ concentration, produced by photolysis of caged Ca2+, triggered exocytosis in squid nerve terminals. This exocytosis was transient in nature, decaying with a time constant of approximately 30 ms. The decay could not be explained by a decline in presynaptic Ca2+ concentration, depletion of synaptic vesicles, or desensitization of postsynaptic receptors. Experiments in which Ca2+ was increased either in a series of steps or continuously at different rates suggested that the decay is caused by adaptation of the exocytotic Ca2+ receptor to higher levels of Ca2+. This adjustable sensitivity to Ca2+ represents a novel property of the triggering mechanism that can be used to evaluate molecular models of exocytosis. Adaptation can limit the amount of transmitter released by a nerve terminal and permit the speed of a presynaptic Ca2+ rise to serve as a critical determinant of synaptic efficacy.

Adaptation, Physiological↗
PubMed1996

Exocytosis: proteins and perturbations.

Exocytosis is the primary means of cellular secretion. Because exocytosis involves fusion between the plasma membrane and the membrane of secretory vesicles, it is likely that proteins on these two membranes, as well as additional proteins in cellular cytoplasm, mediate exocytosis. Although we know much about the proteins of secretory cells, we still have much to learn about how these proteins participate in exocytosis; in no case has an unambiguous exocytotic function been assigned to any of these proteins. To identify the roles of proteins in exocytosis it is necessary to perturb protein function in living secretory cells. We review a number of perturbation strategies and summarize what this approach has taught us about the functional roles of proteins in exocytosis, concluding with a molecular model of protein dynamics during exocytosis.

Animals↗
PubMed1995

Defective glycolysis and calcium signaling underlie impaired insulin secretion in a transgenic mouse.

Pancreatic beta cells from mice that overexpress the Ca(2+)-binding protein calmodulin have a unique secretory defect that leads to chronic hyperglycemia. To further understand the molecular basis underlying this defect, we have studied signaling pathways in these beta cells. Measurements of cytosolic free Ca2+ concentration ([Ca2+]i) using fura-2 or indo-1 revealed a markedly reduced response when glucose was the stimulant. However, eliciting membrane depolarization with 50 mM K+ or the addition of the ATP-sensitive K+ (K+ ATP) channel antagonist tolbutamide restored [Ca2+]i transients to near normal levels. Electrophysiological analysis of the beta cell ion channels revealed that Ca2+ currents, delayed rectifier K+ currents, and K+ATP channel currents were similar in transgenic and nontransgenic cells, suggesting that these ion channels were able to function normally. However, whereas K+ATP channel currents in control cells were reduced by 50% by the presence of high glucose, those in transgenic cells were unaltered. Addition of tolbutamide inhibited this channel and enhanced the secretion of insulin in response to glucose for both control and transgenic cells. As these observations implicated a metabolic defect, glucose utilization, which is an indicator of glucose metabolism and ATP production in beta cells, was measured and found to be reduced by 40% in the transgenic cells. These data support the contention that excessive levels of calmodulin may compromise the ability of the beta cell to metabolize glucose and to modulate the state of the K+ATP channel, resulting in an inadequate control of the membrane potential, which collectively impair [Ca2+]i and thus insulin secretion in response to glucose.

Adenosine Triphosphate↗
PubMed1995

SNAP-mediated protein-protein interactions essential for neurotransmitter release.

The constitutive fusion of transport vesicles with intracellular membranes requires soluble proteins called SNAPs. Certain presynaptic proteins implicated in synaptic vesicle exocytosis also bind SNAPs, suggesting that SNAPs participate in the calcium-regulated membrane fusion events mediating neurotransmitter release. Here we show that injection of recombinant SNAPs into the giant synapse of squid enhances transmitter release. Conversely, injection of peptides designed to mimic the sites at which SNAP interacts with its binding partners inhibits transmitter release downstream of synaptic vesicle docking. A SNAP-dependent protein complex must therefore mediate transmitter release, showing that transmitter release shares a common molecular mechanism with constitutive membrane fusion.

Amino Acid Sequence↗
PubMed1995

Subthreshold synaptic Ca2+ signalling in fine dendrites and spines of cerebellar Purkinje neurons.

The conventional view of synaptic integration is that it results from the simple summation of electrical signals produced by each active synapse innervating a given neuron. However, because synaptic action can go beyond the production of postsynaptic electrical signals, to include intracellular biochemical events such as the generation of second messengers, it is possible that synaptic integration could occur at another level. We have considered this possibility by examining changes in the dendritic concentration of the second messenger, calcium, resulting from subthreshold excitatory synaptic activity in cerebellar Purkinje neurons. We report here clear evidence that such non-electrical synaptic integration occurs and that it takes place in restricted dendritic compartments consisting of spines and adjacent fine dendrites.

Action Potentials↗
PubMed1995

Confocal imaging and local photolysis of caged compounds: dual probes of synaptic function.

Chemical signals generated at synapses are highly limited in both spatial range and time course, so that experiments studying such signals must measure and manipulate them in both these dimensions. We describe an optical system that combines confocal laser scanning microscopy, to measure such signals, with focal photolysis of caged compounds. This system can elevate neurotransmitter and second messenger levels in femtoliter volumes of single dendrites within a millisecond. The method is readily combined with whole-cell patch-clamp measurements of electrical signals in brain slices. In cerebellar Purkinje cells, photolysis of caged IP3 causes spatially restricted intracellular release of Ca2+, and photolysis of a caged Ca2+ compound locally opens Ca(2+)-dependent K+ channels. Furthermore, localized photolysis of the caged neurotransmitter GABA transiently activates GABA receptors. The use of focal uncaging can yield new information about the spatial range of signaling actions at synapses.

Animals↗
PubMed1994

Specific interactions of Mss4 with members of the Rab GTPase subfamily.

Mss4 is a mammalian protein that was identified as a suppressor of a yeast secretory mutant harboring a mutation in the GTPase Sec4 and was found to stimulate GDP release from this protein. We have now performed a biochemical characterization of the Mss4 protein and examined the specificity of its association with mammalian GTPases. Mss4 is primarily a soluble protein with a widespread tissue distribution. Recombinant Mss4 binds GTPases present in tissue extracts, and by a gel overlay assay binds specifically Rab Rab10proteins. We further define the Mss4-GTPase interaction to a subset of Rabs belonging to the same subfamily branch which include Rab1, Rab3, Rab8, Rab10, Sec4 and Ypt1 but not Rab2, Rab4, Rab5, Rab6, Rab9 and Rab11. Accordingly, Mss4 co-precipitates from a brain extract with Rab3a but not Rab5. Mss4 only stimulates GDP release from, and the association of GTP gamma S with, this Rab subset. Recombinant Mss4 and Rab3a form a stable complex in solution that is dissociated with either GDP or GTP gamma S. Injection of Mss4 into the squid giant nerve terminal enhances neurotransmitter release. These results suggest that Mss4 behaves as a guanylnucleotide exchange factor (GEF) for a subset of Rabs to influence distinct vesicular transport steps along the secretory pathway.

Animals↗
PubMed1994

Combining patch-clamp and optical methods in brain slices.

Combining patch-clamp and optical imaging techniques in brain slices offers several advantages for physiological studies of nerve cells. Numerous practical considerations weigh heavily in this design of an apparatus suitable for such combined measurements. These considerations include the thickness of the slices, the type of microscope to be used for imaging and the kind of optical signal to be measured. A system that combine optical and patch-clamp methods can be modified readily to permit studies of intracellular and extracellular signaling pathways via flash photolysis of caged compounds.

Animals↗