Homeostatic control of presynaptic release is triggered by postsynaptic membrane depolarization.

Homeostatic mechanisms regulate synaptic function to maintain nerve and muscle excitation within reasonable physiological limits. The mechanisms that initiate homeostasic changes to synaptic function are not known. We specifically impaired cellular depolarization by expressing the Kir2.1 potassium channel in Drosophila muscle. In Kir2.1-expressing muscle there is a persistent outward potassium current ( approximately 10 nA), decreased muscle input resistance (50-fold), and a hyperpolarized resting potential. Despite impaired muscle excitability, synaptic depolarization of muscle achieves wild-type levels. A quantal analysis demonstrates that increased presynaptic release (quantal content), without a change in quantal size (mEPSC amplitude), compensates for altered muscle excitation. Because morphological synaptic growth is normal, we conclude that a homeostatic increase in presynaptic release compensates for impaired muscle excitability. These data demonstrate that a monitor of muscle membrane depolarization is sufficient to initiate synaptic homeostatic compensation.

Calcium influx via TRP channels is required to maintain PIP2 levels in Drosophila photoreceptors.

The trp (transient receptor potential) gene encodes a Ca2+ channel responsible for the major component of the phospholipase C (PLC) mediated light response in Drosophila. In trp mutants, maintained light leads to response decay and temporary total loss of sensitivity (inactivation). Using genetically targeted PIP2-sensitive inward rectifier channels (Kir2.1) as biosensors, we provide evidence that trp decay reflects depletion of PIP2. Two independent mutations in the PIP2 recycling pathway (rdgB and cds) prevented recovery from inactivation. Abolishing Ca2+ influx in wild-type photoreceptors mimicked inactivation, while raising Ca2+ by blocking Na+/Ca2+ exchange prevented inactivation in trp. The results suggest that Ca2+ influx prevents PIP2 depletion by inhibiting PLC activity and facilitating PIP2 recycling. Without this feedback one photon appears sufficient to deplete the phosphoinositide pool of approximately 4 microvilli.

Altered electrical properties in Drosophila neurons developing without synaptic transmission.

We examine the role of synaptic activity in the development of identified Drosophila embryonic motorneurons. Synaptic activity was blocked by both pan-neuronal expression of tetanus toxin light chain (TeTxLC) and by reduction of acetylcholine (ACh) using a temperature-sensitive allele of choline acetyltransferase (Cha(ts2)). In the absence of synaptic activity, aCC and RP2 motorneurons develop with an apparently normal morphology and retain their capacity to form synapses. However, blockade of synaptic transmission results in significant changes in the electrical phenotype of these neurons. Specifically, increases are seen in both voltage-gated inward Na(+) and voltage-gated outward K(+) currents. Voltage-gated Ca(2+) currents do not change. The changes in conductances appear to promote neuron excitability. In the absence of synaptic activity, the number of action potentials fired by a depolarizing ramp (-60 to +60 mV) is increased and, in addition, the amplitude of the initial action potential fired is also significantly larger. Silencing synaptic input to just aCC, without affecting inputs to other neurons, demonstrates that the capability to respond to changing levels of synaptic excitation is intrinsic to these neurons. The alteration to electrical properties are not permanent, being reversed by restoration of normal synaptic function. Whereas our data suggest that synaptic activity makes little or no contribution to the initial formation of embryonic neural circuits, the electrical development of neurons that constitute these circuits seems to depend on a process that requires synaptic activity.

Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila.

In synaptic transmission, vesicles are proposed to dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). We test this hypothesis in Drosophila strains lacking neural synaptobrevin (n-synaptobrevin) or syntaxin. We showed previously that loss of either protein completely blocks synaptic transmission. Here, we attempt to establish the level of this blockade. Ultrastructurally, vesicles are still targeted to the presynaptic membrane and dock normally at specialized release sites. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of n-synaptobrevin and since vesicle fusion is triggered by hyperosmotic saline in the absence of syntaxin. We conclude that the SNARE hypothesis cannot fully explain the role of these proteins in synaptic transmission. Instead, both proteins play distinct roles downstream of docking.

Characterization of eukaryotic-like kinase activity in Escherichia coli using the gene-protein database.

The gene-protein database was used to obtain the two-dimensional polyacrylamide gel coordinates of proteins phosphorylated in extracts of Escherichia coli including those phosphorylated by eukaryotic-like kinase activities. These suggest that the phosphoproteins correspond to, or co-migrate with, the product of an open reading frame at 1.3 min (Orf80), Enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (PtsI), the tRNA synthetase for histidine (HisS), and proteins involved in the response to carbon starvation and quinone treatment.

Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects.

Tetanus toxin cleaves the synaptic vesicle protein synaptobrevin, and the ensuing loss of neurotransmitter exocytosis has implicated synaptobrevin in this process. To further the study of synaptic function in a genetically tractable organism and to generate a tool to disable neuronal communication for behavioural studies, we have expressed a gene encoding tetanus toxin light chain in Drosophila. Toxin expression in embryonic neurons removes detectable synaptobrevin and eliminates evoked, but not spontaneous, synaptic vesicle release. No other developmental or morphological defects are detected. Correspondingly, only synaptobrevin (n-syb), but not the ubiquitously expressed syb protein, is cleaved by tetanus toxin in vitro. Targeted expression of toxin can produce specific behavioral defects; in one case, the olfactory escape response is reduced.

Nongranular proteolytic enzymes of rat IL-2-activated natural killer cells. II. Purification and identification of rat A-NKP 1 and A-NKP 2 as constituents of the multicatalytic proteinase (proteasome) complex.

We have recently described nongranular, cytosolic, high-molecular-weight trypsin-like (A-NKP 1) and chymotrypsin-like (A-NKP 2) proteases of interleukin-2-activated rat natural killer (A-NK) cells. A functional correlation between the inactivation of A-NKP 2 and the inhibition of rat A-NK cell-mediated cytotoxicity was found. Herein we describe the 6,000-fold purification of A-NKP 2 to apparent homogeneity following: isopycnic sucrose gradient fractionation of postnuclear supernatants, molecular sieve chromatography, and heparin-Sepharose chromatography. We also report the novel finding that A-NKP 2 as well as A-NKP 1, derived from either rat A-NK cells or the rat NK leukemic cell line CRNK-16, are constituents of the multicatalytic proteinase (MCP/proteasome) complexes of these cells. Characteristic biochemical, biophysical, and electron microscopic/ultrastructural similarity to the rat liver proteasome was observed. However, Western blot analysis using polyclonal antibodies to the rat liver proteasome clearly indicated differences in the rat hepatic proteasome and the CRNK-16-derived proteasomal subunits. The identification, characterization, and purification of A-NKP 1 and A-NKP 2, described herein, now allow for further investigation of the potential role of these proteasome components in NK cell function. Moreover, the proteasome of NK and A-NK cells can now be compared and contrasted to the granzymes of lytic granules with respect to their role in cell-mediated cytotoxicity.

Enhanced levels of multicatalytic proteinase mRNAs in Rous sarcoma virus transformed cells.

The multicatalytic proteinase (proteasome; MCP) is a high molecular mass proteinase which is found in all eukaryotic cells. Northern blot analysis of the levels of MCP mRNAs in a Rat-1 fibroblast cell line and in cells transformed with Rous sarcoma virus showed marked increases in the transformed cells. However, the results of immunoblot analysis with anti-MCP antibodies suggested that the MCP protein content of the two cell lines was similar.

A protein kinase C-like activity in Escherichia coli.

The protein kinase C (PKC) family comprises calcium- and phospholipid-dependent kinases whose activity is stimulated by diacylglycerol and tumour-promoting phorbol esters such as 12-tetradecanoyl phorbol-13-acetate (TPA). In the Gram-negative bacterium Escherichia coli, functional similarity to PKC was demonstrated in crude extracts by calcium and phospholipid-dependent, TPA-stimulated phosphorylation of a small number of endogenous substrates. Activity was reduced by sphingosine, a known inhibitor of eukaryotic PKC. Structural similarity to PKC was demonstrated in crude and partially purified bacterial extracts by cross-reactivity with several monoclonal antibodies. This revealed isozyme-specific homology between a protein(s) of relative molecular mass 80-85,000 in E. coli and the alpha- and gamma-isozymes, but probably not the beta-isozyme, of eukaryotic PKC.

Properties of subunits of the multicatalytic proteinase complex revealed by the use of subunit-specific antibodies.

The multicatalytic proteinase (MCP) is a high-molecular-mass non-lysosomal proteinase that gives rise to a characteristic pattern of bands of molecular mass 22-34 kDa on SDS/PAGE gels. Isoelectric-focusing gels of the enzyme purified from rat liver show 16 bands with isoelectric points in the range of pH 5-8.5. Two-dimensional PAGE gels reveal that there are more than the previously reported 13 polypeptides associated with the MCP from rat liver and show a pattern of 15-20 major spots and several minor ones, similar to that of MCP isolated from some other sources. Possible relationships between the different polypeptides were investigated by immunoblot analysis of electrophoretically purified proteinase subunits with affinity-purified subunit-specific antibodies as well as antibodies raised against individual denatured subunits of the complex. The results demonstrate that many of the major polypeptide components of the MCP complex are antigenically distinct. Moreover comparison of immunoreactive material in crude cell extracts with that in purified MCP preparations has shown that the polypeptides are not derived from a smaller number of higher-molecular-mass subunits. Also, individual subunits have the same apparent molecular mass in a variety of rat tissues, suggesting close similarity between MCPs of different tissues. The highest concentrations of MCP subunits occur in liver and kidney. Gel-filtration analysis of crude extracts has demonstrated that MCP polypeptides are also associated with a higher-molecular-mass complex, which may be the 26 S proteinase that has been implicated in the degradation of ubiquitin-protein conjugates.

Components of the multicatalytic proteinase complex.

The multicatalytic proteinase complex is a high molecular weight nonlysosomal proteinase. Kinetic studies of the proteolytic activities of the complex have shown that there are at least three distinct types of catalytic centre, each of which has a different specificity. All of the activities can be inhibited by the serine protease inhibitor 3,4-dichloroisocoumarin. Viewed under the electron microscope, the multicatalytic proteinase purified from rat liver appears to have a hollow cylindrical structure. It is composed of many different types of subunit and on two-dimensional polyacrylamide gels gives rise to a complex pattern of about 20 spots with pI values ranging between 5 and 8.5 and molecular masses between 22 and 34 kDa. Immunoblot analysis has shown that many of the major polypeptides are antigenically distinct. However, there are some relationships between the proteinase polypeptides. For example, although N-terminal sequences of five of the polypeptides are unique, they show considerable sequence similarity suggesting that these proteins are encoded by members of the same gene family. Also, there is some cross-reactivity between certain polypeptides when blots are probed with affinity purified, subunit-specific antisera. In addition to the variety of polypeptide components of the proteinase, a small RNA species (80 nucleotides) can be found associated with the complex even after purification by chromatographic procedures.