Biomedical subjects

K S Krishnan

Publications and source records attributed to K S Krishnan.

At least 19 recordsLinked to original sources

PubMed2007

Mutations in dynamin-related protein result in gross changes in mitochondrial morphology and affect synaptic vesicle recycling at the Drosophila neuromuscular junction.

Mitochondria are the primary source of ATP needed for the steps of the synaptic vesicle cycle. Dynamin-related protein (DRP) is involved in the fission of mitochondria and peroxisomes. To assess the role of mitochondria in synaptic function, we characterized a Drosophila DRP mutant combination that shows an acute temperature-sensitive paralysis. Sequencing of the mutant reveals a single amino acid change in the guanosine triphosphate hydrolysing domain (GTPase domain) of DRP. The synaptic mitochondria in these mutants are remarkably elongated, suggesting a role for DRP in mitochondrial fission in Drosophila. There is a loss of neuronal transmission at restrictive temperatures in electroretinogram (ERG) recordings. Like stress-sensitive B (sesB), a mitochondrial adenosine triphosphate (ATP) translocase mutant we studied earlier for its effects on synaptic vesicle recycling, an allele-specific reduction in the temperature of paralysis of Drosophila synaptic vesicle recycling mutant shibire was seen in the DRP mutant background. These data, in addition to depletion of vesicles observed in electron microscopic sections of photoreceptor synapses at restrictive temperatures, suggest a block in synaptic vesicle recycling due to reduced mitochondrial function.

Amino Acid Sequence↗

PubMed2006

De novo sequencing and disulfide mapping of a bromotryptophan-containing conotoxin by Fourier transform ion cyclotron resonance mass spectrometry.

T-1-family conotoxins belong to the T-superfamily and are composed of 10-17 amino acids. They share a common cysteine framework and disulfide connectivity and exhibit unusual posttranslational modifications, such as tryptophan bromination, glutamic acid carboxylation, and threonine glycosylation. We have isolated and characterized a novel peptide, Mo1274, containing 11 amino acids, that shows the same cysteine pattern, -CC-CC, and disulfide linkage as those of the T-1-family members. The complete sequence, GNWCCSARVCC, in which W denotes bromotryptophan, was derived from MS-based de novo sequencing. The FT-ICR MS/MS techniques of electron capture dissociation (ECD), infrared multiphoton dissociation, and collision-induced dissociation served to detect and localize the tryptophan bromination. The bromine contributes a distinctive isotopic distribution in all fragments that contain bromotryptophan. ECD fragmentation results in the loss of bromine and return to the normal isotopic distribution. Disulfide connectivity of Mo1274, between cysteine pairs 1-3 and 2-4, was determined by mass spectrometry in combination with chemical derivatization employing tris(2-carboxyethyl)phosphine, followed by differential alkylation with N-ethylmaleimide and iodoacetamide. The ECD spectra of the native and partially modified peptide reveal a loss of bromine in a process that requires the presence of a disulfide bond.

Amino Acid Sequence↗

PubMed2006

Characterization of contryphans from Conus loroisii and Conus amadis that target calcium channels.

Distinctly different effects of two closely related contryphans have been demonstrated on voltage-activated Ca(2+) channels. The peptides Lo959 and Am975 were isolated from Conus loroisii, a vermivorous marine snail and Conus amadis, a molluscivore, respectively. The sequences of Lo959 and Am975 were deduced by mass spectrometric sequencing (MALDI-MS/MS) and confirmed by chemical synthesis. The sequences of Lo959, GCP(D)WDPWC-NH(2) and Am975, GCO(D)WDPWC-NH(2) (O: 4-trans-hydroxyproline: Hyp), differ only at residue 3; Pro in Lo959, Hyp in Am975, which is identical to contryphan-P, previously isolated from Conus purpurascens, a piscivore; while Lo959 is a novel peptide. Both Lo959 and Am975 undergo slow conformational interconversion under reverse-phase chromatographic conditions, a characteristic feature of all contryphans reported thus far. Electrophysiological studies performed using dorsal root ganglion neurons reveal that both peptides target high voltage-activated Ca(2+) channels. While Lo959 increases the Ca(2+) current, Am975 causes inhibition. The results establish that subtle sequence effects, which accompany post-translational modifications in Conus peptides, can have dramatic effects on target ion channels.

Amino Acid Sequence↗

PubMed2005

Solution structure of delta-Am2766: a highly hydrophobic delta-conotoxin from Conus amadis that inhibits inactivation of neuronal voltage-gated sodium channels.

The three-dimensional (3D) NMR solution structure (MeOH) of the highly hydrophobic delta-conotoxin delta-Am2766 from the molluscivorous snail Conus amadis has been determined. Fifteen converged structures were obtained on the basis of 262 distance constraints, 25 torsion-angle constraints, and ten constraints based on disulfide linkages and H-bonds. The root-mean-square deviations (rmsd) about the averaged coordinates of the backbone (N, C(alpha), C) and (all) heavy atoms were 0.62+/-0.20 and 1.12+/-0.23 A, respectively. The structures determined are of good stereochemical quality, as evidenced by the high percentage (100%) of backbone dihedral angles that occupy favorable and additionally allowed regions of the Ramachandran map. The structure of delta-Am2766 consists of a triple-stranded antiparallel beta-sheet, and of four turns. The three disulfides form the classical 'inhibitory cysteine knot' motif. So far, only one tertiary structure of a delta-conotoxin has been reported; thus, the tertiary structure of delta-Am2766 is the second such example. Another Conus peptide, Am2735 from C. amadis, has also been purified and sequenced. Am2735 shares 96% sequence identity with delta-Am2766. Unlike delta-Am2766, Am2735 does not inhibit the fast inactivation of Na+ currents in rat brain Na(v)1.2 Na+ channels at concentrations up to 200 nM.

Amino Acid Sequence↗

PubMed2005

Novel peptides of therapeutic promise from Indian Conidae.

Highly structured small peptides are the major toxic constituents of the venom of cone snails, a family of widely distributed predatory marine molluscs. These animals use the venom for rapid prey immobilization. The peptide components in the venom target a wide variety of membrane-bound ion channels and receptors. Many have been found to be highly selective for a diverse range of mammalian ion channels and receptors associated with pain-signaling pathways. Their small size, structural stability, and target specificity make them attractive pharmacologic agents. A select number of laboratories mainly from the United States, Europe, Australia, Israel, and China have been engaged in intense drug discovery programs based on peptides from a few snail species. Coastal India has an estimated 20-30% of the known cone species; however, few serious studies have been reported so far. We have begun a comprehensive program for the identification and characterization of peptides from cone snails found in Indian Coastal waters. This presentation reviews our progress over the last 2 years. As expected from the evolutionary history of these venom components, our search has yielded novel peptides of therapeutic promise from the new species that we have studied.

Amino Acid Sequence↗

PubMed2004

Analysis of conditional paralytic mutants in Drosophila sarco-endoplasmic reticulum calcium ATPase reveals novel mechanisms for regulating membrane excitability.

Individual contributions made by different calcium release and sequestration mechanisms to various aspects of excitable cell physiology are incompletely understood. SERCA, a sarco-endoplasmic reticulum calcium ATPase, being the main agent for calcium uptake into the ER, plays a central role in this process. By isolation and extensive characterization of conditional mutations in the Drosophila SERCA gene, we describe novel roles of this key protein in neuromuscular physiology and enable a genetic analysis of SERCA function. At motor nerve terminals, SERCA inhibition retards calcium sequestration and reduces the amplitude of evoked excitatory junctional currents. This suggests a direct contribution of store-derived calcium in determining the quantal content of evoked release. Conditional paralysis of SERCA mutants is also marked by prolonged neural activity-driven muscle contraction, thus reflecting the phylogenetically conserved role of SERCA in terminating contraction. Further analysis of ionic currents from mutants uncovers SERCA-dependent mechanisms regulating voltage-gated calcium channels and calcium-activated potassium channels that together control muscle excitability. Finally, our identification of dominant loss-of-function mutations in SERCA indicates novel intra- and intermolecular interactions for SERCA in vivo, overlooked by current structural models.

Animals↗

PubMed2004

A novel 13 residue acyclic peptide from the marine snail, Conus monile, targets potassium channels.

A novel 13-residue peptide Mo1659 has been isolated from the venom of a vermivorous cone snail, Conus monile. HPLC fractions of the venom extract yielded an intense UV absorbing fraction with a mass of 1659Da. De novo sequencing using both matrix assisted laser desorption and ionization and electrospray MS/MS methods together with analysis of proteolytic fragments successfully yielded the amino acid sequence, FHGGSWYRFPWGY-NH(2). This was further confirmed by comparison with the chemically synthesized peptide and by conventional Edman sequencing. Mo1659 has an unusual sequence with a preponderance of aromatic residues and the absence of apolar, aliphatic residues like Ala, Val, Leu, and Ile. Mo1659 has no disulfide bridges distinguishing it from the conotoxins and bears no sequence similarity with any of the acyclic peptides isolated thus far from the venom of cone snails. Electrophysiological studies on the effect of Mo1659 on measured currents in dorsal root ganglion neurons suggest that the peptide targets non-inactivating voltage-dependent potassium channels.

Amino Acid Sequence↗

PubMed2003

Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail.

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na(+) channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.

Amino Acid Sequence↗

PubMed2003

Shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes.

We have developed a primary cell culture system derived from embryonic and larval stages of Drosophila. This allows for high-resolution imaging and genetic analyses of endocytic processes. Here, we have investigated endocytic pathways of three types of molecules: an endogenous receptor that binds anionic ligands (ALs), glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP), and markers of the fluid phase in primary hemocytes. We find that the endogenous AL-binding receptor (ALBR) is internalized into Rab5-positive endosomes, whereas the major portion of the fluid phase is taken up into Rab5-negative endosomes; GPI-APs are endocytosed into both classes of endosomes. ALBR and fluid-phase-containing early endosomes subsequently fuse to yield a population of Rab7-positive late endosomes. In primary culture, the endocytic phenotype of ALBR internalization in cells carrying mutations in Drosophila Dynamin (dDyn) at the shibire locus (shits) parallels the temperature-sensitive behavior of shits animals. At the restrictive temperature in shits cells, receptor-bound ALs remain completely surface accessible, localized to clathrin and alpha-adaptin-positive structures. On lowering the temperature, ALs are rapidly sequestered, suggesting a reversible block at a late step in dDyn-dependent endocytosis. By contrast, GPI-AP and fluid-phase endocytosis are quantitatively unaffected at the restrictive temperature in shits hemocytes, demonstrating a constitutive dDyn and Rab5-independent endocytic pathway in Drosophila.

Animals↗

PubMed2003

deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.

Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

Animals↗

PubMed2003

A temperature-sensitive allele of Drosophila sesB reveals acute functions for the mitochondrial adenine nucleotide translocase in synaptic transmission and dynamin regulation.

Rapidly reversible, temperature-sensitive (ts) paralytic mutants of Drosophila have been useful in delineating immediate in vivo functions of molecules involved in synaptic transmission. Here we report isolation and characterization of orangi (org), an enhancer of shibire (shi), a ts paralytic mutant in Drosophila dynamin. org is an allele of the stress sensitive B (sesB) locus that encodes a mitochondrial adenine nucleotide translocase (ANT) and results in a unique ts paralytic behavior that is accompanied by a complete loss of synaptic transmission in the visual system. sesB(org) reduces the restrictive temperature for all shi(ts) alleles tested except for shi(ts1). This characteristic allele-specific interaction of sesB(org) with shi is shared by abnormal wing discs (awd), a gene encoding nucleoside diphosphate kinase (NDK). sesB(org) shows independent synergistic interactions, an observation that is consistent with a shared pathway by which org and awd influence shi function. Genetic and electrophysiological analyses presented here, together with the observation that the sesB(org) mutation reduces biochemically assayed ANT activity, suggest a model in which a continuous mitochondrial ANT-dependent supply of ATP is required to sustain NDK-dependent activation of presynaptic dynamin during a normal range of synaptic activity.

Alleles↗

PubMed2002

Unique biochemical and behavioral alterations in Drosophila shibire(ts1) mutants imply a conformational state affecting dynamin subcellular distribution and synaptic vesicle cycling.

Dynamin is a GTPase protein that is essential for clathrin-mediated endocytosis of synaptic vesicle membranes. The Drosophila dynamin mutation shi(ts1) changes a single residue (G273D) at the boundary of the GTPase domain. In cell fractionation of homogenized fly heads without monovalent cations, all dynamin was in pellet fractions and was minimally susceptible to Triton-X extraction. Addition of Na(+) or K(+) can extract dynamin to the cytosolic (supernatant) fraction. The shi(ts1) mutation reduced the sensitivity of dynamin to salt extraction compared with other temperature-sensitive alleles or wild type. Sensitivity to salt extraction in shi(ts1) was enhanced by GTP and nonhydrolyzable GTP-gammaS. The shi(ts1) mutation may therefore induce a conformational change, involving the GTP binding site, that affects dynamin aggregation. Temperature-sensitive shibire mutations are known to arrest endocytosis at restrictive temperatures, with concomitant accumulation of presynaptic collared pits. Consistent with an effect upon dynamin aggregation, intact shi(ts1) flies recovered much more slowly from heat-induced paralysis than did other temperature-sensitive shibire mutants. Moreover, a genetic mutation that lowers GTP abundance (awd(msf15)), which reduces the paralytic temperature threshold of other temperature-sensitive shibire mutations that lie closer to consensus GTPase motifs, did not reduce the paralytic threshold of shi(ts1). Taken together, the results may link the GTPase domain to conformational shifts that influence aggregation in vitro and endocytosis in vivo, and provide an unexpected point of entry to link the biophysical properties of dynamin to physiological processes at synapses.

Animals↗

PubMed2002

Endophilin is critically required for synapse formation and function in Drosophila melanogaster.

Studies in cell-free systems and the lamprey giant synapse have implicated crucial roles for amphiphysin and endophilin in synaptic transmission. However, null mutants at the amphiphysin locus of Drosophila are viable and have no demonstrable synaptic vesicle-recycling defect. This has necessitated a re-examination of the role of Src homology 3 domain-containing proteins in synaptic vesicle recycling. In this report, we show that endophilin-deficient eye clones in Drosophila have an altered electroretinogram. A characteristic of this defect is its aggravation during heightened visual stimulation. It is shown that endophilin is primarily required in the nervous system. Decreased endophilin activity results in alterations in the neuromuscular junction structure and physiology. Immunofluorescence studies show colocalization of endophilin with dynamin consistent with a possible role in synaptic vesicle recycling.

5' Untranslated Regions↗

PubMed2001

Genetic interaction between shibire and comatose mutations in Drosophila suggest a role for snap-receptor complex assembly and disassembly for maintenance of synaptic vesicle cycling.

NSF is an ATPase required for the fusion of secretory vesicles with plasma membrane. Conditional comatose (Drosophila homolog of N-ethylmaleimide sensitive fusion factor (NSF)) mutations in Drosophila block synaptic transmission at restrictive temperature. Current models hold that NSF-mediated dissociation of SNARE (SNAp REceptor) complexes on mature synaptic vesicles primes them for exocytic release. Paralysis in comt mutants thus reflects defective exocytosis due to buildup of unresolved SNARE complexes. Here, we analyze effects of blocking synaptic vesicle recycling on behavioral, physiological and biochemical phenotypes of comt. Behavioral recovery of comt animals and recovery of comt synapses, as assayed by electroretinograms, after exposure to high temperature is faster if synaptic vesicle recycling is simultaneously blocked using shi(ts) mutants. Concurrently, 7S complex buildup in comt shi double mutants is substantially lower than in comt mutants alone. In addition, we find that 7S complexes can form on presynaptic plasma membrane if NSF is inhibited after synaptic-vesicle depletion. Thus, our experiments demonstrate a need for continuous NSF activity required not only for dissociating cis-SNARE complexes on plasma membrane after exocytosis, but also for maintaining these cis-SNARE complexes in a dissociated state.

Animals↗

PubMed2001

Lethal comatose mutation in Drosophila reveals possible role for NSF in neurogenesis.

NSF is a cytosolic ATPase implicated in a variety of cellular functions including synaptic vesicle exocytosis. Here we report a lethal mutation in the Drosophila homolog of NSF (dNSF1). Lethality staging and rescue experiments with the wild type dNSF1 transgene show that NSF1 is critically required during early larval stages and during late pupariation. Lethality in larval stages is associated with defects in neurogenesis as evidenced by an overall reduction in synapse size and synapse branching. Moreover, escaper adults, though showing abnormal seizure-like paralytic behavior, are normal in terms of synaptic transmission as assayed by electroretinograms. Taken together, these data indicate a role for NSF in neural growth and branching in addition to its documented role in synaptic transmission.

Animals↗

PubMed2001

Drosophila stoned proteins regulate the rate and fidelity of synaptic vesicle internalization.

At an initial step during synaptic vesicle recycling, dynamin and adaptor proteins mediate the endocytosis of synaptic vesicle components from the plasma membrane. StonedA and stonedB, novel synaptic proteins encoded by a single Drosophila gene, have predicted structural similarities to adaptors and other proteins implicated in endocytosis. Here, we test possible roles of the stoned proteins in synaptic vesicle internalization via analyses of third instar larval neuromuscular synapses in two Drosophila stoned (stn) mutants, stn(ts) and stn(8P1). Both mutations reduce presynaptic levels of stonedA and stonedB, although stn(ts) has relatively weak effects. The mutations cause retention of synaptic vesicle proteins on the presynaptic plasma membrane but do not alter the levels or distribution of endocytosis proteins, dynamin, alpha-adaptin, and clathrin. In addition, stn(8P1) mutants exhibit depletion and enlargement of synaptic vesicles. To determine whether these defects arise from altered synaptic vesicle endocytosis or from defects in synaptic vesicle biogenesis, we implemented new methods to assess directly the efficiency of synaptic vesicle recycling and membrane internalization at Drosophila nerve terminals. Behavioral and electrophysiological analyses indicate that stn(ts), an allele with normal evoked release and synaptic vesicle number, enhances defects in synaptic vesicle recycling shown by Drosophila shi(ts) mutants. A dye uptake assay demonstrates that slow synaptic vesicle recycling in stn(ts) is accompanied by a reduced rate of synaptic vesicle internalization after exocytosis. These observations are consistent with a model in which stonedA and stonedB act to facilitate the internalization of synaptic vesicle components from the plasma membrane.

Animals↗

PubMed2001

Nucleoside diphosphate kinase, a source of GTP, is required for dynamin-dependent synaptic vesicle recycling.

Nucleoside diphosphate kinase (NDK), an enzyme encoded by the Drosophila abnormal wing discs (awd) or human nm23 tumor suppressor genes, generates nucleoside triphosphates from respective diphosphates. We demonstrate that NDK regulates synaptic vesicle internalization at the stage where function of the dynamin GTPase is required. awd mutations lower the temperature at which behavioral paralysis, synaptic failure, and blocked membrane internalization occur at dynamin-deficient, shi(ts), mutant nerve terminals. Hypomorphic awd alleles display shi(ts)-like defects. NDK is present at synapses and its enzymatic activity is essential for normal presynaptic function. We suggest a model in which dynamin activity in nerve terminals is highly dependent on NDK-mediated supply of GTP. This connection between NDK and membrane internalization further strengthens an emerging hypothesis that endocytosis, probably of activated growth factor receptors, is an important tumor suppressor activity in vivo.

Alleles↗

PubMed2000

Genetics of anesthetic response: autosomal mutations that render Drosophila resistant to halothane.

Molecular mechanisms of anesthetic action are poorly understood. Genetic approaches to investigate mechanisms of anesthesia, although sparse and rather new, are turning out to be informative and add a new perspective. Before beginning a systematic investigation of anesthesia by this approach, it is necessary to have at hand a large collection of mutations in different loci that alter anesthetic response. We report here the isolation and characterization of six mutant autosomal lines that show a decreased sensitivity to the inhalation of anesthetic halothane. Two of these mutations, Omar(82) and Qajjem(211) are shown to map to separate loci on the third chromosome.

Anesthetics, Inhalation↗