Biomedical subjects

W L Pak

Publications and source records attributed to W L Pak.

At least 19 recordsLinked to original sources

PubMed2003

cGMP-dependent changes in phototaxis: a possible role for the foraging gene in honey bee division of labor.

Division of labor in honey bee colonies is influenced by the foraging gene (Amfor), which encodes a cGMP-dependent protein kinase (PKG). Amfor upregulation in the bee brain is associated with the age-related transition from working in the hive to foraging for food outside, and cGMP treatment (which increases PKG activity) causes precocious foraging. We present two lines of evidence in support of the hypothesis that Amfor affects division of labor by modulating phototaxis. We first show that a subset of worker bees involved in the removal of corpses from the hive had forager-like brain levels of Amfor brain expression despite being middle aged; age-matched food-handlers, who do not leave the hive to perform their job, had low levels of Amfor expression. This finding suggests that occupations that involve working outside the hive are associated with high levels of Amfor in brain. Secondly, foragers were much more positively phototactic than hive bees in a laboratory assay, and cGMP treatment caused a precocious onset of positive phototaxis. The cGMP effect was not due to a general increase in behavioral activity; cGMP treatment had no effect on locomotor activity under either constant darkness or a light:dark regime. The cGMP effect also was not due to changes in circadian rhythmicity; cGMP treatment had no effect on age at onset of locomotor circadian rhythmicity or the period of rhythmicity. The effects of Amfor on phototaxis are not related to peripheral processing; electroretinogram analysis revealed no effect of cGMP treatment on photoreceptor activity and no differences between untreated hive bees and foragers. The cAMP/PKA pathway does not appear to be playing a similar role to cGMP/PKG in the honey bee; cAMP treatment did not affect phototaxis and gene expression analysis revealed task-related differences only for the gene encoding the regulatory subunit, but not the catalytic subunit, of PKA. Our findings implicate one neural process associated with honey bee division of labor that can be affected by naturally occurring changes in the expression of AMFOR:

Aging↗

PubMed2000

Phenotypes of trpl mutants and interactions between the transient receptor potential (TRP) and TRP-like channels in Drosophila.

The trp and trpl genes are thought to encode two classes of light-activated ion channels in Drosophila. A previous report indicated that a null trpl mutant does not display any mutant phenotype. This lack of detectable mutant phenotypes made it difficult to suggest functions for the transient receptor potential-like (TRPL) channel in photoreceptor responses. Here, the properties of trpl photoreceptor responses were studied by using electroretinogram (ERG) and intracellular recording techniques in combination with light stimuli of relatively long durations. Distinct mutant phenotypes were detectable under these conditions. These consisted of a reduced sustained component, oscillations superimposed on the response, a poststimulus hyperpolarization, and altered adaptation properties to dim background light. Comparison of photoreceptor responses obtained from wild type, trp, and trpl showed that the responses obtained from the trp and trpl null mutants did not sum up to that of the wild-type response. To explain the nonlinear summation at the peak of the response, Reuss et al. (1997) proposed that Ca(2+) ions entering through the TRP channel modulate TRP and TRPL channel activities differentially. However, nonlinear summation was present not only at the peak but throughout the duration of response. Two lines of evidence are presented to suggest that, in addition to the interaction proposed by Reuss et al. (1997), there are other forms of interactions between TRP and TRPL channels, probably involving the channel proteins themselves.

Amino Acid Substitution↗

PubMed2000

Novel mechanism of massive photoreceptor degeneration caused by mutations in the trp gene of Drosophila.

The Drosophila trp gene encodes a light-activated Ca(2+) channel subunit, which is a prototypical member of a novel class of channel proteins. Previously identified trp mutants are all recessive, loss-of-function mutants characterized by a transient receptor potential and the total or near-total loss of functional TRP protein. Although retinal degeneration does occur in these mutants, it is relatively mild and slow in onset. We report herein a new mutant, Trp(P365), that does not display the transient receptor potential phenotype and is characterized by a substantial level of the TRP protein and rapid, semi-dominant degeneration of photoreceptors. We show that, in spite of its unusual phenotypes, Trp(P365) is a trp allele because a Trp(P365) transgene induces the mutant phenotype in a wild-type background, and a wild-type trp transgene in a Trp(P365) background suppresses the mutant phenotype. Moreover, amino acid alterations that could cause the Trp(P365) phenotype are found in the transmembrane segment region of the mutant channel protein. Whole-cell recordings clarified the mechanism underlying the retinal degeneration by showing that the TRP channels of Trp(P365) are constitutively active. Although several genes, when mutated, have been shown to cause retinal degeneration in Drosophila, the underlying mechanism has not been identified for any of them. The present studies provide evidence for a specific mechanism for massive degeneration of photoreceptors in Drosophila. Insofar as some human homologs of TRP are highly expressed in the brain, a similar mechanism could be a major contributor to degenerative disorders of the brain.

Amino Acid Sequence↗

PubMed1999

INAF, a protein required for transient receptor potential Ca(2+) channel function.

The trp gene of Drosophila encodes a subunit of a class of Ca(2+)-selective light-activated channels that carry the bulk of the phototransduction current. Transient receptor potential (TRP) homologs have been identified throughout animal phylogeny. In vertebrates, TRP-related channels have been suggested to mediate "store-operated Ca(2+) entry," which is important in Ca(2+) homeostasis in a wide variety of cell types. However, the mechanisms of activation and regulation of the TRP channel are not known. Here, we report on the Drosophila inaF gene, which encodes a highly eye-enriched protein, INAF, that appears to be required for TRP channel function. A null mutation in this gene significantly reduces the amount of the TRP protein and, in addition, specifically affects the TRP channel function so as to nearly shut down its activity. The inaF mutation also dramatically suppresses the severe degeneration caused by a constitutively active mutation in the trp gene. Although the reduction in the amount of the TRP protein may contribute to these phenotypes, several lines of evidence support the view that inaF mutations also more directly affect the TRP channel function, suggesting that the INAF protein may have a regulatory role in the channel function.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Diversification of Drosophila chloride channel gene by multiple posttranscriptional mRNA modifications.

We have identified and analyzed a Drosophila melanogaster gene that encodes a chloride channel subunit (DrosGluCl-alpha) previously shown to function as a glutamate-gated chloride channel in an in vitro expression system. Sequence analysis of several cDNAs corresponding to the gene revealed sequence diversity in their open reading frames at seven specific sites. Site-specific A-to-G variations between cDNA and genomic sequences, consistent with RNA editing, were detected at five nucleotide positions. In addition, sequence variations among cDNA clones consistent with alternative splicing of mRNA were found at two different sites. In the 5' region, two small adjacent exons, containing similar but distinct modular sequences, are alternatively incorporated into the mature mRNA. In the 3' region, alternative splicing generates a variant encoding a protein with four additional amino acids just upstream of the fourth transmembrane domain. Combinations of RNA editing and alternative splicing can lead to extensive diversification of transcripts. These results give the first example of RNA editing in neurotransmitter-gated chloride channel genes or of alternative splicing in a glutamate-gated chloride channel gene of Drosophila.

Alternative Splicing↗

PubMed1998

Selective histamine uptake rescues photo- and mechanoreceptor function of histidine decarboxylase-deficient Drosophila mutant.

In insects, histamine is found both in the peripheral nervous system (PNS) and in the CNS and is known to function as a fast neurotransmitter in photoreceptors that have been shown to express selectively the hdc gene. This gene codes for histidine decarboxylase (HDC), the enzyme for histamine synthesis. Fast neurotransmission requires the efficient removal of the transmitter from the synaptic cleft. Here we identify in Drosophila photo- and mechanoreceptors a histamine uptake mechanism that can restore the function of these receptors in mutants unable to synthesize histamine. When apparent null mutants for the hdc gene imbibe aqueous histamine solution or are genetically "rescued" by a transgene ubiquitously expressing histidine decarboxylase under heat-shock control, sufficient amounts of histamine selectively accumulate in photo- and mechanoreceptors to generate near-normal electrical responses in second-order visual interneurons and qualitatively to restore wild-type visual and mechanosensory behavior. This strongly supports the proposal that histamine functions as a fast neurotransmitter also in a certain class of mechanoreceptors. A set of CNS-intrinsic neurons that in the wild type contain high concentrations of histamine apparently lacks this uptake mechanism. We therefore speculate that histamine of intrinsic neurons may function as a neuromodulator rather than as a fast transmitter.

Animals↗

PubMed1997

Site-directed mutagenesis of highly conserved amino acids in the first cytoplasmic loop of Drosophila Rh1 opsin blocks rhodopsin synthesis in the nascent state.

The cytoplasmic surface of Drosophila melanogaster Rh1 rhodopsin (ninaE) harbours amino acids which are highly conserved among G-protein-coupled receptors. Site-directed mutations which cause Leu81Gln or Asn86Ile amino acid substitutions in the first cytoplasmic loop of the Rh1 opsin protein, are shown to block rhodopsin synthesis in the nascent, glycosylated state from which the mutant opsin is degraded rapidly. In mutants Leu81Gln and Asn86Ile, only 20-30% and <2% respectively, of functional rhodopsins are synthesized and transported to the photoreceptive membrane. Thus, conserved amino acids in opsin's cytoplasmic surface are a critical factor in the interaction of opsin with proteins of the rhodopsin processing machinery. Photoreceptor cells expressing mutant rhodopsins undergo age-dependent degeneration in a recessive manner.

Amino Acid Sequence↗

PubMed1996

Cyclophilin-related protein RanBP2 acts as chaperone for red/green opsin.

Cyclophilins are ubiquitous and abundant proteins that exhibit peptidyl prolyl cis-trans isomerization (PPlase) activity in vitro. Their functions in vivo, however, are not well understood. Two new retinal cyclophilin isoforms, types I and II, are highly expressed in cone photoreceptors of the vertebrate retina. Type-II cyclophilin is identical to RanBP2, a large protein that binds the GTPase Ran. Here we report that two contiguous domains in RanBP2, Ran-binding domain 4 (RBD4) and cyclophilin, act in concert as a chaperone for the opsin molecule of the red/green-sensitive visual pigment of a dichromatic vertebrate. In Drosophila, the cyclophilin NinaA is expressed in all photoreceptors and is required for the expression of only a subset of opsins. The molecular basis of these photoreceptor class-specific effects and the functions of NinaA and other cyclophilins in vivo remain unclear. Unlike NinaA, which forms a stable complex with opsin from retinular cells R1-6, we find that the cyclophilin domain of RanBP2 does not bind opsin directly; rather, it augments and stabilizes the interaction between red/green (R/G) opsin and the RBD4 domain. This involves a cyclophilin-mediated modification of R/G opsin, possibly involving proline isomerization. The RBD4-cyclophilin supradomain of RanBP2, therefore, is a form of vertebrate chaperone of defined substrate specificity, which may be involved in the processing and/or transport of long-wavelength opsin in cone photoreceptor cells.

Amino Acid Isomerases↗

PubMed1996

Molecular, biochemical, and electrophysiological characterization of Drosophila norpA mutants.

Inositol phosphate signaling has been implicated in a wide variety of eukaryotic cellular processes. In Drosophila, the phototransduction cascade is mediated by a phosphoinositide-specific phospholipase C (PLC) encoded by the norpA gene. We have characterized eight norpA mutants by electroretinogram (ERG), Western, molecular, and in vitro PLC activity analyses. ERG responses of the mutants show allele-dependent reductions in amplitudes and retardation in kinetics. The mutants also exhibit allele-dependent reductions in in vitro PLC activity levels and greatly reduced or undetectable NorpA protein levels. Three carry a missense mutation and five carry a nonsense mutation within the norpA coding sequence. In missense mutants, the amino acid substitution occurs at residues highly conserved among PLCs. These substitutions reduce the levels of both the NorpA protein and the PLC activity, with the reduction in PLC activity being greater than can be accounted for simply by the reduction in protein. The effects of the mutations on the amount and activity of the protein are much greater than their effects on the ERG, suggesting an amplification of the transduction signal at the effector (NorpA) protein level. Transgenic flies were generated by germline transformation of a null norpA mutant using a P-element construct containing the wild-type norpA cDNA driven by the ninaE promoter. Transformed flies show rescue of the electrophysiological phenotype in R1-R6 photoreceptors, but not in R7 or R8. The degeneration phenotype of R1-R6 photoreceptors is also rescued.

Alleles↗

PubMed1996

Genetic depletion of histamine from the nervous system of Drosophila eliminates specific visual and mechanosensory behavior.

The role of histamine as a fast neurotransmitter of imaginal insect photoreceptors is firmly established. In adult Drosophila, histamine is also found in mechanosensory receptors of cuticular hair sensilla and in a small number of nonreceptor neurons in head and body ganglia. Here we investigate the function of histamine by immunohistochemical and behavioral analysis of mutants deficient in the hdc gene that codes for histidine decarboxylase. The allele hdcJK910 appears to be a null mutation, as histamine immunoreactivity is almost entirely eliminated. Homozygous flies are blind in various behavioral paradigms. Mutant larvae, on the other hand, show normal photokinetic responses. Thus, adult Drosophila photoreceptors most likely utilize only a single substance, histamine, as a neurotransmitter, whereas larval photoreceptors apparently employ a different transmitter. With the alleles hdcP211, hdcP217, and hdcP218, variable amounts of histamine are found in photoreceptors and mechanoreceptors, but no histamine could be detected in any of the nonreceptor neurons. These mutants show various degrees of visual and mechanosensory impairment, as determined by quantitative behavioral assays. We conclude that histamine is required for normal function of cuticular hair sensilla and for efficient grooming of the body surface. Thus, in Drosophila, histamine represents a major functional neurotransmitter for mechanosensory receptors.

Animals↗

PubMed1996

Drosophila rosA gene, which when mutant causes aberrant photoreceptor oscillation, encodes a novel neurotransmitter transporter homologue.

The Drosophila receptor oscillation A (rosA) mutations, which cause electroretinogram (ERG) defects, including oscillations, were localized to the 24F4-25A2 region of chromosome 2L. Genomic fragments from this region, isolated from bacteriophage P1 clones, included those that detect transcriptional defects in rosA mutants in RNA blot experiments. One of these genomic fragments was used to screen a head cDNA library. The largest cDNA clone (3.6 kb) isolated was shown to rescue a rosA mutant in P element-germline transformation experiments. The ROSA protein deduced from the open reading frame in the 3.6 kb rosA cDNA is 943 amino acids long and is 36-41% identical to members of the superfamily of Na+/Cl(-)-dependent neurotransmitter transporters, with no indication of higher sequence identity to any one subgroup within the superfamily. RNA blot experiments revealed multiple transcripts in various developmental stages, the most abundant one being a 3.7 kb transcript, particularly in the adult head. Tissue in situ experiments identified the rosA transcript to be localized to many tissues, with higher levels of hybridization in the nervous system and digestive tract. The results demonstrate that the rosA gene encodes a novel Na+/Cl(-)-dependent transporter important for normal response properties of the photoreceptor.

Amino Acid Sequence↗

PubMed1995

Retina-specifically expressed novel subtypes of bovine cyclophilin.

The Drosophila ninaA gene encodes photoreceptor-specific cyclophilin thought to play a critical role in rhodopsin folding or transport during its synthesis or maturation in the most abundant subclass of photoreceptors. Cyclophilins comprise a highly conserved family of proteins which are the primary targets of the potent immunosuppressive drug, cyclosporin A (CsA), and which display peptidyl prolyl cis-trans-isomerase (PPIase) activity. In an attempt to identify mammalian cyclophilins with properties similar to the NinaA protein, a probe derived from the ninaA cDNA was used to screen bovine retina cDNA libraries. The screen identified two major alternatively spliced forms of cDNA that would encode proteins containing a region of high homology to other cyclophilins and that are expressed specifically in the retina. These proteins represent a new class of cyclophilins with novel structural features and greatly reduced PPIase and CsA binding activities in comparison to other known cyclophilins. Tissue in situ hybridization and immunolocalization of the proteins showed that the RNA and protein products are expressed in photoreceptors as well s other retinal neurons. However, among photoreceptors, the proteins are found predominantly in cones. Thus, mammalian retinas do contain cyclophilins that are retina-specifically and photoreceptor class-preferentially expressed. The results suggest that, in cones, the main function of these proteins is, like the NinaA protein, to facilitate proper folding or intracellular transport of opsins.

Alternative Splicing↗

PubMed1995

Molecular characterization of two Drosophila guanylate cyclases expressed in the nervous system.

We have isolated, by interspecies hybridization, two classes of Drosophila cDNA each encoding a different guanylate cyclase (GC). One of them encodes an alpha subunit homolog of soluble GC, designated DGC alpha 1, and the other encodes a receptor-type GC, designated DrGC. The dgc alpha 1 cDNA encodes a protein of 676 amino acids and maps to 99B. In situ hybridization to adult tissue sections showed that dgc alpha 1 mRNA is found mainly in the cell bodies of the optic lobe, central brain, and thoracic ganglia. The DGC alpha 1 protein was also localized primarily to the nervous system by immunocytochemical staining, consistent with results of in situ hybridization. However, no detectable expression of this protein was found in the retina. The other class of cDNA, drgc, maps to 76C and encodes a 1525-amino acid protein displaying structural features similar to other known receptor-type guanylate cyclases. However, it has a C-terminal 430 amino acid region that has no homology to any known proteins. drgc RNA is expressed at low levels throughout development and in adult heads and bodies. In situ hybridizations to adult tissue sections showed that drgc mRNA is expressed in a wide range of tissues, including the optic lobe, central brain, thoracic ganglia, digestive tract, and the oocyte.

Amino Acid Sequence↗

PubMed1995

Drosophila in vision research. The Friedenwald Lecture.

A genetic approach involving the use of ERG-defective Drosophila mutants, described in this article, was developed with the explicit goal of elucidating phototransduction and related photoreceptor events at the molecular level. Advances in the past 30 years have made this goal a reality. Although the phototransduction process in Drosophila is not yet fully understood, enormous strides are being made by many investigators now working in this field. In addition to elucidating phototransduction vents in invertebrate photoreceptors, works of these investigators are providing insights into the functional machinery of signaling systems in general. For example, mobilization of Ca2+ and replenishment of intracellular Ca2+ stores after their depletion is a current topic of intense interest (see, for example, references 93, 136 and 137). Study of trp mutants is providing insights into this process. Similarly, the mechanisms of inactivation of G-protein coupled receptors appear to be highly conserved in diverse transduction systems, and inactivation of Drosophila metarhodopsin is proving a valuable model system for this study. The identification of the NinaA protein and its role as chaperon and/or foldase for opsin would not have been possible without the genetic approach described in this article. In fact, study of the NinaA gene is providing fresh insights not only into the rhodopsin maturation process but also into the role of cyclophilins in general. Moreover, recent results suggest that related retina-specific proteins are also present in mammals. As valuable as the NinaA protein has been, it may be only the first of a number of such novel proteins or novel isozymes of known proteins required in the retina to be discovered through this approach. For two of the Drosophila proteins discussed in this article, NorpA and NinaA, related mammalian proteins expressed in photoreceptors have been identified, even though it seemed unlikely that such proteins would have any role in mammalian photoreceptors. A recent report suggests that the Trp protein has human homologs expressed most heavily in the brain (C. Montell, cited in reference 93). It may well be that almost any protein identified in the Drosophila retina has its counterpart(s) in mammals. Discovery of these mammalian homologs of Drosophila proteins will probably spur new lines of investigation in mammalian photoreceptor function. For example, because no role had been assigned to PLC in cone phototransduction, the discovery that NorpA-homologous PLCs are present in cone outer segments raises questions about what role(s) these PLCs might play in signal transduction in cone outer segments.

Amino Acid Isomerases↗

PubMed1994

Bovine phospholipase C highly homologous to the norpA protein of Drosophila is expressed specifically in cones.

The Drosophila norpA gene encodes a phosphatidylinositol-specific phospholipase C (PI-PLC) expressed predominantly in photoreceptors and involved in phototransduction. However, no direct role for a phospholipase C in vertebrate phototransduction has been identified to date. Recently, we reported the isolation and characterization of bovine cDNAs encoding PI-PLC isoforms expressed predominantly in the retina and with higher homology to the NorpA protein than to any other known PI-PLC. Here, we present evidence that the norpA-homologous bovine retinal PI-PLCs, although found in other retinal neurons as well, are found in cones but not in rods. The results suggest that the phototransduction cascade in cones may utilize phospholipase C in addition to phosphodiesterase.

Amino Acid Sequence↗

PubMed1994

Phosrestin I undergoes the earliest light-induced phosphorylation by a calcium/calmodulin-dependent protein kinase in Drosophila photoreceptors.

Activation of PI-PLC initiates two independent branches of protein phosphorylation cascades catalyzed by either PKC or Ca2+/calmodulin-dependent protein kinase (CaMK). We find that phosrestin I (PRI), a Drosophila homolog of vertebrate photoreceptor arrestin, undergoes light-induced phosphorylation on a subsecond time scale which is faster than that of any other protein in vivo. We determine that a CaMK activity is responsible for in vitro PRI phosphorylation at Ser366 in the C-terminal tryptic segment, MetLysSer(P)IleGluGlnHisArg, in which Ser(P) represents phosphoserine366. We also demonstrate that Ser366 is the phosphorylation site of PRI in vivo by identifying the molecular species resulting from in-gel tryptic digestion of purified phospho-PRI using HPLC-electrospray ionization tandem quadrupole mass spectroscopy. From these data, we conclude that the CaMK pathway, not the PKC pathway, is responsible for the earliest protein phosphorylation event following activation of PI-PLC in living Drosophila photoreceptors.

Amino Acid Sequence↗

PubMed1993

Distinctive subtypes of bovine phospholipase C that have preferential expression in the retina and high homology to the norpA gene product of Drosophila.

The Drosophila norpA gene encodes a phospholipase C involved in phototransduction. However, phospholipase C apparently is not directly involved in phototransduction in vertebrate photoreceptors, although light-activated phospholipase C activity has been reported in vertebrate rod outer segments. Conserved regions of norpA cDNA were used to isolate bovine cDNAs that would encode four alternative forms of phospholipase C of the beta class that are highly homologous to the norpA protein and expressed preferentially in the retina. Two of the variants are highly unusual in that they lack much of the N-terminal region present in all other known phospholipases C. The sequence conservation between these proteins and the norpA protein is higher than that between any other known phospholipases C. GTPase sequence motifs found in proteins of the GTPase superfamily are found conserved in all four variants of the bovine retinal protein as well as the norpA protein but not in other phospholipases C. Results suggest that these proteins together with the norpA protein constitute a distinctive subfamily of phospholipases C that are closely related in structure, function, and tissue distribution. Mutations in the norpA gene, in addition to blocking phototransduction, cause light-dependent degeneration of photoreceptors. In view of the strong similarity in structure and tissue distribution, a defect in these proteins may have similar consequences in the mammalian retina.

Amino Acid Sequence↗

PubMed1993

Genetic and molecular identification of a Drosophila histidine decarboxylase gene required in photoreceptor transmitter synthesis.

Drosophila mutants of a single complementation group with defective on-/off-transients of the electroretinogram (ERG) were found to be deficient in synthesis of the photoreceptor transmitter, histamine, in a gene-dosage dependent manner, suggesting that the gene identified by the mutants (hdc) might be the structural gene for Drosophila histidine decarboxylase (HDC). A rat HDC cDNA was used to isolate a Drosophila homolog which shows approximately 60% sequence identity with mammalian HDCs over a region of 476 amino acids. In RNA blots, the Drosophila homolog detects four transcripts that are expressed primarily in the eye and are severely reduced in hdc mutants. The cloned Drosophila cDNA hybridizes to the 46F region of the chromosome, to which hdc mutations have been mapped, and rescues the hdc mutant phenotype in transgenic flies generated by P element-mediated germline transformation. The results thus show that the Drosophila homolog corresponds to the histidine decarboxylase gene, identified by the hdc mutants, and that mutations in the gene disrupt photoreceptor synaptic transmission.

Amino Acid Sequence↗