Effect of substrate on the proliferation of Myxobolus cerebralis in the mitochondrial lineages of the Tubifex tubifex host.

The study goal was to examine the effects of sand and mud on the propagation of Myxobolus cerebralis, the whirling disease agent, in four mitochondrial 16S ribosomal DNA lineages (I, III, V, VI) of its oligochaete host, Tubifex tubifex (Tt). In all the lineage groups held continuously in either substrate (non-shifted) or transferred from sand to mud (shifted), substrate influenced parasite proliferation only in lineage III. Sporogenesis and release of triactinomyxon spores (TAMs) were more prevalent in lineage III Tt in mud compared to sand. Low-infection prevalence and lack of parasite development in lineage I is associated with the greater number of resistant worms and were not affected by substrate type. Substrate did not impact Tt from lineages V and VI that failed to develop any parasite stages in either substrate even after shifting from sand to mud. The relationship between the microbial community in the substrate and parasite proliferation in lineage III was described but not analyzed due to small sample size. Substrate-associated bacteria were hypothesized as essential dietary source for the oligochaete host feeding selectively on fine (mud)-microflora. Progeny was produced by all lineage groups shifted to mud with disparate survival profiles in lineage V and VI and high mortalities in lineage III. Our study demonstrates that substrate type can alter parasite proliferation in lineage III. Conversely, parasite development and infectivity were not altered in lineage V and VI that are refractory to the parasite nor among the more resistant phenotypes (I), regardless of substrate type.

Estimating densities of larval Salmonflies (Pteronarcys californica) through multiple pass removal of post-emergent exuvia in Colorado rivers.

Traditional methods of collecting, sorting, and identifying benthic macroinvertebrate samples are useful for stream biomonitoring and ecological studies, however, these methods are time consuming, expensive, and require taxonomic expertise. Estimating larval densities through collection of post-emergent exuvia can be a practical and time efficient alternative. We evaluated the use of multiple pass depletion techniques of the post-emergent exuvia of Pteronarcys californica to estimate larval densities at ten sites in three Colorado rivers. Exuvia density was highly correlated with both final-instar larval density (R2 = 0.90) and total larval density (R2 = 0.88) and the multiple pass removal technique performed well. Exuvia surveys found P. californica at three low density sites where benthic sampling failed to detect it. At moderate and high density sites the exuvia surveys always produced lower density estimates than benthic surveys. Multiple pass depletion estimates of exuvia proved to be an accurate and efficient technique at estimating larval densities and provided an effective alternative for traditional benthic sampling when objectives are detecting and monitoring P. californica, especially at low density sites.

Elimination of Myxobolus cerebralis in Placer Creek, a Native Cutthroat Trout Stream in Colorado.

Placer Creek, a tributary of Sangre de Cristo Creek in Colorado's San Luis Valley, supported an allopatric core conservation population of native Rio Grande Cutthroat Trout Oncorhynchus clarkii virginalis during much of the 20th century. After the failure of gabion barriers in the late 1990s, Brook Trout Salvelinus fontinalis infected with Myxobolus cerebralis invaded from Sangre de Cristo Creek. By 2005, whirling disease (WD) and competition from Brook Trout reduced Rio Grande Cutthroat Trout numbers to less than 10% of the total trout population. New barriers were constructed in 2006 and the stream was treated with rotenone in 2007 and 2009 to eliminate all fish prior to the reintroduction of Rio Grande Cutthroat Trout. Results of WD research studies in Montana, California, and Colorado indicated it might be possible to break the life cycle of the parasite in some situations. Our management interventions included (1) reducing the fish population in the stream to zero for approximately 14 months, (2) introducing lineage V and VI Tubifex tubifex worms, which are not susceptible to M. cerebralis, and (3) eliminating a small off-channel pond that provided optimal habitat that sustained a localized high-density population of lineage III T. tubifex, the oligochaete host susceptible to M. cerebralis. Electrofishing during the fall of 2009 and spring of 2010 indicated the drainage was devoid of fish. Fry, juvenile, and adult Rio Grande Cutthroat Trout were stocked in September and October of 2010 and 2011. Approximately 975,000 lineage V and VI T. tubifex were introduced into Placer Creek between 2010 and 2012 as possible oligochaete competitors for the lineage III worms. The off-channel pond was filled in, and the surface was reseeded in April 2012. No evidence of M. cerebralis infection was detected among more than 280 Rio Grande Cutthroat Trout tested between July 2012 and July 2016, indicating the parasite had been eradicated from the Placer Creek basin upstream of the barriers.

Accelerated deactivation of Myxobolus cerebralis myxospores by susceptible and non-susceptible Tubifex tubifex.

In the 1990s, the Tubifex tubifex aquatic oligochaete species complex was parsed into 6 separate lineages differing in susceptibility to Myxobolus cerebralis, the myxozoan parasite that can cause whirling disease (WD). Lineage III T. tubifex oligochaetes are highly susceptible to M. cerebralis infection. Lineage I, IV, V and VI oligochaetes are highly resistant or refractory to infection and may function as biological filters by deactivating M. cerebralis myxospores. We designed a 2-phased laboratory experiment using triactinomyxon (TAM) production as the response variable to test that hypothesis. A separate study conducted concurrently demonstrated that M. cerebralis myxospores held in sand and water at temperatures ≤15°C degrade rapidly, becoming almost completely non-viable after 180 d. Those results provided the baseline to assess deactivation of M. cerebralis myxospores by replicates of mixed lineage (I, III, V and VI) and refractory lineage (V and VI) oligochaetes. TAM production was zero among 7 of 8 Lineage V and Lineage VI T. tubifex oligochaete groups exposed to 12500 M. cerebralis myxospores for 15, 45, 90 and 135 d. Among 4 mixed lineage exposure groups, TAM production averaged 14641 compared with 2202495 among 12 groups of Lineage III oligochaetes. Among the 6 unexposed Lineage III experimental groups seeded into original Phase 1 substrates for the 45, 90 and 135 d treatments during the Phase 2 portion of the study, TAM production was reduced by 98.9, 99.9 and 99.9%, respectively, compared with the average for the 15 d exposure groups. These results are congruent with the hypothesis that Lineage V and Lineage VI T. tubifex oligochaetes can deactivate and destroy M. cerebralis myxospores.

Differential Accumulation of Mercury and Selenium in Brown Trout Tissues of a High-Gradient Urbanized Stream in Colorado, USA.

Total mercury (THg) and selenium (Se) were analyzed by Inductively Coupled Plasma Mass Spectrometry in 11 internal and external tissues and stomach contents from 23 brown trout, Salmo trutta, of a 22.9-km reach of a high-gradient stream (upper Fountain Creek) in Colorado, USA, impacted by coal-fired power plants, shale deposits, and urbanization. Trout and water were sampled from four sites ranging from 2335 to 1818 m elevation. Lengths, weights, and ages of fish between pairs of the four sites were not significantly different. The dry weight (dw) to wet weight (ww) conversion factor for each tissue was calculated with egg-ovary highest at 0.379 and epaxial muscle fourth highest at 0.223. THg and Se in stomach contents indicated diet and not ambient water was the major source of Hg and Se bioaccumulated. Mean THg ww in kidney was 40.33 µg/kg, and epaxial muscle second highest at 36.76 µg/kg. None of the tissues exceeded the human critical threshold for Hg. However, all 23 trout had at least one tissue type that exceeded 0.02 mg/kg THg ww for birds, and four trout tissues exceeded 0.1 mg/kg THg ww for mammals, indicating that piscivorous mammals and birds should be monitored. Se concentrations in tissues varied depending on ww or dw listing. Mean Se dw in liver was higher than ovary at the uppermost site and the two lower sites. Liver tissue, in addition to egg-ovary, should be utilized as an indicator tissue for Se toxicity.

Susceptibility to Myxobolus cerebralis among Tubifex tubifex populations from ten major drainage basins in Colorado where Cutthroat Trout are endemic.

Establishment of Myxobolus cerebralis (Mc) resulted in declines of wild Rainbow Trout Oncorhynchus mykiss populations in streams across Colorado during the 1990s. However, the risk for establishment and spread of this parasite into high-elevation habitats occupied by native Cutthroat Trout O. clarkii was unknown. Beginning in 2003, tubificid worms were collected from all major drainages where Cutthroat Trout were endemic and were assayed by quantitative PCR to determine the occurrence and distribution of the various lineages of Tubifex tubifex (Tt) oligochaetes. Over a 5-year period, 40 groups of Tt oligochaetes collected from 27 streams, 3 natural lakes, 2 private ponds, and a reservoir were evaluated for their relative susceptibility to Mc. Exposure groups were drawn from populations of pure lineage III Tt, mixed-lineage populations where one or more of the highly resistant (lineage I) or nonsusceptible lineages (V or VI) were the dominant oligochaete and susceptible lineage III worms were the subdominant worm, or pure lineage VI Tt. Experimental replicates of 250 oligochaetes were exposed to 50 Mc myxospores per worm. The parasite amplification ratio (total triactinomyxons [TAMs] produced / total myxospore exposure) was very high among all pure lineage III Colorado exposure groups, averaging 363 compared with 8.24 among the mixed-lineage exposure groups. Lineage III oligochaetes from Mt. Whitney Hatchery in California, which served as the laboratory standard for comparative purposes, had an average parasite amplification ratio of 933 among 10 exposed replicates over a 5-year period. Lineage I oligochaetes were highly resistant to infection and did not produce any TAMs. Lineages V and VI Tt did not become infected and did not produce any TAMs. These results suggest that the risk of establishment of Mc is high for aquatic habitats in Colorado where Cutthroat Trout and lineage III Tt are sympatric.

Reduced Myxobolus cerebralis actinospore production in a Colorado reservoir may be linked to changes in Tubifex tubifex population structure.

Elucidating the dynamics of a parasitic infection requiring two hosts in a natural ecosystem can be a daunting task. Myxobolus cerebralis (Mc), the myxozoan parasite that causes whirling disease in some salmonids, was detected in the Colorado River upstream of Windy Gap Reservoir (WGR) in 1988. Subsequently, whirling disease was implicated in the decline of wild Rainbow Trout Oncorhynchus mykiss in the river when WGR was identified as a point source of Mc triactinomyxons (TAMs). Between 1997 and 2004, numerous investigations began to elucidate the etiology of Mc in WGR. During this period, Mc TAM production in WGR declined more than 90%. Explanations for the decline have included differences in stream discharge between years, changes in the thermal regime of the lake, severe drought, changes in the fish population structure in WGR, and reductions in the prevalence and severity of Mc infection in salmonids in the Colorado and Fraser rivers upstream of WGR. All of these have been discredited as explanations for the reduced TAM production. In 2005, a new study was conducted to replicate the studies completed in 1998. In this paper, the results of a new real-time polymerase chain reaction assay utilized to quantify the mitochondrial 16S rDNA specific to each of four lineages of Tubifex tubifex in pooled samples of 50 oligochaetes are presented. These results suggest that compared with 1998, the densities of aquatic oligochaetes and T. tubifex have increased, TAM production has been greatly reduced, and the decline is congruent with the dominance of lineages I, V, and VI of T. tubifex-three lineages that are refractory or highly resistant to Mc infection-in the oligochaete population. While it is possible that the resistant lineages function as biofilters that deactivate Mc myxospores, the reason for the decline in TAM production in WGR remains an enigma.

The effect of cohabitation of Tubifex tubifex (Oligochaeta: Tubificidae) populations on infections to Myxobolus cerebralis (Myxozoa: Myxobolidae).

The competitive interactions between susceptible and resistant Tubifex tubifex (Oligochaeta: Tubificidae) exposed to Myxobolus cerebralis (Myxozoa: Myxobolidae) infections were investigated in two laboratory trials. Competition was assessed by the total parasite production over the course of the trials in mixed and pure cultures of M. cerebralis exposed worms, and by the genetic analyses of worms from the control and experimental groups at the beginning and end of the experiments. Mixed cultures of resistant and susceptible worms showed a 70% reduction in production of parasites released when compared with pure cultures of susceptible worms. In studies with laboratory and field-collected oligochaetes the mixed cultures at the end of the cohabitation experiments were dominated by resistant Tubifex from lineage V (HB strain) this strain of Tubifex has a competitive advantage over worms from other lineages. The results of this study suggest that certain species of Tubifex may be dead-end hosts to M. cerebralis by absorbing or inactivating the parasite and may also show greater survival compared to susceptible oligochaetes in certain whirling disease enzootic habitats.

The severity of whirling disease among wild trout corresponds to the differences in genetic composition of Tubifex tubifex populations in central Colorado.

We analyzed the geographic distribution of Tubifex tubifex from various river drainages in central Colorado by genetic screening with specific mitochondrial 16S ribosomal DNA (mt 16S rDNA) markers. Four distinct mt 16S rDNA lineages are evident. The sites varied with respect to land- and water-use practices. All sites represented habitats presumed capable of supporting oligochaetes. At the locations where whirling disease has had the greatest impact on resident rainbow trout, T. tubifex, representing lineages I and III (genotypes known to be susceptible to Mxyobolus cerebralis), were most commonly found. In contrast, at sites less affected by whirling disease, T. tubifex of lineages V and VI that are more resistant to M. cerebralis infections were more abundant. The predominance of resistant T. tubifex worms (lineages V and VI) at low-impact sites supports the conclusion that when these genotypes are in greater abundance, the potential for more severe effects of whirling disease on wild rainbow trout populations may be diminished.

Efficacy of passive sand filtration in reducing exposure of salmonids to the actinospore of Myxobolus cerebralis.

The aquatic oligochaete Tubifex tubifex parasitized by Myxobolus cerebralis releases triactinomyxon (TAM) actinospores that can infect some species of salmonids and cause salmonid whirling disease. Silica sand was tested as a filtration medium for removal of TAMs from water containing the parasite. Laboratory tests indicated sand filtration removed > 99.99% of TAMs. In 2 different field tests, groups of 1 mo old rainbow trout Oncorhynchus mykiss were exposed for 2 wk to filtered and unfiltered water from a spring-fed pond enzootic for M. cerebralis. In November 2000, the exposure dose was estimated as between 3 and 5 TAMs fish(-1). During a March 2001 exposure, the estimated dose was between 286 and 404 TAMs fish(-1). Fish were held for 6 mo post exposure (p.e.) in laboratory aquaria for observation and evidence of clinical signs of whirling disease. We used 4 diagnostic techniques to assess the prevalence and severity of infection by M. cerebralis among fish exposed to filtered and unfiltered water. These included polymerase chain reaction (PCR) for genomic DNA of the parasite, histological evaluation for tissue damage, tissue digestion for quantification of cranial myxospores of the parasite, and total non-sampling mortality that occurred over 6 mo p.e. All diagnostic tests verified that the prevalence and severity of infection was significantly reduced among fish in treatment groups exposed to filtered water compared to those exposed to unfiltered water in both the low-dose and high-dose exposures.

Prevalence and susceptibility of infection to Myxobolus cerebralis, and genetic differences among populations of Tubifex tubifex.

The prevalence of infection and susceptibility of the aquatic oligochaete Tubifex tubifex to Myxobolus cerebralis, was examined in 2 studies on the upper Colorado River, Colorado, USA, where whirling disease occurs in wild trout populations. In the first study, the prevalence of infection ranged from 0.4 to 1.5%, as determined by counting the number of T. tubifex releasing triactinomyxons of M. cerebralis directly following their collection from the field. The susceptibility of those T. tubifex not releasing triactinomyxons was assessed by the number of these oligochaetes releasing triactinomyxons 3 mo following experimental exposures to spores of M. cerebralis. The prevalence of infection following experimental exposures of these T. tubifex ranged from 4.2 to 14.1%. In a second study, all T. tubifex collected at 2 different times directly from the 2 field sites in Colorado were exposed to spores of M. cerebralis. Individual oligochaetes representing those groups of T. tubifex releasing and those groups not releasing triactinomyxons at 3 mo were screened with molecular genetic markers. T. tubifex populations found at the 2 study sites consisted of 4 genetically distinct lineages that varied with respect to their susceptibility to experimental exposure to M. cerebralis. Lineages I and III contained the most oligochaetes susceptible to M. cerebralis and were the most prominent lineages at Windy Gap Reservoir, a site of high infectivity for wild rainbow trout on the upper Colorado River. In contrast, at the Breeze Bridge site which is below Windy Gap Reservoir and where M. cerebralis infections are less severe in wild trout, oligochaetes in lineages V and VI that are resistant to M. cerebralis were more prominent. These results suggest that certain habitats, such as Windy Gap Reservoir, are conducive to large and more homogenous populations of susceptible T. tubifex lineages that may serve as point sources of infection for M. cerebralis. Although not a direct objective of this study, there was no evidence of M. cerebralis infections among any oligochaetes other than those that would be classified as T. tubifex by standard morphological characteristics.

Response of rainbow trout Oncorhynchus mykiss to exposure to Myxobolus cerebralis above and below a point source of infectivity in the upper Colorado River.

We exposed 9 wk old rainbow trout Oncorhynchus mykiss to ambient levels of Myxobolus cerebralis infectious stages at 4 sites of suspected differing infectivity in the Colorado River. Exposure was estimated by periodic filtration of river water at each exposure location. After a 32 d exposure, the fish were held in the Colorado River at a common site for over a year. Resulting infection was evaluated by the presence of clinical signs (whirling behavior, cranial deformity/exophthalmia, and black tail), severity of microscopic lesions, and myxospore counts (8, 10, 12, and 14 mo post-exposure). Two exposure sites that were immediately downstream of Windy Gap Reservoir were much higher in infectivity than the site above the reservoir or the site 26 km downstream of the reservoir. Rainbow trout exposed at those locations showed higher prevalence of clinical signs of whirling disease, more severe histological evidence of infection and higher average myxospore concentrations than those exposed above the reservoir or 26 km below the reservoir. Many more M. cerebralis actinospores were observed from water filtration at the 2 sites immediately below the reservoir compared to the other sites.

The use of passive injectable transponders in fattening lambs from birth to slaughter: effects of injection position, age, and breed.

A total of 1,159 Tiris half-duplex passive injectable transponders (PIT) of 32-mm length were used to study the electronic identification of 618 fattening lambs of two breeds used for different production purposes (Ripollesa, meat breed, n = 271, and Manchega, dairy breed, n = 347). The lambs were s.c. injected in the armpit and the retro-auricular positions at 2, 15, and 30 d of age. All lambs were also tagged with a small plastic ear tag after birth. A group of 76 lambs were injected only in the right armpit and were kept for breeding. The PIT losses, breakages, and electronic failures were evaluated at weekly weight recordings throughout the fattening period using two types of hand-held transceivers. Fattened lambs were harvested in a commercial abattoir between 3 and 4 mo of age when they reached market weight (11 to 12 kg hot carcass weight). The total number of PIT that fell or broke in the slaughtering line, the location method, and the recovery time were recorded. On the farm PIT losses were not affected (P > 0.05) by age at injection, injection position, or breed. Mean losses of PIT and ear tags during the same period were 5% and 6.3%, respectively (P > 0.05). No PIT breakages or failures were observed during the fattening period. Mean recovery of PIT in the abattoir (85.6%) was affected (P < 0.05) by breed and injection position. Losses of PIT in the abattoir were greater (P < 0.05) in the Ripollesa breed (18.4%) than in Manchega (10.0%), and for both breeds losses were greater (P < 0.05) in the retro-auricular than in the armpit positions (18.6 vs 10.8%, respectively). The percentage of PIT broken during slaughtering was low (0.3%). The mean recovery times (18 +/- 2 s) were not affected (P > 0.05) by breed, injection position, or age, thus allowing a harvesting speed of 200 lambs/h on average. In conclusion, the injection of 32-mm PIT into the armpit or the retro-auricular region is not recommended as a practice for the electronic identification of fattening lambs, even though they perform similarly to small plastic ear tags. This is partly a consequence of the PIT losses observed on the farm but mainly because of the difficulties with recovering the PIT in the abattoir. More research will determine whether the use of smaller transponders or injection in other positions could improve their performance in fattening lambs.

Natural variation in light sensitivity of Arabidopsis.

Because plants depend on light for growth, their development and physiology must suit the particular light environment. Plants native to different environments show heritable, apparently adaptive, changes in their response to light. As a first step in unraveling the genetic and molecular basis of these naturally occurring differences, we have characterized intraspecific variation in a light-dependent developmental process-seedling emergence. We examined 141 Arabidopsis thaliana accessions for their response to four light conditions, two hormone conditions and darkness. There was significant variation in all conditions, confirming that Arabidopsis is a rich source of natural genetic diversity. Hierarchical clustering revealed that some accessions had response patterns similar to known photoreceptor mutants, suggesting changes in specific signaling pathways. We found that the unusual far-red response of the Lm-2 accession is due to a single amino-acid change in the phytochrome A (PHYA) protein. This change stabilizes the light-labile PHYA protein in light and causes a 100-fold shift in the threshold for far-red light sensitivity. Purified recombinant Lm-2 PHYA also shows subtle photochemical differences and has a reduced capacity for autophosphorylation. These biochemical changes contrast with previously characterized natural alleles in loci controlling plant development, which result in altered gene expression or loss of gene function.

Heteromeric AMPA receptors assemble with a preferred subunit stoichiometry and spatial arrangement.

AMPA receptors are thought to be a tetrameric assembly of the subunits GluR1-4. We have examined whether two coexpressed subunits (GluR1/2) combine at random to form channels, or preferentially assemble with a specific stoichiometry and spatial configuration. The subunits carried markers controlling ion permeation and desensitization, and these properties were monitored as a function of relative expression level and subunit composition. Homomeric receptors assembled stochastically while heteromeric receptors preferentially formed with a stoichiometry of two GluR1 and two GluR2 subunits, and with identical subunits positioned on opposite sides of the channel pore. This structure will predominate if GluR1 binds to GluR2 more rapidly during receptor assembly than other subunit combinations. The practical outcome of selective heteromeric assembly is a more homogenous receptor population in vivo.

Modes of salmonid MHC class I and II evolution differ from the primate paradigm.

Rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) represent two salmonid genera separated for 15--20 million years. cDNA sequences were determined for the classical MHC class I heavy chain gene UBA and the MHC class II beta-chain gene DAB from 15 rainbow and 10 brown trout. Both genes are highly polymorphic in both species and diploid in expression. The MHC class I alleles comprise several highly divergent lineages that are represented in both species and predate genera separation. The class II alleles are less divergent, highly species specific, and probably arose after genera separation. The striking difference in salmonid MHC class I and class II evolution contrasts with the situation in primates, where lineages of class II alleles have been sustained over longer periods of time relative to class I lineages. The difference may arise because salmonid MHC class I and II genes are not linked, whereas in mammals they are closely linked. A prevalent mechanism for evolving new MHC class I alleles in salmonids is recombination in intron II that shuffles alpha 1 and alpha 2 domains into different combinations.

Interaction of the C-terminal tail region of the metabotropic glutamate receptor 7 with the protein kinase C substrate PICK1.

Group III metabotropic glutamate receptors (mGluRs) are highly enriched in the presynaptic terminals of glutamatergic synapses where they mediate feedback inhibition of neurotransmitter release. Here, we used the yeast two-hybrid system to identify a direct interaction of the C-terminal tail region of mGluR7 with the rat homologue of the protein kinase C substrate PICK1. This interaction is specifically mediated by the very C-terminal amino acids of the receptor and can be reconstituted in human embryonic kidney 293 cells by transfection of full-length mGluR7 and PICK1 cDNAs. Quantitative beta-galactosidase assays revealed that among the different group III mGluRs, mGluR7 is the major PICK1 binding partner although other subfamily members can also interact with PICK1. These data indicate that PDZ domain-containing proteins might contribute to the presynaptic localization of group III mGluRs.

Glycosylation affects agonist binding and signal transduction of the rat somatostatin receptor subtype 3.

The somatostatin receptor subtypes, sst1-sst5, bind their natural ligands, somatostatin-14, somatostatin-28 and cortistatin-17, with high affinity but do not much discriminate between them. Detailed understanding of the interactions between these receptors and their peptide ligands may facilitate the development of selective compounds which are needed to identify the biological functions of individual receptor subtypes. The influence of the amino-terminal domain and of the two putative N-linked glycosylation sites located in this region of rat sst3 was analysed. Biochemical studies in transfected cell lines suggested that the amino-terminus of sst3 is glycosylated at both sites. Mutation of the N-linked glycosylation site, Asn18Thr, had only a small effect on binding properties and inhibition of adenylyl cyclase. The double mutant Asn18Thr/Asn31Thr lacking both glycosylation sites showed a significant reduction in high affinity binding and inhibition of adenylyl cyclase while peptide selectivity was not affected. Truncation of the amino-terminal region by 32 amino acid residues including the two glycosylation sites caused similar but much stronger effects. Immunocytochemical analysis of receptor localisation revealed that the amino-terminal domain but not the carbohydrates appear to be involved in the transport of the receptor polypeptide to the cell surface.

The metabotropic GABAB receptor directly interacts with the activating transcription factor 4.

G protein-coupled receptors regulate gene expression by cellular signaling cascades that target transcription factors and their recognition by specific DNA sequences. In the central nervous system, heteromeric metabotropic gamma-aminobutyric acid type B (GABA(B)) receptors through adenylyl cyclase regulate cAMP levels, which may control transcription factor binding to the cAMP response element. Using yeast-two hybrid screens of rat brain libraries, we now demonstrate that GABA(B) receptors are engaged in a direct and specific interaction with the activating transcription factor 4 (ATF-4), a member of the cAMP response element-binding protein /ATF family. As confirmed by pull-down assays, ATF-4 associates via its conserved basic leucine zipper domain with the C termini of both GABA(B) receptor (GABA(B)R) 1 and GABA(B)R2 at a site which serves to assemble these receptor subunits in heterodimeric complexes. Confocal fluorescence microscopy shows that GABA(B)R and ATF-4 are strongly coclustered in the soma and at the dendritic membrane surface of both cultured hippocampal neurons as well as retinal amacrine cells in vivo. In oocyte coexpression assays short term signaling of GABA(B)Rs via G proteins was only marginally affected by the presence of the transcription factor, but ATF-4 was moderately stimulated in response to receptor activation in in vivo reporter assays. Thus, inhibitory metabotropic GABA(B)Rs may regulate activity-dependent gene expression via a direct interaction with ATF-4.

Neuronal inwardly rectifying K(+) channels differentially couple to PDZ proteins of the PSD-95/SAP90 family.

Several signaling proteins clustered at the postsynaptic density specialization in neurons harbor a conserved C-terminal PDZ domain recognition sequence (X-S/T-X-V/I) that mediates binding to members of the PSD-95/SAP90 protein family. This motif is also present in the C termini of some inwardly rectifying K(+) (Kir) channels. Constitutively active Kir2 channels as well as G protein-gated Kir3 channels, which are fundamental for neuronal excitability, were analyzed as candidates for binding to PSD-95/SAP90 family members. Therefore C termini of Kir2.1(+), Kir2.3(+), Kir2.4(-), Kir3.1(-), Kir3.2(+), Kir3.3(+) and Kir3.4(-) subunits (+, motif present; -, motif absent) were used as baits in the yeast two-hybrid assay to screen for in vivo interaction with PDZ domains 1-3 of PSD-95/SAP90. In contrast to Kir2.1 and Kir2.3, all Kir3 fragments failed to bind PSD-95 in this assay, which was supported by the lack of coimmunoprecipitation and colocalization of the entire proteins in mammalian cells. A detailed analysis of interaction domains demonstrated that the C-terminal motif in Kir3 channels is insufficient for binding PDZ domains. Kir2.1 and Kir2.3 subunits on the other hand coprecipitate with PSD-95. When coexpressed in a bicistronic internal ribosome entry site expression vector in HEK-293 cells macroscopic and elementary current analysis revealed that PSD-95 suppressed the activity of Kir2.3 channels by >50%. This inhibitory action of PSD-95, which predominantly affects the single-channel conductance, is likely attributable to a molecular association with additional internal interaction sites in the Kir2.3 protein.