Truncated recombinant human SP-D attenuates emphysema and type II cell changes in SP-D deficient mice

BACKGROUND: Surfactant protein D (SP-D) deficient mice develop emphysema-like pathology associated with focal accumulations of foamy alveolar macrophages, an excess of surfactant phospholipids in the alveolar space and both hypertrophy and hyperplasia of alveolar type II cells. These findings are associated with a chronic inflammatory state. Treatment of SP-D deficient mice with a truncated recombinant fragment of human SP-D (rfhSP-D) has been shown to decrease the lipidosis and alveolar macrophage accumulation as well as production of proinflammatory chemokines. The aim of this study was to investigate if rfhSP-D treatment reduces the structural abnormalities in parenchymal architecture and type II cells characteristic of SP-D deficiency. METHODS: SP-D knock-out mice, aged 3 weeks, 6 weeks and 9 weeks were treated with rfhSP-D for 9, 6 and 3 weeks, respectively. All mice were sacrificed at age 12 weeks and compared to both PBS treated SP-D deficient and wild-type groups. Lung structure was quantified by design-based stereology at the light and electron microscopic level. Emphasis was put on quantification of emphysema, type II cell changes and intracellular surfactant. Data were analysed with two sided non-parametric Mann-Whitney U-test. MAIN RESULTS: After 3 weeks of treatment, alveolar number was higher and mean alveolar size was smaller compared to saline-treated SP-D knock-out controls. There was no significant difference concerning these indices of pulmonary emphysema within rfhSP-D treated groups. Type II cell number and size were smaller as a consequence of treatment. The total volume of lamellar bodies per type II cell and per lung was smaller after 6 weeks of treatment. CONCLUSION: Treatment of SP-D deficient mice with rfhSP-D leads to a reduction in the degree of emphysema and a correction of type II cell hyperplasia and hypertrophy. This supports the concept that rfhSP-D might become a therapeutic option in diseases that are characterized by decreased SP-D levels in the lung.

Active intestinal calcium transport in the absence of transient receptor potential vanilloid type 6 and calbindin-D9k

To study the role of the epithelial calcium channel transient receptor potential vanilloid type 6 (TRPV6) and the calcium-binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knockout (KO), calbindin-D9k KO, and TRPV6/calbindin-D(9k) double-KO (DKO) mice were generated. TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild-type (WT) mice ( approximately 10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8-fold increase in serum PTH levels (P < 0.05 compared with WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n = 8-22 per group; P > 0.1, WT vs. calbindin-D9k KO, and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Duodenal calcium transport was increased 2.1-fold in the TRPV6/calbindin-D9k DKO mice fed the low-calcium diet (P < 0.05, WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25-Dihydroxyvitamin D3 administration to vitamin D-deficient null mutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4- to 2.0-fold, P < 0.05 compared with vitamin D-deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are essential for vitamin D-induced active intestinal calcium transport.

Antigens of the type-three secretion system of Aeromonas salmonicida subsp. salmonicida prevent protective immunity in rainbow trout

Aeromonas salmonicida subsp. salmonicida is the etiologic agent of furunculosis, a frequent and significant disease of fisheries worldwide. The disease is largely controlled by commercial oil adjuvanted vaccines containing bacterins. However, the mechanisms leading to a protective immune response remain poorly understood. The type-three secretion system (T3SS) plays a central role in virulence of A. salmonicida subsp. salmonicida and thus may have an influence on the immune response of the host. The aim of this study was to evaluate the role of the T3SS antigens in mounting a protective immune response against furunculosis. Rainbow trout were intraperitoneally vaccinated in two independent experiments with bacterins prepared from a wild-type A. salmonicida strain and an isogenic strain carrying a deletion in the T3SS (ΔascV). Fish were challenged with the wt strain eight weeks after vaccination. In both trials, the survival rate of trout vaccinated with the ΔascV strain was significantly higher (23-28%) in comparison to the group vaccinated with the wt strain. High-throughput proteomics analysis of whole bacteria showed the ascV deletion in the mutant strain resulted in lower expression of all the components of the T3SS, several of which have a potential immunosuppressive activity. In a third experiment, fish were vaccinated with recombinant AcrV (homologous to the protective antigen LcrV of Yersinia) or S-layer protein VapA (control). AcrV vaccinated fish were not protected against a challenge while fish vaccinated with VapA were partially protected. The presence of T3SS proteins in the vaccine preparations decreased the level of protection against A. salmonicida infection and that AcrV was not a protective antigen. These results challenge the hypothesis that mounting specific antibodies against T3SS proteins should bring better protection to fish and demonstrate that further investigations are needed to better understand the mechanisms underlying effective immune responses against A. salmonicida infection.

The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors

BACKGROUND Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth. RESULTS Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected. CONCLUSIONS We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.

ROLE OF THE GCN5 HISTONE ACETYLTRANSFERASE IN SPINOCEREBELLAR ATAXIA TYPE 7 AND IN IMMATURE NEURONS

Spinocerebellar Ataxia type 7 (SCA7) is a neurodegenerative disease caused by expansion of a CAG repeat encoding a polyglutamine tract in ATXN7, a component of the SAGA histone acetyltransferase (HAT) complex. Previous studies provided conflicting evidence regarding the effects of polyQ-ATXN7 on the activity of Gcn5, the HAT catalytic subunit of SAGA. Here I showed that reducing Gcn5 expression accelerates both cerebellar and retinal degeneration in a mouse model of SCA7. Deletion of Gcn5 in Purkinje cells in mice expressing wild type Atxn7, however, causes only mild ataxia and does not lead to the early lethality observed in SCA7 mice. Reduced Gcn5 expression strongly enhances retinopathy in SCA7 mice, but does not affect the transcriptional targets of Atxn7, as expression of these genes is not further altered by Gcn5 depletion. These findings demonstrate that loss of Gcn5 functions can contribute to the time of onset and severity of SCA7 phenotypes, but suggest that non-transcriptional functions of SAGA may play a role in neurodegeneration in this disease.

The Salmonella pathogenicity island (SPI)-2 and SPI-1 type III secretion systems allow Salmonella serovar typhimurium to trigger colitis via MyD88-dependent and MyD88-independent mechanisms.

Salmonella typhimurium can colonize the gut, invade intestinal tissues, and cause enterocolitis. In vitro studies suggest different mechanisms leading to mucosal inflammation, including 1) direct modulation of proinflammatory signaling by bacterial type III effector proteins and 2) disruption or penetration of the intestinal epithelium so that penetrating bacteria or bacterial products can trigger innate immunity (i.e., TLR signaling). We studied these mechanisms in vivo using streptomycin-pretreated wild-type and knockout mice including MyD88(-/-) animals lacking an adaptor molecule required for signaling via most TLRs. The Salmonella SPI-1 and the SPI-2 type III secretion systems (TTSS) contributed to inflammation. Mutants that retain only a functional SPI-1 (M556; sseD::aphT) or a SPI-2 TTSS (SB161; DeltainvG) caused attenuated colitis, which reflected distinct aspects of the colitis caused by wild-type S. typhimurium: M556 caused diffuse cecal inflammation that did not require MyD88 signaling. In contrast, SB161 induced focal mucosal inflammation requiring MyD88. M556 but not SB161 was found in intestinal epithelial cells. In the lamina propria, M556 and SB161 appeared to reside in different leukocyte cell populations as indicated by differential CD11c staining. Only the SPI-2-dependent inflammatory pathway required aroA-dependent intracellular growth. Thus, S. typhimurium can use two independent mechanisms to elicit colitis in vivo: SPI-1-dependent and MyD88-independent signaling to epithelial cells and SPI-2-dependent intracellular proliferation in the lamina propria triggering MyD88-dependent innate immune responses.

Chlorophyll breakdown: Pheophorbide a oxygenase is a Rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 gene

Chlorophyll (chl) breakdown during senescence is an integral part of plant development and leads to the accumulation of colorless catabolites. The loss of green pigment is due to an oxygenolytic opening of the porphyrin macrocycle of pheophorbide (pheide) a followed by a reduction to yield a fluorescent chl catabolite. This step is comprised of the interaction of two enzymes, pheide a oxygenase (PaO) and red chl catabolite reductase. PaO activity is found only during senescence, hence PaO seems to be a key regulator of chl catabolism. Whereas red chl catabolite reductase has been cloned, the nature of PaO has remained elusive. Here we report on the identification of the PaO gene of Arabidopsis thaliana (AtPaO). AtPaO is a Rieske-type iron–sulfur cluster-containing enzyme that is identical to Arabidopsis accelerated cell death 1 and homologous to lethal leaf spot 1 (LLS1) of maize. Biochemical properties of recombinant AtPaO were identical to PaO isolated from a natural source. Production of fluorescent chl catabolite-1 required ferredoxin as an electron source and both substrates, pheide a and molecular oxygen. By using a maize lls1 mutant, the in vivo function of PaO, i.e., degradation of pheide a during senescence, could be confirmed. Thus, lls1 leaves stayed green during dark incubation and accumulated pheide a that caused a light-dependent lesion mimic phenotype. Whereas proteins were degraded similarly in wild type and lls1, a chl-binding protein was selectively retained in the mutant. PaO expression correlated positively with senescence, but the enzyme appeared to be post-translationally regulated as well.

Dysregulation of alternative splicing of coagulation factor V results in bleeding disorder, east Texas type and other disorders of hemostasis

The studies completed herein explore different phenotypes related to the genetic defects that predispose individuals to a disruption of normal hemostasis. In the first study, a novel autosomal dominant bleeding disorder, which is characterized by excessive bleeding with trauma or surgery and menorrhagia in affected women, was studied in a large family (16 affected individuals) from east Texas. Affected members had a prolongation of their PT and/or aPTT, but normal clinical coagulation studies. Previous linkage analysis by Kuang et. al. (2001) mapped the defective gene to 1g23-24 (LODmax 7.22), which contains the gene for coagulation factor V (FV). I identified an alteration (A2440G) in the FV gene in exon 13 that segregated with the disease and was not present in 62 controls. Interestingly, this alteration resulted in a 22-fold up-regulation of a novel alternative splicing variant in patients' RNA versus controls. This translated into a similar fold increase in a 250-kDa isoform of FV seen in patients' plasma versus controls. A recombinant of this splicing event exhibited an increased sensitivity to cleavage by activated protein C (APC) that was more striking in the presence of PS. In addition, this novel isoform had increased APC cofactor activity, thus increasing the degradation of FVIIIa. These data indicated that A2440G up-regulates an alternatively spliced transcript of FV, and increases a FV isoform that hinders coagulation as opposed to promoting it like its wild-type counterpart. ^ The second study reports the largest screening to date of African Americans in two independent cohorts for a rare prothrombin variant, C20209T, which is suspected to be associated with thrombotic disease. The Texas Medical Center Genetics Resource (TexGen) Stroke DNA repository revealed 1.67% (Fisher p=0.27) of African American stroke patients were heterozygous for the 20209*T allele. Screening of the Atherosclerosis Risk in Communities Study (ARIC) cohort (n=3470) for the 20209*T allele revealed a population prevalence of 0.58% in individuals of African American descent; however, all associations with thrombotic disease were negative. Analysis of these two independent cohorts revealed that, unlike its neighbor G20210A, the C20209T variant does not increase the risk of thrombotic events in the African American population. ^

THE NEW ROLE OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9: A CONNECTION OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9, APOLIPOPROTEIN B, and AUTOPHAGY

Plasma low-density lipoprotein (LDL) levels are positively correlated with the incidence of coronary artery disease. In the circulation, the plasma LDL clearance is mainly achieved by the uptake via LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a newly discovered gene, playing an important role in LDL metabolism. Gain-of-function mutations of PCSK9 lead to hypercholesterolemia and loss-of-function mutations of PCSK9 are associated with decrease of LDL cholesterol. The effects of PCSK9 on cholesterol levels are the consequence of a strong interaction between the catalytic domain of PCSK9 and epidermal growth factor-like repeat A (EGF-A) domain of LDLR on the cell surface of hepatocytes. This PCSK9/LDLR complex enters the cell via endocytosis, where both PCSK9 and LDLR are removed via the lysosome pathway, resulting in decreased levels of LDLR and accumulation of LDL in the plasma. However, whether this is the exclusive function of PCSK9 on LDL metabolism was challenged by us; we observed PCSK9 interacted with apolipoprotein B (apoB) and increased apoB production, irrespective of the LDLR. ApoB is the primary structure protein of LDL particle and it also serves as the ligand for the LDL receptor. There is ample evidence showing that the levels of apoB are a better indicator for heart disease than either total cholesterol or LDL cholesterol levels. We used a second-generation adenoviral vector to overexpress PCSK9 (Ad-PCSK9) in wild-type C57BL/6 and LDLR deficient mice (Ldlr-/- and Ldlr-/-Apobec1-/-). Our study revealed that overexpression of PCSK9 promoted the production and secretion of apoB in the form of very-low density lipoprotein (VLDL), which is the precursor of LDL, in the 3 mouse models studied (C57BL/6J, Ldlr-/-, and Ldlr-/-Apobec1-/-). The increased apoB production in mice was regulated at post-transcriptional levels, since there was no difference in apoB mRNA levels between mice treated with Ad-PCSK9 and control vector Ad-Null. By using pulse-chase experiment on primary hepatocytes, we showed that overexpression of PCSK9 increased the secretion of apoB, independent of LDLR. In the circulation, we showed that PCSK9 was associated with LDL particles. By using 3 different protein–protein interaction assays of co-immunoprecipitation, mammalian two-hybrid system, and in situ proximity ligation assay, we demonstrated a direct protein–protein interaction between PCSK9 and apoB. The impact of this interaction inhibited the physiological removal process of apoB via autophagosome/lysosome pathway in an LDLR-independent fashion, resulting in increased production and secretion of apoB-containing lipoproteins. The significance of this process was shown in the Pcsk9 knockout mice in the background of Ldlr-/-Apobec1-/- mice (triple knockout mice); in the absence of Pcsk9 (triple knockout mice) the levels of cholesterol, triacylglycerol, and apoB decreased significantly in comparison to that of Ldlr-/-Apobec1-/- mice. Taken together, our study demonstrated a direct intracellular interaction of PCSK9 with apoB, resulting in the inhibition of apoB degradation via the autophagosome/lysosome pathway independent of LDLR. This discovery provides a new concept of the importance of PCSK9 and suggests new approaches for the therapeutic intervention of hyperlipidemia.

Delayed Thrombus Resolution and Fibroproliferative Vascular Wound Healing From Deficiency of Type III Collagen: a Paradoxical Mechanism for Tissue Fragility

Vascular Ehlers-Danlos syndrome is a heritable disease of connective tissue caused by mutations in COL3A1, conferring a tissue deficiency of type III collagen. Cutaneous wounds heal poorly in these patients, and they are susceptible to spontaneous and catastrophic rupture of expansible hollow organs like the gut, uterus, and medium-sized to large arteries, which leads to premature death. Although the predisposition for organ rupture is often attributed to inherent tissue fragility, investigation of arteries from a haploinsufficient Col3a1 mouse model (Col3a1+/-) demonstrates that mutant arteries withstand even supraphysiologic pressures comparably to wild-type vessels. We hypothesize that injury that elicits occlusive thrombi instead unmasks defective thrombus resolution resulting from impaired production of type III collagen, which causes deranged remodeling of matrix, persistent inflammation, and dysregulated behavior by resident myofibroblasts, culminating in the development of penetrating neovascular channels that disrupt the mechanical integrity of the arterial wall. Vascular injury and thrombus formation following ligation of the carotid artery reveals an abnormal persistence and elevated burden of occlusive thrombi at 21 post-operative days in vessels from Col3a1+/- mice, as opposed to near complete resolution and formation of a patent and mature neointima in wild-type mice. At only 14 days, both groups harbor comparable burdens of resolving thrombi, but wild-type mice increase production of type III collagen in actively resolving tissues, while mutant mice do not. Rather, thrombi in mutant mice contain higher burdens of macrophages and proliferative myofibroblasts, which persist through 21 days while wild-type thrombi, inflammatory cells, and proliferation all regress. At the same time that increased macrophage burdens were observed at 14 and 21 days post ligation, the medial layer of mutant arterial walls concurrently harbored a significantly higher incidence of penetrating neovessels compared with those in wild-type mice. To assess whether limited type III collagen production alters myofibroblast behavior, fibroblasts from vEDS patients with COL3A1 missense mutations were seeded into three-dimensional fibrin gel constructs and stimulated with transforming growth factor-β1 to initiate myofibroblast differentiation. Although early signaling events occur similarly in all cell lines, late extracellular matrix- and mechanically-regulated events like transcriptional upregulation of type I and type III collagen secretion are delayed in mutant cultures, while transcription of genes encoding intracellular contractile machinery is increased. Sophisticated imaging of collagen synthesized de novo by resident myofibroblasts visualizes complex matrix reorganization by control cells but only meager remodeling by COL3A1 mutant cells, concordant with their compensatory contraction to maintain tension in the matrix. Finally, administration of immunosuppressive rapamycin to mice following carotid ligation sufficiently halts the initial inflammatory phase of thrombus resolution and fully prevents both myofibroblast migration into the thrombus and the differential development of neovessels between mutant and wild-type mice, suggesting that pathological defects in mutant arteries develop secondarily to myofibroblast dysfunction and chronic inflammatory stimulation, rather than as a manifestation of tissue fragility. Together these data establish evidence that pathological defects in the vessel wall architecture develop in mutant arteries as sequelae to abnormal healing and remodeling responses activated by arterial injury. Thus, these data support the hypothesis that events threatening the integrity of type III collagen-deficient vessels develop not as a result of inherent tissue weakness and fragility at baseline but instead as an episodic byproduct of abnormally persistent granulation tissue and fibroproliferative intravascular remodeling.

The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop

Although infection by primary HIV type 1 (HIV-1) isolates normally requires the functional interaction of the viral envelope protein with both CD4 and the CCR-5 coreceptor, a subset of such isolates also are able to use the distinct CCR-3 receptor. By analyzing the ability of a series of wild-type and chimeric HIV-1 envelope proteins to mediate CCR-3-dependent infection, we have determined that CCR-3 tropism maps to the V1 and V2 variable region of envelope. Although substitution of the V1/V2 region of a CCR-3 tropic envelope into the context of a CCR-5 tropic envelope is both necessary and sufficient to confer CCR-3 tropism, this same substitution has no phenotypic effect when inserted into a CXCR-4 tropic HIV-1 envelope context. However, this latter chimera acquires both CCR-3 and CCR-5 tropism when a CCR-5 tropic V3 loop sequence also is introduced. These data demonstrate that the V1/2 region of envelope can, like the V3 loop region, encode a particular coreceptor requirement and suggest that a functional envelope:CCR-3 interaction may depend on the cooperative interaction of CCR-3 with both the V1/V2 and the V3 region of envelope.

Postsynaptic clustering of γ-aminobutyric acid type A receptors by the γ3 subunit in vivo

Synaptic localization of γ-aminobutyric acid type A (GABAA) receptors is a prerequisite for synaptic inhibitory function, but the mechanism by which different receptor subtypes are localized to postsynaptic sites is poorly understood. The γ2 subunit and the postsynaptic clustering protein gephyrin are required for synaptic localization and function of major GABAA receptor subtypes. We now show that transgenic overexpression of the γ3 subunit in γ2 subunit-deficient mice restores benzodiazepine binding sites, benzodiazepine-modulated whole cell currents, and postsynaptic miniature currents, suggesting the formation of functional, postsynaptic receptors. Moreover, the γ3 subunit can substitute for γ2 in the formation of GABAA receptors that are synaptically clustered and colocalized with gephyrin in vivo. These clusters were formed even in brain regions devoid of endogenous γ3 subunit, indicating that the factors present for clustering of γ2 subunit-containing receptors are sufficient to cluster γ3 subunit-containing receptors. The GABAA receptor and gephyrin-clustering properties of the ectopic γ3 subunit were also observed for the endogenous γ3 subunit, but only in the absence of the γ2 subunit, suggesting that the γ3 subunit is at a competitive disadvantage with the γ2 subunit for clustering of postsynaptic GABAA receptors in wild-type mice.

A point mutation in the γ2 subunit of γ-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity

Benzodiazepines allosterically modulate γ-aminobutyric acid (GABA) evoked chloride currents of γ-aminobutyric acid type A (GABAA) receptors. Coexpression of either rat γ2 or γ3, in combination with α1 and β2 subunits, results both in receptors displaying high [3H]Ro 15-1788 affinity. However, receptors containing a γ3 subunit display a 178-fold reduced affinity to zolpidem as compared with γ2-containing receptors. Eight chimeras between γ2 and γ3 were constructed followed by nine different point mutations in γ2, each to the homologous amino acid residue found in γ3. Chimeric or mutant γ subunits were coexpressed with α1 and β2 in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity. Substitution of a methionine-to-leucine at position 130 of γ2 (γ2M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [3H]Ro 15-1788 remained unchanged. The affinity for diazepam was only decreased by about 2-fold. The same mutation resulted in a 9-fold increase in Cl 218872 affinity. A second mutation (γ2M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and γ2M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved. Dose–response curves for zolpidem and for diazepam showed that the zolpidem but not the diazepam apparent affinity is drastically reduced. The apparent GABA affinity is not significantly affected by the γ2M130L mutation. The identified amino acid residues may define part of the benzodiazepine binding pocket of GABAA receptors. As the modulatory site in the GABAA receptor is homologous to the GABA site, and to all agonist sites of related receptors, γ2M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.

Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca2+ channel activity by cAMP-dependent protein kinase

Preferential phosphorylation of specific proteins by cAMP-dependent protein kinase (PKA) may be mediated in part by the anchoring of PKA to a family of A-kinase anchor proteins (AKAPs) positioned in close proximity to target proteins. This interaction is thought to depend on binding of the type II regulatory (RII) subunits to AKAPs and is essential for PKA-dependent modulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor, the L-type Ca2+ channel, and the KCa channel. We hypothesized that the targeted disruption of the gene for the ubiquitously expressed RIIα subunit would reveal those tissues and signaling events that require anchored PKA. RIIα knockout mice appear normal and healthy. In adult skeletal muscle, RIα protein levels increased to partially compensate for the loss of RIIα. Nonetheless, a reduction in both catalytic (C) subunit protein levels and total kinase activity was observed. Surprisingly, the anchored PKA-dependent potentiation of the L-type Ca2+ channel in RIIα knockout skeletal muscle was unchanged compared with wild type although it was more sensitive to inhibitors of PKA–AKAP interactions. The C subunit colocalized with the L-type Ca2+ channel in transverse tubules in wild-type skeletal muscle and retained this localization in knockout muscle. The RIα subunit was shown to bind AKAPs, although with a 500-fold lower affinity than the RIIα subunit. The potentiation of the L-type Ca2+ channel in RIIα knockout mouse skeletal muscle suggests that, despite a lower affinity for AKAP binding, RIα is capable of physiologically relevant anchoring interactions.

Defective γ-aminobutyric acid type B receptor-activated inwardly rectifying K+ currents in cerebellar granule cells isolated from weaver and Girk2 null mutant mice

Stimulation of inhibitory neurotransmitter receptors, such as γ-aminobutyric acid type B (GABAB) receptors, activates G protein-gated inwardly rectifying K+ channels (GIRK) which, in turn, influence membrane excitability. Seizure activity has been reported in a Girk2 null mutant mouse lacking GIRK2 channels but showing normal cerebellar development as well as in the weaver mouse, which has mutated GIRK2 channels and shows abnormal development. To understand how the function of GIRK2 channels differs in these two mutant mice, we compared the G protein-activated inwardly rectifying K+ currents in cerebellar granule cells isolated from Girk2 null mutant and weaver mutant mice with those from wild-type mice. Activation of GABAB receptors in wild-type granule cells induced an inwardly rectifying K+ current, which was sensitive to pertussis toxin and inhibited by external Ba2+ ions. The amplitude of the GABAB receptor-activated current was severely attenuated in granule cells isolated from both weaver and Girk2 null mutant mice. By contrast, the G protein-gated inwardly rectifying current and possibly the agonist-independent basal current appeared to be less selective for K+ ions in weaver but not Girk2 null mutant granule cells. Our results support the hypothesis that a nonselective current leads to the weaver phenotype. The loss of GABAB receptor-activated GIRK current appears coincident with the absence of GIRK2 channel protein and the reduction of GIRK1 channel protein in the Girk2 null mutant mouse, suggesting that GABAB receptors couple to heteromultimers composed of GIRK1 and GIRK2 channel subunits.