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Biomedical subjects

F Müller

Publications and source records attributed to F Müller.

At least 19 recordsLinked to original sources
PubMed1999

Cytokine network in congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy.

Inflammatory cytokines may play a pathogenic role in the development of congestive heart failure (CHF). Elevated circulating levels of inflammatory cytokines have been reported in CHF, but most studies have focused on only a few cytokine parameters. However, the activity of these cytokines are modulated by soluble cytokine receptors and cytokines with anti-inflammatory activities, and in the present study several of these interacting factors were examined simultaneously in 38 CHF patients with various degrees of heart failure and in 21 healthy controls. Patients with CHF had increased plasma concentrations of tumor necrosis factor (TNF)alpha, interleukin-6, soluble TNF receptors and the soluble interleukin-6 receptor, glycoprotein (gp)130. They also had elevated ratios of TNFalpha/soluble TNF receptors and interleukin-6/soluble gp130 as well as enhanced interleukin-6 bioactivity in serum, suggesting inflammatory net effects. In addition to raised circulating levels of inflammatory cytokines, CHF patients with severe heart failure also had abnormalities in the levels of anti-inflammatory cytokines, with decreased levels of transforming growth factor beta1 and inadequately raised interleukin-10 in relation to the elevated TNFalpha concentrations. This dysbalance between inflammatory and anti-inflammatory cytokines was also found in monocyte supernatants from CHF patients. The abnormalities in the cytokine network were most pronounced in patients with the most severe heart failure, and several of the immunologic parameters, in particular soluble gp130, were correlated with variables reflecting deranged hemodynamic status. The present study analyzing the complexity of the cytokine network in CHF, demonstrates profound disturbances in the levels of both inflammatory and anti-inflammatory mediators with a marked dysbalance favoring inflammatory effects.

Adult↗
PubMed1999

Surface plasmon resonance measurements reveal stable complex formation between p53 and DNA polymerase alpha.

Surface plasmon resonance measurements were used for detecting and quantifying protein-protein interactions between the tumor suppressor protein p53, the SV40 large T antigen (T-ag), the cellular DNA polymerase alpha-primase complex (pol-prim), and the cellular single-strand DNA binding protein RPA. Highly purified p53 protein bound to immobilized T-ag with an apparent binding constant of 2 x 10(8) M(-1). Binding of p53 to RPA was in the same order of magnitude with a binding constant of 4 x 10(8) M(-1), when RPA was coupled to the sensor chip via its smallest subunit, and 1 x 10(8) M(-1), when RPA was coupled via its p70 subunit. Furthermore, p53 bound human DNA polymerase alpha-primase complex (pol-prim) with a K(A) value of 1 x 10(10) m(-1). Both the p68 subunit and the p180 subunit of pol-prim could interact with p53 displaying binding constants of 2 x 10(10) m1(-1) and 5 X 10(9) M(-1), respectively. Complex formation was also observed with a p180/p68 heterodimer, and again with a binding constant similar. Hence, there was no synergistic effect when p53 bound to higher order complexes of pol-prim. A truncated form of p53, consisting of amino acids 1-320, bound pol-prim by four orders of magnitude less efficiently. Therefore, an intact C-terminus of p53 seems to be important for efficient binding to pol-prim. It was also tried to measure complex formation between p53, pol-prim, and T-ag. However there was no evidence for the existence of a ternary complex consisting of T-ag, pol-prim, and p53.

Animals↗
PubMed1999

Increased activation of protein kinase A type I contributes to the T cell deficiency in common variable immunodeficiency.

The molecular mechanisms underlying the T cell dysfunction often present in common variable immunodeficiency (CVI) are not established. cAMP-dependent protein kinase A type I (PKAI) is an important inhibitor of T cell proliferation after Ag stimulation. We therefore investigated the possibility that activation of PKAI may be involved in the development of T cell dysfunction in CVI. An exogenously added PKAI-selective antagonist (Rp-8-Br-cAMPS) induced a significant increase in anti-CD3-stimulated PBMC proliferation in 20 CVI patients compared with no effect in 15 controls. Purified T cells from 7 CVI patients with strictly defined T cell deficiency had elevated endogenous cAMP levels compared with controls. Treatment of T cells from these CVI patients with Rp-8-bromo-cAMP-phosphorothioate markedly improved anti-CD3-stimulated proliferation (up to 3.7-fold), particularly in CD4+ lymphocytes, reaching proliferation levels comparable to control values. No effect of cAMP antagonist on T cell proliferation was seen in controls. In these CVI patients, cAMP antagonist also increased IL-2 production in anti-CD3-stimulated T cells. However, exogenously added IL-2 at concentrations comparable to the achieved increase in IL-2 levels after addition of cAMP antagonist had no effect on T cell proliferation. Furthermore, the stimulatory effects of exogenously added IL-2 at higher concentrations and cAMP antagonist on T cell proliferation were additive. Our findings indicate that increased PKAI activation may be an important molecular basis for the T cell defect in CVI and suggest that the cAMP/PKAI system may be a potential molecular target for immunomodulating therapy in these patients.

Adult↗
PubMed1999

Catalytic triad of microsomal epoxide hydrolase: replacement of Glu404 with Asp leads to a strongly increased turnover rate.

Microsomal epoxide hydrolase (mEH) belongs to the superfamily of alpha/beta-hydrolase fold enzymes. A catalytic triad in the active centre of the enzyme hydrolyses the substrate molecules in a two-step reaction via the intermediate formation of an enzyme-substrate ester. Here we show that the mEH catalytic triad is composed of Asp226, Glu404 and His431. Replacing either of these residues with non-functional amino acids results in a complete loss of activity of the enzyme recombinantly expressed in Saccharomyces cerevisiae. For Glu404 and His431 mutants, their structural integrity was demonstrated by their retained ability to form the substrate ester intermediate, indicating that the lack of enzymic activity is due to an indispensable function of either residue in the hydrolytic step of the enzymic reaction. The role of Asp226 as the catalytic nucleophile driving the formation of the ester intermediate was substantiated by the isolation of a peptide fraction carrying the 14C-labelled substrate after cleavage of the ester intermediate with cyanogen bromide. Sequence analysis revealed that one of the two peptides within this sample harboured Asp226. Surprisingly, the replacement of Glu404 with Asp greatly increased the Vmax of the enzyme with styrene 7,8-oxide (23-fold) and 9, 10-epoxystearic acid (39-fold). The increase in Vmax was paralleled by an increase in Km with both substrates, in line with a selective enhancement of the second, rate-limiting step of the enzymic reaction. Owing to its enhanced catalytic properties, the Glu404-->Asp mutant might represent a versatile tool for the enantioselective bio-organic synthesis of chiral fine chemicals. The question of why all native mEHs analysed so far have a Glu in place of the acidic charge relay residue is discussed.

Amino Acid Sequence↗
PubMed1999

Administration of intravenous immunoglobulin (IVIG) in vivo--down-regulatory effects on the IL-1 system.

Modulation of the cytokine network may be of importance for the beneficial effects of therapy with IVIG seen in a wide range of immune-mediated disorders. In the present study we investigate the effect of IVIG administration in vivo on the IL-1 system in 12 patients with primary hypogammaglobulinaemia. Before IVIG infusion these patients had significantly elevated levels of IL-1alpha and IL-1beta both in plasma and in supernatants from peripheral blood mononuclear cells (PBMC) compared with healthy controls. After one bolus infusion with IVIG (0.4 g/kg) we found a significant change in the profile of the components of the IL-1 system: a marked increase in levels of IL-1 receptor antagonist (IL-1Ra) and neutralizing antibodies against IL-1alpha, a moderate decrease in levels of IL-1alpha, IL-1beta and soluble (s) IL-1 receptor type I and a significant increase in sIL-1 receptor type II levels. These changes were found both in plasma and in PBMC isolated after IVIG administration. Furthermore, pooled serum obtained after IVIG infusion suppressed lipopolysaccharide- and staphylococcal enterotoxin B-stimulated, but not phorbol myristate acetate-stimulated, release of IL-1alpha and IL-1beta from PBMC isolated from healthy controls. Finally, these changes in circulating levels of various IL-1 modulators after IVIG infusion appeared to cause a significantly impaired ability of IL-1 to stimulate PBMC for tumour necrosis factor-alpha release. Our findings suggest that IVIG administration may not only down-regulate the activity in the IL-1 system, but also hamper IL-1 stimulation of PBMC.

Adult↗
PubMed1999

Tumor necrosis factor (TNF) system levels in human immunodeficiency virus-infected patients during highly active antiretroviral therapy: persistent TNF activation is associated with virologic and immunologic treatment failure.

Because persistent tumor necrosis factor (TNF)-alpha activation may play a pathogenic role in human immunodeficiency virus infection, TNF component levels were assessed over 78 weeks in plasma and peripheral blood mononuclear cells (PBMC) during highly active antiretroviral therapy (HAART) in 40 HIV-infected patients. HAART induced a significant decline in plasma levels of TNF-alpha and soluble TNF receptors and was associated with a fall in the abnormally increased unstimulated and a rise in the abnormally low Mycobacterium avium complex-purified-protein derivative-stimulated TNF-alpha released from PBMC. However, concentrations of these TNF components were not normalized. Patients with virologic and immunologic treatment failure after 52 weeks had higher levels of several TNF components than other patients early after initiation of therapy, also during periods with adequate virologic response. Although TNF components significantly decreased during HAART, these results support data indicating that full immunologic normalization is not achieved during such therapy. The persistent activation of the TNF system in a subgroup of persons may be involved in treatment failure.

Adolescent↗
PubMed1999

Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern, and engineering of proteolytic resistance.

Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli. All of the phytases examined are monomeric proteins. While E. coli phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in A. niger, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of A. fumigatus and E. nidulans phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract.

6-Phytase↗
PubMed1999

Decreased bone formative and enhanced resorptive markers in human immunodeficiency virus infection: indication of normalization of the bone-remodeling process during highly active antiretroviral therapy.

As cytokines and 1,25-dihydroxyvitamin D [1,25-(OH)2D] appear to have an important role in bone homeostasis, we examined the possibility that human immunodeficiency virus (HIV)-infected patients, characterized by enhanced levels of proinflammatory cytokines and 1,25-(OH)2D deficiency, have disturbed bone metabolism by analyzing serum markers of bone formation (osteocalcin) and bone resorption (C-telopeptide) in 73 HIV-infected patients. HIV-infected patients with advanced clinical and immunological disease and high viral load were characterized by increased C-telopeptide and particularly by markedly depressed osteocalcin levels. HIV-infected patients had enhanced activation of the TNF system. Serum concentrations of p55 and p75-TNF receptors were negatively correlated with osteocalcin, and p75-TNF receptor was positively correlated with C-telopeptide. HIV-infected patients with advanced disease also had decreased serum concentrations of 1,25-(OH)2D, but this parameter was not correlated with osteocalcin or C-telopeptide. During 24 months with highly active antiretroviral therapy there was a marked rise in serum osteolcalcin levels together with a profound fall in viral load and TNF components and a marked rise in CD4+ T cell counts. Also, there was a shift from no correlation to a significant correlation between osteocalcin and C-telopeptide levels during such therapy. The present study suggests disturbed bone formation and resorption during HIV infection. Our findings indicating synchronization of bone remodeling during highly active antiretroviral therapy may represent a previously unrecognized beneficial effect of such therapy and expand our knowledge of the interactions between cytokines and bone in the bone-remodeling process.

Adult↗
PubMed1999

Anterior organization of the Caenorhabditis elegans embryo by the labial-like Hox gene ceh-13.

The Caenorhabditis elegans lin-39, mab-5 and egl-5 Hox genes specify cell fates along the anterior-posterior body axis of the nematode during postembryonic development, but little is known about Hox gene functions during embryogenesis. Here, we show that the C. elegans labial-like gene ceh-13 is expressed in cells of many different tissues and lineages and that the rostral boundary of its expression domain is anterior to those of the other Hox genes. By transposon-mediated mutagenesis, we isolated a zygotic recessive ceh-13 loss-of-function allele, sw1, that exhibits an embryonic sublethal phenotype. Lineage analyses and immunostainings revealed defects in the organization of the anterior lateral epidermis and anterior body wall muscle cells. The epidermal and mesodermal identity of these cells, however, is correctly specified. ceh-13(sw1) mutant embryos also show fusion and adhesion defects in ectodermal cells. This suggests that ceh-13 plays a role in the anterior organization of the C. elegans embryo and is involved in the regulation of cell affinities.

Amino Acid Sequence↗
PubMed1998

Possible role of interleukin-10 (IL-10) and CD40 ligand expression in the pathogenesis of hypergammaglobulinemia in human immunodeficiency virus infection: modulation of IL-10 and Ig production after intravenous Ig infusion.

The mechanisms leading to polyclonal hypergammaglobulinemia in patients with human immunodeficiency virus (HIV) infection are not well understood. In light of the important role of interleukin-10 (IL-10) and the interaction between CD40 and CD40 ligand in the normal regulation of B-lymphocyte function and Ig production, we examined these parameters in 24 HIV-infected patients. Both plasma IL-10 levels and the percentage of CD4(+) and CD8(+) lymphocytes expressing CD40 ligand were significantly higher in the patients than in the 10 blood donor controls. Serum IgG correlated positively with circulating IL-10 levels and the percentage of CD4(+) lymphocytes expressing CD40 ligand. Furthermore, a single bolus infusion of intravenous Ig (0.4 g/kg) in 8 HIV-infected patients caused a further increase in IL-10 levels in plasma and an increase in both IL-10 and IgG production in peripheral blood mononuclear cell cultures. In another patient group (Wegener's granulomatosis) receiving a single bolus infusion of intravenous Ig, a similar increase in plasma IL-10 levels was found, suggesting that this may be a general effect of intravenous Ig. In patients with HIV infection, our data suggest that a vicious cycle may be operative where high endogenous Ig levels may enhance IL-10 production that, in turn, leads to higher Ig production.

Adult↗
PubMed1998

Elevated levels of serum-soluble CD14 in human immunodeficiency virus type 1 (HIV-1) infection: correlation to disease progression and clinical events.

Soluble (s) CD14, a marker for monocyte/macrophage activation and a mediator of bacterial lipopolysaccharide (LPS) action, was elevated in serum from human immunodeficiency virus type 1 (HIV- 1)-infected individuals (n = 92) compared with seronegative controls. The highest levels were found in patients with advanced clinical and immunological disease. Patients with ongoing clinical events had significantly higher sCD14 levels than symptomatic HIV-1-infected individuals without clinical events, with especially elevated levels in patients infected with Mycobacterium avium complex (MAC). On longitudinal testing of patients (n = 26) with less than 100 x 10(6) CD4 lymphocytes/L at baseline, we found that increasing sCD14 serum concentrations per time unit were associated with death, whereas no differences in CD4 cell number decrease were found between survivors and nonsurvivors. In vitro studies showed that HIV-1 glycoprotein 120 and purified protein derivative (PPD) from M avium (MAC-PPD) stimulated normal monocytes to release sCD14. Furthermore, MAC-PPD induced tumor necrosis factor (TNF) release from monocytes through interactions with CD14 and, importantly, the addition of sCD14 enhanced this MAC-PPD stimulatory effect. Our findings suggest that the CD14 molecule may be involved in the immunopathogenesis of HIV-1 infection, and it is conceivable that serial determination of sCD14 may give useful predictive information concerning disease progression and survival in HIV-1-infected patients.

Adolescent↗
PubMed1998

31P-NMR spectroscopy of human and Paracoccus denitrificans electron transfer flavoproteins, and 13C- and 15N-NMR spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states.

Human and Paracoccus denitrificans wild-type electron transfer flavoproteins have been investigated by 31P-NMR in the oxidised and reduced states. The 31P chemical shifts of the diphosphate moiety of the protein-bound FAD were similar in the proteins and were independent of the redox state. The chemical shifts were remarkably similar to those of ferredoxin-NADP+ reductase and, to a lesser degree, with those of NADPH-cytochrome P-450 reductase. The wild-type human electron transfer apoprotein was reconstituted with [2,4a-13C2]FAD, [4,10a-13C2]FAD, or [U-15N4]FAD. The reconstituted proteins were studied by 13C- and 15N-NMR techniques in the oxidised and reduced states. The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of flavoproteins published in the literature. In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD. The N(1) atom is also hydrogen bonded and, as shown by X-ray data, involves the C'(4)-OH group of FAD. The sp2 hybridisation of N(10) is small compared to other flavoproteins. In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD. The N(1) atom is ionised as observed also in other flavoproteins when investigated by NMR. The intramolecular hydrogen bond between the C'(4)-OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein-bound FAD in both redox states. The N(10) atom in the reduced protein is highly sp3 hybridised in comparison to those of other flavoproteins.

Apoproteins↗
PubMed1998

Calmodulin controls the rod photoreceptor CNG channel through an unconventional binding site in the N-terminus of the beta-subunit.

Calmodulin (CaM) controls the activity of the rod cGMP-gated ion channel by decreasing the apparent cGMP affinity. We have examined the mechanism of this modulation using electrophysiological and biochemical techniques. Heteromeric channels, consisting of alpha- and beta-subunits, display a high CaM sensitivity (EC50 </=5 nM) similar to the native channel. Using surface plasmon resonance spectroscopy, we identified two unconventional CaM-binding sites (CaM1 and CaM2), one in each of the N- and the C-terminal regions of the beta-subunit. Ca2+ co-operatively stimulates binding of CaM to these sites exactly within the range of [Ca2+] occurring during a light response. Deletion of the N-terminal CaM1 site results in channels that are no longer CaM-sensitive, whereas deletion of CaM2 has only minor effects. We discuss different models to explain the high-affinity binding of CaM.

Amino Acid Sequence↗
PubMed1998

Elevated circulating levels of C-C chemokines in patients with congestive heart failure.

BACKGROUND: Immunologic and inflammatory responses appear to play a pathogenic role in the development of congestive heart failure (CHF). Activation and migration of leukocytes to areas of inflammation are important factors in these immunologic responses. Because the C-C chemokines are potent chemoattractants of monocytes and lymphocytes and can modulate other functions of these cells (eg, generation of reactive oxygen species), we measured circulating levels of three C-C chemokines in CHF. METHODS AND RESULTS: Levels of macrophage chemoattractant protein-1 (MCP-1), macrophage inflammatory protein- 1alpha (MIP-1alpha), and RANTES (regulated on activation normally T-cell expressed and secreted) were measured by enzyme immunoassays in 44 patients with CHF and 21 healthy control subjects. CHF patients had significantly elevated levels of all chemokines with the highest levels in New York Heart Association class IV, and MCP-1 and MIP-1alpha levels were significantly inversely correlated with left ventricular ejection fraction. Elevated C-C chemokine levels were found independent of the cause of the heart failure, but MCP-1 levels were particularly raised in patients with coronary artery disease. Studies on cells isolated from peripheral blood suggested that platelets, CD3+ lymphocytes, and in particular, monocytes, might contribute to the elevated C-C chemokine levels in CHF. The increased MCP-1 levels in CHF were correlated with increased monocyte activity reflected in an enhancing effect of serum from CHF patients on O2-generation in monocytes, which was inhibited by neutralizing antibodies against MCP-1. CONCLUSIONS: This first demonstration of increased circulating levels of C-C chemokines in CHF with particularly high levels in patients with severe disease may represent previously unrecognized pathogenic factors in CHF.

Adult↗
PubMed1998

Levels of circulating adhesion molecules in congestive heart failure and after heart transplantation.

Recent reports suggest a role for immunologic and inflammatory processes in the pathogenesis of congestive heart failure (CHF) and accelerated coronary artery disease (CAD) after heart transplantation (HT). The interaction between endothelial cells, leukocytes, and platelets involving various adhesion molecules may be of particular importance. We therefore measured serum levels of soluble(s) vascular cell adhesion molecule-1 (VCAM-1), sP-selectin, and sE-selectin in 34 patients with severe CHF (23 with CAD and 11 with idiopathic dilated cardiomyopathy) and in 20 healthy controls. Twenty of the patients were followed with serial measurements of these circulating adhesion molecules (CAMs) for up to 2 years after HT. Levels of all 3 CAMs were significantly elevated in patients with CHF compared with controls irrespective of the etiology of heart failure, with particularly high concentrations of sVCAM-1. After HT, different patterns in CAMs were found over time. Whereas there was a normalization of sE-selectin levels after HT, concentrations of sVCAM-1 also declined, but without normalization. In contrast, sP-selectin levels were persistently elevated, with the highest concentrations at the end of the study period. The persistent elevation of sP-selectin and the lack of normalization of sVCAM-1 levels were associated with persistently raised serum levels of tumor necrosis factor-alpha, and these findings were not related to either acute episodes of allograft rejection or intercurrent infections. These results support the notion that immunologic and inflammatory processes are important features of CHF. Furthermore, the persistently elevated levels of CAMs and tumor necrosis factor-alpha found up to 2 years after HT may reflect a state of persistent immune activation in these patients, possibly involved in the development of CAD after HT.

Adolescent↗
PubMed1998

Protein phosphatase type-2C isozymes present in vertebrate retinae: purification, characterization, and localization in photoreceptors.

Posttranslational modification of proteins by kinases and phosphatases plays an important role in the regulation of cellular signaling in general and neurochemistry in particular. This also applies to vertebrate photoreceptors where phosphorylation of rhodopsin causes uncoupling from the signal transduction cascade. Functional activity of rhodopsin is restored after substitution of the bleached photopigment 11-cisretinal and by dephosphorylation of the opsin moiety. Phosphatases type-1 and type-2A have been identified in vertebrate retinae. Recently, we have shown by molecular cloning that two isozymes of protein phosphatase type-2C (PP2C, PPM) do exist in retinal tissue. In this report, we have purified PP2Calpha and PP2Cbeta from bovine retinae. Thirty to 40% of PP2C was recovered in the cytosolic fraction. Biochemical properties of native and heterologously expressed recombinant enzymes were similar. Enzymatic activity is strictly dependent on the presence of Mg2+. Addition of Ca2+ ions inhibits Mg2+-sustained activity. Antiserum raised against a C-terminal peptide of PP2Cbeta specifically labeled the outer segments of rod photoreceptor cells. PP2C protein levels were significantly reduced in RCS rats, which develop age-dependent photoreceptor degeneration comparable to the hereditary disease retinitis pigmentosa. Although the retinal substrate(s) remain to be identified, the results suggest that PP2C modulates cellular components of the phototransduction machinery.

Amino Acid Sequence↗
PubMed1998

Phosphorylation of mammalian olfactory cyclic nucleotide-gated channels increases ligand sensitivity.

In vertebrate olfactory sensory neurons, odorant receptors couple the sensory signal to the synthesis of the second messenger cAMP. Cyclic nucleotide-gated (CNG) channels are activated by binding of cAMP and conduct a depolarizing receptor current that leads to electrical excitation of the neuron. The sensitivity of olfactory CNG channels for cAMP can be significantly reduced by binding of calmodulin to a regulatory domain that resides within the N-terminus of the alpha-subunit of the channel. This regulatory domain also contains a consensus phosphorylation sequence for protein kinase C (PKC). We have investigated the effect of channel phosphorylation by PKC and found that phosphorylation increases ligand sensitivity without counteracting modulation of the channel by calmodulin. We have identified the amino acid residue that is phosphorylated by PKC and have localized three isoforms of PKC in olfactory sensory cilia. The results of this study provide information about the control of ligand sensitivity in olfactory CNG channels by an intrinsic regulatory domain, representing both a calmodulin-binding site and a substrate for PKC.

8-Bromo Cyclic Adenosine Monophosphate↗