Biomedical subjects

R Seifert

Publications and source records attributed to R Seifert.

At least 19 recordsLinked to original sources

PubMed2001

Hyperpolarization-activated channels HCN1 and HCN4 mediate responses to sour stimuli.

Sour taste is initiated by protons acting at receptor proteins or channels. In vertebrates, transduction of this taste quality involves several parallel pathways. Here we examine the effects of sour stimuli on taste cells in slices of vallate papilla from rat. From a subset of cells, we identified a hyperpolarization-activated current that was enhanced by sour stimulation at the taste pore. This current resembled Ih found in neurons and cardio-myocytes, a current carried by members of the family of hyperpolarization-activated and cyclic-nucleotide-gated (HCN) channels. We show by in situ hybridization and immunohistochemistry that HCN1 and HCN4 are expressed in a subset of taste cells. By contrast, gustducin, the G-protein involved in bitter and sweet taste, is not expressed in these cells. Lowering extracellular pH causes a dose-dependent flattening of the activation curve of HCN channels and a shift in the voltage of half-maximal activation to more positive voltages. Our results indicate that HCN channels are gated by extracellular protons and may act as receptors for sour taste.

Animals↗

PubMed2001

Defective Gi protein coupling in two formyl peptide receptor mutants associated with localized juvenile periodontitis.

The formyl peptide receptor (FPR) is a prototypical chemoattractant receptor expressed in neutrophils. It is well known that the FPR couples to G(i) proteins to activate phospholipase C, chemotaxis, and cytotoxic cell functions, but the in vivo role of the FPR in man has remained elusive. Recently, F110S and C126W mutations of the FPR have been associated with localized juvenile periodontitis. We studied FPR-F110S and FPR-C126W in comparison with wild-type FPR (FPR-WT) by coexpressing epitope-tagged versions of these receptors with the G protein Galpha(i2)beta(1)gamma(2) in Sf9 insect cells. FPRs were efficiently expressed in Sf9 membranes as assessed by immunoblotting using the beta(2)-adrenoreceptor as a standard. FPR-C126W differed from FPR-WT and FPR-F110S in migration on SDS-polyacrylamide gels and tunicamycin-sensitive glycosylation. FPR-WT efficiently reconstituted high-affinity agonist binding and agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to Galpha(i2)beta(1)gamma(2). In contrast, FPR-F110S only weakly reconstituted agonist-stimulated GTPgammaS binding, and FPR-C126W was completely inefficient. Collectively, our data show almost complete and complete loss of G(i) protein coupling in FPR-F110S and FPR-C126W, respectively. The severe functional defects in FPR-F110S and FPR-C126W contrast with the discrete clinical symptoms associated with these mutations, indicating that loss of FPR function in host defense is, for the most part, readily compensated.

Aggressive Periodontitis↗

PubMed2001

The olfactory G protein G(alphaolf) possesses a lower GDP-affinity and deactivates more rapidly than G(salphashort): consequences for receptor-coupling and adenylyl cyclase activation.

The olfactory G protein G(alphaolf) differs from the short splice variant of G(salpha) (G(salphaS)) in 80 amino acids, but little is known about biochemical differences between G(alphaolf) and G(salphaS). We addressed this question by analyzing fusion proteins of the beta2-adrenoceptor (beta2AR) and G(alphaolf) and G(salphaS), respectively, using Sf9 insect cells as expression system. The fusion ensured defined receptor/G protein stoichiometry and efficient coupling. High-affinity agonist binding studies showed that G(alphaolf) possesses a lower GDP-affinity than G(salphaS) As a result, the agonist-free beta2AR and the beta2AR occupied by partial agonists were more efficient at promoting GDP-dissociation from G(alphaolf) than from G(salphaS) a assessed by guanosine 5'-O-(3-thiotriphosphate) binding, adenylyl cyclase (AC) activity and GTP hydrolysis. Basal AC activity in the absence of GTP was almost sixfold lower in membranes expressing beta2AR-G(alphaolf) than in membranes expressing beta2AR-G(salphaS) at similar levels, reflecting the lower abundance of G(alphaolf-GDP) relative to G(salphaS-GDP). The maximum agonist-stimulated AC activity with beta2AR-G(salphaS) was more than twofold higher than with beta2AR-G(alphaolf), but the relative agonist-stimulation of AC with beta2AR-G(alphaolf) was much greater than with beta2AR-G(salphaS). The difference in maximum AC activity can be explained by more rapid deactivation of G(alphaolf-GTP) by GTP hydrolysis and GTP dissociation relative to G(salphaS-GTP). Taken together, there are biochemical differences between G(alphaolf) and G(salphaS), supporting different roles of these G proteins in vivo.

Adenylyl Cyclases↗

PubMed2001

Distinct interaction of human and guinea pig histamine H2-receptor with guanidine-type agonists.

It is unknown why the potencies and efficacies of long-chained guanidine-type histamine H2-receptor (H2R) agonists are lower at the H2R of human neutrophils than at the H2R of the guinea pig atrium. To elucidate these differences, we analyzed fusion proteins of the human H2R (hH2R) and guinea pig H2R (gpH2R), respectively, and the short splice variant of Gsalpha (GsalphaS) expressed in Sf9 cells. The potencies and efficacies of small H2R agonists in the GTPase assay and the potencies of antagonists at inhibiting histamine-stimulated GTP hydrolysis by hH2R-GsalphaS and gpH2R-GsalphaS were similar. In contrast, the potencies and efficacies of guanidines were lower at hH2R-GsalphaS than at gpH2R-G(salphaS). Guanidines bound to hH2R-GsalphaS with lower affinity than to gpH2R-GsalphaS, and high-affinity binding of guanidines at gpH2R-GsalphaS was more resistant to disruption by GTPgammaS than binding at hH2R-GsalphaS. Molecular modeling suggested that the nonconserved Asp-271 in transmembrane domain 7 of gpH2R (Ala-271 in hH2R) confers high potency to guanidines. This hypothesis was confirmed by Ala-271-->Asp-271 mutation in hH2R-GsalphaS. Intriguingly, the efficacies of guanidines at the Ala-271-->Asp-271 mutant and at hH2R/gpH2R chimeras were lower than at gpH2R. Our model suggests that a Tyr-17/Asp-271 H-bond, present only in gpH2R-GsalphaS but not the other constructs studied, stabilizes the active guanidine-H2R state. Collectively, our data show 1) distinct interaction of H2R species isoforms with guanidines, 2) that a single amino acid in transmembrane domain 7 critically determines guanidine potency, and 3) that an interaction between transmembrane domains 1 and 7 is important for guanidine efficacy.

Alternative Splicing↗

PubMed2001

Molecular diversity of pacemaker ion channels.

Ionic currents activated by hyperpolarization and regulated by cyclic nucleotides were first discovered more than 20 years ago. Recently the molecular identity of the underlying channels has been unveiled. The structural features of the protein sequences are discussed and related to the mechanisms of activation, selectivity for cyclic nucleotides, and ion permeation. Coverage includes a comparison of the biophysical properties of recombinant and native channels and their significance for the physiological functions of these channels.

Animals↗

PubMed2001

Functional differences between full and partial agonists: evidence for ligand-specific receptor conformations.

The interaction of an agonist-bound G-protein-coupled receptor (GPCR) with its cognate G-protein initiates a sequence of experimentally quantifiable changes in both the GPCR and G-protein. These include the release of GDP from G(alpha), the formation of a ternary complex between the nucleotide-free G-protein and the GPCR, which has a high affinity for agonist, followed by the binding of GTP to G(alpha), the dissociation of the GPCR/G-protein complex, and the hydrolysis of GTP. The efficacy of an agonist is a measure of its ability to activate this cascade. It has been proposed that efficacy reflects the ability of the agonist to stabilize the active state of the GPCR. We examined a series of beta(2)-adrenoceptor (beta(2)AR) agonists (weak partial agonists to full agonists) for their efficacy at promoting two different steps of the G-protein activation/deactivation cycle: stabilizing the ternary complex (high-affinity, GTP-sensitive agonist binding), and steady-state GTPase activity. We obtained results for the wild-type beta(2)AR and a constitutively active mutant of the beta(2)AR (beta(2)AR(CAM)) using fusion proteins between the GPCRs and G(salpha) to facilitate GPCR/G-protein interactions. There was no correlation between efficacy of ligands in activating GTPase and their ability to stabilize the ternary complex at beta(2)AR(CAM). Our results suggest that the GPCR state that optimally promotes the GDP release and GTP binding is different from the GPCR state that stabilizes the ternary complex. By strongly stabilizing the ternary complex, certain partial agonists may reduce the rate of G-protein turnover relative to a full agonist.

Adrenergic beta-2 Receptor Agonists↗

PubMed2001

Monovalent anions differentially modulate coupling of the beta2-adrenoceptor to G(s)alpha splice variants.

The beta2-adrenoceptor (beta2AR) fused to the long splice variant of G(s)alpha (G(s)alphaL), but not the beta2AR fused to the short splice variant of G(s)alpha (G(s)alphaS) shows the hallmarks of high constitutive activity, i.e., strong activation of adenylyl cyclase (AC) by GTP and strong inhibition of AC by inverse agonist. These coupling differences are the result of differences in GDP affinity of G(s)alpha splice variants. The aim of this study was to identify experimental variables that differentially affect beta2AR coupling to G(s)alphaS and G(s)alphaL. NaCl substantially reduced agonist-independent AC activation by GTP and inverse agonist inhibition and enhanced agonist stimulation of AC in Sf9 insect cell membranes expressing the beta2AR-G(s)alphaL fusion protein. Salts reduced inverse agonist inhibition and increased agonist stimulation of AC in the order of efficiency NaI approximately KI > NaBr approximately KBr > NaCl approximately LiCl approximately KCl approximately RbCl approximately CsCl approximately choline chloride, indicating that monovalent anions determine salt effects. Salts inhibited guanosine 5'-O-(3-thiotriphosphate)-mediated AC activation by G(salphaL) without beta2AR in the order of efficiency NaI > NaBr > NaCl. NaCl enhanced the affinity of G(s)alphaL for GDP. Salts had much smaller effects on beta2AR ligand regulation of AC in membranes expressing beta2AR-G(s)alphaS than in membranes expressing beta2AR-G(s)alphaL. These data are explained by a model in which anions increase the GDP affinity of G(s)alphaL more efficiently than the GDP affinity of G(s)alphaS, and, thereby, decrease the efficiency of the agonist-free beta2AR and increase the efficiency of the agonist-occupied beta2AR at promoting GDP dissociation from G(s)alphaL. Thus, monovalent anions differentially regulate beta2AR-coupling to G(s)alphaS and G(s)alphaL.

Adenylyl Cyclases↗

PubMed2001

Unmasking different constitutive activity of four chemoattractant receptors using Na+ as universal stabilizer of the inactive (R) state.

Neutrophils express receptors for the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) complement C5a, leukotriene B4 (LTB4) and platelet-activating factor (PAF). The aim of this study was to analyze the constitutive activity of chemoattractant receptors by studying binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to the G-protein Gi alpha 2 beta 1 gamma 2 expressed in Sf9 cells. We used Na+ as modulator of constitutive activity because there are no known inverse agonists for the C5a receptor (C5aR), LTB4 receptor (BLTR) and PAF receptor (PAFR). In the absence of NaCl, PAF and LTB4 exhibited larger relative stimulatory effects on GTP gamma S binding than fMLP and C5a. NaCl showed larger inhibitory effects on basal GTP gamma S binding in membranes expressing the formyl peptide receptor (FPR) and C5aR than in membranes expressing BLTR and PAFR. The order of potency of NaCl at inhibiting basal GTP gamma S binding was FPR > C5aR approximately BLTR > PAFR. As a result of the inhibitory effect of NaCl on basal GTP gamma S binding, the relative stimulatory effects of agonists were increased. By quantitatively analyzing the expression levels of chemoattractant receptors and Gi alpha 2 and the stoichiometry of receptor/G-protein coupling we obtained no evidence for structural instability of constitutively active receptors and catalytical G-protein activation. Taken together, the FPR and C5aR exhibit higher constitutive activity than the BLTR and PAFR. Na+ acts as a universal stabilizer of the inactive (R) state in chemoattractant receptors. The different potencies of NaCl at suppressing basal G-protein activity with different receptors indicate that chemoattractant receptors differ from each other in their Na(+)-affinity.

Antigens, CD↗

PubMed2001

Similar apparent constitutive activity of human histamine H(2)-receptor fused to long and short splice variants of G(salpha).

Fusion proteins allow for the analysis of receptor/G protein coupling under defined conditions. The beta(2)-adrenoceptor (beta(2)AR) fused to the long splice variant of G(salpha) (G(salphaL)) exhibits a higher apparent constitutive activity than the beta(2)-adrenoceptor fused to the short splice variant of G(salpha) (G(salphaS)). Experimentally, this results in higher efficacy and potency of partial agonists and in higher efficacy of inverse agonists at the beta(2)AR fused to G(salphaL) relative to the beta(2)AR fused to G(salphaS), indicating that the agonist-free beta(2)AR and the beta(2)AR occupied by partial agonists promote GDP dissociation from G(salphaL) more efficiently than from G(salphaS). In fact, the GDP affinity of G(salphaS) fused to the beta(2)AR is higher than the GDP affinity of G(salphaL) fused to the beta(2)AR. We asked the question whether the histamine H(2)-receptor (H(2)R) exhibits similar coupling to G(salpha) splice variants as the beta(2)AR. To address this question, we studied H(2)R-G(salpha) fusion proteins expressed in Sf9 cells. In contrast to beta(2)AR-G(salpha) fusion proteins, the potencies and efficacies of partial agonists and the efficacies of inverse agonists were similar at the H(2)R fused to G(salphaL) and G(salphaS) as assessed by guanosine-5'-O-(3-thio)triphosphate binding and/or steady-state GTPase activity. However, the time course analysis of guanosine-5'-O-(3-thio)triphosphate binding indicated that G(salphaS) fused to the H(2)R possesses a higher GDP-affinity than G(salphaL) fused to the H(2)R. Our data show that the H(2)R fused to G(salphaL) and G(salphaS) possesses similar constitutive activity and is insensitive to differences in GDP affinity of G(salpha) splice variants. Thus, GDP affinity of G proteins does not generally determine constitutive activity of receptors.

Alternative Splicing↗

PubMed2000

The effect of pH on beta(2) adrenoceptor function. Evidence for protonation-dependent activation.

The transition of rhodopsin from the inactive to the active state is associated with proton uptake at Glu(134) (1), and recent mutagenesis studies suggest that protonation of the homologous amino acid in the alpha(1B) adrenergic receptor (Asp(142)) may be involved in its mechanism of activation (2). To further explore the role of protonation in G protein-coupled receptor activation, we examined the effects of pH on the rate of ligand-induced conformational change and on receptor-mediated G protein activation for the beta(2) adrenergic receptor (beta(2)AR). The rate of agonist-induced change in the fluorescence of NBD-labeled, purified beta(2)AR was 2-fold greater at pH 6.5 than at pH 8, even though agonist affinity was lower at pH 6.5. This biophysical analysis was corroborated by functional studies; basal (agonist-independent) activation of Galpha(s) by the beta(2)AR was greater at pH 6.5 compared with pH 8.0. Taken together, these results provide evidence that protonation increases basal activity by destabilizing the inactive state of the receptor. In addition, we found that the pH sensitivity of beta(2)AR activation is not abrogated by mutation of Asp(130), which is homologous to the highly conserved acidic amino acids that link protonation to activation of rhodopsin (Glu(134)) and the alpha(1B) adrenergic receptor (Asp(142)).

Animals↗

PubMed2000

Pacemaker oscillations in heart and brain: a key role for hyperpolarization-activated cation channels.

Rhythmic activity of single cells or multicellular networks is a common feature of all organisms. The oscillatory activity is characterized by time intervals of several seconds up to many hours. Cellular rhythms govern the beating of the heart, the swimming behavior of sperm, cycles of sleep and wakefulness, breathing, and the release of hormones. Many neurons in the brain and cardiac cells are characterized by endogenous rhythmic activity, which relies on a complex interplay between several distinct ion channels. In particular, one type of ion channel plays a prominent role in the control of rhythmic electrical activity since it determines the frequency of the oscillations. The activity of the channels is thus setting the "pace" of the oscillations; therefore, these channels are often referred to as "pacemaker" channels. Despite their obvious important physiological function, it was not until recently that genes encoding pacemaker channels have been identified. Because both hyperpolarization and cyclic nucleotides are key elements that control their activity, pacemaker channels have now been designated hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels. The molecular identification of the channels and the upcoming studies on their properties in heterologous systems will certainly enhance our understanding of "pacemaking" in physiological systems. This review gives a brief insight into the physiological importance of these channels and sums up what we have learned since the first cloning of genes succeeded (for recent reviews, see also Clapham 1998; Luthi and McCormick 1998a; Biel et al. 1999; Ludwig, Zong, Hofmann, et al. 1999; Santoro and Tibbs 1999).

Amino Acid Sequence↗

PubMed2000

Molecular analysis of beta(2)-adrenoceptor coupling to G(s)-, G(i)-, and G(q)-proteins.

The beta(2)-adrenoceptor (beta(2)AR) couples to the G-protein G(s) to activate adenylyl cyclase. Intriguingly, several studies have demonstrated that the beta(2)AR can also interact with G-proteins of the G(i)- and G(q)-family. To assess the efficiency of beta(2)AR interaction with various G-protein alpha-subunits (G(xalpha)), we expressed fusion proteins of the beta(2)AR with the long (G(salphaL)) and short (G(salphaS)) splice variants of G(salpha), the G(i)-proteins G(ialpha2) and G(ialpha3), and the G(q)-proteins G(qalpha) and G(16alpha) in Sf9 cells. Fusion proteins provide a rigorous approach for comparing the coupling of a given receptor to G(xalpha) because of the defined 1:1 stoichiometry of receptor and G-protein and the efficient coupling. Here, we show that the beta(2)AR couples to G(s)-, G(i)-, and G(q)-proteins as assessed by ternary complex formation and ligand-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding. The combined analysis of ternary complex formation, GTPgammaS binding, agonist efficacies, and agonist potencies revealed substantial differences in the interaction of the beta(2)AR with the various classes of G-proteins. Comparison of the coupling of the beta(2)AR and formyl peptide receptor to G(ialpha2) revealed receptor-specific differences in the kinetics of GTPgammaS binding. We also detected highly efficient stimulation of GTPgammaS dissociation from G(salphaL), but not from G(qalpha) and G(16alpha), by a beta(2)AR agonist. Moreover, we show that the 1:1 stoichiometry of receptor to G-protein in fusion proteins reflects the in vivo stoichiometry of receptor/G-protein coupling more closely than was previously assumed. Collectively, our data show 1) that the beta(2)AR couples differentially to G(s)-, G(i)-, and G(q)-proteins, 2) that there is ligand-specific coupling of the beta(2)AR to G-proteins, 3) that receptor-specific G-protein conformational states may exist, and 4) that nucleotide dissociation is an important mechanism for G-protein deactivation.

Adrenergic Agonists↗

PubMed1999

Quantitative analysis of formyl peptide receptor coupling to g(i)alpha(1), g(i)alpha(2), and g(i)alpha(3).

The human formyl peptide receptor (FPR) is a prototypical G(i) protein-coupled receptor, but little is known about quantitative aspects of FPR-G(i) protein coupling. To address this issue, we fused the FPR to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to Galpha ensures a defined 1:1 stoichiometry of the signaling partners. By analyzing high affinity agonist binding, the kinetics of agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding and GTP hydrolysis and photolabeling of Galpha, we demonstrate highly efficient coupling of the FPR to fused G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three G(i)alpha isoforms. The K(d) values of high affinity agonist binding were approximately 100-fold lower than the EC(50) (concentration that gives half-maximal stimulation) values of agonist for GTPase activation. Based on the B(max) values of agonist saturation binding and ligand-regulated GTPgammaS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several G(i) proteins. Analysis of agonist saturation binding, ligand-regulated GTPgammaS saturation binding and quantitative immunoblotting with membranes expressing FPR-G(i)alpha fusion proteins and nonfused FPR now reveals that FPR agonist binding greatly underestimates the actual FPR expression level. Our data show the following: (i) the FPR couples to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) with similar efficiency; (ii) the FPR can exist in a state of low agonist affinity that couples efficiently to G proteins; and (iii) in contrast to the previously held view, the FPR appears to activate G(i) proteins linearly and not catalytically.

Animals↗

PubMed1999

Activation of the beta(2)-adrenergic receptor-Galpha(s) complex leads to rapid depalmitoylation and inhibition of repalmitoylation of both the receptor and Galpha(s).

Palmitoylation is unique among lipid modifications in that it is reversible. In recent years, dynamic palmitoylation of G protein alpha subunits and of their cognate receptors has attracted considerable attention. However, very little is known concerning the acylation/deacylation cycle of the proteins in relation to their activity status. In particular, the relative contribution of the activation and desensitization of the signaling unit to the regulation of the receptors and G proteins palmitoylation state is unknown. To address this issue, we took advantage of the fact that a fusion protein composed of the stimulatory alpha subunit of trimeric G protein (Galpha(s)) covalently attached to the beta(2)-adrenergic receptor (beta(2)AR) as a carboxyl-terminal extension (beta(2)AR-Galpha(s)) can be stimulated by agonists but does not undergo rapid inactivation, desensitization, or internalization. When expressed in Sf9 cells, both the receptor and the Galpha(s) moieties of the fusion protein were found to be palmitoylated via thioester linkage. Stimulation with the beta-adrenergic agonist isoproterenol led to a rapid depalmitoylation of both the beta(2)AR and Galpha(s) and inhibited repalmitoylation. The extent of depalmitoylation induced by a series of agonists was correlated (0.99) with their intrinsic efficacy to stimulate the adenylyl cyclase activity. However, forskolin-stimulated cAMP production did not affect the palmitoylation state of beta(2)AR-Galpha(s), indicating that the agonist-promoted depalmitoylation is linked to conformational changes and not to second messenger generation. Given that, upon activation, the fusion protein mimics the activated receptor-G protein complex but cannot undergo desensitization, the data demonstrate that early steps in the activation process lead to the depalmitoylation of both receptor and G protein and that repalmitoylation requires later events that cannot be accommodated by the activated fusion protein.

Animals↗

PubMed1999

Restricting the mobility of Gs alpha: impact on receptor and effector coupling.

The alpha-subunit of the stimulatory G protein, Gs, has been shown to dissociate from the plasma membrane into the cytosol following activation by G protein-coupled receptors (GPCR) in some experimental systems. This dissociation may involve depalmitoylation of an amino-terminal cysteine residue. However, the functional significance of this dissociation is not known. To investigate the functional consequence of Gs alpha dissociation, we constructed a membrane-tethered Gs alpha (tetGs alpha), expressed it in Sf9 insect cells, and examined its ability to couple with the beta(2) adrenoceptor and to activate adenylyl cyclase. Compared to wild-type Gs alpha, tetGs alpha coupled much more efficiently to the beta 2 adrenoceptor and the D1 dopamine receptor as determined by agonist-stimulated GTP gamma S binding and GTPase activity. The high coupling efficiency was abolished when Gs )alpha was proteolytically cleaved from the membrane tether. The membrane tether did not prevent the coupling of tetGS alpha to adenylyl cyclase. These results demonstrate that regulating the mobility of Gs alpha relative to the plasma membrane, through fatty acylation or perhaps interactions with cytoskeletal proteins, could have a significant impact on receptor-G protein coupling. Furthermore, by enabling the use of more direct measures of receptor-G protein coupling (GTPase activity, GTP gamma S binding), tetGS alpha can facilitate the study for receptor-G protein interactions.

Adenylyl Cyclases↗

PubMed1999

Interaction of the retinal G-protein transducin with uracil nucleotides.

Little is known about the interaction of pyrimidine nucleotides with G-proteins. Here we report that under experimental conditions that exclude transphosphorylation reactions, nucleoside 5'-triphosphates inhibited transducin-catalyzed GTP hydrolysis in the order of potency guanosine 5'-[gamma-thio]triphosphate > GTP > guanosine 5'-[beta,gamma-imido]triphosphate > uridine 5'-[gamma-thio]triphosphate > UTP > CTP. Nucleoside 5'-diphosphates inhibited GTP hydrolysis in the order of potency GDP approximately guanosine 5'-[beta-thio]thiodiphosphate > uridine 5'-[beta-thio]diphosphate >> UDP (no effect). UTP inhibited GTP hydrolysis competitively, indicative for nucleotide binding to the same site. Uracil nucleotides had a distinct activity profile with respect to disruption of the transitory complex between photoexcited rhodopsin and nucleotide-free transducin. We conclude that (i) uracil nucleotides bind to transducin-alpha with lower affinity than the corresponding guanine nucleotides, (ii) phosphorothioate modification of uracil nucleotides increases their affinity for transducin, and (iii) uracil nucleotides induce conformational changes in G-proteins that are different from the conformational changes induced by guanine nucleotides.

Animals↗

PubMed1999

Molecular characterization of a slowly gating human hyperpolarization-activated channel predominantly expressed in thalamus, heart, and testis.

Rhythmic activity of neurons and heart cells is endowed by pacemaker channels that are activated by hyperpolarization and directly regulated by cyclic nucleotides (termed HCN channels). These channels constitute a multigene family, and it is assumed that the properties of each member are adjusted to fit its particular function in the cell in which it resides. Here we report the molecular and functional characterization of a human subtype hHCN4. hHCN4 transcripts are expressed in heart, brain, and testis. Within the brain, the thalamus is the predominant area of hHCN4 expression. Heterologous expression of hHCN4 produces channels of unusually slow kinetics of activation and inactivation. The mean potential of half-maximal activation (V(1/2)) was -75.2 mV. cAMP shifted V(1/2) by 11 mV to more positive values. The hHCN4 gene was mapped to chromosome band 15q24-q25. The characteristic expression pattern and the sluggish gating suggest that hHCN4 controls the rhythmic activity in both thalamocortical neurons and pacemaker cells of the heart.

Amino Acid Sequence↗

PubMed1999

Molecular determinants of a Ca2+-binding site in the pore of cyclic nucleotide-gated channels: S5/S6 segments control affinity of intrapore glutamates.

Cyclic nucleotide-gated (CNG) channels play an important role in Ca2+ signaling in many cells. CNG channels from various tissues differ profoundly in their Ca2+ permeation properties. Using the voltage-dependent Ca2+ blockage of monovalent current in wild-type channels, chimeric constructs and point mutants, we have identified structural elements that determine the distinctively different interaction of Ca2+ with CNG channels from rod and cone photoreceptors and olfactory neurons. Segments S5 and S6 and the extracellular linkers flanking the pore region are the only structural elements that account for the differences between channels. Ca2+ blockage is strongly modulated by external pH. The different pH dependence of blockage suggests that the pKa of intrapore glutamates and their protonation pattern differ among channels. The results support the hypothesis that the S5-pore-S6 module, by providing a characteristic electrostatic environment, determines the protonation state of pore glutamates and thereby controls Ca2+ affinity and permeation in each channel type.

Amino Acid Sequence↗