UMLS:C0043167 - FACTA Search

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Query: UMLS:C0043167 (pertussis)
19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In heart, G-protein-activated channels are complexes of two homologous proteins, GIRK1 and GIRK4. Expression of either protein alone results in barely active or non-active channels, making it difficult to assess the individual contribution of each subunit to the channel complex. The residue Phe137, located within the H5 region of GIRK1, is critical to the synergy between GIRK1 and GIRK4 (Chan, K. W., Sui, J. L., Vivaudou, M., and Logothetis, D. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14193-14198). By modifying this residue or the matching residue of GIRK4, Ser143, we have been able to generate mutant proteins that produced large inwardly rectifying, G-protein-modulated currents when expressed alone in Xenopus oocytes. The enhanced activity of the heterologous expression of each of two active mutants, GIRK1(F137S) and GIRK4(S143T), was not caused by association with an endogenous oocyte channel subunit, and these mutants did not display apparent differences in the ability to localize to the cell surface compared with their wild-type counterparts. When these functional mutant channels were compared individually with wild-type heteromeric channels, they responded with only small differences to a number of maneuvers involving coexpression with muscarinic receptors, G-protein betagamma subunits, wild-type or mutated G-protein alpha subunits, and active protomers of pertussis toxin. These experiments, which confirmed the crucial, though not exclusive, role of Gbetagamma in regulating channel activity, demonstrated that GIRK1(F137S) and GIRK4(S143T), and by extrapolation their wild-type counterparts, interact in a qualitatively similar way with G-protein subunits. These findings suggest that functionally important sites of interaction with G-proteins are likely to be located within the homologous regions of GIRK1 and GIRK4 rather than within the divergent terminal regions. They also raise the question of the functional advantage of a heteromeric over homomeric design for G-protein-gated channels.
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PMID:Probing the G-protein regulation of GIRK1 and GIRK4, the two subunits of the KACh channel, using functional homomeric mutants. 939 92

We examined the ability of rat Y1, Y2 and Y4 neuropeptide Y (NPY) receptors to regulate K+ and Ca++ channels expressed in Xenopus oocytes and HEK 293 cells, respectively. Stimulation of all three receptors with NPY or related peptides activated inwardly rectifying K+ currents resulting from the expression of rat GIRK1/CIR in frog oocytes. These effects were inhibited by pertussis toxin treatment. The effects of activating Y1 receptors were antagonized competitively by BIBP3226, SR120819A and GW1229. The effects of Y2 receptor activation were not blocked by these drugs, and the effects of Y4 receptor activation were only blocked by GW1229. Activation of all three NPY receptors also inhibited human alpha-1B Ca++ channels stably expressed in HEK293 cells. The effects of agonists at all three receptors were blocked by pertussis toxin treatment and were voltage dependent. Activation of all three types of NPY receptors produced much smaller inhibition of human alpha-1E Ca++ channels also stably expressed in HEK293 cells. These results suggest that NPY receptors can regulate K+ and Ca++ channels and that these effects may be responsible for the observed effects of NPY on neuronal excitability and synaptic transmission.
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PMID:Regulation of K+ and Ca++ channels by a family of neuropeptide Y receptors. 945 7

1. G protein-gated inwardly rectifying K+ (GIRK) channels were heterologously expressed in rat superior cervical ganglion (SCG) neurons by intranuclear microinjection. The properties of GIRK channels and their coupling to native receptors were characterized using the whole-cell patch-clamp technique. 2. Following coinjection of either GIRK1-2 or GIRK1-4 cDNA, application of noradrenaline (NA) produced large inwardly rectifying K+ currents. Injection of cDNA encoding individual GIRK subunits produced only small and inconsistent NA-activated inward currents. Current arising from the native expression of GIRK channels in SCG neurons was not observed. 3. NA-mediated activation of GIRK channels was abolished by pertussis toxin (PTX) pretreatment, indicating coupling via G proteins of the Gi/Go subfamily. Conversely, vasoactive intestinal peptide (VIP) activated GIRK channel currents via a cholera toxin-sensitive pathway suggesting coupling through Galphas. Pretreatment of neurons with PTX caused a significant increase in amplitude of the VIP-mediated GIRK channel currents when compared with untreated cells. 4. Application of adenosine, prostaglandin E2 and somatostatin resulted in activation of GIRK channel currents. Activation of m1 muscarinic acetylcholine receptors (i.e. application of oxotremorine M to PTX-treated neurons) failed to elicit overt GIRK channel currents. 5. GIRK channel overexpression decreased basal Ca2+ channel facilitation significantly when compared with uninjected neurons. Furthermore, the NA-mediated inhibition of Ca2+ channels was significantly attenuated. 6. In summary, the ability to heterologously express GIRK channels in adult sympathetic neurons allows the experimental alteration of receptor-G protein-effector stoichiometry. Such studies may increase our understanding of the mechanisms underlying ion channel modulation by G proteins in a neuronal environment.
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PMID:Heterologous expression and coupling of G protein-gated inwardly rectifying K+ channels in adult rat sympathetic neurons. 982 16

1. G protein-regulated inward rectifier K+ (GIRK) channels were over-expressed in dissociated rat superior cervical sympathetic (SCG) neurones by co-transfecting green fluorescent protein (GFP)-, GIRK1- and GIRK2-expressing plasmids using the biolistic technique. Membrane currents were subsequently recorded with whole-cell patch electrodes. 2. Co-transfected cells had larger Ba2+-sensitive inwardly rectifying currents and 13 mV more negative resting potentials (in 3 mM [K+]o) than non-transfected cells, or cells transfected with GIRK1 or GIRK2 alone. 3. Carbachol (CCh, 1-30 microM) increased the inwardly rectifying current in 70 % of GIRK1+ GIRK2-transfected cells by 261 +/- 53 % (n = 6, CCh 30 microM) at -120 mV, but had no effect in non-transfected cells or in cells transfected with GIRK1 or GIRK2 alone. Pertussis toxin prevented the effect of carbachol but had no effect on basal currents. 4. The effect of CCh was antagonized by 6 nM tripitramine but not by 100 nM pirenzepine, consistent with activation of endogenous M2 muscarinic acetylcholine receptors. 5. In contrast, inhibition of the voltage-activated Ca2+ current by CCh was antagonized by 100 nM pirenzepine but not by 6 nM tripitramine, indicating that it was mediated by M4 muscarinic acetylcholine receptors. 6. We conclude that endogenous M2 and M4 muscarinic receptors selectively couple to GIRK currents and Ca2+ currents respectively, with negligible cross-talk.
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PMID:Selective activation of heterologously expressed G protein-gated K+ channels by M2 muscarinic receptors in rat sympathetic neurones. 1006 93

1. The activation of G protein-regulated inward rectifying potassium (GIRK) channels is modulated by G protein-coupled receptors (GPCRs) via the G protein betagamma subunits and is accelerated by regulators of G protein signalling (RGS). In the present study we investigated the ratio dependence of receptor-mediated activation and deactivation and the influence of new members of the RGS protein family on GIRK currents by coexpressing the recombinant protein subunits in Xenopus oocytes and further analysis of the whole cell currents. 2. The activation of GIRK channels by the muscarinic acetylcholine receptor M2 (M2 mAChR) is strongly dependent on the ratio of receptor to channel in Xenopus oocytes. The increase and on-rate of the amplified current is affected by this ratio. An excess of receptor over channel is necessary for current amplification, while the reverse excess of channel over receptor abolishes the effect. 3. The speed of receptor-mediated activation of GIRK currents is accelerated for a high ratio of receptor to channel, while the time of deactivation is independent of this ratio. 4. Coexpression of RGS2, 5 and 8 accelerates the speed for ACh-mediated activation and deactivation of GIRK1/2 and GIRK1/4 currents. Thereby the receptor/channel/RGS ratio determines the amount of current amplification. 5. Bordetella pertussis toxin completely abolished ACh-mediated current amplification of GIRK channels coexpressed with or without RGS2. 6. Two single point mutations in the RGS2 protein (RGS2(N109S) and RGS2(L180F)) reduced the acceleration of current amplification after ACh application on GIRK1/4 channels compared with RGS2 wild-type protein.
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PMID:New roles for RGS2, 5 and 8 on the ratio-dependent modulation of recombinant GIRK channels expressed in Xenopus oocytes. 1033 86

Blood plasma and serum contain factors that activate inwardly rectifying GIRK1/GIRK4 K+ channels in atrial myocytes via one or more non-atropine-sensitive receptors coupled to pertussis-toxin-sensitive G-proteins. This channel is also the target of muscarinic M(2) receptors activated by the physiological release of acetylcholine from parasympathetic nerve endings. By using a combination of HPLC and TLC techniques with matrix-assisted laser desorption ionization-time-of-flight MS, we purified and identified sphingosine 1-phosphate (SPP) and sphingosylphosphocholine (SPC) as the plasma and serum factors responsible for activating the inwardly rectifying K+ channel (I(K)). With the use of MS the concentration of SPC was estimated at 50 nM in plasma and 130 nM in serum; those concentrations exceeded the 1.5 nM EC(50) measured in guinea-pig atrial myocytes. With the use of reverse-transcriptase-mediated PCR and/or Western blot analysis, we detected Edg1, Edg3, Edg5 and Edg8 as well as OGR1 sphingolipid receptor transcripts and/or proteins. In perfused guinea-pig hearts, SPC exerted a negative chronotropic effect with a threshold concentration of 1 microM. SPC was completely removed after perfusion through the coronary circulation at a concentration of 10 microM. On the basis of their constitutive presence in plasma, the expression of specific receptors, and a mechanism of ligand inactivation, we propose that SPP and SPC might have a physiologically relevant role in the regulation of the heart.
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PMID:Sphingosylphosphocholine is a naturally occurring lipid mediator in blood plasma: a possible role in regulating cardiac function via sphingolipid receptors. 1125 63

Local anesthetics, commonly used for treating cardiac arrhythmias, pain, and seizures, are best known for their inhibitory effects on voltage-gated Na(+) channels. Cardiovascular and central nervous system toxicity are unwanted side-effects from local anesthetics that cannot be attributed to the inhibition of only Na(+) channels. Here, we report that extracellular application of the membrane-permeant local anesthetic bupivacaine selectively inhibited G protein-gated inwardly rectifying K(+) channels (GIRK:Kir3) but not other families of inwardly rectifying K(+) channels (ROMK:Kir1 and IRK:Kir2). Bupivacaine inhibited GIRK channels within seconds of application, regardless of whether channels were activated through the muscarinic receptor or directly via coexpressed G protein G(beta)gamma subunits. Bupivacaine also inhibited alcohol-induced GIRK currents in the absence of functional pertussis toxin-sensitive G proteins. The mutated GIRK1 and GIRK2 (GIRK1/2) channels containing the high-affinity phosphatidylinositol 4,5-bisphosphate (PIP(2)) domain from IRK1, on the other hand, showed dramatically less inhibition with bupivacaine. Surprisingly, GIRK1/2 channels with high affinity for PIP(2) were inhibited by ethanol, like IRK1 channels. We propose that membrane-permeant local anesthetics inhibit GIRK channels by antagonizing the interaction of PIP(2) with the channel, which is essential for G(beta)gamma and ethanol activation of GIRK channels.
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PMID:Mechanism underlying bupivacaine inhibition of G protein-gated inwardly rectifying K+ channels. 1135 68

Dopamine (DA) is a physiological regulator of PRL secretion, exerting tonic inhibitory control. DA activates an inward rectifier K(+) (IRK) channel in rat lactotropes, causing membrane hyperpolarization and inhibition of Ca(2+)-dependent action potentials. Both the activation of this effector K(+) channel and the inhibition of PRL release are mediated by D(2)-type receptor activation and pertussis toxin- sensitive G proteins. To study the molecular basis of this physiologically relevant channel, a homology-based PCR approach was employed to identify members of the IRK channel family expressed in the anterior pituitary gland. Nondegenerate primers corresponding to regions specific for IRK channels known to be G protein activated (GIRKs; gene subfamily Kir 3.0) were synthesized and used in the PCR with reverse transcribed female rat anterior pituitary messenger RNA as the template. PCR products of predicted sizes for Kir 3.1, 3.2, and 3.4 were consistently observed by ethidium bromide staining after 16 amplification cycles. The identities of the products were confirmed by subcloning and sequencing. Expression of each of these gene products in anterior pituitary was confirmed by Northern blot analysis. Functional analysis of the GIRK proteins was performed in the heterologous expression system, Xenopus laevis oocytes. Macroscopic K(+) currents were examined in oocytes injected with different combinations of Kir 3.0 complementary RNA (cRNA) and G protein subunit (beta(1)gamma(2)) cRNA. The current-voltage relationships demonstrated strong inward rectification for each individual and pairwise combination of GIRK channel subunits. Oocytes coinjected with any pair of GIRK subunit cRNA exhibited significantly larger inward K(+) currents than oocytes injected with only one GIRK channel subtype. Ligand-dependent activation of only one of the GIRK combinations (GIRK1 and GIRK4) was observed when channel subunits were coexpressed with the D(2) receptor in Xenopus oocytes. Dose-response data fit to a Michaelis-Menten equation gave an apparent K(d) similar to that for DA binding in anterior pituitary tissue. GIRK1 and GIRK4 proteins were coimmunoprecipitated from anterior pituitary lysates, confirming the presence of native GIRK1/GIRK4 oligomers in this tissue. These data indicate that GIRK1 and GIRK4 are excellent candidate subunits for the D(2)-activated, G protein-gated channel in pituitary lactotropes, where they play a critical role in excitation-secretion coupling.
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PMID:Identification of G protein-coupled, inward rectifier potassium channel gene products from the rat anterior pituitary gland. 1141 1

1. cDNA constructs coding for a yellow-emitting green fluorescent protein (GFP) mutant fused to the N-terminus of the G-protein subunit beta 1 (YFP-beta 1) and a cyan-emitting GFP mutant fused to the N-terminus of the G-protein subunit gamma 2 (CFP-gamma 2) were heterologously expressed in rat superior cervical ganglion (SCG) neurons following intranuclear injection of the tagged subunits. The ability of the tagged subunits to modulate effectors, form a heterotrimer and couple to receptors was characterized using the whole-cell patch-clamp technique. Fluorescent resonance energy transfer (FRET) was also measured to determine the protein-protein interaction between the two fusion proteins. 2. Similar to co-expression of untagged beta 1/gamma 2, co-expression of YFP-beta 1/gamma 2, beta 1/CFP-gamma 2, or YFP-beta 1/CFP-gamma 2 resulted in a significant increase in basal N-type Ca(2+) channel facilitation when compared to uninjected neurons. Furthermore, the noradrenaline (NA)-mediated inhibition of Ca(2+) channels was significantly attenuated. 3. Co-expression of YFP-beta 1/CFP-gamma 2 with G-protein-gated inwardly rectifying K(+) channels (GIRK1 and GIRK4) resulted in tonic GIRK currents that were blocked by Ba(2+). 4. The ability of the tagged subunits to form heterotrimers was tested by co-injecting either tagged or untagged G beta 1 and G gamma 2 with excess G alpha(oA) cDNA. Under these conditions, the NA-mediated Ca(2+) current inhibition was significantly decreased when compared to uninjected neurons. 5. Coupling to the alpha 2-adrenergic receptor was reconstituted in neurons expressing pertussis toxin (PTX)-insensitive G alpha(oA) and either tagged or untagged G beta 1 gamma 2 subunits. Application of NA to PTX-treated cells resulted in a voltage-dependent inhibition of N-type Ca(2+) currents. 6. FRET measurements in the SCG revealed an in vivo interaction between YFP-beta 1 and CFP-gamma 2. Co-expression of untagged beta 1 significantly decreased the interaction between the two fusion proteins. 7. In summary, the attachment of GFP mutants to the N-terminus of G beta 1 or G gamma 2 does not qualitatively impair their ability to form a heterotrimer, modulate effectors (N-type Ca(2+) and GIRK channels), or couple to receptors.
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PMID:Functional expression and FRET analysis of green fluorescent proteins fused to G-protein subunits in rat sympathetic neurons. 1174 47

To investigate the effects of adenosine on endogenous Xenopus oocyte receptors, we analysed defolliculated oocytes injected with mRNAs for the G protein-activated inwardly rectifying K(+) (GIRK) channels. In oocytes injected with mRNAs for either GIRK1/GIRK2 or GIRK1/GIRK4 subunits, application of adenosine or ATP reversibly induced inward K(+) currents, although ATP was less potent than adenosine. The responses were attenuated by caffeine, a non-selective adenosine receptor antagonist. Furthermore, in uninjected oocytes from the same donor, adenosine produced no significant current. The endogenous receptor was activated by two selective A(1) adenosine receptor agonists, N(6)-cyclopentyladenosine (CPA) and N(6)-cyclohexyladenosine (CHA), and antagonized by a selective A(1) adenosine receptor antagonist, 1,3-dipropyl-8-cyclopenylxanthine (DPCPX) at moderate nanomolar concentrations, but insensitive to micromolar concentrations of selective A(2A) and A(3) adenosine receptor agonists, 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680) and N(6)-(3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine (IB-MECA), respectively. However, the pharmacological characteristics of the receptor were different from those of the cloned Xenopus A(1) adenosine receptor and previously proposed adenosine receptors. The adenosine-induced GIRK currents were abolished by injection of pertussis toxin and CPA inhibited forskolin-stimulated cyclic AMP accumulation. We conclude that an adenosine receptor on the Xenopus oocyte membrane can activate GIRK channels and inhibit adenylyl cyclase via G(i/o) proteins. Moreover, our results suggest the existence of an endogenous adenosine receptor with the unique pharmacological characteristics. As the receptor was activated by nanomolar concentrations of adenosine, which is a normal constituent of extracellular fluid, the receptor may be involved in some effects through the G(i/o) protein signalling pathways in ovarian physiology.
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PMID:Functional characterization of an endogenous Xenopus oocyte adenosine receptor. 1181 66

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