A cell cycle alteration precedes apoptosis of granule cell precursors in the weaver mouse cerebellum.
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A missense mutation in the gene coding for the G-protein-activated inwardly rectifying potassium (GIRK) channel, GIRK2, is responsible for apoptosis in the external germinal layer (EGL) of the cerebellum and a nonapoptotic death of midbrain dopaminergic neurons in the weaver (wv) mouse. Failure of axonogenesis and migration are considered to be the primary consequences of GIRK2 channel malfunction in the cerebellum. We investigated whether a disruption of the cell cycle precedes the failure of migration and axonogenesis and leads to massive apoptosis. To this end, immunohistochemistry and immunoblotting for PCNA, Cdk4, cyclin D, cyclin A, and the Cdk inhibitor p27/kip1, as well as in situ end-labeling for apoptotic DNA fragmentation, were applied to cerebella of P7-P21+/+, wv/+, and wv/wv mice. In +/+ and wv/+ mice, the expression of cell cycle proteins was limited to the outer, premigratory zone of the EGL. Antibodies to p27, a marker of cell differentiation, gave a reverse staining pattern. Due to migration delay, patches of p27-positive cells persisted in the outer EGL in P21 wv/+ mice. On the contrary, marked cell cycle up-regulation and absence of p27 occurred throughout the EGL at all ages in wv/wv mice, indicating an inability to switch off the cell cycle. Mitotic index evaluation showed that cell cycle activation was unrelated to proliferative events. Cell cycle proteins were not expressed in the substantia nigra, suggesting that nonapoptotic death of mature dopaminergic neurons is not preceded by abortive cell cycle re-entry. Our data show that abnormalities of the cell cycle in wv/wv cerebellum represent a major and early consequence of GIRK2 channel malfunction and may strongly influence the susceptibility of EGL cells to apoptosis. These observations may help in understanding the pathogenesis of human neurological channelopathies. (+info)
Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1).
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To identify possible ligands of the orphan somatostatin-like receptor 1 (SLC-1), rat brain extracts were analyzed by using the functional expression system of Xenopus oocytes injected with cRNAs encoding SLC-1 and G protein-gated inwardly rectifying potassium channels (GIRK). A strong inward current was observed with crude rat brain extracts which upon further purification by cation exchange chromatography and high performance liquid chromatography (HPLC) yielded two peptides with a high agonist activity. Mass spectrometry and partial peptide sequencing revealed that one peptide is identical with the neuropeptide melanin concentrating hormone (MCH), the other represents a truncated version of MCH lacking the three N-terminal amino acid residues. Xenopus oocytes expressing the MCH receptor responded to nM concentrations of synthetic MCH not only by the activation of GIRK-mediated currents but also by the induction of Ca(2+) dependent chloride currents mediated by phospholipase C. This indicates that the MCH receptor can couple either to the G(i)- or G(q)-mediated signal transduction pathway, suggesting that MCH may serve for a number of distinct brain functions including food uptake behavior. (+info)
Rescue of cerebellar granule cells from death in weaver NR1 double mutants.
(11/480)
The weaver mutation results in the extensive death of midline cerebellar granule cells. The mutation consists of a single base pair substitution of the gene encoding the G-protein-activated inwardly rectifying potassium channel protein, GIRK2. The functional consequences of this mutation are still in dispute. In this study we demonstrate the in vivo and in vitro rescue of weaver granule cells when NR1 NMDA subunits are eliminated in weaver NR1 double mutants. This rescue of weaver granule cells provides evidence that wvGIRK2 alone is not sufficient to cause granule cell death. (+info)
Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction.
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Opiate analgesia, tolerance, and addiction are mediated by drug-induced activation of the mu opioid receptor. A fundamental question in addiction biology is why exogenous opiate drugs have a high liability for inducing tolerance and addiction while native ligands do not. Studies indicate that highly addictive opiate drugs such as morphine are deficient in their ability to induce the desensitization and endocytosis of receptors. Here, we demonstrate that this regulatory mechanism reveals an independent functional property of opiate drugs that can be distinguished from previously established agonist properties. Moreover, this property correlates with agonist propensity to promote physiological tolerance, suggesting a fundamental revision of our understanding of the role of receptor endocytosis in the biology of opiate drug action and addiction. (+info)
The inwardly rectifying K(+) channel subunit GIRK1 rescues the GIRK2 weaver phenotype.
(13/480)
The weaver (wv) gene has been identified as a glycine to serine substitution at residue 156 in the H5 region of inwardly rectifying K(+) channel, GIRK2. The mutation is permissive for the expression of homotetrameric channels that are nonselective for cations and G-protein-independent. Coexpression of GIRK2wv with GIRK1, GIRK2, or GIRK3 in Xenopus oocytes along with expression of subunit combinations linked as dimers and tetramers was used to investigate the effects of the pore mutation on channel selectivity and gating as a function of relative subunit position and number within a heterotetrameric complex. GIRK1 formed functional, K(+) selective channels with GIRK2 and GIRK3. Coexpression of GIRK2wv with GIRK1 gave rise to a component of K(+)-selective, G-protein-dependent current. Currents resulting from coexpression of GIRK2wv with GIRK2 or GIRK3 were weaver-like. Current from dimers of GIRK1-GIRK2wv, GIRK2-GIRK2wv, and GIRK3-GIRK2wv was phenotypically similar to that obtained from coexpression of monomers. Linked tetramers containing GIRK1 and GIRK2wv in an alternating array gave rise to wild-type, K(+)-selective currents. When two mutant subunits were arranged adjacently in a tetramer, currents were weaver-like. These results support the hypothesis that in specific channel stoichiometries, GIRK1 rescues the weaver phenotype and suggests a basis for the selective neuronal vulnerability that is observed in the weaver mouse. (+info)
The weaver mouse gain-of-function phenotype of dopaminergic midbrain neurons is determined by coactivation of wvGirk2 and K-ATP channels.
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The phenotype of substantia nigra (SN) neurons in homozygous weaver (wv/wv) mice was studied by combining patch-clamp and single-cell RT-multiplex PCR techniques in midbrain slices of 14-d-old mice. In contrast to GABAergic SN neurons, which were unaffected in homozygous weaver mice (wv/wv), dopaminergic SN neurons possessed a dramatically altered phenotype with a depolarized membrane potential and complete loss of spontaneous pacemaker activity. The gain-of-function phenotype was mediated by a large, nonselective membrane conductance exclusively present in (wv/wv) dopaminergic SN neurons. This constitutively activated conductance displayed a sensitivity to external QX-314 (IC(50) = 10.6 microM) very similar to that of heterologously expressed wvGirk2 channels and was not further activated by G-protein stimulation. Single-cell Girk1-4 expression profiling suggested that homomeric Girk2 channels were present in most dopaminergic SN neurons, whereas Girk2 was always coexpressed with other Girk family members in GABAergic SN neurons. Surprisingly, acute QX-314 inhibition of wvGirk2 channels did not induce wild-type-like pacemaker activity but instead caused membrane hyperpolarization. Additional application of a blocker of ATP-sensitive potassium channels (100 microM tolbutamide) induced wild-type-like pacemaker activity. We conclude that the gain-of-function weaver phenotype of dopaminergic substantia nigra neurons is mediated by coactivation of wvGirk2 and SUR1/Kir6. 2-mediated ATP-sensitive K(+) channels. We also show that in contrast to wild-type neurons, all (wv/wv) dopaminergic SN neurons expressed calbindin, a calcium-binding protein that marks dopaminergic SN neurons resistant to neurodegeneration. The identification of two ion channels that in concert determine the weaver phenotype of surviving calbindin-positive dopaminergic SN neurons will help to understand the molecular mechanisms of selective neurodegeneration of dopaminergic SN neurons in the weaver mouse and might be important in Parkinson's disease. (+info)
Synergistic activation of G protein-gated inwardly rectifying potassium channels by the betagamma subunits of G proteins and Na(+) and Mg(2+) ions.
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Native and recombinant G protein-gated inwardly rectifying potassium (GIRK) channels are directly activated by the betagamma subunits of GTP-binding (G) proteins. The presence of phosphatidylinositol-bis-phosphate (PIP(2)) is required for G protein activation. Formation (via hydrolysis of ATP) of endogenous PIP(2) or application of exogenous PIP(2) increases the mean open time of GIRK channels and sensitizes them to gating by internal Na(+) ions. In the present study, we show that the activity of ATP- or PIP(2)-modified channels could also be stimulated by intracellular Mg(2+) ions. In addition, Mg(2+) ions reduced the single-channel conductance of GIRK channels, independently of their gating ability. Both Na(+) and Mg(2+) ions exert their gating effects independently of each other or of the activation by the G(betagamma) subunits. At high levels of PIP(2), synergistic interactions among Na(+), Mg(2+), and G(betagamma) subunits resulted in severalfold stimulated levels of channel activity. Changes in ionic concentrations and/or G protein subunits in the local environment of these K(+) channels could provide a rapid amplification mechanism for generation of graded activity, thereby adjusting the level of excitability of the cells. (+info)
Molecular mechanism for sodium-dependent activation of G protein-gated K+ channels.
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1. G protein-gated inwardly rectifying K+ (GIRK) channels are activated independently by Gbetagamma and internal Na+ via mechanisms requiring phosphatidylinositol phosphates. An aspartate (Asp) at position 226 in GIRK2 is crucial for Na+-dependent activation of GIRK1-GIRK2 heteromeric channels. We expressed wild-type and mutant GIRK1-GIRK2 channels in Xenopus oocytes and tested the effects of Na+ and neutralizing Asp226 on the functional interactions of the channels with phosphatidylinositol 4, 5-bisphosphate (PIP2). 2. The rate of inhibition of GIRK1-GIRK2 currents by application of anti-PIP2 antibody to inside-out membrane patches was slowed > 2-fold by the D226N mutation in GIRK2 and by increasing internal [Na+]. The reverse mutation in GIRK1 (N217D) increased the rate of inhibition. 3. The dose-response relationship for activation by purified PIP2 was shifted to lower concentrations in the presence of 20 mM Na+. 4. Three synthetic isoforms of PIP2, PI(4,5)P2, PI(3,4)P2 and PI(3,5)P2, activated GIRK channels with similar potencies. 5. We conclude that Na+ directly interacts with Asp226 of GIRK2 to reduce the negative electrostatic potential and promote the functional interaction of the channels with PIP2. (+info)