Specific inhibition of the rat ligand-gated ion channel P2X3 function via methoxyethoxy-modified phosphorothioated antisense oligonucleotides.

P2X3 is one receptor of a family of seven ligand-gated ion channels responding to purines. Increasing evidence indicates its involvement in neuronal signaling and in pain. However, there is currently no selective inhibitor known for this subtype. In order to obtain such a specific inhibitor, a variety of antisense oligonucleotides (ASO) against rat P2X3 was tested, and dose-dependent, sequence-specific downregulation of the rat P2X3 receptor (expressed in a Chinese hamster ovary cell line [CHO-K1]) on the mRNA, protein, and functional levels was observed. Using real-time quantitative PCR, a dose-dependent downregulation of P2X3 mRNA by ASO, as compared with untreated and mismatch controls, was demonstrated. Subsequently, downregulation by the two most potent ASO was confirmed at the protein level by Western blot. Sequence specificity was shown by titration of mismatches to the original selected oligonucleotide, and this correlated with progressive loss of P2X3 inhibition. The functional response of the P2X3 receptor was examined using whole-cell voltage clamping. Upon application of 10 microM of a nonspecific agonist, alpha,beta-methylene-ATP (alphabeta meATP), pretreatment with increasing amounts of the most active ASO 5037 correlated with a decrease in depolarization. The ability to specifically downregulate the P2X3 receptor by ASO treatment will allow investigation of the biologic role of this receptor in neuronal tissues and eventually in in vivo models of chronic pain.

C-terminal interaction is essential for surface trafficking but not for heteromeric assembly of GABA(b) receptors.

Assembly of fully functional GABA(B) receptors requires heteromerization of the GABA(B(1)) and GABA(B(2)) subunits. It is thought that GABA(B(1)) and GABA(B(2)) undergo coiled-coil dimerization in their cytoplasmic C termini and that assembly is necessary to overcome GABA(B(1)) retention in the endoplasmatic reticulum (ER). We investigated the mechanism underlying GABA(B(1)) trafficking to the cell surface. We identified a signal, RSRR, proximal to the coiled-coil domain of GABA(B(1)) that when deleted or mutagenized allows for surface delivery in the absence of GABA(B(2)). A similar motif, RXR, was recently shown to function as an ER retention/retrieval (ERR/R) signal in K(ATP) channels, demonstrating that G-protein-coupled receptors (GPCRs) and ion channels use common mechanisms to control surface trafficking. A C-terminal fragment of GABA(B(2)) is able to mask the RSRR signal and to direct the GABA(B(1)) monomer to the cell surface, where it is functionally inert. This indicates that in the heteromer, GABA(B(2)) participates in coupling to the G-protein. Mutagenesis of the C-terminal coiled-coil domains in GABA(B(1)) and GABA(B(2)) supports the possibility that their interaction is involved in shielding the ERR/R signal. However, assembly of heteromeric GABA(B) receptors is possible in the absence of the C-terminal domains, indicating that coiled-coil interaction is not necessary for function. Rather than guaranteeing heterodimerization, as previously assumed, the coiled-coil structure appears to be important for export of the receptor complex from the secretory apparatus.

Cultured rat microglia express functional beta-chemokine receptors.

We have investigated the functional expression of the beta-chemokine receptors CCR1 to 5 in cultured rat microglia. RT-PCR analysis revealed constitutive expression of CCR1, CCR2 and CCR5 mRNA. The beta-chemokines MCP-1 (1-30 nM) as well as RANTES and MIP-1alpha (100-1000 nM) evoked calcium transients in control and LPS-treated microglia. Whereas, the response to MCP-1 was dependent on extracellular calcium the response to RANTES was not. The effect of MCP-1 but not that of RANTES was inhibited by the calcium-induced calcium release inhibitor ryanodine. Calcium responses to MCP-1- and RANTES were observed in distinct populations of microglia.

Functional expression of the fractalkine (CX3C) receptor and its regulation by lipopolysaccharide in rat microglia.

Functional expression of CX3CR1, a recently discovered receptor for the chemokine fractalkine, was investigated in cultured rat microglia. Reverse transcriptase polymerase chain reaction (PCR) experiments show abundant expression of fractalkine receptor mRNA in microglia. mRNA expression of fractalkine was undetectable in astrocytes and microglia but was very strong in cortical neurons. Incubation of microglia with lipopolysaccharide (100 ng/ml) transiently suppressed expression of fractalkine receptor mRNA. Fractalkine induced a concentration-dependent (10(-10)-10(-8) M) and, at high concentrations, oscillatory mobilization of intracellular Ca2+ in microglia The concentration-response curve of fractalkine was shifted to the right after 12 h incubation with lipopolysaccharide. It is concluded that treatment with endotoxin downregulates expression of fractalkine receptor mRNA in rat microglia and suppresses the functional response to fractalkine.

Modulation by calcineurin of 5-HT3 receptor function in NG108-15 neuroblastoma x glioma cells.

1. We have investigated the mechanism of regulation of 5-HT3 receptor channel sensitivity in voltage-clamped (-80 mV) NG108-15 neuroblastoma cells. 2. The 5-HT-induced inward current activated rapidly. The fast onset was followed by a biphasic decay which was characterized by two time constants, tau 1 (1.1 +/- 0.21s) and tau 2 (8.9 +/- 1.6s), respectively. Brief applications of 5-HT, applied at 2 min intervals, induced a decrease in the amplitude of the 5-HT3 receptor-mediated peak inward currents. 3. Buffering of intracellular calcium with the calcium chelator BAPTA (10 mM) instead of EGTA (10 mM) attenuated the 5-HT-induced loss of responsiveness of 5-HT3 receptors. Omission of calcium from the extracellular medium yielded a similar attenuation of loss of responsiveness. 4. Inclusion of the protein kinase inhibitor, staurosporine (1 microM) or of okadaic acid (1 microM), an inhibitor of protein phosphatases 1 and 2A, in the intracellular buffer solution did not affect 5-HT3 receptor sensitivity. 5. Injection of cyclosporin A-cyclophilin A complex (20 nM), which potently inhibits calcineurin, did not affect the time constants of the biphasic decay of the 5-HT response tau 1 (1.4 +/- 0.28s) and tau 2 (11.3 +/- 1.7s). The complex, however, prevented the loss of 5-HT3, receptor responsiveness upon repeated application of 5-HT. A similar, but weaker effect was observed after intracellular application of the autoinhibitory peptide domain of calcineurin (1 microM). 6. The recovery of desensitized 5-HT3 receptors upon a second application of 5-HT (1 microM) showed a half-life time (tau 1/2) of 2.6 +/- 0.12 min in control cells which was reduced to 1.6 +/- 0.09 min in cells treated with cyclosporin A-cyclophilin A (20 nM) complex. 7. We conclude that calcineurin does not affect the fast decay of the 5-HT3 receptor response but may be involved in a slower process which regulates channel activity.

The amyloid precursor protein fragment His 657-Lys 676 inhibits noradrenaline- and enkephaline-induced suppression of voltage sensitive calcium currents in NG108-15 hybrid cells.

We have investigated the effects of the C-terminal amyloid precursor protein fragment His 657-Lys 676 upon calcium currents in NG108-15 neuroblastoma x glioma hybrid cells. The amyloid precursor protein fragment His 657-Lys 676 (1-10 microM) did not affect calcium currents per se, but clearly blocked the calcium current suppression mediated by both adrenergic alpha 2B- and opioid delta receptors in a concentration-dependent manner. The reverse amyloid precursor protein fragment Lys 676-His 657 and the shorter amyloid precursor protein fragment Gly 659-Lys 676 did not affect calcium current suppression by adrenergic alpha 2B- and opioid delta receptors. The similar interaction of C-terminal amyloid precursor protein with adrenergic alpha 2B- and opioid delta receptors suggest that the effect occurs downstream of the receptor, possibly via the GTP binding protein Go.