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The open state of M(Kv7.2/7.3) potassium channels is maintained by membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). They can be closed on stimulating receptors that induce PI(4,5)P2 hydrolysis. In sympathetic neurons, closure induced by stimulating M1-muscarinic acetylcholine receptors (mAChRs) has been attributed to depletion of PI(4,5)P2, whereas closure by bradykinin B2-receptors (B2-BKRs) appears to result from formation of IP3 and release of Ca2+, implying that BKR stimulation does not deplete PI(4,5)P2. We have used a fluorescently tagged PI(4,5)P2-binding construct, the C-domain of the protein tubby, mutated to increase sensitivity to PI(4,5)P2 changes (tubby-R332H-cYFP), to provide an on-line read-out of PI(4,5)P2 changes in single living sympathetic neurons after receptor stimulation. We find that the mAChR agonist, oxotremorine-M (oxo-M), produces a near-complete translocation of tubby-R332H-cYFP into the cytoplasm, whereas bradykinin (BK) produced about one third as much translocation. However, translocation by BK was increased to equal that produced by oxo-M when synthesis of PI(4,5)P2 was inhibited by wortmannin. Further, wortmannin ‘rescued’ M-current inhibition by BK after Ca2+-dependent inhibition was reduced by thapsigargin. These results provide the first direct support for the view that BK accelerates PI(4,5)P2 synthesis in these neurons, and show that the mechanism of BKR-induced inhibition can be switched from Ca2+ dependent to PI(4,5)P2 dependent when PI(4,5)P2 synthesis is inhibited. [ABSTRACT FROM AUTHOR]

M-type (Kv7) potassium channels are closed by Gq/11 G-protein-coupled receptors. Several membrane- or channel-associated molecules have been suggested to contribute to this effect, including depletion of phosphatidylinositol-4,5-bisphosphate (PIP2 ) and activation of Ca2+/calmodulin and protein kinase C. To facilitate further study of these pathways in intact neurons, we have devised novel membranetargeted probes that can be applied from the outside of the neuron, by attaching a palmitoyl group to site-directed peptides ("palpeptides") (cf. Covic et al., 2002a,b). A palpeptide incorporating the 10-residue C terminus of Gαq/11 reduced Gq/11-mediated M-current inhibition in sympathetic neurons by the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine-M but not Go-mediated inhibition of the N-type Ca2+ current by norepinephrine. Instead, the latter was inhibited by the corresponding Go palpeptide. A PIP2 palpeptide, based on the putative PIP2 binding domain of the Kv7.2 channel, inhibited M current (IC50 = ∼1.5 µM) and enhanced inhibition by oxotremorine-M. Inhibition could not be attributed to activation of mAChRs, calcium influx, or block of M channels but was antagonized by intracellular diC8-PIP2 (dioctanoyl-phosphatidylinositol-4,5-bisphosphate), suggesting that it disrupted PIP2--M channel gating. A fluorescently tagged PIP2 palpeptide was highly targeted to the plasma membrane but did not accumulate in the cytoplasm. We suggest that these palpeptides are anchored in the plasma membrane via the palmitoyl group, such that the peptide moiety can interact with target molecules on the inner face of the membrane. The G-protein-replicating palpeptides were sequence specific and probably compete with the receptor for the cognate G-protein. The PIP2 palpeptide was not sequence specific so probably interacts electrostatically with anionic PIP2 head groups. [ABSTRACT FROM AUTHOR]

Quinidine and Ba2+, non-selective K+-channel blockers, have previously been shown to inhibit antigen-induced mediator (β-hexosaminidase) release from RBL-2H3 cells, a mucosal-type mast cell line. We therefore used selective blockers of Ca2+-activated and other K+ channels to determine if there was a role for these channels in antigen-induced mediator release. Charybdotoxin and cetiedil dose-dependently inhibited β-hexosaminidase release with IC50 values of 133 nM and 84 μM, respectively. Charybdotoxin also inhibited the repolarization phase of the antigen-induced biphasic change in the membrane potential (IC50 84 nM), antigen-stimulated 86Rb+-efflux and increase in free intracellular calcium, [Ca2+]i. Iberiotoxin, margatoxin, apamin and tetraethylammonium had no effect on β-hexosaminidase release. These results suggest that K+ conductances play a significant role in mediator release from RBL-2H3, that these conductances are of the intermediate conductance Ca2+-activated K+ channel (IKCa) type, and that they are somewhat similar to those which have been described in red blood cells, though they are much less sensitive to clotrimazole. [Copyright &y& Elsevier]

Spectrin is a cytoskeletal protein that plays a role in formation of the specialized plasma membrane domains. However, little is known of the molecular mechanism that regulates responses of spectrin to extracellular stimuli, such as activation of G-protein-coupled receptor (GPCR). We have found that α II spectrin is a component of the Gαq/11-associated protein complex in CHO cells stably expressing the M1 muscarinic receptor, and investigated the effect of activation of GPCR on the cellular localization of yellow-fluorescent-protein-tagged α II spectrin. Stimulation of Gαq/11-coupled M1 muscarinic receptor triggered reversible redistribution of α II spectrin following a rise in intracellular Ca2+ concentration. This redistribution, accompanied by non-apoptotic membrane blebbing, required an intact actin cytoskeleton and was dependent on activation of phospholipase C, protein kinase C, and Rho-associated kinase ROCK. Muscarinic-agonist-induced spectrin remodeling appeared particularly active at localized domains, which is clear contrast to that caused by constitutive activation of ROCK and to global rearrangement of the spectrin lattice caused by changes in osmotic pressure. These results suggest a role for spectrin in providing a dynamic and reversible signaling platform to the specific domains of the plasma membrane in response to stimulation of GPCR. [ABSTRACT FROM AUTHOR]

Complex regional pain syndrome (CRPS) is thought to have an auto-immune component. One such target recently proposed from the effects of auto-immune IgGs on Ca 2 + transients in cardiac myocytes and cell lines is the α1-adrenoceptor. We have tested whether such IgGs exerted comparable effects on nociceptive sensory neurons isolated from rat dorsal root ganglia. Depolarisation-induced [Ca 2 + ] i transients were generated by applying 30 mM KCl for 2 min and monitored by Fura-2 fluorescence imaging. No IgGs tested (including 3 from CRPS patients) had any significant effect on these [Ca 2 + ] i transients. However, IgG from one CRPS patient consistently and significantly reduced the K + -induced response of cells that had been pre-incubated for 24 h with a mixture of inflammatory mediators (1 μM histamine, 5-hydroxytryptamine, bradykinin and PGE2). Since this pre-incubation also appeared to induce a comparable inhibitory response to the α1-agonist phenylephrine, this is compatible with the α1-adrenoceptor as a target for CRPS auto-immunity. A mechanism whereby this might enhance pain is suggested. [ABSTRACT FROM AUTHOR]

M-channels are voltage-gated K+ channels that regulate the excitability of many neurons. They are composed of Kv7 (KCNQ) family subunits, usually Kv7.2 + Kv7.3. Native M-channels and expressed Kv7.2 + 7.3 channels are inhibited by stimulating Gq/11-coupled receptors – prototypically the M1 muscarinic acetylcholine receptor (M1-mAChR). The channels require membrane phosphatidylinositol-4,5-bisphosphate (PIP2) to open and the effects of mAChR stimulation result primarily from the reduction in membrane PIP2 levels following Gq/phospholipase C-catalysed PIP2 hydrolysis. However, in sympathetic neurons, M-current inhibition by bradykinin appears to be mediated through the release and action of intracellular Ca2+ by inositol-1,4,5-trisphosphate (IP3), a product of PIP2 hydrolysis, rather than by PIP2 depletion. We have therefore compared the effects of bradykinin and oxotremorine-M (a muscarinic agonist) on membrane PIP2 in sympathetic neurons using a fluorescently tagged mutated C-domain of the PIP2 binding probe, ‘tubby’. In concentrations producing equal M-current inhibition, bradykinin produced about one-quarter of the reduction in PIP2 produced by oxotremorine-M, but equal reduction when PIP2 synthesis was blocked with wortmannin. Likewise, wortmannin restored bradykinin-induced M-current inhibition when Ca2+ release was prevented with thapsigargin. Thus, inhibition by bradykinin can use product (IP3/Ca2+)-dependent or substrate (PIP2) dependent mechanisms, depending on Ca2+ availability and PIP2 synthesis rates. [ABSTRACT FROM AUTHOR]

The relationship between receptor-induced membrane phosphatidylinositol-4′5′-bisphosphate (PiP2) hydrolysis and M-current inhibition was assessed in single-dissociated rat sympathetic neurons by simultaneous or parallel recording of membrane current and membrane-to-cytosol translocation of the fluorescent PIP2/inositol 1,4,5-trisphosphate (IP3)-binding peptide green fluorescent protein-tagged pleckstrin homology domain of phospholipase C (GFP-PLCδ-PH). The muscarinic receptor agonist oxotremorine-M produced parallel time- and concentration-dependent M-current inhibition and GFP-PLCδ-PH translocation; bradykinin also produced parallel time-dependent inhibition and translocation. Phosphatidylinositol-4-phosphate-5-kinase (PI5-K) overexpression reduced both M-current inhibition and GFP-PLCδ-PH translocation by both oxotremorine-M and bradykinin. These effects were partly reversed by wortmannin, which inhibits phosphatidylinositol-4-kinase (PI4-K). PI5-K overexpression also reduced the inhibitory action of oxotremorine-M on PIP2-gated G-protein-gated inward rectifier (Kir3.1/3.2) channels; bradykinin did not inhibit these channels. Overexpression of neuronal calcium sensor- 1 protein (NCS- 1), which increases PI4-K activity, did not affect responses to oxotremorine-M but reduced both fluorescence translocation and M-current inhibition by bradykinin. Using an intracellular IP3 membrane fluorescence-displacement assay, initial mean concentrations of membrane [PIP2] were estimated at 261 µM (95% confidence limit; 192-381 µM), rising to 693 µM (417-1153 µM) in neurons overexpressing PI5-K. Changes in membrane [PIP2] during application of oxotremorine-M were calculated from fluorescence data. The results, taken in conjunction with previous data for KCNQ2/3 (Kv7.2/Kv7.3) channel gating by PIP2 (Zhang et al., 2003), accorded with the hy. [ABSTRACT FROM AUTHOR]

Presents a study that demonstrated the presence of KCNQ/M in nociceptive sensory systems and its significance for pain therapy. Feature of neuropathic pain; Expression of KCNQ subtypes in dorsal root ganglian neurons; Effect of retigabine on electrically evoked dorsal horn responses.