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Endothelial barrier disruption is a hallmark of tissue injury, edema, and inflammation. Vascular endothelial cells express the G protein-coupled receptor (GPCR) protease acctivated receptor 1 (PAR1) and the ion channel transient receptor potential vanilloid 4 (TRPV4), and these signaling proteins are known to respond to inflammatory conditions and promote edema through remodeling of cell-cell junctions and modulation of endothelial barriers. It has previously been established that signaling initiated by the related protease activated receptor 2 (PAR2) is enhanced by TRPV4 in sensory neurons and that this functional interaction plays a critical role in the development of neurogenic inflammation and nociception. Here, we investigated the PAR1-TRPV4 axis, to determine if TRPV4 plays a similar role in the control of edema mediated by thrombin-induced signaling. Using Evans Blue permeation and retention as an indication of increased vascular permeability in vivo, we showed that TRPV4 contributes to PAR1-induced vascular hyperpermeability in the airways and upper gastrointestinal tract of mice. TRPV4 contributes to sustained PAR1-induced Ca 2+ signaling in recombinant cell systems and to PAR1-dependent endothelial junction remodeling in vitro. This study supports the role of GPCR-TRP channel functional interactions in inflammatory-associated changes to vascular function and indicates that TRPV4 is a signaling effector for multiple PAR family members.

Background and Purpose: Protease-activated receptor 2 (PAR2) is expressed on nociceptive neurons, and can sensitize transient receptor potential (TRP) ion channels to amplify neurogenic inflammation and pain. The mechanisms by which this occurs are not fully understood. PAR2 causes receptor-operated activation of TRPV4 channels and TRPV4 null mice have attenuated PAR2-stimulated neurogenic inflammation and mechanical hyperalgesia. Here we investigate the intracellular signalling mechanisms underlying PAR2-induced TRPV4 channel activation and pain., Experimental Approach: Responses of non-transfected and TRPV4-transfected HEK293 cells to agonists of PAR2 (trypsin and SLIGRL) and TRPV4 channels (GSK1016790A) were determined using calcium imaging. Inhibitors of TRPV4 channels (HC067047), sarcoendoplasmic reticulum calcium transport ATPase (thapsigargin), Gαq (UBO-QIC), tyrosine kinases (bafetinib and dasatinib) or PI3 kinases (wortmannin and LY294002) were used to investigate signalling mechanisms. In vivo effects of tyrosine kinase inhibitors on PAR2 -induced mechanical hyperalgesia were assessed in mice., Key Results: In non-transfected HEK293 cells, PAR2 activation transiently increased intracellular calcium ([Ca(2+) ]i ). Functional expression of TRPV4 channels caused a sustained increase of [Ca(2+) ]i , inhibited by HC067047, bafetinib and wortmannin; but not by thapsigargin, UBO-QIC, dasatinib or LY294002. Bafetinib but not dasatinib inhibited PAR2-induced mechanical hyperalgesia in vivo., Conclusions and Implications: This study supports a role for tyrosine kinases in PAR2-mediated receptor-operated gating of TRPV4 channels, independent of Gαq stimulation. The ability of a tyrosine kinase inhibitor to diminish PAR2-induced activation of TRPV4 channels and consequent mechanical hyperalgesia identifies bafetinib (which is in development in oncology) as a potential novel analgesic therapy., (© 2014 The British Pharmacological Society.)

The KCNQ2 gene product, Kv7.2, is a subunit of the M-channel, a low-threshold voltage-gated K(+) channel that regulates mammalian and human neuronal excitability. Spontaneous mutations one of the KCNQ2 genes cause disorders of neural excitability such as Benign Familial Neonatal Seizures. However there appear to be no reports in which both human KCNQ2 genes are mutated. We therefore asked what happens to M-channel function when both KCNQ2 genes are disrupted. We addressed this using sympathetic neurons isolated from mice in which the KCNQ2 gene was truncated at a position corresponding to the second transmembrane domain of the Kv7.2 protein. Since homozygote KCNQ2-/- mice die postnatally, experiments were largely restricted to neurons from late embryos. Quantitative PCR revealed an absence of KCNQ2 mRNA in ganglia from KCNQ2-/- embryos but 100-120% increase of KCNQ3 and KCNQ5 mRNAs; KCNQ2+/- ganglia showed ∼30% less KCNQ2 mRNA than wild-type (+/+) ganglia but 40-50% more KCNQ3 and KCNQ5 mRNA. Neurons from KCNQ2-/- embryos showed a complete absence of M-current, even after applying the Kv7 channel enhancer, retigabine. Neurons from heterozygote KCNQ2+/- embryos had ∼60% reduced M-current. In contrast, M-currents in neurons from adult KCNQ2+/- mice were no smaller than those in neurons from wild-type mice. Measurements of tetraethylammonium block did not indicate an increased expression of Kv7.5-containing subunits, implying a compensatory increase in Kv7.2 expression from the remaining KCNQ2 gene. We conclude that mouse embryonic M-channels have an absolute requirement for Kv7.2 subunits for functionality, that the reduced M-channel activity in heterozygote KCNQ2+/- mouse embryos results primarily from a gene-dosage effect, and that there is a compensatory increase in Kv7.2 expression in adult mice.

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR(2)), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR(2) regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR(2) agonist stimulated a transient increase in [Ca(2+)](i). TRPV4 expression led to a markedly sustained increase in [Ca(2+)](i). Removal of extracellular Ca(2+) and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A(2) and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR(2) generates arachidonic acid-derived lipid mediators, such as 5',6'-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR(2)-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR(2) was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR(2) signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR(2)-stimulated neurogenic inflammation. Thus, PAR(2) activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR(2).

Circulating ghrelin reduces blood pressure, but the mechanism for this action is unknown. This study investigated whether ghrelin has direct vasodilator effects mediated through the growth hormone secretagogue receptor 1a (GHSR1a) and whether ghrelin reduces sympathetic nerve activity. Mice expressing enhanced green fluorescent protein under control of the promoter for growth hormone secretagogue receptor (GHSR) and RT-PCR were used to locate sites of receptor expression. Effects of ghrelin and the nonpeptide GHSR1a agonist capromorelin on rat arteries and on transmission in sympathetic ganglia were measured in vitro. In addition, rat blood pressure and sympathetic nerve activity responses to ghrelin were determined in vivo. In reporter mice, expression of GHSR was revealed at sites where it has been previously demonstrated (hypothalamic neurons, renal tubules, sympathetic preganglionic neurons) but not in any artery studied, including mesenteric, cerebral, and coronary arteries. In rat, RT-PCR detected GHSR1a mRNA expression in spinal cord and kidney but not in the aorta or in mesenteric arteries. Moreover, the aorta and mesenteric arteries from rats were not dilated by ghrelin or capromorelin at concentrations >100 times their EC(50) determined in cells transfected with human or rat GHSR1a. These agonists did not affect transmission from preganglionic sympathetic neurons that express GHSR1a. Intravenous application of ghrelin lowered blood pressure and decreased splanchnic nerve activity. It is concluded that the blood pressure reduction to ghrelin occurs concomitantly with a decrease in sympathetic nerve activity and is not caused by direct actions on blood vessels or by inhibition of transmission in sympathetic ganglia.

The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.

Basal forebrain (BF) neurons provide the principal cholinergic drive to the hippocampus and cortex. Their degeneration is associated with the cognitive defects of Alzheimer's disease. Immunohistochemical studies suggest that some of these neurons contain glutamate, so might also release it. To test this, we made microisland cultures of single BF neurons from 12- to 14-d-old rats. Over 1-8 weeks in culture, neuronal processes made autaptic connections onto the neuron. In 34 of 36 cells tested, a somatically generated action potential was followed by a short-latency EPSC that was blocked by 1 mM kynurenic acid, showing that they released glutamate. To test whether the same neuron also released acetylcholine, we placed a voltage-clamped rat myoball expressing nicotinic receptors in contact with a neurite. In six of six neurons tested, the glutamatergic EPSC was accompanied by a nicotinic (hexamethonium-sensitive) myoball current. Stimulation of the M2-muscarinic presynaptic receptors (characterized using tripitramine and pirenzepine) produced a parallel inhibition of autaptic glutamatergic and myoball nicotinic responses; metabotropic glutamate receptor stimulation produced similar but less consistent and weaker effects. Atropine enhanced the glutamatergic EPSCs during repetitive stimulation by 25 +/- 6%; the anti-cholinesterase neostigmine reduced the train EPSCs by 37 +/- 6%. Hence, synaptically released acetylcholine exerted a negative-feedback inhibition of coreleased glutamate. We conclude that most cholinergic basal forebrain neurons are capable of releasing glutamate as a cotransmitter and that the release of both transmitters is subject to simultaneous feedback inhibition by synaptically released acetylcholine. This has implications for BF neuron function and for the use of cholinesterase inhibitors in Alzheimer's disease.

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-PLCdelta-PH). The muscarinic receptor agonist oxotremorine-M produced parallel time- and concentration-dependent M-current inhibition and GFP-PLCdelta-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-PLCdelta-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 microm (95% confidence limit; 192-381 microm), rising to 693 microm (417-1153 microm) 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 hypothesis that the inhibitory action of oxotremorine-M on M current resulted from depletion of PIP2. The effects of bradykinin require additional components of action, which might involve IP3-induced Ca2+ release and consequent M-channel inhibition (as proposed previously) and stimulation of PIP2 synthesis by Ca2+-dependent activation of NCS-1.

A new class of Conus peptides, the I-superfamily of conotoxins, has been characterized using biochemical, electrophysiological and molecular genetic methods. Peptides in this superfamily have a novel pattern of eight Cys residues. Five peptides that elicited excitatory symptomatology, r11a, r11b, r11c, r11d and r11e, were purified from Conus radiatus venom; four were tested on amphibian peripheral axons and shown to elicit repetitive action potentials, consistent with being members of the 'lightning-strike cabal' of toxins that effect instant immobilization of fish prey. A parallel analysis of Conus cDNA clones revealed a new class of conotoxin genes that was particularly enriched (with 18 identified paralogues) in a Conus radiatus venom duct library; several C. radiatus clones encoded the excitatory peptides directly characterized from venom. The remarkable diversity of related I-superfamily peptides within a single Conus species is unprecedented. When combined with the excitatory effects observed on peripheral circuitry, this unexpected diversity suggests a corresponding molecular complexity of the targeted signaling components in peripheral axons; the I-conotoxin superfamily should provide a rich lode of pharmacological tools for dissecting and understanding these. Thus, the I-superfamily conotoxins promise to provide a significant new technology platform for dissecting the molecular components of axons.

Antisense approaches are increasingly used to dissect signaling pathways linking cell surface receptors to intracellular effectors. Here we used a recombinant adenovirus to deliver G-protein alpha(q) antisense into rat superior cervical ganglion (SCG) neurons and neuronal cell lines to dissect G(alpha)(q)-mediated signaling pathways in these cells. This approach was compared with other G(alpha)(q) gene knockdown strategies, namely, antisense plasmid and knockout mice. Infection with adenovirus expressing G(alpha)(q) antisense (G(alpha)(q)AS AdV) selectively decreased immunoreactivity for the G(alpha)(q) protein. Expression of other G(alpha) protein subunits, such as G(alpha)(oA/B,) was unaltered. Consistent with this, modulation of Ca(2+) currents by the G(alpha)(q)-coupled M(1) muscarinic receptor was severely impaired in neurons infected with G(alpha)(q)AS AdV whereas modulation via the G(alpha)(oA)-coupled M(4) muscarinic receptor was unchanged. In agreement, activation of phospholipase C and consequent mobilization of intracellular Ca(2+) by UTP receptors was lost in NG108-15 cells infected with G(alpha)(q)AS AdV but not in cells infected with the control GFP-expressing adenovirus. Results obtained with this recombinant AdV strategy qualitatively and quantitatively replicated results obtained using SCG neurons microinjected with G(alpha)(q) antisense plasmids or SCG neurons from G(alpha)(q) knockout mice. This combined antisense/recombinant adenoviral approach can therefore be useful for dissecting signal transduction mechanisms in SCG and other neurons.

M(1) muscarinic (M(1)AChRs) and B(2) bradykinin (B(2)Rs) receptors are two PLCbeta-coupled receptors that mobilize Ca(2+) in nonexcitable cells. In many neurons, however, B(2)Rs but not M(1)AChRs mobilize intracellular Ca(2+). We have studied the membrane organization and dynamics underlying this coupling specificity by using Trp channels as biosensors for real-time detection of PLCbeta products. We found that, in sympathetic neurons, although both receptors rapidly produced DAG and InsP(3) as messengers, only InsP(3) formed by B(2)Rs has the ability to activate IP(3)Rs. This exclusive coupling results from spatially restricted complexes linking B(2)Rs to IP(3)Rs, a missing partnership for M(1)AChRs. These complexes allow fast and localized rises of InsP(3), necessary to activate the low-affinity neuronal IP(3)R. Thus, these signaling microdomains are of critical importance for the induction of selective responses, discriminating proinflammatory information associated with B(2)Rs from cholinergic neurotransmission.

Retigabine [D-23129; N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester] is a novel anticonvulsant compound that is now in clinical phase II development. It has previously been shown to enhance currents generated by KCNQ2/3 K(+) channels when expressed in Chinese hamster ovary (CHO) cells (Main et al., 2000; Wickenden et al., 2000). In the present study, we have compared the actions of retigabine on KCNQ2/3 currents with those on currents generated by other members of the KCNQ family (homomeric KCNQ1, KCNQ2, KCNQ3, and KCNQ4 channels) expressed in CHO cells and on the native M current in rat sympathetic neurons [thought to be generated by KCNQ2/3 channels (Wang et al., 1998)]. Retigabine produced a hyperpolarizing shift of the activation curves for KCNQ2/3, KCNQ2, KCNQ3, and KCNQ4 currents with differential potencies in the following order: KCNQ3 > KCNQ2/3 > KCNQ2 > KCNQ4, as measured either by the maximum hyperpolarizing shift in the activation curves or by the EC(50) values. In contrast, retigabine did not enhance cardiac KCNQ1 currents. Retigabine also produced a hyperpolarizing shift in the activation curve for native M channels in rat sympathetic neurons. The retigabine-induced current was inhibited by muscarinic receptor stimulation, with similar agonist potency but 25% reduced maximum effect. In unclamped neurons, retigabine produced a hyperpolarization and reduced the number of action potentials produced by depolarizing current injections, without change in action potential configuration.

Although many G-protein-coupled neurotransmitter receptors are potentially capable of modulating both voltage-dependent Ca(2+) channels (I(Ca)) and G-protein-gated K(+) channels (I(GIRK)), there is a substantial degree of selectivity in the coupling to one or other of these channels in neurons. Thus, in rat superior cervical ganglion (SCG) neurons, M(2) muscarinic acetylcholine receptors (mAChRs) selectively activate I(GIRK) whereas M(4) mAChRs selectively inhibit I(Ca). One source of selectivity might be that the two receptors couple preferentially to different G-proteins. Using antisense depletion methods, we found that M(2) mAChR-induced activation of I(GIRK) is mediated by G(i) whereas M(4) mAChR-induced inhibition of I(Ca) is mediated by G(oA). Experiments with the beta gamma-sequestering peptides alpha-transducin and beta ARK1(C-ter) indicate that, although both effects are mediated by G-protein beta gamma subunits, the endogenous subunits involved in I(GIRK) inhibition differ from those involved in I(Ca) inhibition. However, this pathway divergence does not result from any fundamental selectivity in receptor-G-protein-channel coupling because both I(GIRK) and I(Ca) modulation can be rescued by heterologously expressed G(i) or G(o) proteins after the endogenously coupled alpha-subunits have been inactivated with Pertussis toxin (PTX). We suggest instead that the divergence in the pathways activated by the endogenous mAChRs results from a differential topographical arrangement of receptor, G-protein and ion channel.

Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (I(K(M))), which can be inhibited by activation of M(1) muscarinic receptors (M(1) mAChR) and bradykinin (BK) B(2) receptors. Inhibition by the M(1) mAChR agonist oxotremorine methiodide (Oxo-M) is mediated, at least in part, by the pertussis toxin-insensitive G-protein Galpha(q) (Caulfield et al., 1994; Haley et al., 1998a), whereas BK inhibition involves Galpha(q) and/or Galpha(11) (Jones et al., 1995). Galpha(q) and Galpha(11) can stimulate phospholipase C-beta (PLC-beta), raising the possibility that PLC is involved in I(K(M)) inhibition by Oxo-M and BK. RT-PCR and antibody staining confirmed the presence of PLC-beta1, -beta2, -beta3, and -beta4 in rat SCG. We have tested the role of two PLC isoforms (PLC-beta1 and PLC-beta4) using antisense-expression constructs. Antisense constructs, consisting of the cytomegalovirus promoter driving antisense cRNA corresponding to the 3'-untranslated regions of PLC-beta1 and PLC-beta4, were injected into the nucleus of dissociated SCG neurons. Injected cells showed reduced antibody staining for the relevant PLC-beta isoform when compared to uninjected cells 48 hr later. BK inhibition of I(K(M)) was significantly reduced 48 hr after injection of the PLC-beta4, but not the PLC-beta1, antisense-encoding plasmid. Neither PLC-beta antisense altered M(1) mAChR inhibition by Oxo-M. These data support the conclusion of Cruzblanca et al. (1998) that BK, but not M(1) mAChR, inhibition of I(K(M)) involves PLC and extends this finding by indicating that PLC-beta4 is involved.

Voltage-gated Ca2+ channels participate in dendritic integration, yet functional properties of Ca2+ channels and mechanisms of their modulation by neurotransmitters in dendrites are unknown. Here we report how pharmacologically identified Ca2+ channels behave in different neural compartments. Whole-cell and cell-attached patch-clamp recordings were made on both cell bodies and electrically isolated dendrites of sympathetic neurons. We found not only that Ca2+ channel populations differentially contribute to somatic and dendritic currents but also that families of Ca2+ channels display gating properties and neurotransmitter modulation that depend on channel compartmentalization. By comparison with their somatic counterparts, dendritic N-type Ca2+ currents were hypersensitive to neurotransmitters and G proteins. Single-channel analysis showed that dendrites express a unique N-type channel that has enhanced interaction with Gbetagamma. Thus Ca2+ channels in dendrites seem to be specialized elements with unique regulatory mechanisms.

Activation of M(1) muscarinic acetylcholine receptors (M(1) mAChR) inhibits M-type potassium currents (I(K(M))) and N-type calcium currents (I(Ca)) in mammalian sympathetic ganglia. Previous antisense experiments suggested that, in rat superior cervical ganglion (SCG) neurons, both effects were partly mediated by the G-protein Galpha(q) (Delmas et al., 1998a; Haley et al., 1998a), but did not eliminate a contribution by other pertussis toxin (PTX)-insensitive G-proteins. We have tested this further using mice deficient in the Galpha(q) gene. PTX-insensitive M(1) mAChR inhibition of I(Ca) was strongly reduced in Galpha(q) -/- mouse SCG neurons and was fully restored by acute overexpression of Galpha(q). In contrast, M(1) mAChR inhibition of I(K(M)) persisted in Galpha(q)-/- mouse SCG cells. However, unlike rat SCG neurons, muscarinic inhibition of I(K(M)) was partly PTX-sensitive. Residual (PTX-insensitive) I(K(M)) inhibition was slightly reduced in Galpha(q) -/- neurons, and the remaining response was then suppressed by anti-Galpha(q/11) antibodies. Bradykinin (BK) also inhibits I(K(M)) in rat SCG neurons via a PTX-insensitive G-protein (G(q) and/or G(11); Jones et al., 1995). In mouse SCG neurons, I(K(M)) inhibition by BK was fully PTX-resistant. It was unchanged in Galpha(q) -/- mice but was abolished by anti-Galpha(q/11) antibody. We conclude that, in mouse SCG neurons (1) M(1) mAChR inhibition of I(Ca) is mediated principally by G(q), (2) M(1) mAChR inhibition of I(K(M)) is mediated partly by G(q), more substantially by G(11), and partly by a PTX-sensitive G-protein(s), and (3) BK-induced inhibition of I(K(M)) is mediated wholly by G(11).

1. KCNQ1-4 potassium channels were expressed in mammalian Chinese hamster ovary (CHO) cells stably transfected with M1 muscarinic acetylcholine receptors and currents were recorded using the whole-cell perforated patch technique and cell-attached patch recording. 2. Stimulation of M1 receptors by 10 microM oxotremorine-M (Oxo-M) strongly reduced (to 0-10%) currents produced by KCNQ1-4 subunits expressed individually and also those produced by KCNQ2 + KCNQ3 and KCNQ1 + KCNE1 heteromers, which are thought to generate neuronal M-currents (IK,M) and cardiac slow delayed rectifier currents (IK,s), respectively. 3. The activity of KCNQ2 + KCNQ3, KCNQ2 and KCNQ3 channels recorded with cell-attached pipettes was strongly and reversibly reduced by Oxo-M applied to the extra-patch membrane. 4. It is concluded that M1 receptors couple to all known KCNQ subunits and that inhibition of KCNQ2 + KCNQ3 channels, like that of native M-channels, requires a diffusible second messenger.

In Shaker-group potassium channels the presence of a tyrosine residue, just downstream of the pore signature sequence GYG, determines sensitivity to tetraethylammonium (TEA). The KCNQ family of channels has a variety of amino acid residues in the equivalent position. We studied the effect of TEA on currents generated by KCNQ homomers and heteromers expressed in CHO cells. We used wild-type KCNQ1-4 channels and heteromeric KCNQ2/3 channels incorporating either wild-type KCNQ3 subunits or a mutated KCNQ3 in which tyrosine replaced threonine at position 323 (mutant T323Y). IC50 values were (mM): KCNQ1, 5.0; KCNQ2, 0.3; KCNQ3, > 30; KCNQ4, 3.0; KCNQ2 + KCNQ3, 3.8; and KCNQ2 + KCNQ3(T323Y), 0.5. While the high TEA sensitivity of KCNQ2 may be conferred by a tyrosine residue lacking in the other channels, the intermediate TEA sensitivity of KCNQ1 and KCNQ4 implies that other residues are also important in determining TEA block of the KCNQ channels.

The purification, characterization, and synthesis of conantokin-R (Con-R), an N-methyl-D-aspartate (NMDA) receptor peptide antagonist from the venom of Conus radiatus, are described. With the use of well defined animal seizure models, Con-R was found to possess an anticonvulsant profile superior to that of ifenprodil and dizocilpine (MK-801). With voltage-clamp recording of Xenopus oocytes expressing heteromeric NMDA receptors from cloned NR1 and NR2 subunit RNAs, Con-R exhibited the following order of preference for NR2 subunits: NR2B approximately NR2A > NR2C >> NR2D. Con-R was without effect on oocytes expressing the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR1 or the kainate receptor subunit GluR6. In mouse cortical neurons voltage-clamped at -60 mV, Con-R application produced a slowly developing block of inward currents evoked by 10 microM NMDA and 1 microM glycine (IC(50) = 350 nM). At 3 microM, Con-R did not affect gamma-aminobutyric acid- or kainate-evoked currents. Con-R prevented sound-induced tonic extension seizures in the Frings audiogenic seizure-susceptible mice at i.c.v. doses below toxic levels. It was also effective at nontoxic doses in CF#1 mice against tonic extension seizures induced by threshold (15 mA) and maximal (50 mA) stimulation, and it partially blocked clonic seizures induced by s.c. pentylenetetrazol. In contrast, MK-801 and ifenprodil were effective only at doses approaching (audiogenic seizures) or exceeding (electrical and pentylenetetrazol seizures) those required to produce significant behavioral impairment. These results indicate that the subtype selectivity and other properties of Con-R afford a distinct advantage over the noncompetitive NMDA antagonists MK-801 and ifenprodil. Con-R is a useful new pharmacological agent for differentiation between the anticonvulsant and toxic effects of NMDA antagonists.

The potassium M current was originally identified in sympathetic ganglion cells, and analogous currents have been reported in some central neurons and also in some neural cell lines. It has recently been suggested that the M channel in sympathetic neurons comprises a heteromultimer of KCNQ2 and KCNQ3 (Wang et al., 1998) but it is unclear whether all other M-like currents are generated by these channels. Here we report that the M-like current previously described in NG108-15 mouse neuroblastoma x rat glioma cells has two components, "fast" and "slow", that may be differentiated kinetically and pharmacologically. We provide evidence from PCR analysis and expression studies to indicate that these two components are mediated by two distinct molecular species of K(+) channel: the fast component resembles that in sympathetic ganglia and is probably carried by KCNQ2/3 channels, whereas the slow component appears to be carried by merg1a channels. Thus, the channels generating M-like currents in different cells may be heterogeneous in molecular composition.

1. Using perforated-patch recordings, we have examined the part played by endogenous G-protein subunits in the alpha2-adrenoceptor-mediated inhibition of N-type Ca2+ currents in sympathetic neurones. 2. Two components of ICa inhibition by noradrenaline were recorded: a prominent, high affinity and voltage-dependent pertussis toxin (PTX)-sensitive pathway and a minor, low affinity and mostly voltage-insensitive PTX-resistant pathway. 3. PTX-sensitive inhibition was reduced by microinjection of antibodies against either GalphaoA,B or Galphai1,2. The voltage-dependent fraction of inhibition was reduced by anti-Galphao but not by anti-Galphai antibody. 4. Antisense depletion of GalphaoA led to a marked reduction of noradrenaline-induced inhibition and voltage dependence. By contrast, Galphai depletion attenuated noradrenergic modulation without affecting the voltage dependence. 5. Expression of the betagamma-binding agents beta-adrenergic receptor kinase 1 (C-terminus, betaARK1C-ter) or Galphai1 with a Cys3 to Ser mutation partially prevented noradrenergic inhibition while alpha-transducin abolished it. Residual inhibition was mostly voltage independent in cells expressing betaARK1C-ter but was strongly reversed by depolarization in Galphai1 Cys3Ser-expressing cells. 6. Expression of the PTX-resistant Galphai1 Cys351Ile mutant in cells treated with PTX restored alpha2-adrenoceptor inhibition. This restored inhibition was weakly reversed by depolarization. Both the degree and voltage dependence of inhibition were correlated with the level of expression of the Galphai1 Cys351Ile subunit. 7. Our findings identify betagamma dimers associated with GalphaoA and Galphai as mediators of the PTX-sensitive alpha2-adrenoceptor-mediated inhibition of N-type Ca2+ channels. Different betagamma combinations may account for the differential voltage-dependent effects of Go and Gi on ICa.

Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (IK(M)), which can be inhibited by activation of M1 muscarinic receptors. This inhibition occurs via pertussis toxin-insensitive G-proteins belonging to the Galphaq family (Caulfield et al., 1994 ). We have used DNA plasmids encoding antisense sequences against the 3' untranslated regions of Galpha subunits (antisense plasmids) to investigate the specific G-protein subunits involved in muscarinic inhibition of IK(M). These antisense plasmids specifically reduced levels of the target G-protein 48 hr after intranuclear injection. In cells depleted of Galphaq, muscarinic inhibition of IK(M) was attenuated compared both with uninjected neurons and with neurons injected with an inappropriate GalphaoA antisense plasmid. In contrast, depletion of Galpha11 protein did not alter IK(M) inhibition. To determine whether the alpha or beta gamma subunits of the G-protein mediated this inhibition, we have overexpressed the C terminus of beta adrenergic receptor kinase 1 (betaARK1), which binds free beta gamma subunits. betaARK1 did not reduce muscarinic inhibition of IK(M) at a concentration of plasmid that can reduce beta gamma-mediated inhibition of calcium current (). Also, expression of beta1gamma2 dimers did not alter the IK(M) density in SCG neurons. In contrast, IK(M) was virtually abolished in cells expressing GTPase-deficient, constitutively active forms of Galphaq and Galpha11. These data suggest that Galphaq is the principal mediator of muscarinic IK(M) inhibition in rat SCG neurons and that this more likely results from an effect of the alpha subunit than the beta gamma subunits of the Gq heterotrimer.

One postsynaptic action of the transmitter acetylcholine in sympathetic ganglia is to inhibit somatic N-type Ca2+ currents: this reduces Ca2+-activated K+ currents and facilitates high-frequency spiking. Previous experiments on rat superior cervical ganglion neurons have revealed two distinct pathways for this inhibitory action: a rapid, voltage-dependent inhibition through activation of M4 muscarinic acetylcholine receptors (mAChRs), and a slower, voltage-independent inhibition via M1 mAChRs [Hille (1994) Trends in Neurosci., 17, 531-536]. We have analysed the mechanistic basis for this divergence at the level of the individual G-proteins and their alpha and betagamma subunits, using a combination of site-directed antibody injection, plasmid-driven antisense RNA expression, overexpression of selected constitutively active subunits, and antagonism of endogenously liberated betagamma subunits by over-expression of Dy-binding P-adrenergic receptor kinase 1 (PARK1) peptide. The results indicate that: (i) M4 mAChR-induced inhibition is mediated by GoA; (ii) a and Py subunits released from the activated GoA heterotrimer produce separate voltage-insensitive and voltage-sensitive components of inhibition, respectively; and (iii) voltage-insensitive M1 mAChR-induced inhibition is likely to be mediated by the alpha subunit of Gq. Hence, Ca2+ current inhibition results from the concerted, but independent actions of three different G-protein subunits.

1. Using whole-cell and perforated-patch recordings, we have examined the part played by endogenous G-protein beta gamma subunits in neurotransmitter-mediated inhibition of N-type Ca2+ channel current (ICa) in dissociated rat superior cervical sympathetic neurones. 2. Expression of the C-terminus domain of beta-adrenergic receptor kinase 1 (beta ARK1), which contains the consensus motif (QXXER) for binding G beta gamma, reduced the fast (pertussis toxin (PTX)-sensitive) and voltage-dependent inhibition of ICa by noradrenaline and somatostatin, but not the slow (PTX-insensitive) and voltage-independent inhibition induced by angiotensin II. beta ARK1 peptide reduced GTP-gamma-S-induced voltage-dependent and PTX-sensitive inhibition of ICa but not GTP-gamma-S-mediated voltage-independent inhibition. 3. Overexpression of G beta 1 gamma 2, which mimicked the voltage-dependent inhibition by reducing ICa density and enhancing basal facilitation, occluded the voltage-dependent noradrenaline- and somatostatin-mediated inhibitions but not the inhibition mediated by angiotensin II. 4. Co-expression of the C-terminus of beta ARK1 with beta 1 and gamma 2 subunits prevented the effects of G beta gamma dimers on basal Ca2+ channel behaviour in a manner consistent with the sequestering of G beta gamma. 5. The expression of the C-terminus of beta ARK1 slowed down reinhibition kinetics of ICa following conditioning depolarizations and induced long-lasting facilitation by cumulatively sequestering beta gamma subunits. 6. Our findings identify endogenous G beta gamma as the mediator of the voltage-dependent, PTX-sensitive inhibition of ICa induced by both noradrenaline and somatostatin but not the voltage-independent. PTX-insensitive inhibition by angiotensin II. They also support the view that voltage-dependent inhibition results from a direct G beta gamma-Ca2+ channel interaction.

We report a novel post-translational modification involving halogenation of tryptophan in peptides recovered from the venom of carnivorous marine cone snails (Conus). The residue, L-6-bromotryptophan, was identified in the sequence of a heptapeptide, isolated from Conus imperialis, a worm-hunting cone. This peptide does not elicit gross behavioral symptoms when injected centrally or peripherally in mice. L-6-Bromotryptophan was also identified in a 33-amino acid peptide from Conus radiatus; this peptide has been shown to induce a sleep-like state in mice of all ages and is referred to as bromosleeper peptide. The sequences of the two peptides and were determined using a combination of mass spectrometry, amino acid, and chemical sequence analyses, where Pca = pyroglutamic acid, Hyp = hydroxyproline, Gla = gamma-carboxyglutamate, and Trp* = L-6-bromotryptophan. The precise structure and stereochemistry of the modified residue were determined as L-6-bromotryptophan by synthesis, co-elution, and enzymatic hydrolysis experiments. To our knowledge this is the first documentation of tryptophan residues in peptides/proteins being modified in a eukaryotic system and the first report of halogenation of tryptophan in vivo.

The receptor subtype and transduction mechanisms involved in the regulation of various neuronal ionic currents are reviewed, with some recent observations on sympathetic neurons, hippocampal cell membranes and basal forebrain cells.

A 2.7 kb clone encoding the partial (about 66%) sequence of a phosphoinositide-specific phospholipase C (PLC) was isolated from a cDNA library constructed from channel catfish (Ictalurus punctatus) olfactory rosettes. The clone, designated 30c7, was completely sequenced by automated DNA sequencing and was found to share significant homology with rat and bovine PLCs of the delta 1 isotype. In situ hybridization showed that 30c7 transcripts were expressed in a small subpopulation of olfactory neurons, as well as in other cell types in the olfactory epithelium. Polymerase chain reaction (PCR) analysis indicated that the enzyme was also expressed in several additional tissues, including brain, gill, heart, liver and skeletal muscle. These results suggest that the PLC encoded by clone 30c7 is expressed in several tissues and therefore may have a role in mediating transduction events in diverse tissues as well as in a small group of olfactory neurons.

Olfactory signal transduction in a number of species has been shown to be mediated by heterotrimeric GTP-binding proteins (G-proteins). The expression of different G-proteins in channel catfish (Ictalurus punctatus) olfactory epithelium was investigated using antibodies to both the alpha and beta subunits of G-proteins. Based on Western blotting and immunohistochemical data, the following G-protein subunits were identified in the olfactory epithelium: Gs/G(olf), Gi1, Gi2, Gq and G beta. Immunohistochemical results indicated that all of these G-proteins, encompassing three G-protein subfamilies, were expressed in the dendrites and cilia of olfactory receptor neurons. These findings suggest that different G-protein subunits may mediate multiple signal transduction pathways in the catfish olfactory system, i.e. G(olf)/Gs, may mediate odorant activation of adenylyl cyclase while Gi and G beta may mediate odorant activation of phospholipase C.

The expression of three phosphoinositide-specific phospholipase C (PLC) isotypes in rat olfactory epithelium was investigated using monoclonal antibodies. In intact animals, PLC beta 1 was not expressed in the olfactory epithelium but was found in glands below the epithelium. However, following unilateral olfactory bulbectomy (OBX), PLC beta 1 was expressed in the dentrites of some olfactory receptor neurons, primarily in the endoturbinates on the unoperated side. PLC gamma 1 immunoreactivity was found in the apices of sustentacular cells and in glands below the epithelium. PLC delta 1 immunoreactivity was found in the glands and in the perinuclear region and dendrites of some receptor neurons. Since none of the PLC isotypes studied were expressed in the cilia of receptor neurons, the results suggest that another PLC isotype is likely to be involved in mediating olfactory transduction.

Conus venoms contain a remarkable diversity of pharmacologically active small peptides. Their targets are ion channels and receptors in the neuromuscular system. The venom of Conus geographus contains high-affinity peptides that act on voltage-sensitive calcium channels, sodium channels, N-methyl-D-aspartate (NMDA) receptors, acetylcholine receptors, and vasopressin receptors; many more peptides with still uncharacterized receptor targets are present in this venom. It now seems that the Conus species (approximately 500 in number) will each use a distinctive assortment of peptides and that the pharmacological diversity in Conus venoms may be ultimately comparable to that of plant alkaloids or secondary metabolites of microorganisms. The cone snails may generate this diverse spectrum of venom peptides by a "fold-lock-cut" synthetic pathway. These peptides are specific enough to discriminate effectively between closely related receptor subtypes and can be used for structure-function correlations.