Effects of serum immunoglobulins from patients with complex regional pain syndrome (CRPS) on depolarisation-induced calcium transients in isolated dorsal root ganglion (DRG) neurons.

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 24h 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.

The actions of Pasteurella multocida toxin on neuronal cells.

Pasteurella multocida toxin (PMT) activates the G-proteins Gαi(1₋3), Gα(q), Gα11, Gα12 and Gα13 by deamidation of specific glutamine residues. A number of these alpha subunits have signalling roles in neurones. Hence we studied the action of this toxin on rat superior cervical ganglion (SCG) neurones and NG108-15 neuronal cells. Both Gα(q) and Gα11 could be identified in SCGs with immunocytochemistry. PMT had no direct action on Kv7 or Cav2 channels in SCGs. However PMT treatment enhanced muscarinic receptor mediated inhibition of M-current (Kv7.2 + 7. 3) as measured by a 19-fold leftward shift in the oxotremorine-M concentration-inhibition curve. Agonists of other receptors, such as bradykinin or angiotensin, that inhibit M-current did not produce this effect. However the amount of PIP2 hydrolysis could be enhanced by PMT for all three agonists. In a transduction system in SCGs that is unlikely to be affected by PMT, Go mediated inhibition of calcium current, PMT was ineffective whereas the response was blocked by pertussis toxin as expected. M1 muscarinic receptor evoked calcium mobilisation in transformed NG108-15 cells was enhanced by PMT. The calcium rises evoked by uridine triphosphate acting on endogenous P2Y2 receptors in NG108-15 cells were enhanced by PMT. The time and concentration dependence of the PMT effect was different for the resting calcium compared to the calcium rise produced by activation of P2Y2 receptors. PMT's action on these neuronal cells would suggest that if it got into the brain, symptoms of a hyperexcitable nature would be seen, such as seizures.

Effects of KCNQ2 gene truncation on M-type Kv7 potassium currents.

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.

Kv7/M-type potassium channels in rat skin keratinocytes.

Skin keratinocytes fulfil important signalling and protective functions. Immunocytochemical experiments revealed the unexpected presence of immunoreactivity for the M-type potassium channel subunit Kv7.2 in the keratinocyte layer of intact rat paw skin and in keratinocytes isolated from the skin of 1-day-old rats and cultured in vitro for 3-10 days. Application of the M-channel enhancer retigabine (3-10 µM) to isolated cultured rat keratinocytes: (a) increased outward membrane currents recorded under voltage clamp, (b) produced ~3 mV hyperpolarization at rest, (c) enhanced ~3-fold the release of ATP induced by the TRPV3 agonist carvacrol (1 mM) and (d) increased the amplitude of the carvacrol-induced intracellular Ca(2+) transient measured with Fura-2. The effect of retigabine on ATP release was prevented by the M-channel blocking agent XE991. We conclude that rat skin keratinocytes possess M-channels that, when activated, can modify their physiological properties, with potential significance for their sensory and other biological functions.

Functional significance of M-type potassium channels in nociceptive cutaneous sensory endings.

M-channels carry slowly activating potassium currents that regulate excitability in a variety of central and peripheral neurons. Functional M-channels and their Kv7 channel correlates are expressed throughout the somatosensory nervous system where they may play an important role in controlling sensory nerve activity. Here we show that Kv7.2 immunoreactivity is expressed in the peripheral terminals of nociceptive primary afferents. Electrophysiological recordings from single afferents in vitro showed that block of M-channels by 3 µM XE991 sensitized Aδ- but not C-fibers to noxious heat stimulation and induced spontaneous, ongoing activity at 32°C in many Aδ-fibers. These observations were extended in vivo: intraplantar injection of XE991 selectively enhanced the response of deep dorsal horn (DH) neurons to peripheral mid-range mechanical and higher range thermal stimuli, consistent with a selective effect on Aδ-fiber peripheral terminals. These results demonstrate an important physiological role of M-channels in controlling nociceptive Aδ-fiber responses and provide a rationale for the nocifensive behaviors that arise following intraplantar injection of the M-channel blocker XE991.

Structural requirements of membrane phospholipids for M-type potassium channel activation and binding.

M-channels are voltage-gated potassium channels that regulate cell excitability. They are heterotetrameric assemblies of Kv7.2 and Kv7.3 subunits. Their opening requires the presence of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)). However, the specificity of PI(4,5)P(2) as a binding and activating ligand is unknown. Here, we tested the ability of different phosphoinositides and lipid phosphates to activate or bind to M-channel proteins. Activation of functional channels was measured in membrane patches isolated from cells coexpressing Kv7.2 and Kv7.3 subunits. Channels were activated to similar extents (maximum open probability of ∼0.8 at 0 mV) by 0.1-300 µM dioctanoyl homologs of the three endogenous phosphoinositides, PI(4)P, PI(4,5)P(2), and PI(3,4,5)P(3), with sensitivity increasing with increasing numbers of phosphates. Non-acylated inositol phosphates had no effect up to 100 µM. Channels were also activated with increasing efficacy by 1-300 µM concentrations of the monoacyl monophosphates fingolimod phosphate, sphingosine 1-phosphate, and lysophosphatidic acid but not by phosphate-free fingolimod or sphingosine or by phosphate-masked phosphatidylcholine or phosphatidylglycerol. An overlay assay confirmed that a fusion protein containing the full-length C terminus of Kv7.2 could bind to a broad range of phosphoinositides and phospholipids. A mutated Kv7.2 C-terminal construct with reduced sensitivity to PI(4,5)P showed significantly less binding to most polyphosphoinositides. We concluded that M-channels bind to, and are activated by, a wide range of lipid phosphates, with a minimum requirement for an acyl chain and a phosphate headgroup. In this, they more closely resemble inwardly rectifying Kir6.2 potassium channels than the more PI(4,5)P(2)-specific Kir2 channels. Notwithstanding, the data also support the view that the main endogenous activator of M-channels is PI(4,5)P(2).