The prime task of nociceptors is the transformation of noxious stimuli into action potentials that are propagated along the neurites of nociceptive neurons from the periphery to the spinal cord. This ...function of nociceptors relies on the coordinated operation of a variety of ion channels. In this review, we summarize how members of nine different families of ion channels expressed in sensory neurons contribute to nociception. Furthermore, data on 35 different types of G protein coupled receptors are presented, activation of which controls the gating of the aforementioned ion channels. These receptors are not only targeted by more than 20 separate endogenous modulators, but can also be affected by pharmacotherapeutic agents. Thereby, this review provides information on how ion channel modulation via G protein coupled receptors in nociceptors can be exploited to provide improved analgesic therapy.
Paroxysmal depolarization shifts (PDS) have been described by epileptologists for the first time several decades ago, but controversy still exists to date regarding their role in epilepsy. In ...addition to the initial view of a lack of such a role, seemingly opposing hypotheses on epileptogenic and anti-ictogenic effects of PDS have emerged. Hence, PDS may provide novel targets for epilepsy therapy. Evidence for the roles of PDS has often been obtained from investigations of the multi-unit correlate of PDS, an electrographic spike termed "interictal" because of its occurrence during seizure-free periods of epilepsy patients. Meanwhile, interictal spikes have been found to be associated with neuronal diseases other than epilepsy, e.g., Alzheimer's disease, which may indicate a broader implication of PDS in neuropathologies. In this article, we give an introduction to PDS and review evidence that links PDS to pro- as well as anti-epileptic mechanisms, and to other types of neuronal dysfunction. The perturbation of neuronal membrane voltage and of intracellular Ca
that comes with PDS offers many conceivable pathomechanisms of neuronal dysfunction. Out of these, the operation of L-type voltage-gated calcium channels, which play a major role in coupling excitation to long-lasting neuronal changes, is addressed in detail.
Serotonin (5HT) is a constituent of the so-called “inflammatory soup” that sensitizes nociceptors during inflammation. Nevertheless, receptors and signaling mechanisms that mediate an excitation of ...dorsal root ganglion (DRG) neurons by 5HT remained controversial. Therefore, capsaicin-sensitive nociceptive neurons dissociated from rat DRGs were used to investigate effects of 5HT on membrane excitability and currents through ligand- as well as voltage-gated ion channels. In 58% of the neurons tested, 5HT increased action potential firing, an effect that was abolished by the 5HT2 receptor antagonist ritanserin, but not by the 5HT3 antagonist tropisetron. Unlike other algogenic mediators, such as PGE2 and bradykinin, 5HT did not affect currents through TTX-resistant Na+ channels or Kv7 K+ channels. In all neurons investigated, 5HT potentiated capsaicin-evoked currents through TRPV1 channels, an effect that was attenuated by antagonists at 5HT2A (4 F 4 PP), 5HT2B (SB 204741), as well as 5HT2C (RS 102221) receptors. 5HT triggered slowly arising inward Cl− currents in 53% of the neurons. This effect was antagonized by the 5HT2C receptor blocker only, and the current was prevented by an inhibitor of Ca2+-activated chloride channels (CaCC). The 5HT-induced increase in action potential firing was also abolished by this CaCC blocker and by the TRPV1 inhibitor capsazepine. Amongst the subtype selective 5HT2 antagonists, only RS 102221 (5HT2C-selectively) counteracted the rise in action potential firing elicited by 5HT. These results show that 5HT excites DRG neurons mainly via 5HT2C receptors which concomitantly mediate a sensitization of TRPV1 channels and an opening of CaCCs.
•5HT sensitizes TRPV1 channels in DRG neurons via 5HT2A, 5HT2B, as well as 5HT2C receptors.•5HT triggers inward currents through Ca2+-activated Cl- channels of DRG neurons via 5HT2C, but not 5HT2A or 2B receptors.•5HT causes an increase in action potential firing in DRG neurons that involves TRPV1 and Ca2+-activated Cl− channels.•The increase in action potential firing in DRG neurons caused by 5HT involves 5HT2C, but not 5HT2A or 2B receptors.•5HT2C rather than 5HT2A or 2B receptors mediate the excitation of DRG neurons by 5HT.
Many drugs used in cardiovascular therapy, such as angiotensin receptor antagonists and beta-blockers, may exert at least some of their actions through effects on the sympathetic nervous system, and ...this also holds true for e.g., P2Y
12
antagonists. A new target at the horizon of cardiovascular drugs is the P2Y
6
receptor which contributes to the development of arteriosclerosis and hypertension. To learn whether P2Y
6
receptors in the sympathetic nervous system might contribute to actions of respective receptor ligands, responses of sympathetic neurons to P2Y
6
receptor activation were analyzed in primary cell culture. UDP in a concentration dependent manner caused membrane depolarization and enhanced numbers of action potentials fired in response to current injections. The excitatory action was antagonized by the P2Y
6
receptor antagonist MRS2578, but not by the P2Y
2
antagonist AR-C118925XX. UDP raised intracellular Ca
2+
in the same range of concentrations as it enhanced excitability and elicited inward currents under conditions that favor Cl
−
conductances, and these were reduced by a blocker of Ca
2+
-activated Cl
−
channels, CaCCInh-A01. In addition, UDP inhibited currents through K
V
7 channels. The increase in numbers of action potentials caused by UDP was not altered by the K
V
7 channel blocker linopirdine, but was enhanced in low extracellular Cl
−
and was reduced by CaCCInh-A01 and by an inhibitor of phospholipase C. Moreover, UDP enhanced release of previously incorporated 3H noradrenaline, and this was augmented in low extracellular Cl
−
and by linopirdine, but attenuated by CaCCInh-A01. Together, these results reveal sympathoexcitatory actions of P2Y
6
receptor activation involving Ca
2+
-activated Cl
−
channels.
Introduction:
In addition to members of the family of Na
+
/Cl
−
dependent monoamine transporters, organic cation transporters (OCTs), in particular OCT3, as well as the plasma membrane monoamine ...transporter (PMAT) may contribute to neuronal reuptake of according neurotransmitters. As opposed to the numerous blockers of monoamine transporters, only a very limited number of specific blockers of OCT3 and PMAT are available. In fact, decynium-22 is the only blocking agent with micromolar affinities for both transport proteins, and this molecule is frequently used to establish roles of OCT3 and/or PMAT as targets for antidepressant drugs and psychostimulants, respectively.
Methods/Results:
To test for a function of these transporters in the sympathetic nervous system, uptake and release of 3H1-methyl-4-phenylpyridinium (MPP
+
) was investigated in primary cultures of rat superior cervical ganglia. Uptake was reduced by cocaine or desipramine, blockers of the noradrenaline transporter, by about 70% and by corticosterone or β-estradiol, blockers of OCT3, by about 30%; decynium-22 achieved complete inhibition of uptake with half maximal effects at 3 μM. Depolarization dependent release was enhanced by corticosterone or β-estradiol, but reduced by decynium-22. As the latter effect is unlikely to be related to actions at OCT3 and/or PMAT, electrophysiological recordings were performed to reveal that decynium-22 inhibits action potential firing and currents through voltage activated calcium channels in superior cervical ganglion neurons.
Discussion:
These results demonstrate that decynium-22 can impair exocytotic neurotransmitter release by interfering with several types of ion channels. Such transporter-independent effects of decynium-22 that my interfere with basic neuronal functions need to be considered when interpreting results obtained with decynium-22 as prototypic inhibitor of transmitter reuptake via OCT3 and/or PMAT.
Amphetamine abuse is a major public health concern for which there is currently no effective treatment. To develop effective treatments, the mechanisms by which amphetamine produces its abuse-related ...effects need to be fully understood. It is well known that amphetamine exerts its actions by targeting high-affinity transporters for monoamines, in particular the cocaine-sensitive dopamine transporter. Organic cation transporter 3 (OCT3) has recently been found to play an important role in regulating monoamine signaling. However, whether OCT3 contributes to the actions of amphetamine is unclear. We found that OCT3 is expressed in dopamine neurons. Then, applying a combination of in vivo, ex vivo, and in vitro approaches, we revealed that a substantial component of amphetamine's actions is OCT3-dependent and cocaine insensitive. Our findings support OCT3 as a new player in the actions of amphetamine and encourage investigation of this transporter as a potential new target for the treatment of psychostimulant abuse.
The mechanism of acetaminophen (APAP) analgesia is at least partially unknown. Previously, we showed that the APAP metabolite N-acetyl-p-benzoquinone imine (NAPQI) activated Kv7 channels in neurons ...in vitro, and this activation of Kv7 channels dampened neuronal firing. Here, the effect of the Kv7 channel blocker XE991 on APAP-induced analgesia was investigated in vivo. APAP had no effect on naive animals. Induction of inflammation with λ-carrageenan lowered mechanical and thermal thresholds. Systemic treatment with APAP reduced mechanical hyperalgesia, and co-application of XE991 reduced APAP's analgesic effect on mechanical pain. In a second experiment, the analgesic effect of systemic APAP was not antagonized by intrathecal XE991 application. Analysis of liver samples revealed APAP and glutathione-coupled APAP indicative of metabolization. However, there were no relevant levels of these metabolites in cerebrospinal fluid, suggesting no relevant APAP metabolite formation in the CNS. In summary, the results support an analgesic action of APAP by activating Kv7 channels at a peripheral site through formation of the metabolite NAPQI.
The release of transmitters at sympathoeffector junctions is not constant, but subject to modulation by a plethora of different mechanisms. In this respect, presynaptic receptors located on the ...sympathetic axon terminals are of utmost importance, because they are activated by exogenous agonists and by endogenous neurotransmitters. In the latter case, the transmitters that activate the presynaptic receptors of a nerve terminal may be released either from the very same nerve ending or from a different axon terminal, and the receptors involved are auto- and heteroreceptors, respectively. In terms of their structural and functional features, receptors of sympathetic axon terminals can be categorized as either ionotropic (transmitter-gated ion channels) or metabotropic (most commonly G protein-coupled) receptors. This review summarizes results on more than 30 different metabotropic and four different ionotropic receptors that have been found to control the amount of transmitter being released from sympathetic neurons. Each of these receptors may not only stimulate, facilitate, and reduce sympathetic transmitter release, respectively, but also interact with the functions of other receptors present on the same axonal varicosity. This provides a multitude of mechanisms that regulate the amount of sympathetic transmitter output. Accordingly, a sophisticated cross-talk within and between extra- and intracellular signals is integrated at axon terminals to adapt the strength of sympathoeffector transmission to a given situation. This will not only determine the function of the sympathetic nervous system in health and disease, but also therapeutic and untoward effects of drugs that bind to the presynaptic receptors in sympathetically innervated tissues.
Key points
Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is a key regulator of many membrane proteins, including voltage‐gated Kv7.2 channels.
In this study, we identified the residues in five ...phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2‐binding domains in Kv7.2.
Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2.
Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors.
Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2, thereby ensuring the tight regulation of the channel via G protein‐coupled receptors.
The function of numerous ion channels is tightly controlled by G protein‐coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol‐4,5‐bisphosphate (PIP2). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP2 and through phosphorylation. Using liquid chromatography‐coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2‐binding domains. To evaluate the effect of phosphorylation on PIP2‐mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A5 mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP2 depletion via the voltage‐sensitive phosphatase Dr‐VSP than were wild‐type channels. In vitro phosphorylation assays with the purified C‐terminus of Kv7.2 revealed that CDK5, p38 MAPK, CaMKIIα and PKA were able to phosphorylate the five serines. Inhibition of these protein kinases reduced the sensitivity of wild‐type but not mutant Kv7.2 channels towards PIP2 depletion via Dr‐VSP. In superior cervical ganglion neurons, the protein kinase inhibitors attenuated Kv7 current regulation via M1 receptors, but left unaltered the control by B2 receptors. Our results revealed that the phosphorylation status of serines located within a putative PIP2‐binding domain determined the phospholipid sensitivity of Kv7.2 channels and supported GPCR‐mediated channel regulation.
Key points
Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is a key regulator of many membrane proteins, including voltage‐gated Kv7.2 channels.
In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2‐binding domains in Kv7.2.
Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2.
Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors.
Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2, thereby ensuring the tight regulation of the channel via G protein‐coupled receptors.
Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP ₂) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters ...(SERTs); however, SERT regulation by PIP ₂ has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP ₂ by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP ₂-synthesis. We tested the effects of the verified scarcity of PIP ₂ on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP ₂ in pull-down assays. On decreased PIP ₂ availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP ₂ reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP ₂. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP ₂-dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP ₂ binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.
PDF
