Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate.

Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP2) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP2 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 PIP2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP2-synthesis. We tested the effects of the verified scarcity of PIP2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP2 in pull-down assays. On decreased PIP2 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 PIP2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP2. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP2-dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP2 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.

Dynamic interplay of excitatory and inhibitory coupling modes of neuronal L-type calcium channels.

L-type voltage-gated calcium channels (LTCCs) have long been considered as crucial regulators of neuronal excitability. This role is thought to rely largely on coupling of LTCC-mediated Ca(2+) influx to Ca(2+)-dependent conductances, namely Ca(2+)-dependent K(+) (K(Ca)) channels and nonspecific cation (CAN) channels, which mediate afterhyperpolarizations (AHPs) and afterdepolarizations (ADPs), respectively. However, in which manner LTCCs, K(Ca) channels, and CAN channels co-operate remained scarcely known. In this study, we examined how activation of LTCCs affects neuronal depolarizations and analyzed the contribution of Ca(2+)-dependent potassium- and cation-conductances. With the use of hippocampal neurons in primary culture, pulsed current-injections were applied in the presence of tetrodotoxin (TTX) for stepwise depolarization and the availability of LTCCs was modulated by BAY K 8644 and isradipine. By varying pulse length and current strength, we found that weak depolarizing stimuli tend to be enhanced by LTCC activation, whereas in the course of stronger depolarizations LTCCs counteract excitation. Both effect modes appear to involve the same channels that mediate ADP and AHP, respectively. Indeed, ADPs were activated at lower stimulation levels than AHPs. In the absence of TTX, activation of LTCCs prolonged or shortened burst firing, depending on the initial burst duration, and invariably augmented brief unprovoked (such as excitatory postsynaptic potentials) and provoked electrical events. Hence, regulation of membrane excitability by LTCCs involves synchronous activity of both excitatory and inhibitory Ca(2+)-activated ion channels. The overall enhancing or dampening effect of LTCC stimulation on excitability does not only depend on the relative abundance of the respective coupling partner but also on the stimulus intensity.

P2Y1 receptors mediate an activation of neuronal calcium-dependent K+ channels.

Molecularly defined P2Y receptor subtypes are known to regulate the functions of neurons through an inhibition of K(V)7 K(+) and Ca(V)2 Ca(2+) channels and via an activation or inhibition of Kir3 channels. Here, we searched for additional neuronal ion channels as targets for P2Y receptors. Rat P2Y(1) receptors were expressed in PC12 cells via an inducible expression system, and the effects of nucleotides on membrane currents and intracellular Ca(2+) were investigated. At a membrane potential of 30 mV, ADP induced transient outward currents in a concentration-dependent manner with half-maximal effects at 4 µm. These currents had reversal potentials close to the K(+) equilibrium potential and changed direction when extracellular Na(+) was largely replaced by K(+), but remained unaltered when extracellular Cl() was changed. Currents were abolished by P2Y(1) antagonists and by blockade of phospholipase C. ADP also caused rises in intracellular Ca(2+), and ADP-evoked currents were abolished when inositol trisphosphate-sensitive Ca(2+) stores were depleted. Blockers of K(Ca)2, but not those of K(Ca)1.1 or K(Ca)3.1, channels largely reduced ADP-evoked currents. In hippocampal neurons, ADP also triggered outward currents at 30 mV which were attenuated by P2Y(1) antagonists, depletion of Ca(2+) stores, or a blocker of K(Ca)2 channels. These results demonstrate that activation of neuronal P2Y(1) receptors may gate Ca(2+)-dependent K(+) (K(Ca)2) channels via phospholipase C-dependent increases in intracellular Ca(2+) and thereby define an additional class of neuronal ion channels as novel effectors for P2Y receptors. This mechanism may form the basis for the control of synaptic plasticity via P2Y(1) receptors.

Raised activity of L-type calcium channels renders neurons prone to form paroxysmal depolarization shifts.

Neuronal L-type voltage-gated calcium channels (LTCCs) are involved in several physiological functions, but increased activity of LTCCs has been linked to pathology. Due to the coupling of LTCC-mediated Ca(2+) influx to Ca(2+)-dependent conductances, such as KCa or non-specific cation channels, LTCCs act as important regulators of neuronal excitability. Augmentation of after-hyperpolarizations may be one mechanism that shows how elevated LTCC activity can lead to neurological malfunctions. However, little is known about other impacts on electrical discharge activity. We used pharmacological up-regulation of LTCCs to address this issue on primary rat hippocampal neurons. Potentiation of LTCCs with Bay K8644 enhanced excitatory postsynaptic potentials to various degrees and eventually resulted in paroxysmal depolarization shifts (PDS). Under conditions of disturbed Ca(2+) homeostasis, PDS were evoked frequently upon LTCC potentiation. Exposing the neurons to oxidative stress using hydrogen peroxide also induced LTCC-dependent PDS. Hence, raising LTCC activity had unidirectional effects on brief electrical signals and increased the likeliness of epileptiform events. However, long-lasting seizure-like activity induced by various pharmacological means was affected by Bay K8644 in a bimodal manner, with increases in one group of neurons and decreases in another group. In each group, isradipine exerted the opposite effect. This suggests that therapeutic reduction in LTCC activity may have little beneficial or even adverse effects on long-lasting abnormal discharge activities. However, our data identify enhanced activity of LTCCs as one precipitating cause of PDS. Because evidence is continuously accumulating that PDS represent important elements in neuropathogenesis, LTCCs may provide valuable targets for neuroprophylactic therapy.

Concomitant facilitation of GABAA receptors and KV7 channels by the non-opioid analgesic flupirtine.

BACKGROUND AND PURPOSE: Flupirtine is a non-opioid analgesic that has been in clinical use for more than 20 years. It is characterized as a selective neuronal potassium channel opener (SNEPCO). Nevertheless, its mechanisms of action remain controversial and are the purpose of this study. EXPERIMENTAL APPROACH: Effects of flupirtine on native and recombinant voltage- and ligand-gated ion channels were explored in patch-clamp experiments using the following experimental systems: recombinant K(IR)3 and K(V)7 channels and α3ß4 nicotinic acetylcholine receptors expressed in tsA 201 cells; native voltage-gated Na(+), Ca(2+), inward rectifier K(+), K(V)7 K(+), and TRPV1 channels, as well as GABA(A), glycine, and ionotropic glutamate receptors expressed in rat dorsal root ganglion, dorsal horn and hippocampal neurons. KEY RESULTS: Therapeutic flupirtine concentrations (≤10 µM) did not affect voltage-gated Na(+) or Ca(2+) channels, inward rectifier K(+) channels, nicotinic acetylcholine receptors, glycine or ionotropic glutamate receptors. Flupirtine shifted the gating of K(V)7 K(+) channels to more negative potentials and the gating of GABA(A) receptors to lower GABA concentrations. These latter effects were more pronounced in dorsal root ganglion and dorsal horn neurons than in hippocampal neurons. In dorsal root ganglion and dorsal horn neurons, the facilitatory effect of therapeutic flupirtine concentrations on K(V)7 channels and GABA(A) receptors was comparable, whereas in hippocampal neurons the effects on K(V)7 channels were more pronounced. CONCLUSIONS AND IMPLICATIONS: These results indicate that flupirtine exerts its analgesic action by acting on both GABA(A) receptors and K(V)7 channels.

Biosynthesis of truncated N-linked oligosaccharides results from non-orthologous hexosaminidase-mediated mechanisms in nematodes, plants, and insects.

In many invertebrates and plants, the N-glycosylation profile is dominated by truncated paucimannosidic N-glycans, i.e. glycans consisting of a simple trimannosylchitobiosyl core often modified by core fucose residues. Even though they lack antennal N-acetylglucosamine residues, the biosynthesis of these glycans requires the sequential action of GlcNAc transferase I, Golgi mannosidase II, and, finally, beta-N-acetylglucosaminidases. In Drosophila, the recently characterized enzyme encoded by the fused lobes (fdl) gene specifically removes the non-reducing N-acetylglucosamine residue from the alpha1,3-antenna of N-glycans. In the present study, we examined the products of five beta-N-acetylhexosaminidase genes from Caenorhabditis elegans (hex-1 to hex-5, corresponding to reading frames T14F9.3, C14C11.3, Y39A1C.4, Y51F10.5, and Y70D2A.2) in addition to three from Arabidopsis thaliana (AtHEX1, AtHEX2, and AtHEX3, corresponding to reading frames At1g65590, At3g55260, and At1g05590). Based on homology, the Caenorhabditis HEX-1 and all three Arabidopsis enzymes are members of the same sub-family as the aforementioned Drosophila fused lobes enzyme but either act as chitotriosidases or non-specifically remove N-acetylglucosamine from both N-glycan antennae. The other four Caenorhabditis enzymes are members of a distinct sub-family; nevertheless, two of these enzymes displayed the same alpha1,3-antennal specificity as the fused lobes enzyme. Furthermore, a deletion of part of the Caenorhabditis hex-2 gene drastically reduces the native N-glycan-specific hexosaminidase activity in mutant worm extracts and results in a shift in the N-glycan profile, which is a demonstration of its in vivo enzymatic relevance. Based on these data, it is hypothesized that the genetic origin of paucimannosidic glycans in nematodes, plants, and insects involves highly divergent members of the same hexosaminidase gene family.