Alexander K. Filippov

INTRACELLULAR RETENTION OF RECOMBINANT GABAB RECEPTORS

γ-Aminobutyric acid type B (GABAB) receptors mediate the transmission of slow and prolonged inhibitory signals in the central nervous system. Two splice variants of GABAB receptors, GABABR1a and GABABR1b, were recently cloned from a mouse cortical and cerebellar cDNA library. As predicted, these receptors belong to the G protein-coupled receptor superfamily. We have used epitope-tagged versions of GABABR1a receptors to study the cellular distribution of these proteins in a variety of non-neuronal and neuronal cell types. Here we report that recombinant GABAB receptors fail to reach the cell surface when expressed in heterologous systems and are retained in the endoplasmic reticulum when introduced into COS cells. In addition, we prove that recombinant GABAB receptors are excluded from the cell surface when overexpressed in ganglion neurons and we further demonstrate that they fail to activate in superior cervical ganglion neurons. Together our observations suggest that recombinant GABAB receptors require additional information for functional targeting to the plasma membrane.

Relationship between Membrane Phosphatidylinositol-4,5-Bisphosphate and Receptor-Mediated Inhibition of Native Neuronal M Channels

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 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.

The P2Y1 receptor closes the N-type Ca2+ channel in neurones, with both adenosine triphosphates and diphosphates as potent agonists

The rat P2Y1 nucleotide receptor, the P2Y subtype abundant in the brain, was heterologously expressed in rat superior cervical ganglion neurones by micro‐injection of the receptor cRNA or cDNA. ADP inhibited the N‐type Ca2+ current by 64%, with EC50 8.2 nM, an action blocked competitively by the P2Y1 receptor antagonist adenosine 3′, 5′‐bis‐phosphate (Ki 0.7 μM). 2‐Methylthio‐ADP inhibited the Ca2+ current likewise, but with EC50 0.57 nM, giving the highest potency reported therewith for P2Y1. Significantly, ATP and 2‐methylthio‐ATP were also agonists, the latter again at a very high potency (EC50 2.5 nM). We propose that this neuronal receptor, when present in brain at a high density as at synapses, can respond to very low concentrations of ATP and ADP as agonists, and that this would result in inhibition of N‐type Ca2+ currents and hence can reduce transmitter release or increase neuronal excitability.

Dual coupling of heterologously-expressed rat P2Y6 nucleotide receptors to N-type Ca2+ and M-type K+ currents in rat sympathetic neurones.

The P2Y6 receptor is a uridine nucleotide‐specific G protein‐linked receptor previously reported to stimulate the phosphoinositide (PI) pathway. We have investigated its effect in neurones, by micro‐injecting its cRNA into dissociated rat sympathetic neurones and recording responses of N‐type Ca2+ (ICa(N)) and M‐type K+ (IK(M)) currents. In P2Y6 cRNA‐injected neurones, UDP or UTP produced a voltage‐dependent inhibition of ICa(N) by ∼53% in whole‐cell (disrupted‐patch) mode and by ∼73% in perforated‐patch mode; no inhibition occurred in control cells. Mean IC50 values (whole‐cell) were: UDP, 5.9±0.3 nM; UTP, 20±1 nM. ATP and ADP (1 μM) had no significant effect. Pertussis toxin (PTX) substantially (∼60%) reduced UTP‐mediated inhibition in disrupted patch mode but not in perforated‐patch mode. Uridine nucleotides also inhibited IK(M) in P2Y6 cRNA‐injected cells (by up to 71% at 10 μM UTP; perforated‐patch). Mean IC50 values were: UDP, 30±3 nM; UTP, 115±12 nM. ATP (10 μM) again had no effect. No significant inhibition occurred in control cells. Inhibition was PTX‐resistant. Thus, the P2Y6 receptor, like the P2Y2 subtype studied in this system, couples to both of these two neuronal ion channels through at least two different G proteins. However, the P2Y6 receptor displays a much higher sensitivity to its agonists than the P2Y2 receptor in this expression system and higher than previously reported using other expression methods. The very high sensitivity to both UDP and UTP suggests that it might be preferentially activated by any locally released uridine nucleotides.

Inhibition of potassium and calcium currents in neurones by molecularly-defined P2Y receptors

Messenger RNAs and cDNAs for individual cloned P2Y(1), P2Y2 and P2Y(6) nucleotide receptors have been expressed by micro-injection into dissociated rat superior cervical sympathetic neurones and the effects of stimulating the expressed receptors on voltage-activated N-type Ca(2+) currents and M-type K(+) currents recorded. Both currents were reduced by stimulating all three receptors, with the following mean IC(50) values: P2Y(1) (agonist: ADP) - I(K(M)) 6.9 nM, I(Ca) 8.2 nM; P2Y(2) (agonist: UTP) - I(K(M)) 1.5 microM, I(Ca) 0.5 microM; P2Y(6) (agonist: UDP) - I(K(M)) 30 nM, I(Ca) 5.9 nM. Inhibition of I(K(M)) was voltage-independent and insensitive to Pertussis toxin; inhibition of I(Ca) showed both voltage-sensitive and insensitive, and Pertussis toxin-sensitive and insensitive components. It is concluded that these P2Y receptors can couple to more than one G protein and thereby modulate more than one ion channel. It is suggested that these effects on K(M) and Ca(N) channels may induce both postsynaptic excitory and presynaptic inhibitory responses.

Coupling of the nucleotide P2Y4 receptor to neuronal ion channels

G protein‐linked P2Y nucleotide receptors are known commonly to stimulate the phosphoinositide signalling pathway. However, we have previously demonstrated that the cloned P2Y2, P2Y6 and P2Y1 receptors couple to neuronal N‐type Ca2+ channels and to M‐type K+ channels. Here we investigate the coupling of recombinant, neuronally expressed rat‐ and human P2Y4 receptors (rP2Y4, hP2Y4) to those channels. Rat sympathetic neurones were nuclear‐injected with a P2Y4 cDNA plasmid. A subsequent activation of rP2Y4 or hP2Y4 by UTP (100 μM) in whole‐cell (ruptured‐patch) mode produced only about 12% inhibition of the N‐type Ca2+ current (ICa(N)). Surprisingly, in perforated patch mode, UTP produced much more inhibition of ICa(N) (maximally 51%), with an IC50 value of 273 nM. This inhibition was voltage‐dependent and was blocked by co‐expression of the βγ‐binding transducin Gα‐subunit. Pertussis toxin (PTX) pretreatment also suppressed ICa(N) inhibition. UTP inhibited the M‐current, recorded in perforated patch mode, by (maximally) 52%, with IC50 values of 21 nM for rP2Y4 and 28 nM for hP2Y4. This inhibition was not affected by PTX pretreatment. With rP2Y4, ATP inhibited the M‐current (IC50 524 nM, 26 times weaker than UTP), whereas ATP had no agonist activity at hP2Y4. This suggests a difference in agonist binding site between rP2Y4 and hP2Y4. We conclude that, in contrast to other nucleotide receptors studied, the P2Y4 receptor couples much more effectively to M‐type K+ channels than to Ca2+ channels. Coupling to the Ca2+ channels involves the βγ‐subunits of Gi/o‐proteins and requires a diffusible intracellular component that is lost in ruptured‐patch recording.

Activation of P2Y1 Nucleotide Receptors Induces Inhibition of the M-Type K+ Current in Rat Hippocampal Pyramidal Neurons

We have shown previously that stimulation of heterologously expressed P2Y1 nucleotide receptors inhibits M-type K+ currents in sympathetic neurons. We now report that activation of endogenous P2Y1 receptors induces inhibition of the M-current in rat CA1/CA3 hippocampal pyramidal cells in primary neuron cultures. The P2Y1 agonist adenosine 5′-[β-thio]diphosphate trilithium salt (ADPβS) inhibited M-current by up to 52% with an IC50 of 84 nm. The hydrolyzable agonist ADP (10 μm) produced 32% inhibition, whereas the metabotropic glutamate receptor 1/5 agonist DHPG [(S)-3,5-dihydroxyphenylglycine] (10 μm) inhibited M-current by 44%. The M-channel blocker XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride] produced 73% inhibition at 3 μm; neither ADPβS nor ADP produced additional inhibition in the presence of XE991. The effect of ADPβS was prevented by a specific P2Y1 antagonist, MRS 2179 (2′-deoxy-N′-methyladenosine-3′,5′-bisphosphate tetra-ammonium salt) (30 μm). Inhibition of the M-current by ADPβS was accompanied by increased neuronal firing in response to injected current pulses. The neurons responding to ADPβS were judged to be pyramidal cells on the basis of (1) morphology, (2) firing characteristics, and (3) their distinctive staining for the pyramidal cell marker neurogranin. Strong immunostaining for P2Y1 receptors was shown in most cells in these cultures: 74% of the cells were positive for both P2Y1 and neurogranin, whereas 16% were only P2Y1 positive. These results show the presence of functional M-current-inhibitory P2Y1 receptors on hippocampal pyramidal neurons, as predicted from their effects when expressed in sympathetic neurons. However, the mechanism of inhibition in the two cell types seems to differ because, unlike nucleotide-mediated M-current inhibition in sympathetic neurons, that in hippocampal neurons did not appear to result from raised intracellular calcium

Inhibition by heterologously-expressed P2Y2 nucleotide receptors of N-type calcium currents in rat sympathetic neurones

The P2Y2 nucleotide receptor has previously been shown to stimulate phosphoinositide breakdown. We now show that, when P2Y2 receptors are heterologously expressed by cRNA injection into dissociated rat sympathetic neurones, activation of these receptors by uridine 5′‐triphosphate (UTP) or adenosine 5′‐triphosphate (ATP) inhibits the N‐type voltage‐gated calcium current by ∼65%, with an IC50 of 0.5 μM. Thus, the same molecular species of nucleotide receptor can link to two different effector pathways.

M-type K + current inhibition by a toxin from the scorpion Buthus eupeus

A number of invertebrate venoms have been tested for effects on M‐type K+ currents (I K(M)) in differentiated mouse neuroblastoma X rat glioma NG108‐15 cells. Among the venoms tested, Buthus eupeus scorpion venom reversibly inhibited I K(M) by ∼ 44% at 50 μg/ml. Inhibition was not due to activation of bradykinin or nucleotide (pyrymidine) receptors. On venom fractionation, a polypeptide of 4 kDa was purified that inhibited I K(M) by ∼45% with an IC50 of ∼;33 nM. Neither the crude venom nor the purified polypeptide affected the Ca2+ current or the delayed rectifier K+ current. While the crude venom prolonged the Na+ current, the polypeptide did not. Thus, the 4 kDa Buthus eupeus polypeptide appears to be a selective inhibitor of I K(M) in NG108‐15 cells.

The Scaffold Protein NHERF2 Determines the Coupling of P2Y1 Nucleotide and mGluR5 Glutamate Receptor to Different Ion Channels in Neurons

Expressed metabotropic group 1 glutamate mGluR5 receptors and nucleotide P2Y1 receptors (P2Y1Rs) show promiscuous ion channel coupling in sympathetic neurons: their stimulation inhibits M-type [Kv7, K(M)] potassium currents and N-type (CaV2.2) calcium currents (Kammermeier and Ikeda, 1999; Brown et al., 2000). These effects are mediated by Gq and Gi/o G-proteins, respectively. Via their C-terminal tetrapeptide, these receptors also bind to the PDZ domain of the scaffold protein NHERF2, which enhances their coupling to Gq-mediated Ca2+ signaling (Fam et al., 2005; Paquet et al., 2006b). We investigated whether NHERF2 could modulate coupling to neuronal ion channels. We find that coexpression of NHERF2 in sympathetic neurons (by intranuclear cDNA injections) does not affect the extent of M-type potassium current inhibition produced by either receptor but strongly reduced CaV2.2 inhibition by both P2Y1R and mGluR5 activation. NHERF2 expression had no significant effect on CaV2.2 inhibition by norepinephrine (via α2-adrenoceptors, which do not bind NHERF2), nor on CaV2.2 inhibition produced by an expressed P2Y1R lacking the NHERF2-binding DTSL motif. Thus, NHERF2 selectively restricts downstream coupling of mGluR5 and P2Y1Rs in neurons to Gq-mediated responses such as M-current inhibition. Differential distribution of NHERF2 in neurons may therefore determine coupling of mGluR5 receptors and P2Y1 receptors to calcium channels.