Hana Cho

Phosphatidylinositol 4,5-Bisphosphate Is Acting as a Signal Molecule in α1-Adrenergic Pathway via the Modulation of Acetylcholine-activated K+ Channels in Mouse Atrial Myocytes

We have investigated the effect of α1-adrenergic agonist phenylephrine (PE) on acetylcholine-activated K+ currents (I KACh). I KACh was recorded in mouse atrial myocytes using the patch clamp technique.I KACh was activated by 10 μm ACh and the current decreased by 44.27 ± 2.38% (n = 12) during 4 min due to ACh-induced desensitization. When PE was applied with ACh, the extent of desensitization was markedly increased to 69.34 ± 2.22% (n = 9), indicating the presence of PE-induced desensitization. I KAChwas fully recovered from desensitization after a 6-min washout. PE-induced desensitization of I KACh was not affected by protein kinase C inhibitor, calphostin C, but abolished by phospholipase C (PLC) inhibitor, neomycin. When phophatidylinositol 4,5-bisphosphate (PIP2) replenishment was blocked by wortmannin (an inhibitor of phophatidylinositol 3-kinase and phophatidylinositol 4-kinase), desensitization ofI KACh in the presence of PE was further increased (97.25 ± 7.63%, n = 6). Furthermore, the recovery from PE-induced desensitization was inhibited, and the amplitude of I KACh at the second exposure after washout was reduced to 19.65 ± 2.61% (n = 6) of the preceding level. These data suggest that the KAChchannel is modulated by PE through PLC stimulation and depletion of PIP2.

Receptor-specific inhibition of GABAB-activated K+ currents by muscarinic and metabotropic glutamate receptors in immature rat hippocampus

It has been shown that the activation of Gq‐coupled receptors (GqPCRs) in cardiac myocytes inhibits the G protein‐gated inwardly rectifying K+ current (IGIRK) via receptor‐specific depletion of phosphatidylinositol 4,5‐bisphosphate (PIP2). In this study, we investigated the mechanism of the receptor‐mediated regulation of IGIRK in acutely isolated hippocampal CA1 neurons by the muscarinic receptor agonist, carbachol (CCh), and the group I metabotropic glutamate receptor (mGluR) agonist, 3,5‐dihydroxyphenylglycine (DHPG). IGIRK was activated by the GABAB receptor agonist, baclofen. When baclofen was repetitively applied at intervals of 2–3 min, the amplitude of the second IGIRK was 92.3 ± 1.7% of the first IGIRK in control. Pretreatment of neurons with CCh or DHPG prior to the second application of baclofen caused a reduction in the amplitude of the second IGIRK to 54.8 ± 1.3% and 51.4 ± 0.6%, respectively. In PLCβ1 knockout mice, the effect of CCh on IGIRK was significantly reduced, whereas the effect of DHPG remained unchanged. The CCh‐mediated inhibition of IGIRK was almost completely abolished by PKC inhibitors and pipette solutions containing BAPTA. The DHPG‐mediated inhibition of IGIRK was attenuated by the inhibition of phospholipase A2 (PLA2), or the sequestration of arachidonic acid. We confirmed that DHPG eliminated the inhibition of IGIRK by arachidonic acid. These results indicate that muscarinic inhibition of IGIRK is mediated by the PLC/PKC signalling pathway, while group I mGluR inhibition of IGIRK occurs via the PLA2‐dependent production of arachidonic acid. These results present a novel receptor‐specific mechanism for crosstalk between GqPCRs and GABAB receptors.

Inhibition of acetylcholine-activated K+ currents by U73122 is mediated by the inhibition of PIP2-channel interaction

We have investigated the effect of U73122, a specific inhibitor of phospholipase C (PLC), on acetylcholine‐activated K+ currents (IKACh) in mouse atrial myocytes. In perforated patch clamp mode, IKACh was activated by 10u2003μM acetylcholine. When atrial myocytes were pretreated with U73122 or U73343, IKACh was inhibited dose‐dependently (half‐maximal inhibition at 0.12±0.0085 and 0.16±0.0176u2003μM, respectively). The current‐voltage relationships for IKACh in the absence and in the presence of U73122 showed that the inhibition occurred uniformly from −120 to +40u2003mV, indicating a voltage‐independent inhibition. When U73122 was applied after IKACh reached steady‐state, a gradual decrease in IKACh was observed. The time course of the current decrease was well fitted to a single exponential, and the rate constant was proportional to the concentration of U73122. When KACh channels were directly activated by adding 1u2003mM GTPγS to the bath solution in inside‐out patches, U73122 (1u2003μM) decreased the open probability significantly without change in mean open time. When KACh channels were activated independently of G‐protein activation by 20u2003mM Na+, open probability was also inhibited by U73122. Voltage‐activated K+ currents and inward rectifying K+ currents were not affected by U73122. These findings show that inhibition by U73122 and U73343 of KACh channels occurs at a level downstream of the action of Gβγ or Na+ on channel activation. The interference with phosphatidylinositol 4,5‐bisphosphate (PIP2)‐channel interaction can be suggested as a most plausible mechanism.

Acetylcholine-induced phosphatidylinositol 4,5-bisphosphate depletion does not cause short-term desensitization of G protein-gated inwardly rectifying K+ current in mouse atrial myocytes.

Depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) induced by phenylephrine or endothelin causes the inhibition of acetylcholine-activated K+ current (IKACh) in atrial myocytes. In the present study, we have investigated the hypothesis that muscarinic receptor induced PIP2 depletion also causes inhibition of IKACh, resulting in desensitization. We confirmed the expression of Gq-coupled muscarinic receptors in mouse atrial myocytes using reverse transcriptase-polymerase chain reaction. The involvement of M1 and M3receptors in desensitization is examined using specific antagonists, 4-DAMP and pirenzepine, but they significantly reduced peak IKACh, implying nonspecific M2 blockade. When ACh-induced phosphoinositide depletion was specifically inhibited using PLCβ1 knock-out mice, the extent of desensitization during 4 min was 47.5 ± 3.2%, which was not different from that in wild type (46.8 ± 2.1%). Phenylephrine-induced phosphoinositide hydrolysis and phenylephrine-induced inhibition of IKACh were not affected by PLCβ1 knock-out. To facilitate PIP2depletion, replenishment of PIP2 was blocked by wortmannin. Wortmannin did not affect the desensitization and the recovery from desensitization. These results suggest that PIP2 depletion by acetylcholine does not contribute to short-term desensitization of IKACh. The differential regulation of IKACh by different phospholipase C-linked receptors may imply that receptor co-localization is required for PIP2 to act as a signaling molecule.

Inhibition of acetylcholine-activated K+ current by chelerythrine and bisindolylmaleimide I in atrial myocytes from mice

The effects of the protein kinase C inhibitors chelerythrine and bisindolylmaleimide I on acetylcholine-activated K+ currents (I(KACh)) were examined in atrial myocytes of mice, using the patch clamp technique. Chelerythrine and bisindolylmaleimide I inhibited I(KACh) in a reversible and dose-dependent manner. Half-maximal effective concentrations were 0.49+/-0.01 microM for chelerythrine and 98.69+/-12.68 nM for bisindolylmaleimide I. However, I(KACh) was not affected either by calphostin C, which is also known as a protein kinase C inhibitor, or by a protein kinase C activator, phorbol 12,13-dibutyrate. When K(ACh) channels were activated directly by adding 1 mM GTPgammaS to the bath solution in inside-out patches, chelerythrine (10 microM) decreased the open probability from 0.043+/-0.01 to 0.014+/-0.007 (n=5), but bisindolylmaleimide I did not affect the channel activity. From these results, it is concluded that both chelerythrine and bisindolylmaleimide I inhibit K(ACh) channels independently of protein kinase C inhibition, but the level of inhibition is different.

Serotonin contracts the rat mesenteric artery by inhibiting 4-aminopyridine-sensitive Kv channels via the 5-HT2A receptor and Src tyrosine kinase.

Serotonin (5-hydroxytryptamine (5-HT)) is a neurotransmitter that regulates a variety of functions in the nervous, gastrointestinal and cardiovascular systems. Despite such importance, 5-HT signaling pathways are not entirely clear. We demonstrated previously that 4-aminopyridine (4-AP)-sensitive voltage-gated K+ (Kv) channels determine the resting membrane potential of arterial smooth muscle cells and that the Kv channels are inhibited by 5-HT, which depolarizes the membranes. Therefore, we hypothesized that 5-HT contracts arteries by inhibiting Kv channels. Here we studied 5-HT signaling and the detailed role of Kv currents in rat mesenteric arteries using patch-clamp and isometric tension measurements. Our data showed that inhibiting 4-AP-sensitive Kv channels contracted arterial rings, whereas inhibiting Ca2+-activated K+, inward rectifier K+ and ATP-sensitive K+ channels had little effect on arterial contraction, indicating a central role of Kv channels in the regulation of resting arterial tone. 5-HT-induced arterial contraction decreased significantly in the presence of high KCl or the voltage-gated Ca2+ channel (VGCC) inhibitor nifedipine, indicating that membrane depolarization and the consequent activation of VGCCs mediate the 5-HT-induced vasoconstriction. The effects of 5-HT on Kv currents and arterial contraction were markedly prevented by the 5-HT2A receptor antagonists ketanserin and spiperone. Consistently, α-methyl 5-HT, a 5-HT2 receptor agonist, mimicked the 5-HT action on Kv channels. Pretreatment with a Src tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, prevented both the 5-HT-mediated vasoconstriction and Kv current inhibition. Our data suggest that 4-AP-sensitive Kv channels are the primary regulator of the resting tone in rat mesenteric arteries. 5-HT constricts the arteries by inhibiting Kv channels via the 5-HT2A receptor and Src tyrosine kinase pathway.

Cholesterol inhibits M-type K+ channels via protein kinase C-dependent phosphorylation in sympathetic neurons.

M-type (KCNQ) potassium channels play an important role in regulating the action potential firing in neurons. Here, we investigated the effect of cholesterol on M current in superior cervical ganglion (SCG) sympathetic neurons, using the patch clamp technique. M current was inhibited in a dose-dependent manner by cholesterol loading with a methyl-β-cyclodextrin-cholesterol complex. This effect was prevented when membrane cholesterol level was restored by including empty methyl-β-cyclodextrin in the pipette solution. Dialysis of cells with AMP-PNP instead of ATP prevented cholesterol action on M currents. Protein kinase C (PKC) inhibitor, calphostin C, abolished cholesterol-induced inhibition whereas the PKC activator, PDBu, mimicked the inhibition of M currents by cholesterol. The in vitro kinase assay showed that KCNQ2 subunits of M channel can be phosphorylated by PKC. A KCNQ2 mutant that is defective in phosphorylation by PKC failed to show current inhibition not only by PDBu but also by cholesterol. These results indicate that cholesterol-induced inhibition of M currents is mediated by PKC phosphorylation. The inhibition of M currents by PDBu and cholesterol was completely blocked by PIP2 loading, indicating that the decrease in PIP2-channel interaction underlies M channel inhibition by PKC-mediated phosphorylation. We conclude that cholesterol specifically regulates M currents in SCG neurons via PKC activation.

Agonist-induced Localization of Gq-coupled Receptors and G Protein-gated Inwardly Rectifying K+ (GIRK) Channels to Caveolae Determines Receptor Specificity of Phosphatidylinositol 4,5-Bisphosphate Signaling

G protein-gated inwardly rectifying K+ (GIRK) channels are parasympathetic effectors in cardiac myocytes that act as points of integration of signals from diverse pathways. Neurotransmitters and hormones acting on the Gq protein regulate GIRK channels by phosphatidylinositol 4,5-bisphosphate (PIP2) depletion. In previous studies, we found that endothelin-1, but not bradykinin, inhibited GIRK channels, even though both of them hydrolyze PIP2 in cardiac myocytes, showing receptor specificity. The present study assessed whether the spatial organization of the PIP2 signal into caveolar microdomains underlies the specificity of PIP2-mediated signaling. Using biochemical analysis, we examined the localization of GIRK and Gq protein-coupled receptors (GqPCRs) in mouse atrial myocytes. Agonist stimulation induced a transient co-localization of GIRK channels with endothelin receptors in the caveolae, excluding bradykinin receptors. Such redistribution was eliminated by caveolar disruption with methyl-β-cyclodextrin (MβCD). Patch clamp studies showed that the specific response of GIRK channels to GqPCR agonists was abolished by MβCD, indicating the functional significance of the caveolae-dependent spatial organization. To assess whether low PIP2 mobility is essential for PIP2-mediated signaling, we blocked the cytoskeletal restriction of PIP2 diffusion by latrunculin B. This abolished the GIRK channel regulation by GqPCRs without affecting their targeting to caveolae. These data suggest that without the hindered diffusion of PIP2 from microdomains, PIP2 loses its signaling efficacy. Taken together, these data suggest that specific targeting combined with restricted diffusion of PIP2 allows the PIP2 signal to be compartmentalized to the targets localized closely to the GqPCRs, enabling cells to discriminate between identical PIP2 signaling that is triggered by different receptors.

Coexistence of glutamatergic spine synapses and shaft synapses in substantia nigra dopamine neurons.

Dopamine neurons of the substantia nigra have long been believed to have multiple aspiny dendrites which receive many glutamatergic synaptic inputs from several regions of the brain. But, here, using high-resolution two-photon confocal microscopy in the mouse brain slices, we found a substantial number of common dendritic spines in the nigral dopamine neurons including thin, mushroom, and stubby types of spines. However, the number of dendritic spines of the dopamine neurons was approximately five times lower than that of CA1 pyramidal neurons. Immunostaining and morphological analysis revealed that glutamatergic shaft synapses were present two times more than spine synapses. Using local two-photon glutamate uncaging techniques, we confirmed that shaft synapses and spine synapses had both AMPA and NMDA receptors, but the AMPA/NMDA current ratios differed. The evoked postsynaptic potentials of spine synapses showed lower amplitudes but longer half-widths than those of shaft synapses. Therefore, we provide the first evidence that the midbrain dopamine neurons have two morphologically and functionally distinct types of glutamatergic synapses, spine synapses and shaft synapses, on the same dendrite. This peculiar organization could be a new basis for unraveling many physiological and pathological functions of the midbrain dopamine neurons.

Cholesterol regulates HERG K+ channel activation by increasing phospholipase C β1 expression.

Human ether-a-go-go-related gene (HERG) K+ channel underlies the rapidly activating delayed rectifier K+ conductance (IKr) during normal cardiac repolarization. Also, it may regulate excitability in many neuronal cells. Recently, we showed that enrichment of cell membrane with cholesterol inhibits HERG channels by reducing the levels of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] due to the activation of phospholipase C (PLC). In this study, we further explored the effect of cholesterol enrichment on HERG channel kinetics. When membrane cholesterol level was mildly increased in human embryonic kidney (HEK) 293 cells expressing HERG channel, the inactivation and deactivation kinetics of HERG current were not affected, but the activation rate was significantly decelerated at all voltages tested. The application of PtdIns(4,5)P2 or inhibitor for PLC prevented the effect of cholesterol enrichment, while the presence of antibody against PtdIns(4,5)P2 in pipette solution mimicked the effect of cholesterol enrichment. These results indicate that the effect of cholesterol enrichment on HERG channel is due to the depletion of PtdIns(4,5)P2. We also found that cholesterol enrichment significantly increases the expression of β1 and β3 isoforms of PLC (PLCβ1, PLCβ3) in the membrane. Since the effects of cholesterol enrichment on HERG channel were prevented by inhibiting transcription or by inhibiting PLCβ1 expression, we conclude that increased PLCβ1 expression leads to the deceleration of HERG channel activation rate via downregulation of PtdIns(4,5)P2. These results confirm a crosstalk between two plasma membrane-enriched lipids, cholesterol and PtdIns(4,5)P2, in the regulation of HERG channels.