Signal transduction pathway for the substance P-induced inhibition of rat Kir3 (GIRK) channel

Certain transmitters inhibit Kir3 (GIRK) channels, resulting in neuronal excitation. We analysed signalling mechanisms for substance P (SP)-induced Kir3 inhibition in relation to the role of phosphatidylinositol 4,5-bisphosphate (PIP₂). SP rapidly – with a half-time of ∼10 s with intracellular GTPγS and ∼14 s with intracellular GTP – inhibits a robustly activated Kir3.1/Kir3.2 current. A mutant Kir3 channel, Kir3.1(M223L)/Kir3.2(I234L), which has a stronger binding to PIP₂ than does the wild type Kir3.1/Kir3.2, is inhibited by SP as rapidly as the wild type Kir3.1/Kir3.2. This result contradicts the idea that Kir3 inhibition originates from the depletion of PIP₂. A Kir2.1 (IRK1) mutant, Kir2.1(R218Q), despite having a weaker binding to PIP₂ than wild type Kir3.1/Kir3.2, shows a SP-induced inhibition slower than the wild type Kir3.1/Kir3.2 channel, again conflicting with the PIP₂ theory of channel inhibition. Co-immunoprecipitation reveals that Gα_q binds with Kir3.2, but not with Kir2.2 or Kir2.1. These functional results and co-immunoprecipitation data suggest that G_q activation rapidly inhibits Kir3 (but not Kir2), possibly by direct binding of Gα_q to the channel.     

G-protein-coupled receptor oligomers: two or more for what? Lessons from mGlu and GABAB receptors

G-protein-coupled receptors (GPCRs) are key players in the precise tuning of intercellullar communication. In the brain, both major neurotransmitters, glutamate and GABA, act on specific GPCRs [the metabotropic glutamate (mGlu) and GABA_B receptors] to modulate synaptic transmission. These receptors are encoded by the largest gene family, and have been found to associate into both homo- and hetero-oligomers, which increases the complexity of this cell communication system. Here we show that dimerization is required for mGlu and GABA_B receptors to function, since the activation process requires a relative movement between the subunits to occur. We will also show that, in contrast to the mGlu receptors, which form strict dimers, the GABA_B receptors assemble into larger complexes, both in transfected cells and in the brain, resulting in a decreased G-protein coupling efficacy. We propose that GABA_B receptor oligomerization offers a way to increase the possibility of modulating receptor signalling and activity, allowing the same receptor protein to have specific properties in neurons at different locations.     

Protein kinase C modulation of recombinant ATP-sensitive K+ channels composed of Kir6.1 and/or Kir6.2 expressed with SUR2B

The molecular identity of smooth muscle ATP-sensitive K⁺ channels (K_ATP) is not established with certainty. Patch clamp methods were employed to determine if recombinant K_ATP channels composed of Kir6.1 and SUR2B subunits expressed by human embryonic kidney (HEK293) cells share an identical modulation by protein kinase C (PKC) with the vascular K_NDP subtype of K_ATP channel. The open probability of Kir6.1/SUR2B channels was determined before and after sequential exposure to pinacidil (50 μM) and the combination of pinacidil and phorbol 12,13-dibutyrate (PdBu; 50 n m ). Treatment with PdBu caused a decline in channel activity, but this was not seen with an inactive phorbol ester, 4α-phorbol 12,13-didecanoate (PdDe; 50 n m ). Angiotensin II (0.1 μM) induced a similar inhibition of Kir6.1/SUR2B channels in cells expressing angiotensin AT₁ receptors. The effects of PdBu and angiotensin II were blocked by the PKC inhibitor, chelerythrine (3 μM). Purified PKC inhibited Kir6.1/SUR2B activity (in 0.5 m m ATP/ 0.5 m m ADP), and the inhibition was blocked by a specific peptide inhibitor of PKC, PKC(19-31). In contrast, PdBu increased the activity of recombinant K_ATP channels composed of Kir6.2 and SUR2B, or the combination of Kir6.1, Kir6.2 and SUR2B subunits. The results indicate that the modulation by PKC of Kir6.1/SUR2B, but not Kir6.2/SUR2B or Kir6.1-Kir6.2/SUR2B channel gating mimics that of native vascular K_NDP channels. Physiological inhibition of vascular K_ATP current by vasoconstrictors which utilize intracellular signalling cascades involving PKC is concluded to involve the modulation of K_NDP channel complexes composed of four Kir6.1 and their associated SUR2B subunits.     

Rapid changes in shear stress induce dissociation of a Gαq/11–platelet endothelial cell adhesion molecule-1 complex

It has been recently shown that endothelial platelet endothelial cell adhesion molecule-1 (PECAM-1) expression is pro-atherogenic. PECAM-1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane-bound G proteins Gα_q/11 also mediate flow-induced responses. Here, we investigated whether PECAM-1 and Gα_q/11 could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co-localization of Gα_q/11 and PECAM-1 at the cell–cell junction in the atheroprotected section of mouse aortae. In contrast, Gα_q/11 was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM-1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the Gα_q/11–PECAM-1 complex within 30 s and a partial relocalization of the Gα_q/11 staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow-induced Gα_q/11–PECAM-1 dissociation. These results allow us to conclude that Gα_q/11–PECAM-1 forms a mechanosensitive complex and its localization suggests the Gα_q/11–PECAM-1 complex is a critical mediator of vascular diseases.     

Neuronal two-pore-domain potassium channels and their regulation by G protein-coupled receptors

Leak potassium currents in the nervous system are often carried through two-pore-domain potassium (K2P) channels. These channels are regulated by a number of different G protein-coupled receptor (GPCR) pathways. The TASK subfamily of K2P channels are inhibited following activation of the G protein Gα_q. The mechanism(s) that transduce this inhibition have yet to be established but there is evidence to support a role of phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis products, depletion of PIP₂ itself from the membrane, or a direct action of activated Gα_q on TASK channels. It seems possible that more than one pathway may act in parallel to transduce inhibition. By contrast, TRESK channels are stimulated following activation of Gα_q. This is due to stimulation of the protein phosphatase, calcineurin, which dephosphorylates TRESK channels and enhances their activity. TREK channels are the most widely regulated of the K2P channel subfamilies being inhibited following activation of Gα_q and Gα_s but enhanced following activation of Gα_i. The multiple pathways activated and the apparent promiscuous coupling of at least some K2P channel types to different G protein regulatory pathways suggests that the excitability of neurons that express K2P channels will be profoundly sensitive to variations in GPCR activity.     

GABA Mediates Autoreceptor Feedback Inhibition in the Rat Carotid Body Via Presynaptic GABAB Receptors and TASK-1

Background K⁺ channels exert control over neuronal excitability by regulating resting potential and input resistance. Here, we show that GABA_B receptor-mediated activation of a background K⁺ conductance modulates transmission at rat carotid body chemosensory synapses in vitro . Carotid body chemoreceptor (type I) cells expressed GABA_B(1) and GABA_B(2) subunits as well as endogenous GABA. The GABA_B receptor agonist baclofen activated an anandamide- and Ba²⁺-sensitive TASK-1-like background K⁺ conductance in chemoreceptor cell clusters, but was without effect on voltage-gated Ca²⁺ channels. Hydroxysaclofen (50 μ m ), 5-aminovaleric acid (100 μ m ) and CGP 55845 (100 n m ), selective GABA_B receptor blockers, potentiated the hypoxia-induced receptor potential; this effect was abolished by pre-treatment with pertussis toxin (PTX; 500 ng ml^-1), an inhibitor of G_i, or by H-89 (50 μ m ), a selective inhibitor of protein kinase A. The protein kinase C inhibitor chelerythrine chloride (100 μ m ) was without effect on this potentiation. GABA_B receptor blockers also caused depolarisation of type I cells in clusters, and enhanced spike discharge in spontaneously firing cells. In functional co-cultures of type I clusters and petrosal sensory neurones, GABA_B receptor blockers potentiated hypoxia-induced postsynaptic chemosensory responses mediated by the fast-acting transmitters ACh and ATP. Thus GABA_B receptor-mediated activation of TASK-1 or a related channel provides a presynaptic autoregulatory feedback mechanism that modulates fast synaptic transmission in the rat carotid body.     

Functional effects of naturally occurring KCNJ11 mutations causing neonatal diabetes on cloned cardiac KATP channels

ATP-sensitive K⁺ (K_ATP) channels are hetero-octamers of inwardly rectifying K⁺ channel (Kir6.2) and sulphonylurea receptor subunits (SUR1 in pancreatic β-cells, SUR2A in heart). Heterozygous gain-of-function mutations in Kir6.2 cause neonatal diabetes, which may be accompanied by epilepsy and developmental delay. However, despite the importance of K_ATP channels in the heart, patients have no obvious cardiac problems. We examined the effects of adenine nucleotides on K_ATP channels containing wild-type or mutant (Q52R, R201H) Kir6.2 plus either SUR1 or SUR2A. In the absence of Mg²⁺, both mutations reduced ATP inhibition of SUR1- and SUR2A-containing channels to similar extents, but when Mg²⁺ was present ATP blocked mutant channels containing SUR1 much less than SUR2A channels. Mg-nucleotide activation of SUR1, but not SUR2A, channels was markedly increased by the R201H mutation. Both mutations also increased resting whole-cell K_ATP currents through heterozygous SUR1-containing channels to a greater extent than for heterozygous SUR2A-containing channels. The greater ATP inhibition of mutant Kir6.2/SUR2A than of Kir6.2/SUR1 can explain why gain-of-function Kir6.2 mutations manifest effects in brain and β-cells but not in the heart.     

G protein-independent neuromodulatory action of adenosine on metabotropic glutamate signalling in mouse cerebellar Purkinje cells

Adenosine receptors (ARs) are G protein-coupled receptors (GPCRs) mediating the neuromodulatory actions of adenosine that influence emotional, cognitive, motor, and other functions in the central nervous system (CNS). Previous studies show complex formation between ARs and metabotropic glutamate receptors (mGluRs) in heterologous systems and close colocalization of ARs and mGluRs in several central neurons. Here we explored the possibility of intimate functional interplay between G_i/o protein-coupled A₁-subtype AR (A1R) and type-1 mGluR (mGluR1) naturally occurring in cerebellar Purkinje cells. Using a perforated-patch voltage-clamp technique, we found that both synthetic and endogenous agonists for A1R induced continuous depression of a mGluR1-coupled inward current. A1R agonists also depressed mGluR1-coupled intracellular Ca²⁺ mobilization monitored by fluorometry. A1R indeed mediated this depression because genetic depletion of A1R abolished it. Surprisingly, A1R agonist-induced depression persisted after blockade of G_i/o protein. The depression appeared to involve neither the cAMP-protein kinase A cascade downstream of the alpha subunits of G_i/o and G_s proteins, nor cytoplasmic Ca²⁺ that is suggested to be regulated by the beta-gamma subunit complex of G_i/o protein. Moreover, A1R did not appear to affect G_q protein which mediates the mGluR1-coupled responses. These findings suggest that A1R modulates mGluR1 signalling without the aid of the major G proteins. In this respect, the A1R-mediated depression of mGluR1 signalling shown here is clearly distinguished from the A1R-mediated neuronal responses described so far. These findings demonstrate a novel neuromodulatory action of adenosine in central neurons.     

Remodelling of the SUR–Kir6.2 interface of the KATP channel upon ATP binding revealed by the conformational blocker rhodamine 123

ATP-sensitive K⁺ channels (K_ATP channels) are metabolic sensors formed by association of a K⁺ channel, Kir6, and an ATP-binding cassette (ABC) protein, SUR, which allosterically regulates channel gating in response to nucleotides and pharmaceutical openers and blockers. How nucleotide binding to SUR translates into modulation of Kir6 gating remains largely unknown. To address this issue, we have used a novel conformational K_ATP channel inhibitor, rhodamine 123 (Rho123) which targets the Kir6 subunit in a SUR-dependent manner. Rho123 blocked SUR-less Kir6.2 channels with an affinity of ∼1 μ m , regardless of the presence of nucleotides, but it had no effect on channels formed by the association of Kir6.2 and the N-terminal transmembrane domain TMD0 of SUR. Rho123 blocked SUR + Kir6.2 channels with the same affinity as Kir6.2 but this effect was antagonized by ATP. Protection from Rho123 block by ATP was due to direct binding of ATP to SUR and did not entail hydrolysis because it was not mimicked by AMP, did not require Mg²⁺ and was reduced by mutations in the nucleotide-binding domains of SUR. These results suggest that Rho123 binds at the TMD0–Kir6.2 interface and that binding of ATP to SUR triggers a change in the structure of the contact zone between Kir6.2 and domain TMD0 of SUR that causes masking of the Rho123 site on Kir6.2.     

Differential dissociation of G protein heterotrimers

Signalling by heterotrimeric G proteins is often isoform-specific, meaning certain effectors are regulated exclusively by one family of heterotrimers. For example, in excitable cells inwardly rectifying potassium (GIRK) channels are activated by Gβγ dimers derived specifically from G_i/o heterotrimers. Since all active heterotrimers are thought to dissociate and release free Gβγ dimers, it is unclear why these channels respond primarily to dimers released by G_i/o heterotrimers. We reconstituted GIRK channel activation in cells where we could quantify heterotrimer expression at the plasma membrane, GIRK channel activation, and heterotrimer dissociation. We find that G_oA heterotrimers are more effective activators of GIRK channels than G_s heterotrimers when comparable amounts of each are available. We also find that active G_oA heterotrimers dissociate more readily than active G_s heterotrimers. Differential dissociation may thus provide a simple explanation for Gα-specific activation of GIRK channels and other Gβγ-sensitive effectors.     

Gene targeting approach to clarification of ion channel function: studies of Kir6.x null mice

ATP-sensitive potassium (K_ATP) channels are present in many tissues, including pancreatic β-cells, heart, skeletal muscle, vascular smooth muscle and brain, in which they couple the cell metabolic state to membrane potential. K_ATP channels are hetero-octameric proteins composed of the pore-forming subunits Kir6.x (Kir6.1 or Kir6.2) of the inwardly rectifying K⁺ channel family and the regulatory subunits SURx (SUR1, SUR2A or SUR2B), the receptor of the sulphonylureas widely used in treatment of type 2 diabetes mellitus. Different combinations of Kir6.x and SURx comprise K_ATP channels with distinct electrophysiological and pharmacological properties, but their physiological functions in the various tissues are unclear. Our studies of Kir6.2 null (knockout) and Kir6.1 null mice have shown that K_ATP channels are critical metabolic sensors in protection against acute metabolic stress such as hyperglycaemia, hypoglycaemia, ischaemia and hypoxia.     

Enhanced G protein-dependent modulation of excitatory synaptic transmission in the cerebellum of the Ca2+ channel-mutant mouse, tottering

Tottering , a mouse model for absence epilepsy and cerebellar ataxia, carries a mutation in the gene encoding class A (P/Q-type) Ca²⁺ channels, the dominant exocytotic Ca²⁺ channel at most synapses in the mammalian central nervous system. Comparing tottering to wild-type mice, we have studied glutamatergic transmission between parallel fibres and Purkinje cells in cerebellar slices. Results from biochemical assays and electrical field recordings demonstrate that glutamate release from parallel fibre terminals of the tottering mouse is controlled largely by class B Ca²⁺ channels (N-type), in contrast to the P/Q-channels that dominate release from wild-type terminals. Since N-channels, in a variety of assays, are more effectively inhibited by G proteins than are P/Q-channels, we tested whether synaptic transmission between parallel fibres and Purkinje cells in tottering mice was more susceptible to inhibitory modulation by G protein-coupled receptors than in their wild-type counterparts. GABA_B receptors and α₂-adrenergic receptors (activated by bath application of transmitters) produced a three- to fivefold more potent inhibition of transmission in tottering than in wild-type synapses. This increased modulation is likely to be important for cerebellar transmission in vivo , since heterosynaptic depression, produced by activating GABAergic interneurones, greatly prolonged GABA_B receptor-mediated presynaptic inhibition in tottering as compared to wild-type slices. We propose that this enhanced modulation shifts the balance of synaptic input to Purkinje cells in favour of inhibition, reducing Purkinje cell output from the cerebellum, and may contribute to the aberrant motor phenotype that is characteristic of this mutant animal.     

CaV3.2 is the major molecular substrate for redox regulation of T-type Ca2+ channels in the rat and mouse thalamus

Although T-type Ca²⁺ channels in the thalamus play a crucial role in determining neuronal excitability and are involved in sensory processing and pathophysiology of epilepsy, little is known about the molecular mechanisms involved in their regulation. Here, we report that reducing agents, including endogenous sulfur-containing amino acid l -cysteine, selectively enhance native T-type currents in reticular thalamic (nRT) neurons and recombinant Ca_V3.2 (α1H) currents, but not native and recombinant Ca_V3.1 (α1G)- and Ca_V3.3 (α1I)-based currents. Consistent with this data, T-type currents of nRT neurons from transgenic mice lacking Ca_V3.2 channel expression were not modulated by reducing agents. In contrast, oxidizing agents inhibited all native and recombinant T-type currents non-selectively. Thus, our findings directly demonstrate that Ca_V3.2 channels are the main molecular substrate for redox regulation of neuronal T-type channels. In addition, because thalamic T-type channels generate low-threshold Ca²⁺ spikes that directly correlate with burst firing in these neurons, differential redox regulation of these channels may have an important function in controlling cellular excitability in physiological and pathological conditions and fine-tuning of the flow of sensory information into the central nervous system.     

Intracellular Na+ inhibits voltage-dependent N-type Ca2+ channels by a G protein βγ subunit-dependent mechanism

N-type  voltage-dependent  Ca²⁺ channels (N-VDCCs) play important roles in neurotransmitter release and certain postsynaptic phenomena. These channels are modulated by a number of intracellular factors, notably by Gβγ subunits of G proteins, which inhibit N-VDCCs in a voltage-dependent (VD) manner. Here we show that an increase in intracellular Na⁺ concentration inhibits N-VDCCs  in hippocampal pyramidal neurones and in Xenopus oocytes. In acutely dissociated hippocampal neurones, Ba²⁺ current via N-VDCCs was inhibited by Na⁺ influx caused by the activation of NMDA receptor channels. In Xenopus oocytes expressing N-VDCCs, Ba²⁺ currents were inhibited by Na⁺ influx and enhanced by depletion of Na⁺, after incubation in a Na⁺-free extracellular solution. The Na⁺-induced inhibition was accompanied by the development of  VD facilitation, a hallmark of a Gβγ-dependent process. Na⁺-induced regulation of N-VDCCs is Gβγ dependent, as suggested by the blocking of Na⁺ effects by Gβγ scavengers and by excess Gβγ, and may be mediated by the Na⁺-induced dissociation of Gαβγ heterotrimers. N-VDCCs may be novel effectors of Na⁺ion, regulated by the Na⁺ concentration via Gβγ.     

A mutation in the ATP-binding site of the Kir6.2 subunit of the KATP channel alters coupling with the SUR2A subunit

Mutations in the pore-forming subunit of the ATP-sensitive K⁺ (K_ATP) channel Kir6.2 cause neonatal diabetes. Understanding the molecular mechanism of action of these mutations has provided valuable insight into the relationship between the structure and function of the K_ATP channel. When Kir6.2 containing a mutation (F333I) in the putative ATP-binding site is coexpressed with the cardiac type of regulatory K_ATP channel subunit, SUR2A, the channel sensitivity to ATP inhibition is reduced and the intrinsic open probability ( P _o ) is increased. However, the extent of macroscopic current activation by MgADP was unaffected. Here we examine rundown and MgADP activation of wild-type and Kir6.2-F333I/SUR2A channels using single-channel recording, noise analysis and spectral analysis. We also compare the effect of mutating the adjacent residue, G334, on rundown and MgADP activation. All three approaches indicated that rundown of Kir6.2-F333I/SUR2A channels is due to a reduction in the number of active channels in the patch and that MgADP reactivation involves recruitment of inactive channels. In contrast, rundown and MgADP reactivation of wild-type and Kir6.2-G334D/SUR2A channels, and of Kir6.2-F333I/SUR1 channels, involve a gradual change in P _o . Our results suggest that F333 in Kir6.2 interacts functionally with SUR2A to modulate channel rundown and MgADP activation. This interaction is fairly specific as it is not disturbed when the adjacent residue (G334) is mutated. It is also not a consequence of the enhanced P _o of Kir6.2-F333I/SUR2A channels, as it is not found for other mutant channels with high P _o (Kir6.2-I296L/SUR2A).     

Pyridine nucleotide regulation of the KATP channel Kir6.2/SUR1 expressed in Xenopus oocytes

The pancreatic β-cell type of ATP-sensitive potassium (K_ATP) channel (Kir6.2/SUR1) is inhibited by intracellular ATP and ADP, which bind to the Kir6.2 subunit, and is activated by Mg-nucleotide interaction with the regulatory sulphonylurea receptor subunits (SUR1). The nicotinamide adenine dinucleotides NAD and NADP consist of an ADP molecule with a ribose group and a nicotinamide moiety attached to the terminal phosphate. Both these molecules block native K_ATP channels in pancreatic β-cells at concentrations above 500 μM, and activate them at lower concentrations. We therefore investigated whether NAD and NADP interact with both Kir6.2 and SUR1 subunits of the K_ATP channel by comparing the potency of these agents on recombinant Kir6.2ΔC and Kir6.2/SUR1 channels expressed in Xenopus oocytes. Our results show that, at physiological concentrations, NAD and NADP interact with the nucleotide inhibitory site of Kir6.2 to inhibit Kir6.2/SUR1 currents. They may therefore contribute to the resting level of channel inhibition in the intact cell. Importantly, our data also reveal that this interaction is dependent on the presence of SUR1, which may act by increasing the width of the nucleotide-binding pocket of Kir6.2.     

GABAB receptor activation desensitizes postsynaptic GABAB and A1 adenosine responses in rat hippocampal neurones

Whole-cell recordings of EPSCs and G-protein-activated inwardly rectifying (GIRK) currents were made from cultured hippocampal neurones to determine the effect of long-term agonist treatment on the presynaptic and postsynaptic responses mediated by GABA_B receptors (GABA_BRs). GABA_BR-mediated presynaptic inhibition was unaffected by agonist (baclofen) treatment for up to 48 h, and was desensitized by about one-half after 96 h. In contrast, GABA_BR-mediated GIRK currents were desensitized by a similar amount after only 2 h of agonist treatment. In addition, presynaptic inhibition mediated by A₁ adenosine receptors (A₁Rs) was unaffected by prolonged GABA_BR activation, whereas A₁R-mediated GIRK currents were desensitized. Desensitization of postsynaptic GABA_BR and A₁R responses was blocked by the GABA_BR antagonist (1-(S)-3,4-dichlorophenylethyl)amino-2-(S) hydroxypropyl-p-benzyl-phosphonic acid (CGP 55845A), but not by the A₁R antagonist cyclopentyldipropylxanthine (DPCPX). GIRK current amplitude could be partially restored after baclofen treatment by either coapplication of baclofen and adenosine, or intracellular infusion of the non-hydrolysable GTP analog 5'-guanylylimidodiphosphate (Gpp(NH)p). Short-term (4-24 h) baclofen treatment also significantly desensitized the inhibition of postsynaptic voltage-gated calcium channels by activation of GABA_BRs or A₁Rs. These results show that responses mediated by GABA_BRs and A₁Rs desensitize differently in presynaptic and postsynaptic compartments, and demonstrate the heterologous desensitization of postsynaptic A1R responses.     

Molecular, pharmacological and functional properties of GABA(A) receptors in anterior pituitary cells

Anterior pituitary cells express γ-aminobutyric acid (GABA)-A receptor-channels, but their structure, distribution within the secretory cell types, and nature of action have not been clarified. Here we addressed these questions using cultured anterior pituitary cells from postpubertal female rats and immortalized αT3-1 and GH₃ cells. Our results show that mRNAs for all GABA_A receptor subunits are expressed in pituitary cells and that α1/β1 subunit proteins are present in all secretory cells. In voltage-clamped gramicidin-perforated cells, GABA induced dose-dependent increases in current amplitude that were inhibited by bicuculline and picrotoxin and facilitated by diazepam and zolpidem in a concentration-dependent manner. In intact cells, GABA and the GABA_A receptor agonist muscimol caused a rapid and transient increase in intracellular calcium, whereas the GABA_B receptor agonist baclofen was ineffective, suggesting that chloride-mediated depolarization activates voltage-gated calcium channels. Consistent with this finding, RT-PCR analysis indicated high expression of NKCC1, but not KCC2 cation/chloride transporter mRNAs in pituitary cells. Furthermore, the GABA_A channel reversal potential for chloride ions was positive to the baseline membrane potential in most cells and the activation of ion channels by GABA resulted in depolarization of cells and modulation of spontaneous electrical activity. These results indicate that secretory pituitary cells express functional GABA_A receptor-channels that are depolarizing.     

Dopamine selectively reduces GABAB transmission onto dopaminergic neurones by an unconventional presynaptic action

The functioning of midbrain dopaminergic neurones is closely involved in mental processes and movement. In particular the modulation of the inhibitory inputs on these cells might be crucial in controlling firing activity and dopamine (DA) release in the brain. Here, we report a concentration-dependent depressant action of dopamine on the GABA_B IPSPs intracellularly recorded from dopaminergic neurones. Such effect was observed in spite of the presence of D₁/D₂ dopamine receptor antagonists. A reduction of the GABA_B IPSPs was also caused by noradrenaline (norepinephrine) and by l -β-3,4-dihydroxyphenylalanine ( l -DOPA), which is metabolically transformed into DA. The DA-induced depression of the IPSPs was partially antagonised by the α2 antagonists yohimbine and phentolamine. DA did not change the postsynaptic effects of the GABA_B agonist baclofen, suggesting a presynaptic site of action. Furthermore, DA did not modulate the GABA_A-mediated IPSP. The DA-induced depression of the GABA_B IPSP occluded the depression produced by serotonin and was not antagonized by serotonin antagonists. The DA- and 5-HT-induced depression of the GABA_B IPSP persisted when calcium and potassium currents were reduced in to the presynaptic terminals. These results describe an unconventional presynaptic, D₁ and D₂ independent action of DA on the GABA_B IPSP. This might have a principal role in determining therapeutic/side effects of l -DOPA and antipsychotics and could be also involved in drug abuse.     

A cytosolic factor that inhibits KATP channels expressed in Xenopus oocytes by impairing Mg-nucleotide activation by SUR1

ATP-sensitive K⁺ (K_ATP) channels couple cell metabolism to cell electrical activity. Wild-type (Kir6.2/SUR1) K_ATP channels heterologously expressed in Xenopus oocytes give rise to very small inward currents in cell-attached patches. A large increase in the current is observed on patch excision into zero ATP solution. This is presumably due to loss of intracellular ATP leading to unblock of K_ATP channels. In contrast, channels containing Kir6.2 mutations associated with reduced ATP-sensitivity display non-zero cell-attached currents. Unexpectedly, these cell-attached currents are significantly smaller (by ∼40%) than those observed when excised patches are exposed to physiological ATP concentrations (1–10 m m ). Cramming the patch back into the oocyte cytoplasm restores mutant K_ATP current amplitude to that measured in the cell-attached mode. This implies that the magnitude of the cell-attached current is regulated not only by intracellular ATP but also by another cytoplasmic factor/s. This factor seems to require the nucleotide-binding domains of SUR1 to be effective. Thus a mutant Kir6.2 (Kir6.2ΔC-I296L) expressed in the absence of SUR1 exhibited currents of similar magnitude in cell-attached patches as in inside-out patches exposed to 10 m m MgATP. Similar results were found when Kir6.2-I296L was coexpressed with an SUR1 mutant that is insensitive to MgADP or MgATP activation. This suggests the oocyte contains a cytoplasmic factor that reduces nucleotide binding/hydrolysis at the NBDs of SUR1. In conclusion, our results reveal a novel regulatory mechanism for the K_ATP channel. This was not evident for wild-type channels because of their high sensitivity to block by ATP.