IRK1 Channel Research Articles

Endoplasmic Reticulum Calcium Mediates Drosophila Wing Development.

The temporal dynamics of morphogen presentation impacts transcriptional responses and tissue patterning. However, the mechanisms controlling morphogen release are far from clear. We found that inwardly rectifying potassium (Irk) channels regulate endogenous transient increases in intracellular calcium and bone morphogenetic protein (BMP/Dpp) release for Drosophila wing development. Inhibition of Irk channels reduces BMP/Dpp signaling, and ultimately disrupts wing morphology. Ion channels impact development of several tissues and organisms in which BMP signaling is essential. In neurons and pancreatic beta cells, Irk channels modulate membrane potential to affect intracellular Ca++ to control secretion of neurotransmitters and insulin. Based on Irk activity in neurons, we hypothesized that electrical activity controls endoplasmic reticulum (ER) Ca++ release into the cytoplasm to regulate the release of BMP. To test this hypothesis, we reduced expression of four proteins that control ER calcium, Stromal interaction molecule 1 (Stim), Calcium release-activated calcium channel protein 1 (Orai), SarcoEndoplasmic Reticulum Calcium ATPase (SERCA), small conductance calcium-activated potassium channel (SK), and Bestrophin 2 (Best2) using RNAi and documented wing phenotypes. We use live imaging to study calcium and Dpp release within pupal wings and larval wing discs. Additionally, we employed immunohistochemistry to characterize Small Mothers Against Decapentaplegic (SMAD) phosphorylation downstream of the BMP/Dpp pathway following RNAi knockdown. We found that reduced Stim and SERCA function decreases amplitude and frequency of endogenous calcium transients in the wing disc and reduced BMP/Dpp release. Our results suggest control of ER calcium homeostasis is required for BMP/Dpp release, and Drosophila wing development.

Deep Domain Insertion Profiling is a Window into Inward Rectifier K+ Channel Dynamics and Gating

Inward rectifier K+ (IRK) channels arose early in cellular life as the combination of a pore and an immunoglobulin (Ig)-like domain. This architecture is conserved throughout the IRK family and enables regulation by different allosteric ligands (lipids, ATP, G proteins). Functional diversity in light of architectural conservation suggests that the required signal transduction functions for different ligands already existed within ancestral IRK channels. The term ‘latent allosteric capacity’ describes how new functions can emerge from conformational flexibility that arise from the combination of specific domains.

Inwardly rectifying potassium channels influence Drosophila wing morphogenesis by regulating Dpp release.

Loss of embryonic ion channel function leads to morphological defects, but the underlying reason for these defects remains elusive. Here, we show that inwardly rectifying potassium (Irk) channels regulate release of the Drosophila bone morphogenetic protein Dpp in the developing fly wing and that this is necessary for developmental signaling. Inhibition of Irk channels decreases the incidence of distinct Dpp-GFP release events above baseline fluorescence while leading to a broader distribution of Dpp-GFP. Work by others in different cell types has shown that Irk channels regulate peptide release by modulating membrane potential and calcium levels. We found calcium transients in the developing wing, and inhibition of Irk channels reduces the duration and amplitude of calcium transients. Depolarization with high extracellular potassium evokes Dpp release. Taken together, our data implicate Irk channels as a requirement for regulated release of Dpp, highlighting the importance of the temporal pattern of Dpp presentation for morphogenesis of the wing.

Analysis of G-protein-activated inward rectifying K+ (GIRK) channel currents upon GABAB receptor activation in rat supraoptic neurons

While magnocellular neurons in the supraoptic nucleus (SON) possess rich Gi/o-mediated mechanisms, molecular and cellular properties of G-protein-activated inwardly rectifying K+ (GIRK) channels have been controversial. Here, properties of GIRK channels are examined by RT-PCR and whole-cell patch-clamp techniques in rat SON neurons. Patch clamp experiments showed that the selective GABAB agonist, baclofen, enhanced currents in a high K+ condition. The baclofen-enhanced currents exhibited evident inward rectification and were blocked by the selective GABAB antagonist, CGP55845A, the IRK channel blocker, Ba2+, and the selective GIRK channel blocker, tertiapin, indicating that baclofen activates GIRK channels via GABAB receptors. The GIRK currents were abolished by N-ethylmaleimide pretreatment, and prolonged by GTPγS inclusion in the patch pipette, suggesting that Gi/o proteins are involved. RT-PCR analysis revealed mRNAs for all four GIRK 1–4 channels and for both GABABR1 and GABABR2 receptors in rat SON. However, the concentration-dependency of the baclofen-induced activation of GIRK currents had an EC50 of 110µM, which is about 100 times higher than that of baclofen-induced inhibition of voltage-dependent Ca2+ channels. Moreover, baclofen caused no significant changes in the membrane potential and the firing rate. These results suggest that although GIRK channels can be activated by GABAB receptors via the Gi/o pathway, this occurs at high agonist concentrations, and thus may not be a physiological mechanism regulating the function of SON neurons. This property that the membrane potential receives little influence from GIRK currents seems to be uncommon for CNS neurons possessing rich Gi/o-coupled receptors, and could be a special feature of rat SON neurons.

IRK-1 Potassium Channels Mediate Peptidergic Inhibition ofCaenorhabditis elegansSerotonin Neurons via a GoSignaling Pathway

To identify molecular mechanisms that function in G-protein signaling, we have performed molecular genetic studies of a simple behavior of the nematode Caenorhabditis elegans, egg laying, which is driven by a pair of serotonergic neurons, the hermaphrodite-specific neurons (HSNs). The activity of the HSNs is regulated by the G(o)-coupled receptor EGL-6, which mediates inhibition of the HSNs by neuropeptides. We report here that this inhibition requires one of three inwardly rectifying K(+) channels encoded by the C. elegans genome: IRK-1. Using ChannelRhodopsin-2-mediated stimulation of HSNs, we observed roles for egl-6 and irk-1 in regulating the excitability of HSNs. Although irk-1 is required for inhibition of HSNs by EGL-6 signaling, we found that other G(o) signaling pathways that inhibit HSNs involve irk-1 little or not at all. These findings suggest that the neuropeptide receptor EGL-6 regulates the potassium channel IRK-1 via a dedicated pool of G(o) not involved in other G(o)-mediated signaling. We conclude that G-protein-coupled receptors that signal through the same G-protein in the same cell might activate distinct effectors and that specific coupling of a G-protein-coupled receptor to its effectors can be determined by factors other than its associated G-proteins.

An inwardly rectifying K+ channel is required for patterning

Mutations that disrupt function of the human inwardly rectifying potassium channel KIR2.1 are associated with the craniofacial and digital defects of Andersen-Tawil Syndrome, but the contribution of Kir channels to development is undefined. Deletion of mouse Kir2.1 also causes cleft palate and digital defects. These defects are strikingly similar to phenotypes that result from disrupted TGFβ/BMP signaling. We use Drosophila melanogaster to show that a Kir2.1 homolog, Irk2, affects development by disrupting BMP signaling. Phenotypes of irk2 deficient lines, a mutant irk2 allele, irk2 siRNA and expression of a dominant-negative Irk2 subunit (Irk2DN) all demonstrate that Irk2 function is necessary for development of the adult wing. Compromised Irk2 function causes wing-patterning defects similar to those found when signaling through a Drosophila BMP homolog, Decapentaplegic (Dpp), is disrupted. To determine whether Irk2 plays a role in the Dpp pathway, we generated flies in which both Irk2 and Dpp functions are reduced. Irk2DN phenotypes are enhanced by decreased Dpp signaling. In wild-type flies, Dpp signaling can be detected in stripes along the anterior/posterior boundary of the larval imaginal wing disc. Reducing function of Irk2 with siRNA, an irk2 deletion, or expression of Irk2DN reduces the Dpp signal in the wing disc. As Irk channels contribute to Dpp signaling in flies, a similar role for Kir2.1 in BMP signaling may explain the morphological defects of Andersen-Tawil Syndrome and the Kir2.1 knockout mouse.

Gαi and Gβγ Jointly Regulate the Conformations of a Gβγ Effector, the Neuronal G Protein-activated K+ Channel (GIRK)

Stable complexes among G proteins and effectors are an emerging concept in cell signaling. The prototypical G betagamma effector G protein-activated K(+) channel (GIRK; Kir3) physically interacts with G betagamma but also with G alpha(i/o). Whether and how G alpha(i/o) subunits regulate GIRK in vivo is unclear. We studied triple interactions among GIRK subunits 1 and 2, G alpha(i3) and G betagamma. We used in vitro protein interaction assays and in vivo intramolecular Förster resonance energy transfer (i-FRET) between fluorophores attached to N and C termini of either GIRK1 or GIRK2 subunit. We demonstrate, for the first time, that G betagamma and G alpha(i3) distinctly and interdependently alter the conformational states of the heterotetrameric GIRK1/2 channel. Biochemical experiments show that G betagamma greatly enhances the binding of GIRK1 subunit to G alpha(i3)(GDP) and, unexpectedly, to G alpha(i3)(GTP). i-FRET showed that both G alpha(i3) and G betagamma induced distinct conformational changes in GIRK1 and GIRK2. Moreover, GIRK1 and GIRK2 subunits assumed unique, distinct conformations when coexpressed with a "constitutively active" G alpha(i3) mutant and G betagamma together. These conformations differ from those assumed by GIRK1 or GIRK2 after separate coexpression of either G alpha(i3) or G betagamma. Both biochemical and i-FRET data suggest that GIRK acts as the nucleator of the GIRK-G alpha-G betagamma signaling complex and mediates allosteric interactions between G alpha(i)(GTP) and G betagamma. Our findings imply that G alpha(i/o) and the G alpha(i) betagamma heterotrimer can regulate a G betagamma effector both before and after activation by neurotransmitters.

Identification of the Alcohol Activation Site in GIRK Channels

In addition to G proteins, ethanol can activate G protein-gated inwardly rectifying K (GIRK) channels. The mechanism underlying GIRK channel activation by alcohol is not well understood. Based on a crystal structure of a related IRK1 channel which contains the alcohol (2-methyl,2-4-pentanediol- MPD) bound to a cytoplasmic hydrophobic pocket, we used structure-based mutagenesis and patch-clamp electrophysiology to investigate the role of the homologous alcohol pocket in GIRK2 channels. In HEK293T cells transfected with GIRK2 cDNA, both ethanol and MPD activated GIRK2 channels. Replacing a conserved Leucine (L257) in this pocket with a bulkier Tyrosine or Tryptophan led to significant attenuation or loss of alcohol-dependent activation of GIRK2 channels, suggesting these larger hydrophobic side-chains filled the pocket. Based on structure and functional evidence, we conclude that this hydrophobic pocket is the site for alcohol activation of GIRK channels. We hypothesized that tethering a hydrophobic group near the pocket might mimic alcohol mediated activation of the channel. To test this idea, we introduced a S246C mutation in a Cysteine-less GIRK2 channel and examined the effect of bath applied MTS-Benzene. Application of 10 micromolar MTS-Benzene dramatically increased the size of basal GIRK currents by 336+66% n=5. This rapid activation was reversed by application of reducing agent DTT (10 mM), indicating a disulfide bond had formed. In addition to the change in basal current, MTS modification of S246C channel altered the rank order for alcohol activation -with significantly less activation by the larger alcohol MPD. These results suggest that attachment of a bulky hydrophobic amino acid near the hydrophobic alcohol-binding pocket can produce sustained activation of the channel by associating with the activation site. These experiments provide a launching point to study molecular events at this hydrophobic pocket that lead to activation of GIRK channels.

Construction Of Functional N-type Ca2+ Channels (Cav2.2) With Accessible External Epitope Tags Suitable For Live Cell Labeling

Recent studies of voltage-gated Ca2+ channels indicate that mechanisms other than alterations in gating contribute to modulation. For example, internalization of receptor/channel complexes (e.g., ORL1) or formation of non-conducting species (e.g., RGK proteins), potentially contribute to CaV2.2 function. An essential technique for investigating these phenomena is the ability to specifically label fully functional CaV2.2 α-subunits (CACNA1B) in the plasma membrane of living cells. Towards this end, we attempted a systematic insertion of a hemagglutinin (HA) epitope tag into each of the 12 predicted extracellular loops of the rat CaV2.2 α-subunit. The first round of mutagenesis resulted in the addition of sites to 1E1 (domain 1, extracellular loop 1), 1E2, 2E2, 3E1, 3E2 and 4E3. A sequence coding for a short flexible linker plus two contiguous copies of the hemagglutinin epitope was ligated into each site. The resulting constructs were electroporated into HEK293 cells along with plasmids encoding Ca2+ channel β2a and α2δ subunits, and the K+ channel IRK1. Robust “rim type” immunofluorescent labeling (in living cells) was detected for 4 of 6 clones (1E2, 3E1, 3E2 and 4E3). Expression of these constructs in HEK293 cells produced channels with electrophysiological properties similar to wildtype as determined by whole-cell patch-clamp with 10 mM Ca2+ as the charge carrier. Average peak currents were (in nA) -5.4±0.7 for the control and -3.8±0.7, -1.2±0.2, -3.1±0.7 and -4.0±0.7 for 1E2, 3E1, 3E2 and 4E3, respectively. The IV curves and individual current trajectories for each clone were superficially similar to the wildtype. These clones should provide powerful tools for the study of trafficking and modulatory mechanisms of CaV2.2. Additionally, the tagging strategy may be applicable to additional members of the CaV and NaV families of voltage-gated channels.

Unique Role For The -5′ Position In The Carboxyl Terminus Of GIRK3 Channel In Determining Binding Specificity To The PDZ Domain Of Sorting Nexin 27

We recently established that sorting nexin 27 (SNX27) regulates trafficking of neuronal GIRK2/3 but not the closely related IRK1 channels. This regulation is achieved by the interaction of the C-terminal PDZ binding motif (-SKV) of GIRK3 with the PDZ domain of SNX27. We also found that IRK1(-SEI) binds to PSD95-PDZ2 but not to SNX27-PDZ. Due to the similarity in the PDZ binding motif between IRK1 and GIRK3, we hypothesized that amino acids in the -4′ and -5′ position are important for determining PDZ binding specificity. Using in vitro binding assays, we discovered that exchanging the these amino acids, IRK1(-ESESKV) and GIRK3(-RRESKV), reversed the binding specificity, suggesting a critical role for -5′ glutamate for binding SNX27 and -5′ arginine for binding PSD95. Further mutagenesis in IRK1 revealed that aspartate or glutamine substitution at -5′ position, IRK1(-DRESKV) and IRK1(-QRESKV), promotes binding to SNX27-PDZ. Surprisingly, substitution with the large, hydrophobic tryptophan enhanced SNX27 binding more than the wild-type glutamate; IRK1(-WRESKV) was 5±1.3 fold greater than IRK1(-ESESKV). We next investigated the consequence of altering the PDZ binding specificity using a clustering assay. CFP-tagged IRK1 or GIRK3 were co-expressed with YFP-tagged PSD95 or SNX27 in COS7 cells. Using TIRF microscopy to visualize proteins at or near the cell membrane, we observed that mutant IRK1(-ESESKV) and wild-type GIRK3(-ESESKV) partially co-localized with SNX27 but did not form clusters. By contrast, mutant GIRK3(-RRESKV) and IRK1(-RRESKV) co-clustered with PSD95. We then used X-ray crystallography to solve the crystal structure of SNX27-PDZ complexed with ESESKV and are now using the structural data to correlate with binding data. These studies provide new details into the specificity of PDZ binding among class I PDZ binding motifs

Evidence for a Discrete Alcohol Pocket Mediating GIRK Channel Activation

Alcohols can activate G protein-gated inwardly rectifying K (GIRK) channels but the molecular mechanism is not well understood. To investigate the possibility of a physical alcohol pocket located in the cytoplasmic domain of GIRK channels, we used a crystal structure of related IRK1 channel containing a bound alcohol (2-methyl, 2-4-pentanediol - MPD) and structure-based mutagenesis of GIRK2 and GIRK4 channels combined with patch-clamp electrophysiology. In transiently transfected HEK293 cells, both wild-type GIRK2 and GIRK4 channels were activated by 100 mM ethanol, MPD and 1-Propanol. Replacing a conserved Leucine (GIRK2-L257 / GIRK4-L252) in the betaD-betaE region of the cytoplasmic domain with bulkier Tyrosine or Tryptophan led to significant attenuation or loss of alcohol-dependent activation for both GIRK2 and GIRK4 channels. Constitutively open channels, such as IRK1 and GIRK2-PIP2 (engineered to bind PIP2 with high affinity), on the other hand, were inhibited by ethanol, 1-propanol, 1-butanol and MPD. Mutating the homologous Leucine in IRK1 (L257) or in GIRK2-PIP2 channels did not alter the sensitivity to inhibition by these alcohols, suggesting a second site in the channel is involved in inhibition by alcohols. Consistent with this, mutagenesis of the extracellular pore-helix of GIRK4 and GIRK2-PIP2 channels reduced the sensitivity to alcohol-mediated inhibition. Interestingly, mutation of the conserved Leucine (L257/L252) in the betaD-betaE domain also disrupted G protein-dependent activation, suggesting a common mechanism of activation by alcohols and G-proteins. Using our data and an analysis of high-resolution structures of inwardly rectifying K channels, we propose a novel model for alcohol activation of GIRK channels that is mediated by the cytoplasmic hydrophobic alcohol pocket.

Regulation of neural KCNQ channels: signalling pathways, structural motifs and functional implications

Neural M-type (KCNQ/Kv7) K(+) channels control somatic excitability, bursting and neurotransmitter release throughout the nervous system. Their activity is regulated by multiple signalling pathways. In superior cervical ganglion sympathetic neurons, muscarinic M(1), angiotensin II AT(1), bradykinin B(2) and purinergic P2Y agonists suppress M current (I(M)). Probes of PLC activity show agonists of all four receptors to induce robust PIP(2) hydrolysis. We have grouped these receptors into two related modes of action. One mode involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in the membrane, whose interaction with the channels is thought necessary for their function. The other involves IP(3)-mediated intracellular Ca(2+) signals that stimulate PIP(2) synthesis, preventing its depletion, and suppress I(M) via calmodulin. Carbon-fibre amperometry can evaluate the effect of M channel activity on release of neurotransmitter. Consistent with the dominant role of M current in control of neuronal discharge, M channel openers, or blockers, reduced or augmented the evoked release of noradrenaline neurotransmitter from superior cervical ganglion (SCG) neurons, respectively. We seek to localize the subdomains on the channels critical to their regulation by PIP(2). Based on single-channel recordings from chimeras between high-PIP(2) affinity KCNQ3 and low-PIP(2) affinity KCNQ4 channels, we focus on a 57-residue domain within the carboxy-terminus that is a possible PIP(2) binding site. Homology modelling of this domain using the published structure of IRK1 channels as a template predicts a structure very similar to an analogous region in IRK1 channels, and shows a cluster of basic residues in the KCNQ2 domain to correspond to those implicated in PIP(2) regulation of Kir channels. We discuss some important issues dealing with these topics.

Depolarization activates the phosphoinositide phosphatase Ci‐VSP, as detected in Xenopus oocytes coexpressing sensors of PIP2

Voltage-evoked signals play critical roles in neural activities, muscle contraction and exocytosis. Ciona voltage-sensor containing phosphatase (Ci-VSP) consists of the transmembrane voltage sensor domain (VSD) and a cytoplasmic domain of phosphoinositide phosphatase, homologous to phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Previous experiments utilizing potassium channels as the sensor for phosphoinositides have demonstrated that phosphatase activities of Ci-VSP are voltage dependent. However, it still remained unclear whether enzyme activity is activated by depolarization or hyperpolarization. Further, a large gap in voltage dependency was found between the charge movement of the VSD and potassium channel-reporting phosphatase activities. In this study, voltage-dependent dynamics of phosphoinositides mediated by Ci-VSP were examined by confocal imaging and electrical measurements in Xenopus oocytes. Imaging of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) using green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains from phospholipase C delta subunit (PLC-delta) showed that PtdIns(4,5)P(2) concentration is reduced during depolarization. In the presence of Ci-VSP, IRK1 channels with higher sensitivity to phosphoinositide than GIRK2 channels decreased their magnitude during depolarization over 0 mV, indicating that the PtdIns(4,5)P(2) level is reduced upon depolarization. KCNQ2/3 channels coexpressed with Ci-VSP exhibited voltage-dependent decay of the outward current that became sharper with higher depolarization in a voltage range up to 100 mV. These results indicate that Ci-VSP has an activity that depletes PtdIns(4,5)P(2) unlike PTEN and that depolarization-activated voltage sensor movement is translated into activation of phosphatase activity.

Functional Aspects of Evolution in IRK Channel Genomic Genes

Functional Aspects of Evolution in IRK Channel Genomic Genes

Characterization of Inward-Rectifier K+ Channel Inhibition by Antiarrhythmic Piperazine

Strong inward-rectifier K(+) (Kir) channels play a significant role in shaping the cardiac action potential: they help produce its long plateau and accelerate its rate of repolarization. Consequently, genetic deletion of the gene encoding the strongly rectifying K(+) channel IRK1 (Kir2.1) prolongs the cardiac action potential in mice. In principle, broadening the action potential lengthens the refractory period, which may in turn be antiarrhythmogenic. Interestingly, previous studies showed that piperazine, an inexpensive and safe anthelmintic, both inhibits IRK1 channels and is antiarrhythmic in some animal preparations. This potential pharmacological benefit motivated us to further characterize the energetic, kinetic, and molecular properties of IRK1 inhibition by piperazine. We show how its blocking characteristics, in particular, its shallow voltage dependence, allow piperazine to be effective even in the presence of high-affinity polyamine blockers. We also examine the channel selectivity of piperazine and its molecular determinants.

Regulation of Kir channels by intracellular pH and extracellular K(+): mechanisms of coupling.

ROMK channels are regulated by internal pH (pHi) and extracellular K+ (K+ o). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H+, increasing the apparent pKa for inhibition by ∼0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K+ o, consistent with coupling between the responses to pHi and K+ o. The ion selectivity sequence of the activation of the channel by cations was K+ ≅ Rb+ > NH4 + >> Na+, similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K+ from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K+ o. We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K+ o between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K+ to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba2+ block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H+. We conclude that decreasing pHI increases the sensitivity of ROMK2 channels to K+ o by altering the properties of the selectivity filter.

Dopamine modulates inwardly rectifying potassium currents in medial prefrontal cortex pyramidal neurons.

Dopamine (DA) modulation of excitability in medial prefrontal cortex (mPFC) pyramidal neurons has attracted considerable attention because of the involvement of mPFC DA in several neuronal disorders. Here, we focused on DA modulation of inwardly rectifying K(+) current (IRKC) in pyramidal neurons acutely dissociated from rat mPFC. A Cs(+)-sensitive whole-cell IRKC was elicited by hyperpolarizing voltage steps from a holding potential of -50 mV. DA (20 microm) reduced IRKC amplitude, as did selective stimulation of DA D(1) or D(2) class receptors (D(1)Rs and D(2)Rs). D(1)Rs activate, whereas D(2)Rs inhibit, the adenylyl cyclase-cAMP-protein kinase A (PKA) signaling pathway. Suppression of IRKC by D(2)R stimulation was attributable to decreased PKA activity because similar inhibition was observed with PKA inhibitors, whereas enhancing PKA activity increased IRKC. This suggests that the DA D(1)R suppression of IRKC occurred through a PKA phosphorylation-independent process. Using outside-out patches of mPFC pyramidal neurons, which preclude involvement of cytosolic signaling molecules, we observed a Cs(+)-sensitive macroscopic IRKC that was suppressed by the membrane-permeable cyclic nucleotide Sp-cAMP but was unaffected by non-nucleotide modulators of PKA, suggesting direct interactions of the cyclic nucleotides with IRK channels. Our results indicate that DA suppresses IRKC through two mechanisms: D(1)R activation of cAMP and direct interactions of the nucleotide with IRK channels and D(2)R-mediated dephosphorylation of IRK channels. The DA modulation of IRKC indicates that ambient DA would tend to increase responsiveness to excitatory inputs when PFC neurons are near the resting membrane potential and may provide a mechanism by which DA impacts higher cognitive function.

5-hydroxytryptamine 1a-likereceptor activation in the bed nucleus of the stria terminalis: Electrophysiological and behavioral studies

The anteriorlateral bed nucleus of the stria terminalis (BNST AL) and the serotonergic system are believed to modulate behavioral responses to stressful and/or anxiogenic stimuli. However, although the BNST AL receives heavy serotonergic innervation, the functional significance of this input is not known. Data obtained from in vitro whole-cell patch clamp recording in the rat BNST slice show that exogenous application of 5-hydroxytryptamine (5-HT) evoked a heterogeneous response in BNST AL neurons. The principal action of 5-HT in this region was inhibitory, evoking a membrane hyperpolarization (5-HT Hyp) and a concomitant reduction in input resistance in the majority of neurons tested. The broad-spectrum 5-HT 1 agonist, 5-carboxamindotryptamine (5-CT), but not R(±)8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), mimicked the 5-HT Hyp response in the BNST. Moreover, the outward current mediating 5-HT Hyp was inwardly rectifying and sensitive to the G protein activated inwardly rectifying K + (G IRK) channel blocker, tertiapin-Q. In the CNS 5-HT 1A receptors are thought to couple to G IRK channels, suggesting that 5-HT Hyp in BNST AL neurons was mediated by activation of 5-HT 1A-like receptors. This was confirmed by the blockade of both 5-HT Hyp and 5-CT Hyp by the specific 5-HT 1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N-2-pyridinylcyclohexanecarboxamide maleate salt (WAY100635 200nM). Furthermore, an in vivo examination of the functional consequences of 5-HT 1A-like induced inhibition of BNST neurons revealed that infusion of 5-CT into the BNST significantly reduced the acoustic startle response, without affecting the general motor activity of the animals. These data point to the possibility that 5-HT 1A mediated inhibition of the BNST AL could contribute to an anxiolytic action. Hence, we propose that in response to stressful stimuli, enhanced levels of 5-HT in the BNST AL plays a critical homeostatic role in feedback inhibition of the anxiogenic response to these stimuli.

Inhibition of G protein-activated inwardly rectifying K+ channels by fluoxetine (Prozac).

1. The effects of fluoxetine, a commonly used antidepressant drug, on G protein-activated inwardly rectifying K(+) channels (GIRK, Kir3) were investigated using Xenopus oocyte expression assays. 2. In oocytes injected with mRNAs for GIRK1/GIRK2, GIRK2 or GIRK1/GIRK4 subunits, fluoxetine reversibly reduced inward currents through the basal GIRK activity. The inhibition by fluoxetine showed a concentration-dependence, a weak voltage-dependence and a slight time-dependence with a predominant effect on the instantaneous current elicited by voltage pulses and followed by slight further inhibition. Furthermore, in oocytes expressing GIRK1/2 channels and the cloned Xenopus A(1) adenosine receptor, GIRK current responses activated by the receptor were inhibited by fluoxetine. In contrast, ROMK1 and IRK1 channels in other Kir channel subfamilies were insensitive to fluoxetine. 3. The inhibitory effect on GIRK channels was not obtained by intracellularly applied fluoxetine, and not affected by extracellular pH, which changed the proportion of the uncharged to protonated fluoxetine, suggesting that fluoxetine inhibits GIRK channels from the extracellular side. 4. The GIRK currents induced by ethanol were also attenuated in the presence of fluoxetine. 5. We demonstrate that fluoxetine, at low micromolar concentrations, inhibits GIRK channels that play an important role in the inhibitory regulation of neuronal excitability in most brain regions and the heart rate through activation of various G-protein-coupled receptors. The present results suggest that inhibition of GIRK channels by fluoxetine may contribute to some of its therapeutic effects and adverse side effects, particularly seizures in overdose, observed in clinical practice.

IRK1 inward rectifier K(+) channels exhibit no intrinsic rectification.

In intact cells the depolarization-induced outward IRK1 currents undergo profound relaxation so that the steady-state macroscopic I-V curve exhibits strong inward rectification. A modest degree of rectification persists after the membrane patches were perfused with artificial solutions devoid of Mg2+ and polyamines, which has been interpreted as a reflection of intrinsic channel gating and led to the view that inward rectification results from enhancement of the intrinsic gating by intracellular cations rather than simple pore block. Furthermore, IRK1 exhibits significant extracellular K+-sensitive relaxation of its inward current, a feature that has been likened to the C-type inactivation observed in the voltage-activated Shaker K+ channels. We found that both these current relaxations can be accounted for by impurities in some common constituents of recording solutions, such as residual hydroxyethylpiperazine in HEPES and ethylenediamine in EDTA. Therefore, inherently, IRK1 channels are essentially ohmic at the macroscopic level, and the voltage jump–induced current relaxations do not reflect IRK1 gating but the unusually high affinity of its pore for cations. Furthermore, our study helps define the optimal experimental conditions for studying IRK1.