Ezequiel Marron Fernandez de Velasco

New insights into the therapeutic potential of Girk channels

G protein-dependent signaling pathways control the activity of excitable cells of the nervous system and heart, and are the targets of neurotransmitters, clinically relevant drugs, and drugs of abuse. G protein-gated inwardly rectifying potassium (K(+)) (Girk/Kir3) channels are a key effector in inhibitory signaling pathways. Girk-dependent signaling contributes to nociception and analgesia, reward-related behavior, mood, cognition, and heart-rate regulation, and has been linked to epilepsy, Down syndrome, addiction, and arrhythmias. We discuss recent advances in our understanding of Girk channel structure, organization in signaling complexes, and plasticity, as well as progress on the development of subunit-selective Girk modulators. These findings offer new hope for the selective manipulation of Girk channels to treat a variety of debilitating afflictions.

Repeated Cocaine Weakens GABAB-Girk Signaling in Layer 5/6 Pyramidal Neurons in the Prelimbic Cortex

Repeated cocaine exposure triggers adaptations in layer 5/6 glutamatergic neurons in the medial prefrontal cortex (mPFC) that promote behavioral sensitization and drug-seeking behavior. While suppression of metabotropic inhibitory signaling has been implicated in these behaviors, underlying mechanisms are unknown. Here, we show that Girk/K(IR)3 channels mediate most of the GABA(B) receptor (GABA(B)R)-dependent inhibition of layer 5/6 pyramidal neurons in the mPFC and that repeated cocaine suppresses this pathway. This adaptation was selective for GABA(B)R-dependent Girk signaling in layer 5/6 pyramidal neurons of the prelimbic cortex (PrLC) and involved a D₁/₅ dopamine receptor- and phosphorylation-dependent internalization of GABA(B)R and Girk channels. Persistent suppression of Girk signaling in layer 5/6 of the dorsal mPFC enhanced cocaine-induced locomotor activity and occluded behavioral sensitization. Thus, the cocaine-induced suppression of GABA(B)R-Girk signaling in layer 5/6 pyramidal neurons of the prelimbic cortex appears to represent an early adaptation critical for promoting addiction-related behavior.

A Rapid Cytoplasmic Mechanism for PI3 Kinase Regulation by the Nuclear Thyroid Hormone Receptor, TRβ, and Genetic Evidence for Its Role in the Maturation of Mouse Hippocampal Synapses In Vivo

Several rapid physiological effects of thyroid hormone on mammalian cells in vitro have been shown to be mediated by the phosphatidylinositol 3-kinase (PI3K), but the molecular mechanism of PI3K regulation by nuclear zinc finger receptor proteins for thyroid hormone and its relevance to brain development in vivo have not been elucidated. Here we show that, in the absence of hormone, the thyroid hormone receptor TRβ forms a cytoplasmic complex with the p85 subunit of PI3K and the Src family tyrosine kinase, Lyn, which depends on two canonical phosphotyrosine motifs in the second zinc finger of TRβ that are not conserved in TRα. When hormone is added, TRβ dissociates and moves to the nucleus, and phosphatidylinositol (3, 4, 5)-trisphosphate production goes up rapidly. Mutating either tyrosine to a phenylalanine prevents rapid signaling through PI3K but does not prevent the hormone-dependent transcription of genes with a thyroid hormone response element. When the rapid signaling mechanism was blocked chronically throughout development in mice by a targeted point mutation in both alleles of Thrb, circulating hormone levels, TRβ expression, and direct gene regulation by TRβ in the pituitary and liver were all unaffected. However, the mutation significantly impaired maturation and plasticity of the Schaffer collateral synapses on CA1 pyramidal neurons in the postnatal hippocampus. Thus, phosphotyrosine-dependent association of TRβ with PI3K provides a potential mechanism for integrating regulation of development and metabolism by thyroid hormone and receptor tyrosine kinases.

New kappa opioid receptor from zebrafish Danio rerio.

A cDNA that encodes a kappa opioid receptor like from zebrafish (ZFOR3) has been cloned and characterized. The encoded protein is 377 residues long and presents 70% identity with the mammalian kappa receptors, although less homology is found in the amino- and carboxyl-terminus as well as in the extracellular loops. In situ hybridization studies have revealed that ZFOR3 mRNA is highly expressed in particular brain areas that coincide with the expression of the kappa opioid receptor in other species. When ZFOR3 is stably expressed in HEK293 cells, [(3)H]-diprenorphine binds with high affinity (K(D)=1.05+/-0.26 nM), being this value on the same range as those reported for mammalian kappa opioid receptors. On the other hand, the selective agonist for mammalian kappa receptors U69,593 does not bind to ZFOR3. [(3)H]-diprenorphine binding is readily displaced by the peptidic ligand dynorphin A and by the non-endogenous compounds bremazocine, naloxone and morphine, although with different affinities. Our results demonstrate that ZFOR3 is a unique model to study the kappa opioid receptor functionality.

Mechanisms underlying the activation of G-protein–gated inwardly rectifying K+ (GIRK) channels by the novel anxiolytic drug, ML297

Significance Many neurotransmitters dampen excitability in the heart and brain by activating G-protein–gated inwardly rectifying K+ (GIRK) channels. The lack of selective pharmacological tools for GIRK channels has hindered investigations into their physiological and pathophysiological relevance. Here, we examined the mechanisms underlying the activation of GIRK channels by ML297, the prototypical member of a new family of small molecule GIRK channel modulators. ML297 activates GIRK channels via a unique mechanism that requires two amino acids specific to the GIRK1 subunit. In addition, ML297 reduces anxiety-related behavior in mice, in a GIRK1-dependent manner, without triggering sedation or addiction-related behavior. Thus, ML297 is a new tool for probing the therapeutic potential of GIRK channel modulation, which may benefit individuals with anxiety-related disorders. ML297 was recently identified as a potent and selective small molecule agonist of G-protein–gated inwardly rectifying K+ (GIRK/Kir3) channels. Here, we show ML297 selectively activates recombinant neuronal GIRK channels containing the GIRK1 subunit in a manner that requires phosphatidylinositol-4,5-bisphosphate (PIP2), but is otherwise distinct from receptor-induced, G-protein–dependent channel activation. Two amino acids unique to the pore helix (F137) and second membrane-spanning (D173) domain of GIRK1 were identified as necessary and sufficient for the selective activation of GIRK channels by ML297. Further investigation into the behavioral effects of ML297 revealed that in addition to its known antiseizure efficacy, ML297 decreases anxiety-related behavior without sedative or addictive liabilities. Importantly, the anxiolytic effect of ML297 was lost in mice lacking GIRK1. Thus, activation of GIRK1-containing channels by ML297 or derivatives may represent a new approach to the treatment of seizure and/or anxiety disorders.

GIRK channels modulate opioid-induced motor activity in a cell type- and subunit-dependent manner

G-protein-gated inwardly rectifying K+ (GIRK/Kir3) channel activation underlies key physiological effects of opioids, including analgesia and dependence. GIRK channel activation has also been implicated in the opioid-induced inhibition of midbrain GABA neurons and consequent disinhibition of dopamine (DA) neurons in the ventral tegmental area (VTA). Drug-induced disinhibition of VTA DA neurons has been linked to reward-related behaviors and underlies opioid-induced motor activation. Here, we demonstrate that mouse VTA GABA neurons express a GIRK channel formed by GIRK1 and GIRK2 subunits. Nevertheless, neither constitutive genetic ablation of Girk1 or Girk2, nor the selective ablation of GIRK channels in GABA neurons, diminished morphine-induced motor activity in mice. Moreover, direct activation of GIRK channels in midbrain GABA neurons did not enhance motor activity. In contrast, genetic manipulations that selectively enhanced or suppressed GIRK channel function in midbrain DA neurons correlated with decreased and increased sensitivity, respectively, to the motor-stimulatory effect of systemic morphine. Collectively, these data support the contention that the unique GIRK channel subtype in VTA DA neurons, the GIRK2/GIRK3 heteromer, regulates the sensitivity of the mouse mesolimbic DA system to drugs with addictive potential.

Selective Ablation of GIRK Channels in Dopamine Neurons Alters Behavioral Effects of Cocaine in Mice

The increase in dopamine (DA) neurotransmission stimulated by in vivo cocaine exposure is tempered by G protein-dependent inhibitory feedback mechanisms in DA neurons of the ventral tegmental area (VTA). G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels mediate the direct inhibitory effect of GABAB receptor (GABABR) and D2 DA receptor (D2R) activation in VTA DA neurons. Here we examined the effect of the DA neuron-specific loss of GIRK channels on D2R-dependent regulation of VTA DA neuron excitability and on cocaine-induced, reward-related behaviors. Selective ablation of Girk2 in DA neurons did not alter the baseline excitability of VTA DA neurons but significantly reduced the magnitude of D2R-dependent inhibitory somatodendritic currents and blunted the impact of D2R activation on spontaneous activity and neuronal excitability. Mice lacking GIRK channels in DA neurons exhibited increased locomotor activation in response to acute cocaine administration and an altered locomotor sensitization profile, as well as increased responding for and intake of cocaine in an intravenous self-administration test. These mice, however, showed unaltered cocaine-induced conditioned place preference. Collectively, our data suggest that feedback inhibition to VTA DA neurons, mediated by GIRK channel activation, tempers the locomotor stimulatory effect of cocaine while also modulating the reinforcing effect of cocaine in an operant-based self-administration task.

Sex differences in GABABR-GIRK signaling in layer 5/6 pyramidal neurons of the mouse prelimbic cortex

The medial prefrontal cortex (mPFC) has been implicated in multiple disorders characterized by clear sex differences, including schizophrenia, attention deficit hyperactivity disorder, post-traumatic stress disorder, depression, and drug addiction. These sex differences likely represent underlying differences in connectivity and/or the balance of neuronal excitability within the mPFC. Recently, we demonstrated that signaling via the metabotropic γ-aminobutyric acid receptor (GABABR) and G protein-gated inwardly-rectifying K(+) (GIRK/Kir3) channels modulates the excitability of the key output neurons of the mPFC, the layer 5/6 pyramidal neurons. Here, we report a sex difference in the GABABR-GIRK signaling pathway in these neurons. Specifically, GABABR-dependent GIRK currents recorded in the prelimbic region of the mPFC were larger in adolescent male mice than in female counterparts. Interestingly, this sex difference was not observed in layer 5/6 pyramidal neurons of the adjacent infralimbic cortex, nor was it seen in young adult mice. The sex difference in GABABR-GIRK signaling is not attributable to different expression levels of signaling pathway components, but rather to a phosphorylation-dependent trafficking mechanism. Thus, sex differences related to some diseases associated with altered mPFC function may be explained in part by sex differences in GIRK-dependent signaling in mPFC pyramidal neurons.

G Protein-Gated K+ Channel Ablation in Forebrain Pyramidal Neurons Selectively Impairs Fear Learning

BACKGROUND Cognitive dysfunction occurs in many debilitating conditions including Alzheimers disease, Down syndrome, schizophrenia, and mood disorders. The dorsal hippocampus is a critical locus of cognitive processes linked to spatial and contextual learning. G protein-gated inwardly rectifying potassium ion (GIRK/Kir3) channels, which mediate the postsynaptic inhibitory effect of many neurotransmitters, have been implicated in hippocampal-dependent cognition. Available evidence, however, derives primarily from constitutive gain-of-function models that lack cellular specificity. METHODS We used constitutive and neuron-specific gene ablation models targeting an integral subunit of neuronal GIRK channels (GIRK2) to probe the impact of GIRK channels on associative learning and memory. RESULTS Constitutive Girk2-/- mice exhibited a striking deficit in hippocampal-dependent (contextual) and hippocampal-independent (cue) fear conditioning. Mice lacking GIRK2 in gamma-aminobutyric acid neurons (GAD-Cre:Girk2flox/flox mice) exhibited a clear deficit in GIRK-dependent signaling in dorsal hippocampal gamma-aminobutyric acid neurons but no evident behavioral phenotype. Mice lacking GIRK2 in forebrain pyramidal neurons (CaMKII-Cre(+):Girk2flox/flox mice) exhibited diminished GIRK-dependent signaling in dorsal, but not ventral, hippocampal pyramidal neurons. CaMKII-Cre(+):Girk2flox/flox mice also displayed a selective impairment in contextual fear conditioning, as both cue fear and spatial learning were intact in these mice. Finally, loss of GIRK2 in forebrain pyramidal neurons correlated with enhanced long-term depression and blunted depotentiation of long-term potentiation at the Schaffer collateral/cornu ammonis 1 synapse in the dorsal hippocampus. CONCLUSIONS Our data suggest that GIRK channels in dorsal hippocampal pyramidal neurons are necessary for normal learning involving aversive stimuli and support the contention that dysregulation of GIRK-dependent signaling may underlie cognitive dysfunction in some disorders.

Bioactivity studies on atypical natural opioid hexapeptides processed from proenkephalin (PENK) precursor polypeptides

Endogenous opioids are derived from four related polypeptide precursors: proenkephalin (PENK), prodynorphin (PDYN), pronociceptin (PNOC) and proopiomelanocortin (POMC). In mammals PENK encodes for four copy of Met-enkephalin, one octapeptide Met-enkephalin-Arg-Gly-Leu, one heptapeptide Met-enkephalin-Arg-Phe and a single copy of Leu-enkephalin. Our detailed bioinformatic search on the existing PENK sequences revealed several atypical hexapeptide Met-enkephalins in different vertebrate animals. They are located either in the second enkephalin unit or in the seventh enkephalin core position at the C-terminus. Altogether four different hexapeptide sequences were obtained representing eleven animal species: Met-enkephalin-Arg(6) (YGGFMR) in the bird zebra finch, Met-enkephalin-Asp(6) (YGGFMD), Met-enkephalin-Ile(6) (YGGFMI) in zebrafish; and Met-enkephalin-Ser(6) (YGGFMS) in two pufferfish species. All novel peptides were chemically synthesized and studied in receptor binding and G-protein activation assays performed on rat brain membranes. The four novel enkephalins were equipotent in stimulating G-proteins. Affinities of the peptides determined by equilibrium competition assays in receptor binding experiments were statistically different. At the MOP receptors the highest affinity (Ki 4nM) was obtained with the zebra finch peptide Met-enkephalin-Arg(6). The pufferfish Met-enkephalin-Ser(6) exhibited the highest affinity (Ki 6.7nM) at the DOP receptor. Phylogenetic neuropeptide libraries, defined here as a collection of mutationally different species variants of orthologous and paralogous peptide sequences, represent the natural molecular diversity of the neuropeptides. Such libraries can provide a wide range of structural information establishing comparative functional analyses. Since DNA sequencing data are rapidly increasing, more development in the natural peptide library concept is expected.