Thomas V. McDonald

Pharmacological Rescue of Synaptic Plasticity, Courtship Behavior, and Mushroom Body Defects in a Drosophila Model of Fragile X Syndrome

Fragile X syndrome is a leading heritable cause of mental retardation that results from the loss of FMR1 gene function. A Drosophila model for Fragile X syndrome, based on the loss of dfmr1 activity, exhibits phenotypes that bear similarity to Fragile X-related symptoms. Herein, we demonstrate that treatment with metabotropic glutamate receptor (mGluR) antagonists or lithium can rescue courtship and mushroom body defects observed in these flies. Furthermore, we demonstrate that dfmr1 mutants display cognitive deficits in experience-dependent modification of courtship behavior, and treatment with mGluR antagonists or lithium restores these memory defects. These findings implicate enhanced mGluR signaling as the underlying cause of the cognitive, as well as some of the behavioral and neuronal, phenotypes observed in the Drosophila Fragile X model. They also raise the possibility that compounds having similar effects on metabotropic glutamate receptors may ameliorate cognitive and behavioral defects observed in Fragile X patients.

A minK-HERG complex regulates the cardiac potassium current I(Kr).

MinK is a widely expressed protein of relative molecular mass ∼15K that forms potassium channels by aggregation with other membrane proteins. MinK governs ion channel activation, regulation by second messengers,, and the function and structure of the ion conduction pathway,. Association of minK with a channel protein known as KvLQT1 produces a voltage-gated outward K+ current (IsK) resembling the slow cardiac repolarization current (IKs),. HERG, a human homologue of the ether-a-go-go gene of the fruitfly Drosophila melanogaster, encodes a protein that produces the rapidly activating cardiac delayed rectifier (IKr),. These two potassium currents, IKs and IKr, provide the principal repolarizing currents in cardiac myocytes for the termination of action potentials,. Although heterologously expressed HERG channels are largely indistinguishable from native cardiac IKr, a role for minK in this current is suggested by the diminished IKr in an atrial tumour line subjected to minK antisense suppression. Here we show that HERG and minK form a stable complex, and that this heteromultimerization regulates IKr activity. MinK, through the formation of heteromeric channel complexes, is thus central to the control of the heart rate and rhythm.

Cyclic AMP regulates the HERG K+ channel by dual pathways

Lethal cardiac arrhythmias are a hallmark of the hereditary Long QT syndrome (LQTS), a disease produced by mutations of cardiac ion channels [1]. Often these arrhythmias are stress-induced, suggesting a relationship between beta-adrenergic activation of adenylate cyclase and cAMP-dependent alteration of one or more of the ion channels involved in LQTS. Second messengers modulate ion channel activity either by direct interaction or through intermediary kinases and phosphatases. Here we show that the second messenger cAMP regulates the K(+) channel mutated in the LQT2 form of LQTS, HERG [2], both directly and indirectly. Activation of cAMP-dependent protein kinase (PKA) causes phosphorylation of HERG accompanied by a rapid reduction in current amplitude, acceleration of voltage-dependent deactivation, and depolarizing shift in voltage-dependent activation. In a parallel pathway, cAMP directly binds to the HERG protein with the opposing effect of a hyperpolarizing shift in voltage-dependent activation. The summation of cAMP-mediated effects is a net diminution of the effective current, but when HERG is complexed with with the K(+) channel accessory proteins MiRP1 or minK, the stimulatory effects of cAMP are favored. These findings provide a direct link between stress and arrhythmia by a unique mechanism where a single second messenger exerts complex regulation of an ion channel via two distinct pathways.

KCNE1 Binds to the KCNQ1 Pore to Regulate Potassium Channel Activity

Potassium channels control the resting membrane potential and excitability of biological tissues. Many voltage-gated potassium channels are controlled through interactions with accessory subunits of the KCNE family through mechanisms still not known. Gating of mammalian channel KCNQ1 is dramatically regulated by KCNE subunits. We have found that multiple segments of the channel pore structure bind to the accessory protein KCNE1. The sites that confer KCNE1 binding are necessary for the functional interaction, and all sites must be present in the channel together for proper regulation by the accessory subunit. Specific gating control is localized to a single site of interaction between the ion channel and accessory subunit. Thus, direct physical interaction with the ion channel pore is the basis of KCNE1 regulation of K+ channels.

14‐3‐3 amplifies and prolongs adrenergic stimulation of HERG K + channel activity

Acute stress provokes lethal cardiac arrhythmias in the hereditary long QT syndrome. Here we provide a novel molecular mechanism linking β‐adrenergic signaling and altered human ether‐a‐go‐go related gene (HERG) channel activity. Stress stimulates β‐adrenergic receptors, leading to cAMP elevations that can regulate HERG K+ channels both directly and via phosphorylation by cAMP‐dependent protein kinase (PKA). We show that HERG associates with 14‐3‐3ϵ to potentiate cAMP/PKA effects upon HERG. The binding of 14‐3‐3 occurs simultaneously at the N‐ and C‐termini of the HERG channel. 14‐3‐3 accelerates and enhances HERG activation, an effect that requires PKA phosphorylation of HERG and dimerization of 14‐3‐3. The interaction also stabilizes the lifetime of the PKA‐phosphorylated state of the channel by shielding the phosphates from cellular phosphatases. The net result is a prolongation of the effect of adrenergic stimulation upon HERG activity. Thus, 14‐3‐3 interactions with HERG may provide a unique mechanism for plasticity in the control of membrane excitability and cardiac rhythm.

Pharmacological reversal of synaptic plasticity deficits in the mouse model of fragile X syndrome by group II mGluR antagonist or lithium treatment.

Fragile X syndrome is the leading single gene cause of intellectual disabilities. Treatment of a Drosophila model of Fragile X syndrome with metabotropic glutamate receptor (mGluR) antagonists or lithium rescues social and cognitive impairments. A hallmark feature of the Fragile X mouse model is enhanced mGluR-dependent long-term depression (LTD) at Schaffer collateral to CA1 pyramidal synapses of the hippocampus. Here we examine the effects of chronic treatment of Fragile X mice in vivo with lithium or a group II mGluR antagonist on mGluR-LTD at CA1 synapses. We find that long-term lithium treatment initiated during development (5-6 weeks of age) and continued throughout the lifetime of the Fragile X mice until 9-11 months of age restores normal mGluR-LTD. Additionally, chronic short-term treatment beginning in adult Fragile X mice (8 weeks of age) with either lithium or an mGluR antagonist is also able to restore normal mGluR-LTD. Translating the findings of successful pharmacologic intervention from the Drosophila model into the mouse model of Fragile X syndrome is an important advance, in that this identifies and validates these targets as potential therapeutic interventions for the treatment of individuals afflicted with Fragile X syndrome.

Voltage-Gated Potassium Channels: Regulation by Accessory Subunits

Voltage-gated potassium channels regulate cell membrane potential and excitability in neurons and other cell types. A precise control of neuronal action potential patterns underlies the basic functioning of the central and peripheral nervous system. This control relies on the adaptability of potassium channel activities. The functional diversity of potassium currents, however, far exceeds the considerable molecular diversity of this class of genes. Potassium current diversity contributes to the specificity of neuronal firing patterns and may be achieved by regulated transcription, RNA splicing, and posttranslational modifications. Another mechanism for regulation of potassium channel activity is through association with interacting proteins and accessory subunits. Here the authors highlight recent work that addresses this growing area of exploration and discuss areas of future investigation.

KCNE Regulation of KvLQT1 Channels: Structure–Function Correlates

K(+) channels play a central role in determining resting membrane potential and cellular excitability. There is growing recognition that the channels exist not as independent units but as macromolecular complexes able to integrate a plethora of cellular signals to fine-tune channel activities. Interaction of K(+) channels with accessory proteins and subunits is increasingly reported as providing mechanisms for channels to respond to a variety of stimuli beyond just changes in membrane potential. One such association is that between some voltage-gated K(+) channels and the proteins encoded by the KCNE family of genes. The significance of these interactions is manifest in reports of genetic disorders such as the Long QT Syndrome linked to KCNE mutations and proarrhythmic drug susceptibilities from KCNE polymorphisms. The mechanism by which KCNE-encoded proteins control channel behavior is an emerging story. This article reviews some of the recent work addressing the prototypical KCNE-channel interaction between minK and KvLQT1.

Age-dependent cognitive impairment in a Drosophila Fragile X model and its pharmacological rescue

Fragile X syndrome afflicts 1 in 2,500 individuals and is the leading heritable cause of mental retardation worldwide. The overriding clinical manifestation of this disease is mild to severe cognitive impairment. Age-dependent cognitive decline has been identified in Fragile X patients, although it has not been fully characterized nor examined in animal models. A Drosophila model of this disease has been shown to display phenotypes bearing similarity to Fragile X symptoms. Most notably, we previously identified naive courtship and memory deficits in young adults with this model that appear to be due to enhanced metabotropic glutamate receptor (mGluR) signaling. Herein we have examined age-related cognitive decline in the Drosophila Fragile X model and found an age-dependent loss of learning during training. We demonstrate that treatment with mGluR antagonists or lithium can prevent this age-dependent cognitive impairment. We also show that treatment with mGluR antagonists or lithium during development alone displays differential efficacy in its ability to rescue naive courtship, learning during training and memory in aged flies. Furthermore, we show that continuous treatment during aging effectively rescues all of these phenotypes. These results indicate that the Drosophila model recapitulates the age-dependent cognitive decline observed in humans. This places Fragile X in a category with several other diseases that result in age-dependent cognitive decline. This demonstrates a role for the Drosophila Fragile X Mental Retardation Protein (dFMR1) in neuronal physiology with regard to cognition during the aging process. Our results indicate that misregulation of mGluR activity may be causative of this age onset decline and strengthens the possibility that mGluR antagonists and lithium may be potential pharmacologic compounds for counteracting several Fragile X symptoms.

Phosphatidylinositol 4,5-bisphosphate interactions with the HERG K+ channel

Regulation of ion channel activity plays a central role in controlling heart rate, rhythm, and contractility responses to cardiovascular demands. Dynamic beat-to-beat regulation of ion channels is precisely adjusted by autonomic stimulation of cardiac G protein-coupled receptors. The rapidly activating delayed rectifier K+ current (IKr) is produced by the channel that is encoded by human ether-a-gogo-related gene (HERG) and is essential for the proper repolarization of the cardiac myocyte at the end of each action potential. Reduction of IKr via HERG mutations or drug block can lead to lethal cardiac tachyarrhythmias. Autonomic regulation of HERG channels is an area of active investigation with the emerging picture of a complex interplay of signal transduction events, including kinases, second messengers, and protein–protein interactions. A recently described pathway for regulation of HERG is through channel interaction with the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). Changes in cellular PIP2 concentrations may occur with Gq-coupled receptor activation. Here, we review the evidence for PIP2–HERG interactions, its potential biological significance, and unfilled gaps in our understanding of this regulatory mechanism.