Johanna Nilsson

Truncation of the Shaker‐like voltage‐gated potassium channel, Kv1.1, causes megencephaly

The megencephaly mouse, mceph/mceph, displays dramatically increased brain volume and hypertrophic brain cells. Despite overall enlargement, the mceph/mceph brain appears structurally normal, without oedema, hydrocephaly or leukodystrophy, and with only minor astrocytosis. Furthermore, it presents striking disturbances in expression of trophic and neuromodulating factors within the hippocampus and cortex. Using a positional cloning approach we have identified the mceph mutation. We show that mceph/mceph mice carry an 11‐base‐pair deletion in the gene encoding the Shaker‐like voltage‐gated potassium channel subtype 1, Kcna1. The mutation leads to a frame shift and the predicted MCEPH protein is truncated at amino acid 230 (out of 495), terminating with six aberrant amino acids. The expression of Kcna1 mRNA is increased in the mceph/mceph brain. However, the C‐terminal domains of the corresponding Kv1.1 protein are absent. The putative MCEPH protein retains only the N‐terminal domains for channel assembly and may congregate nonfunctional complexes of multiple Shaker‐like subunits. Indeed, whereas Kcna2 and Kcna3 mRNA expression is normal, the mceph/mceph hippocampus displays decreased amounts of Kv1.2 and Kv1.3 proteins, suggesting interactions at the protein level. We show that mceph/mceph mice have disturbed brain electrophysiology and experience recurrent behavioural seizures, in agreement with the abnormal electrical brain activity found in Shaker mutants. However, in contrast to the commonly demonstrated epilepsy‐induced neurodegeneration, we find that the mceph mutation leads to seizures with a concomitant increase in brain size, without overt neural atrophy.

Mechanisms of Anesthesia: Towards Integrating Network, Cellular, and Molecular Level Modeling

The mechanisms of anesthesia are surprisingly little understood. The present article summarizes current knowledge about the function of general anesthetics at different organization levels of the nervous system. It argues that a consensus view can be constructed, assuming that general anesthetics modulate the activity of ion channels, the main targets being GABA and NMDA channels and possibly voltage-gated and background channels, thereby hyperpolarizing neurons in thalamocortical loops, which lead to disruption of coherent oscillatory activity in the cortex. Two computational cases are used to illustrate the possible importance of molecular level effects on cellular level activity. Subtle differences in the mechanism of ion channel block can be shown to cause considerable differences in the modification of the oscillatory activity in a single neuron, and consequently in an associated network. Finally, the relation between the anesthesia problem and the classical consciousness problem is discussed, and some consequences of introducing the phenomenon of degeneracy into the picture are pointed out.

On the opening of voltage-gated ion channels

Voltage-gated ion channels are key players in fast neuronal signalling. Detailed knowledge about channel gating is essential for our understanding of channel function in general and of drug action of channels in particular. Despite a number of recent atomic channel structures, the opening of voltage-gated channels is the subject of heated debates. Here we will discuss two of the controversies: one concerning the mechanism of opening and closing the pore, and the other concerning the location and movement of the voltage sensor. The channels were originally suggested to open at a conserved proline rich sequence (PVP) at the intracellular end of the transmembrane segment 6 (S6). The crystallization of a channel in the open state instead suggested an opening involving a conserved glycine hinge located in the middle portion of S6. Based on pharmacological studies, autodocking and molecular dynamics simulations we have found support for the PVP-bend model. The voltage sensor, transmembrane segment 4 (S4), was originally suggested to be buried in the channel protein, undergoing a helical-screw-like motion to open the channel. A recent crystallographic study suggested that S4 is located in the periphery, facing lipid, and undergoing a paddle-like motion to open the channel. We have found experimental evidence for a novel helical-screw model; with the voltage sensor moving in a screw-like fashion but being located in the periphery of the channel. This model opens up for understanding how lipophilic drugs and toxins directly affect the voltage sensor.

Coenzyme Q10 prevents peripheral neuropathy and attenuates neuron loss in the db-/db- mouse, a type 2 diabetes model.

Diabetic peripheral neuropathy (DPN) is the most common complication in both type 1 and type 2 diabetes. Here we studied some phenotypic features of a well-established animal model of type 2 diabetes, the leptin receptor-deficient db−/db− mouse, and also the effect of long-term (6 mo) treatment with coenzyme Q10 (CoQ10), an endogenous antioxidant. Diabetic mice at 8 mo of age exhibited loss of sensation, hypoalgesia (an increase in mechanical threshold), and decreases in mechanical hyperalgesia, cold allodynia, and sciatic nerve conduction velocity. All these changes were virtually completely absent after the 6-mo, daily CoQ10 treatment in db−/db− mice when started at 7 wk of age. There was a 33% neuronal loss in the lumbar 5 dorsal root ganglia (DRGs) of the db−/db− mouse versus controls at 8 mo of age, which was significantly attenuated by CoQ10. There was no difference in neuron number in 5/6-wk-old mice between diabetic and control mice. We observed a strong down-regulation of phospholipase C (PLC) β3 in the DRGs of diabetic mice at 8 mo of age, a key molecule in pain signaling, and this effect was also blocked by the 6-mo CoQ10 treatment. Many of the phenotypic, neurochemical regulations encountered in lumbar DRGs in standard models of peripheral nerve injury were not observed in diabetic mice at 8 mo of age. These results suggest that reactive oxygen species and reduced PLCβ3 expression may contribute to the sensory deficits in the late-stage diabetic db−/db− mouse, and that early long-term administration of the antioxidant CoQ10 may represent a promising therapeutic strategy for type 2 diabetes neuropathy.

A truncated Kv1.1 protein in the brain of the megencephaly mouse: expression and interaction

BackgroundThe megencephaly mouse, mceph/mceph, is epileptic and displays a dramatically increased brain volume and neuronal count. The responsible mutation was recently revealed to be an eleven base pair deletion, leading to a frame shift, in the gene encoding the potassium channel Kv1.1. The predicted MCEPH protein is truncated at amino acid 230 out of 495. Truncated proteins are usually not expressed since nonsense mRNAs are most often degraded. However, high Kv1.1 mRNA levels in mceph/mceph brain indicated that it escaped this control mechanism. Therefore, we hypothesized that the truncated Kv1.1 would be expressed and dysregulate other Kv1 subunits in the mceph/mceph mice.ResultsWe found that the MCEPH protein is expressed in the brain of mceph/mceph mice. MCEPH was found to lack mature (Golgi) glycosylation, but to be core glycosylated and trapped in the endoplasmic reticulum (ER). Interactions between MCEPH and other Kv1 subunits were studied in cell culture, Xenopus oocytes and the brain. MCEPH can form tetramers with Kv1.1 in cell culture and has a dominant negative effect on Kv1.2 and Kv1.3 currents in oocytes. However, it does not retain Kv1.2 in the ER of neurons.ConclusionThe megencephaly mice express a truncated Kv1.1 in the brain, and constitute a unique tool to study Kv1.1 trafficking relevant for understanding epilepsy, ataxia and pathologic brain overgrowth.

Cocaine produces D2R-mediated conformational changes in the adenosine A2AR-dopamine D2R heteromer

Adenosine A(2A) receptors (A(2A)Rs) and dopamine D(2) receptors (D(2)Rs) form constitutive heteromers in living cells and exhibit a strong functional antagonistic interaction. Recent findings give neurochemical evidence that extended cocaine self-administration in the rat give rise to an up-regulation of functional A(2A)Rs in the nucleus accumbens that return to baseline expression levels during cocaine withdrawal. In the present work, the acute in vitro effects of a concentration of cocaine known to fully block the dopamine (DA) transporter without exerting any toxic actions were investigated on A(2A)R and D(2L)R formed heteromers in transiently co-transfected HEK-293T cells. In vitro treatment of cocaine was found to produce changes in D(2)R homodimers and in A(2A)R-D(2)R heterodimers detected through bioluminescent energy transfer (BRET). Cocaine was found to produce a time- and concentration-dependent reduction in the BRET(max) between A(2A)R-D(2L)R heterodimers and D(2L)R homodimers, but not A(2A)R homodimers, indicating its effect on D(2)R. Cocaine was evaluated with regard to D(2)R binding using a human D(2L)R stable expressing CHO cell line and was found to produce an increase in the affinity of hD(2L)R for DA. At the level of G protein-coupling, cocaine produced a small, but significant increase in DA-stimulated binding of GTPgammaS. However, cocaine failed to modulate D(2)R agonist-induced inhibition of cAMP in stable hD(2L)R CHO cells or the gating of GIRK channels in oocytes. Taken together, these results indicate a direct and specific effect of a moderate concentration of cocaine on the DA D(2L)R, that results in enhanced agonist recognition, G protein-coupling and an altered conformational state of D(2)R homodimers and A(2A)R-D(2)R heterodimers.

Voltage-dependence of the human dopamine D2 receptor

The dopamine D2 receptor plays a critical role in activity‐dependent synaptic plasticity in the striatum, and regulates the transitions between different states of electrical activity. The D2 receptor is the main target for antipsychotics, and its affinity towards dopamine has been shown to be increased in psychotic patients. Recently, voltage‐sensitivity has been reported for the ligand binding and G protein‐coupling properties of some neurotransmitter receptors, raising the question whether the D2 receptor is also regulated by voltage. Our present electrophysiology data from Xenopus oocytes indicate that the D2 receptor is indeed voltage‐sensitive. Comparing concentration–response relationships for the activation of G protein‐coupled inward rectifier potassium (GIRK) channels via D2 receptor stimulation by quinpirole or dopamine at −80 and at +40 mV revealed rightward shifts upon depolarisation of nearly tenfold, for both agonists. Our results are likely to bear relevance to the function of the D2 receptor in gating synaptic input and in regulating plasticity. Synapse 62:476–480, 2008.

Voltage-sensitivity at the human dopamine D2S receptor is agonist-specific.

Recently, we and others have shown that agonist potencies at some, but not all, G protein-coupled receptors are voltage-sensitive. Several of those studies employed electrophysiology assays in Xenopus oocytes with G protein-coupled potassium channels as a readout. Using this assay, we have now obtained evidence that voltage-sensitivity at the dopamine D(2S) receptor is agonist-specific. Whereas the potency of dopamine at the D(2S) receptor is decreased by depolarization, the potencies of beta-phenethylamine, p- and m-tyramine are voltage-insensitive. Furthermore, both monohydroxylated and non-hydroxylated N,N-dipropyl-2-aminotetralin compounds are voltage-sensitive. Differential activation of G protein subtypes or differential ratios between effector and active G protein do not underlie this agonist-selective voltage-sensitivity. This is the first demonstration of voltage-sensitive and voltage-insensitive behaviour of different agonists acting via the same receptor.

Mechanisms of bupivacaine action on Na+ and K+ channels in myelinated axons of Xenopus laevis

The local anaesthetic bupivacaine has recently been proposed to inhibit Na+ channels indirectly by making the resting potential less negative. To test this hypothesis we analysed the effects of bupivacaine on voltage and current clamped nodes of Ranvier. Contrary to the hypothesis, the leak current and the resting potential were unaffected. The Na+ and K+ channels were, however, affected at relatively low concentrations (33 microM). Steady-state activation curves were decreased without notable shift effects, whereas the Na+ inactivation curve was decreased and shifted in negative direction. The effect on the Na+ current was tentatively explained by a single-site, state-dependent binding model (Kd = 44 microM), while that on the K+ current was explained by two population-specific mechanisms, one open-state dependent (Kd = 550 microM) and one state independent (Kd = 59 microM). The binding stoichiometry was higher than 1:1 for the main sites of action. In conclusion, bupivacaine exerts its main anaesthetic action on myelinated nerve axons by a direct modification of Na+ channels.

Typical and atypical antipsychotics do not differ markedly in their reversibility of antagonism of the dopamine D2 receptor.

It has been suggested that the favorable side-effect profiles of atypical antipsychotics (e.g. clozapine and amisulpride) are related to their ∼100-fold faster dissociation from dopamine D2 receptors (D2R) compared with typical antipsychotics (e.g. haloperidol and chlorpromazine). Fast dissociation would entail rapidly reversible antagonism; however, this has not been thoroughly studied using functional assays. We compared the reversibilities of D2R antagonism by 17 compounds using an electrophysiological method to measure dopamine-evoked potassium channel activation via D2R. Varying rates and amplitudes of D2R response recovery were observed following antagonist removal. Whereas recovery rates differed 15-fold between atypical drugs, recovery from clozapine and amisulpride antagonism was, unexpectedly, less than twofold faster than from chlorpromazine. The recovery amplitude correlated with calculated water solubility and lipid/water distribution coefficients, suggesting variable drug partitioning into cell membranes. Our data do not support the notion that the rate of reversibility of D2R antagonism is what distinguishes atypical from typical antipsychotics.