Murine startle mutant Nmf11 affects the structural stability of the glycine receptor and increases deactivation.

KEY POINTS: Hyperekplexia or startle disease is a serious neurological condition affecting newborn children and usually involves dysfunctional glycinergic neurotransmission. Glycine receptors (GlyRs) are major mediators of inhibition in the spinal cord and brainstem. A missense mutation, replacing asparagine (N) with lysine (K), at position 46 in the GlyR α1 subunit induced hyperekplexia following a reduction in the potency of the transmitter glycine; this resulted from a rapid deactivation of the agonist current at mutant GlyRs. These effects of N46K were rescued by mutating a juxtaposed residue, N61 on binding Loop D, suggesting these two asparagines may interact. Asparagine 46 is considered to be important for the structural stability of the subunit interface and glycine binding site, and its mutation represents a new mechanism by which GlyR dysfunction induces startle disease. ABSTRACT: Dysfunctional glycinergic inhibitory transmission underlies the debilitating neurological condition, hyperekplexia, which is characterised by exaggerated startle reflexes, muscle hypertonia and apnoea. Here we investigated the N46K missense mutation in the GlyR α1 subunit gene found in the ethylnitrosourea (ENU) murine mutant, Nmf11, which causes reduced body size, evoked tremor, seizures, muscle stiffness, and morbidity by postnatal day 21. Introducing the N46K mutation into recombinant GlyR α1 homomeric receptors, expressed in HEK cells, reduced the potencies of glycine, ß-alanine and taurine by 9-, 6- and 3-fold respectively, and that of the competitive antagonist strychnine by 15-fold. Replacing N46 with hydrophobic, charged or polar residues revealed that the amide moiety of asparagine was crucial for GlyR activation. Co-mutating N61, located on a neighbouring ß loop to N46, rescued the wild-type phenotype depending on the amino acid charge. Single-channel recording identified that burst length for the N46K mutant was reduced and fast agonist application revealed faster glycine deactivation times for the N46K mutant compared with the WT receptor. Overall, these data are consistent with N46 ensuring correct alignment of the α1 subunit interface by interaction with juxtaposed residues to preserve the structural integrity of the glycine binding site. This represents a new mechanism by which GlyR dysfunction induces startle disease.

Serotonin receptor 1A-modulated phosphorylation of glycine receptor α3 controls breathing in mice.

Rhythmic breathing movements originate from a dispersed neuronal network in the medulla and pons. Here, we demonstrate that rhythmic activity of this respiratory network is affected by the phosphorylation status of the inhibitory glycine receptor α3 subtype (GlyRα3), which controls glutamatergic and glycinergic neuronal discharges, subject to serotonergic modulation. Serotonin receptor type 1A-specific (5-HTR1A-specific) modulation directly induced dephosphorylation of GlyRα3 receptors, which augmented inhibitory glycine-activated chloride currents in HEK293 cells coexpressing 5-HTR1A and GlyRα3. The 5-HTR1A-GlyRα3 signaling pathway was distinct from opioid receptor signaling and efficiently counteracted opioid-induced depression of breathing and consequential apnea in mice. Paradoxically, this rescue of breathing originated from enhanced glycinergic synaptic inhibition of glutamatergic and glycinergic neurons and caused disinhibition of their target neurons. Together, these effects changed respiratory phase alternations and ensured rhythmic breathing in vivo. GlyRα3-deficient mice had an irregular respiratory rhythm under baseline conditions, and systemic 5-HTR1A activation failed to remedy opioid-induced respiratory depression in these mice. Delineation of this 5-HTR1A-GlyRα3 signaling pathway offers a mechanistic basis for pharmacological treatment of opioid-induced apnea and other breathing disturbances caused by disorders of inhibitory synaptic transmission, such as hyperekplexia, hypoxia/ischemia, and brainstem infarction.

A Selective Role for alpha3 Subunit Glycine Receptors in Inflammatory Pain.

GlyR alpha3 has previously been found to play a critical role in pain hypersensitivity following spinal PGE(2) injection, complete Freund's adjuvant (CFA) and zymosan induced peripheral inflammation. In this study, although all models displayed typical phenotypic behaviours, no significant differences were observed when comparing the pain behaviours of Glra3(-/-) and wild-type littermates following the injection of capsaicin, carrageenan, kaolin/carrageenan or monosodium iodoacetate, models of rheumatoid and osteoarthritis, respectively. However, clear differences were observed following CFA injection (p < 0.01). No significant differences were observed in the pain behaviours of Glra3(-/-) and wild-type littermates following experimentally induced neuropathic pain (partial sciatic nerve ligation). Similarly, Glra3(-/-) and wild-type littermates displayed indistinguishable visceromotor responses to colorectal distension (a model of visceral pain) and in vivo spinal cord dorsal horn electrophysiology revealed no differences in responses to multimodal suprathreshold stimuli, intensities which equate to higher pain scores such as those reported in the clinic. These data suggest that apart from its clear role in CFA- and zymosan-induced pain sensitisation, hypersensitivity associated with other models of inflammation, neuropathy and visceral disturbances involves mechanisms other than the EP2 receptor - GlyR alpha3 pathway.

An unexpected role for TASK-3 potassium channels in network oscillations with implications for sleep mechanisms and anesthetic action.

TASK channels are acid-sensitive and anesthetic-activated members of the family of two-pore-domain potassium channels. We have made the surprising discovery that the genetic ablation of TASK-3 channels eliminates a specific type of theta oscillation in the cortical electroencephalogram (EEG) resembling type II theta (4-9 Hz), which is thought to be important in processing sensory stimuli before initiating motor activity. In contrast, ablation of TASK-1 channels has no effect on theta oscillations. Despite the absence of type II theta oscillations in the TASK-3 knockout (KO) mice, the related type I theta, which has certain neuronal pathways in common and is involved in exploratory behavior, is unaffected. In addition to the absence of type II theta oscillations, the TASK-3 KO animals show marked alterations in both anesthetic sensitivity and natural sleep behavior. Their sensitivity to halothane, a potent activator of TASK channels, is greatly reduced, whereas their sensitivity to cyclopropane, which does not activate TASK-3 channels, is unchanged. The TASK-3 KO animals exhibit a slower progression from their waking to sleeping states and, during their sleeping period, their sleep episodes as well as their REM theta oscillations are more fragmented. These results imply a previously unexpected role for TASK-3 channels in the cellular mechanisms underlying these behaviors and suggest that endogenous modulators of these channels may regulate theta oscillations.

Determinants of the anesthetic sensitivity of two-pore domain acid-sensitive potassium channels: molecular cloning of an anesthetic-activated potassium channel from Lymnaea stagnalis.

Certain two-pore domain K(+) channels are plausible targets for volatile general anesthetics, yet little is known at the molecular level about how these simple agents cause channel activation. The first anesthetic-activated K(+) current I(K(An)) that was characterized was discovered in the mollusk Lymnaea stagnalis and is remarkable for both its sensitivity to general anesthetics and its stereoselective responses to anesthetic enantiomers (Franks, N. P., and Lieb, W. R. (1988) Nature 333, 662-664 and Franks, N. P., and Lieb, W. R. (1991) Science 254, 427-430). Here we report the molecular cloning of a two-pore domain K(+) channel LyTASK from L. stagnalis and show that, when expressed in HEK-293 cells, it displays the same biophysical characteristics as the anesthetic-activated K(+) current I(K(An)). Sequence analysis and functional properties show it to be a member of the TASK family of channels with approximately 47% identity at the amino acid level when compared with human TASK-1 and TASK-3. By using chimeric channel constructs and site-directed mutagenesis we have identified the specific amino acid 159 to be a critical determinant of anesthetic sensitivity, which, when mutated to alanine, essentially eliminates anesthetic activation in the human channels and greatly reduces activation in LyTASK. The L159A mutation in LyTASK disrupts the stereoselective response to isoflurane while having no effect on the pH sensitivity of the channel, suggesting this critical amino acid may form part of an anesthetic binding site.

The effects of hypoxia on the modulation of human TREK-1 potassium channels.

Two-pore-domain potassium channels are a family of ion channels that are widely believed to play an important role in maintaining and regulating neuronal excitability. It has been shown that they can be modulated by an extraordinarily diverse range of endogenous and exogenous factors. One particular member of the family, TREK-1 (also known as KCNK2), is activated by increasing temperature, membrane stretch and internal acidosis, but is also sensitive to the presence of certain polyunsaturated fatty acids (such as arachidonic acid), neuroprotectants (such as riluzole) and volatile and gaseous general anaesthetics (such as halothane and nitrous oxide). It has recently been reported that TREK-1 channels are also affected by oxygen concentrations, and that at the levels of hypoxia that occur in the normal human brain, the channels greatly change their properties and, for example, lose their ability to be modulated by arachidonic acid and internal acidosis. These reports seriously challenge the idea that TREK-1 is a target for general anaesthetics and neuroprotectants. However, in this report we show that TREK-1 is not oxygen sensitive, and its ability to be activated by anaesthetics, arachidonic acid and internal acidosis remains unaltered under conditions of hypoxia. We further show that the protocol used by previous workers to prepare hypoxic solutions of arachidonic acid results in the removal of the compound from solution.