A new family of glutamate-gated chloride channels in parasitic sea louse Caligus rogercresseyi: A subunit refractory to activation by ivermectin is dominant in heteromeric assemblies.

Sea louse ectoparasitosis is a major threat to fish aquaculture. Avermectins such as ivermectin and emamectin have been effectively used against sea louse infestation, but the emergence of resistance has limited their use. A better understanding of the molecular targets of avermectins is essential to the development of novel treatment strategies or new, more effective drugs. Avermectins are known to act by inhibiting neurotransmission through allosteric activation of glutamate-gated chloride channels (GluCls). We have investigated the GluCl subunit present in Caligus rogercresseyi, a sea louse affecting aquaculture in the Southern hemisphere. We identify four new subunits, CrGluCl-B to CrGluCl-E, and characterise them functionally. CrGluCl-A (previously reported as CrGluClα), CrGluCl-B and CrGluCl-C all function as glutamate channel receptors with different sensitivities to the agonist, but in contrast to subunit -A and -C, CrGluCl-B is not activated by ivermectin but is rather antagonised by the drug. CrGluCl-D channel appears active in the absence of any stimulation by glutamate or ivermectin and CrGluCl-E does not exhibit any activity. Notably, the expression of CrGluCl-B with either -A or -C subunits gives rise to receptors unresponsive to ivermectin and showing altered response to glutamate, suggesting that coexpression has led to the preferential formation of heteromers to which the presence of CrGluCl-B confers the property of ivermectin-activation refractoriness. Furthermore, there was evidence for heteromer formation with novel properties only when coexpressing pairs E/C and D/B CrGluCl subtypes. Site-directed mutagenesis shows that three transmembrane domain residues contribute to the lack of activation by ivermectin, most crucially Gln 15' in M2, with mutation Q15'T (the residue present in ivermectin-activated subunits A and C) conferring ivermectin activation to CrGluCl-B. The differential response to avermectin of these Caligus rogercresseyi GluClsubunits, which are highly conserved in the Northern hemisphere sea louse Lepeophtheirus salmonis, could have an influence on the response of these parasites to treatment with macrocyclic lactones. They could serve as molecular markers to assess susceptibility to existing treatments and might be useful molecular targets in the search for novel antiparasitic drugs.

Normal vision and development in mice with low functional expression of Kir7.1 in heterozygosis for a blindness-producing mutation inactivating the channel.

K+ channel Kir7.1 expressed at the apical membrane of the retinal pigment epithelium (RPE) plays an essential role in retinal function. An isoleucine-to-threonine mutation at position 120 of the protein is responsible for blindness-causing vitreo-retinal dystrophy. We have studied the molecular mechanism of action of Kir7.1-I120T in vitro by heterologous expression and in vivo in CRISPR-generated knockin mice. Full-size Kir7.1-I120T reaches the plasma membrane but lacks any activity. Analysis of Kir7.1 and the I120T mutant in mixed transfection experiments, and that of tandem tetrameric constructs made by combining wild type (WT) and mutant protomers, leads us to conclude that they do not form heterotetramers in vitro. Homozygous I120T/I120T mice show cleft palate and tracheomalacia and do not survive beyond P0, whereas heterozygous WT/I120T develop normally. Membrane conductance of RPE cells isolated from WT/WT and heterozygous WT/I120T mice is dominated by Kir7.1 current. Using Rb+ as a charge carrier, we demonstrate that the Kir7.1 current of WT/I120T RPE cells corresponds to approximately 50% of that in cells from WT/WT animals, in direct proportion to WT gene dosage. This suggests a lack of compensatory effects or interference from the mutated allele product, an interpretation consistent with results obtained using WT/- hemizygous mouse. Electroretinography and behavioral tests also show normal vision in WT/I120T animals. The hypomorphic ion channel phenotype of heterozygous Kir7.1-I120T mutants is therefore compatible with normal development and retinal function. The lack of detrimental effect of this degree of functional deficit might explain the recessive nature of Kir7.1 mutations causing human eye disease.NEW & NOTEWORTHY Human retinal pigment epithelium K+ channel Kir7.1 is affected by generally recessive mutations leading to blindness. We investigate one such mutation, isoleucine-to-threonine at position 120, both in vitro and in vivo in knockin mice. The mutated channel is inactive and in heterozygosis gives a hypomorphic phenotype with normal retinal function. Mutant channels do not interfere with wild-type Kir7.1 channels which are expressed concomitantly without hindrance, providing an explanation for the recessive nature of the disease.

Approaches to supratentorial brain tumours in children.

The differential diagnosis of supratentorial brain tumours in children can be challenging, especially considering the recent changes to the WHO classification of CNS tumours published in 2021. Many new tumour types have been proposed which frequently present in children and young adults and their imaging features are currently being described by the neuroradiology community. The purpose of this article is to provide guidance to residents and fellows new to the field of paediatric neuroradiology on how to evaluate an MRI of a patient with a newly diagnosed supratentorial tumour. Six different approaches are discussed including: 1. Tumour types, briefly discussing the main changes to the recent WHO classification of CNS tumours, 2. Patient age and its influence on incidence rates of specific tumour types, 3. Growth patterns, 4. Tumour location and how defining the correct location helps in narrowing down the differential diagnoses and 5. Imaging features of the tumour on DWI, SWI, FLAIR and post contrast sequences.

Neuroimaging Findings in Fetal Hemimegalencephaly: Case Study and Review.

BACKGROUND: Limited information exists in the prenatal literature regarding the neuroimaging features of fetal hemimegalencephaly. SUMMARY: This report describes ultrasound and magnetic resonance imaging (MRI) findings in a second-trimester fetus with an isolated, severe form of hemimegalencephaly. The most prominent imaging findings included unilateral enlarged cerebral hemisphere and ipsilateral ventriculomegaly causing cerebral asymmetry, midline shift, and macrocephaly. Abnormal cortical development imaging signs were also evident. A literature review encompassing 23 reports describing 36 cases, including ours, is presented. KEY MESSAGES: Characteristic ultrasound findings for the diagnosis of hemimegalencephaly are not always apparent prenatally. Asymmetric ventriculomegaly emerges as the most common but nonspecific presenting feature during routine second- or third-trimester ultrasound scans. Subsequent high-resolution prenatal neurosonography and fetal MRI facilitate definitive prenatal diagnosis, showcasting associated features primarily related to cortical migration, differentiation, and maturation. Postnatally, the prognosis is poor due to intractable seizures, hemiplegia, and progressive neurodevelopmental delay.

Role of Fetal Magnetic Resonance Imaging in Differentiating Isolated Septal Agenesis from Septo-Optic Dysplasia: Case Study and Review.

INTRODUCTION: The detection of absent septi pellucidi (ASP) during obstetric ultrasound is a rare event. However, the clinical implications of this finding are significant. ASP can be associated with severe central nervous system anomalies such as holoprosencephaly, agenesis/dysgenesis of the corpus callosum, schizencephaly, severe ventriculomegaly, and open neural tube defects. In such cases, the prognosis is poor. When no such anomalies are identified, isolated ASP usually carries a good prognosis. However, some fetuses thought to have isolated ASP actually have septo-optic dysplasia (SOD), which is associated with optic nerve hypoplasia, hypothalamic-pituitary dysfunction, and developmental delay. CASE PRESENTATION: A case in which fetal 3.0 Tesla magnetic resonance imaging (MRI) was considered crucial to definitively diagnose isolated ASP is presented. A review of the literature was conducted and analyzed to determine the role of MRI in the evaluation of fetuses with ASP, with special consideration on the differential diagnosis between isolated ASP and SOD. CONCLUSION: Differentiating isolated ASP from SOD is imperative for adequate prenatal counseling. Unfortunately, making a prenatal diagnosis of SOD requires visualization and evaluation of the fetal optic nerves, chiasm, and pituitary gland, which is very demanding and not always possible using ultrasound. Fetal MRI has the potential of obtaining high-quality images of the fetal brain, and therefore this technique can be used for establishing the differential diagnosis in utero.

Ultrasound and fetal magnetic resonance imaging: Clinical performance in the prenatal diagnosis of orofacial clefts and mandibular abnormalities.

Cleft lip, with or without cleft palate, is the most common congenital craniofacial anomaly and the second most common birth defect worldwide. Micrognathia is a rare facial malformation characterized by small, underdeveloped mandible and frequently associated with retrognathia. Second- and third-trimester prenatal ultrasound is the standard modality for screening and identification of fetal orofacial abnormalities, with a detection rate in the low-risk population ranging from 0% to 73% for all types of cleft. The prenatal ultrasonography detection can also be performed during the first trimester of pregnancy. Given the potential limitations of obstetric ultrasound for examining the fetal face, such as suboptimal fetal position, shadowing from the surrounding bones, reduce amniotic fluid around the face, interposition of fetal limbs, umbilical cord and placenta, and maternal habitus/abdominal scars, the use of adjunct imaging modalities can enhance prenatal diagnosis of craniofacial anomalies in at-risk pregnancies. Fetal magnetic resonance imaging (MRI) is a potentially useful second-line investigation for the prenatal diagnosis of orofacial malformations with a pooled sensitivity of 97%. In this review, we discuss the role of ultrasound and fetal MRI in the prenatal assessment of abnormalities of the upper lip, palate, and mandible.

Molecular aspects of structure, gating, and physiology of pH-sensitive background K2P and Kir K+-transport channels.

K(+) channels fulfill roles spanning from the control of excitability to the regulation of transepithelial transport. Here we review two groups of K(+) channels, pH-regulated K2P channels and the transport group of Kir channels. After considering advances in the molecular aspects of their gating based on structural and functional studies, we examine their participation in certain chosen physiological and pathophysiological scenarios. Crystal structures of K2P and Kir channels reveal rather unique features with important consequences for the gating mechanisms. Important tasks of these channels are discussed in kidney physiology and disease, K(+) homeostasis in the brain by Kir channel-equipped glia, and central functions in the hearing mechanism in the inner ear and in acid secretion by parietal cells in the stomach. K2P channels fulfill a crucial part in central chemoreception probably by virtue of their pH sensitivity and are central to adrenal secretion of aldosterone. Finally, some unorthodox behaviors of the selectivity filters of K2P channels might explain their normal and pathological functions. Although a great deal has been learned about structure, molecular details of gating, and physiological functions of K2P and Kir K(+)-transport channels, this has been only scratching at the surface. More molecular and animal studies are clearly needed to deepen our knowledge.

Prenatal intracranial hypotension syndrome in congenital cephalocele: Insights into pathophysiologic mechanisms and importance of defect coverage.

OBJECTIVE: To compare specific fetal cranial and cerebral findings indicative of prenatal intracranial hypotension (PICH) syndrome between open and closed cephaloceles. METHODS: Two groups of fetuses with cephalocele who underwent magnetic resonance imaging scan were selected based on the defect coverage: covered ("closed cephalocele" group, n = 4) and uncovered by skin ("open cephalocele" group, n = 8). The presence of signs associated with PICH syndrome was evaluated and findings compared between groups. RESULTS: Cases from the open cephalocele group showed statistically significant differences in comparison with the closed cephalocele group regarding reduction of the interpeduncular angle (p = 0.006), reduced amount of cerebrospinal fluid in the subarachnoid space (p = 0.01), collapsed/severely reduced fourth ventricle (p = 0.03), cephalocele content (p = 0.03), and identification of a vector traction (p = 0.03). There were no differences in the presence of cerebellar tonsillar descent, obstructive ventriculomegaly, and presence of the lemon sign. In comparison with gestational age-matched controls, only the biparietal diameter but not the head circumference was significantly smaller in both the open and closed cephalocele groups (p < 0.05). CONCLUSION: Congenital cephaloceles present distinct imaging behavior depending on the defect coverage. Therefore, the classification of cephalocele should include, in addition to location and content, the presence or absence of skin fully covering the defect.

Heat sensitization of Escherichia coli by the natural antimicrobials vanillin and emulsified citral in blended carrot-orange juice.

Efforts have been focusing on the way to overcome the impact of heating on food quality while achieving the desired shelf life. In this sense, the thermosensitization of E. coli using the natural antimicrobials vanillin (V; 0.8 and 1.0 g/L) or/and emulsified citral (C; 0.012 and 0.025 g/L) was assessed at 58 and 60 °C in blended carrot-orange juice (pH 4.0; 9.0°Brix). All combined treatments exceeded the inactivation achieved by the single thermal treatments in half the time. The inactivation of the binary treatments (V or C + heating) at 58 °C was 3.84-0.62 log-cycles more effective than the control, particularly with vanillin. Ternary treatments (V + C + heating) at 58 °C increased the microbial reduction approximately 30%; however, at 60 °C no further inactivation was observed, suggesting the thermal effect prevailed. This was verified by the higher b Weibullian parameter and the narrower frequency distributions. The selected treatments 1.0 V + 0.012C at 58 and 60 °C were challenged against the pathogenic E. coli O157:H7 and found to be effective. Additionally, the microbiota of the juice was maintained at acceptable levels during storage (4 °C). In conclusion, there was an increase in the heat sensitivity of E. coli due to the natural antimicrobials, particularly vanillin at 58 °C. Therefore, reducing the intensity of the thermal processing will lead to clean label, high-quality juices, while addressing food safety requirements.

Graft survival after kidney transplantation with standard versus prolonged kidney procurement time.

BACKGROUND: During kidney procurement, after ice removal, kidneys located in the retroperitoneum are at risk for rewarming owing to the time taken to retrieve other abdominal and thoracic organs, which may lead to poorer outcomes. The purpose of this study was to evaluate the impact of prolonged kidney procurement time (PKP) on outcomes of kidney transplantation performed at the Queen Elizabeth II Health Sciences Centre, Halifax, Nova Scotia, Canada. METHODS: We retrospectively reviewed the cases of all adult (age ≥ 18 yr) patients who underwent kidney transplantation at the Queen Elizabeth II Health Sciences Centre between Jan. 1, 2010, and Dec. 31, 2015. We included all patients who received kidney transplants from deceased donors with a minimum follow-up period of 3 years. We defined PKP as more than 65 minutes from aortic cross-clamp to final organ extraction, and standard procurement time (SP) as 65 minutes or less. RESULTS: Among the 455 transplantation procedures performed during the study period, we reviewed the cases of 145 patients who received kidneys from Nova Scotian donors and were followed in Nova Scotia. No statistically significant differences were seen in outcomes between kidney-only (n = 46) and multiorgan (n = 99) procurement, although more organs from kidney-only donors than multiorgan donors had a Kidney Donor Profile Index score greater than 50% (32 [69.6%] v. 48 [48.5%], p < 0.01). Compared to the SP group (n = 115), the PKP group (n = 30) had a higher rate of 30-day graft loss (6.7% v. 0.0%, p < 0.01), a higher incidence of de novo formation of donor-specific antibodies (3 [10.0%] v. 1 [0.9%], p < 0.01) and a lower 5-year graft survival rate (90.0% v. 97.4%, p = 0.03). Left kidneys remained 11 minutes longer on the donor than right kidneys when multiorgan procurement was performed (p < 0.01), and their 5-year survival rate was significantly lower than that of right kidneys (p = 0.03). CONCLUSION: Procurement times longer than 65 minutes may be associated with poorer outcomes after kidney transplantation. Measures to reduce kidney exposure to rewarming during procurement may improve long-term outcomes.

Altered phosphatidylinositol regulation of mutant inwardly rectifying K+ Kir7.1 channels associated with inherited retinal degeneration disease.

KEY POINTS: Kir7.1 K+ channel expressed in retinal pigment epithelium is mutated in inherited retinal degeneration diseases. We study Kir7.1 in heterologous expression to test the hypothesis that pathological R162 mutation to neutral amino acids results in loss of a crucial site that binds PI(4,5)P2 . Although R162W mutation inactivates Kir7.1, changes to smaller volume (e.g. Gln) amino acids are tolerated or even enhance function (Ala or Cys). Chemical modification of Kir7.1-R162C confirms that large residues of the size of Trp are incompatible with normal channel function even if positively charged. In addition to R162, K164 (and possibly K159) forms a binding site for the phosphoinositide and is essential for channel activity. R162 substitution with a large, neutral side chain like Trp exerts a dominant negative effect on Kir7.1 activity such that less than one fifth of the full activity is expected in a cell expressing the same amount of mutant and wild-type channels. ABSTRACT: Mutations in the Kir7.1 K+ channel, highly expressed in retinal pigment epithelium, have been linked to inherited retinal degeneration diseases. Examples are mutations changing Arg 162 to Trp in snowflake vitreoretinal degeneration (SVD) and Gln in retinitis pigmentosa. R162 is believed to be part of a site that binds PI(4,5)P2 and stabilises the open state. We have tested the hypothesis that R162 mutation to neutral amino acids will result in the loss of this crucial interaction to the detriment of channel function. Our findings indicate that although R612W mutation inactivates Kir7.1, changes to smaller volume (e.g. Gln) amino acids are tolerated or even enhance function (Ala or Cys). Cys chemical modification of Kir7.1-R162C confirms that large residues of the size of Trp are incompatible with normal channel function even if positively charged. Experiments titrating the levels of plasma membrane PI(4,5)P2 with voltage-dependent phosphatase DrVSP reveal that, in addition to R162, K164 (and possibly K159) forms a binding site for the phosphoinositide and ensures channel activity. Finally, the use of a concatemeric approach shows that substitution of R162 with a large, neutral side chain mimicking a Trp residue exerts a dominant negative effect on Kir7.1 activity such that less than one fifth of the full activity is expected in heterozygous cells carrying the SVD mutation. Our results suggest that if mutations in the human KCNJ13 gene resulting in the neutralisation of R162 and Kir7.1 malfunction led to retinal degeneration diseases, their severity might depend on the nature of the side chain of the replacing amino acid.

Modelling the effects of ephaptic coupling on selectivity and response patterns during artificial stimulation of peripheral nerves.

Artificial electrical stimulation of peripheral nerves for sensory feedback restoration can greatly benefit from computational models for simulation-based neural implant design in order to reduce the trial-and-error approach usually taken, thus potentially significantly reducing research and development costs and time. To this end, we built a computational model of a peripheral nerve trunk in which the interstitial space between the fibers and the tissues was modelled using a resistor network, thus enabling distance-dependent ephaptic coupling between myelinated axons and between fascicles as well. We used the model to simulate a) the stimulation of a nerve trunk model with a cuff electrode, and b) the propagation of action potentials along the axons. Results were used to investigate the effect of ephaptic interactions on recruitment and selectivity stemming from artificial (i.e., neural implant) stimulation and on the relative timing between action potentials during propagation. Ephaptic coupling was found to increase the number of fibers that are activated by artificial stimulation, thus reducing the artificial currents required for axonal recruitment, and it was found to reduce and shift the range of optimal stimulation amplitudes for maximum inter-fascicular selectivity. During propagation, while fibers of similar diameters tended to lock their action potentials and reduce their conduction velocities, as expected from previous knowledge on bundles of identical axons, the presence of many other fibers of different diameters was found to make their interactions weaker and unstable.

Interpeduncular angle: A new parameter for assessing intracranial hypotension in fetuses with spinal dysraphism.

OBJECTIVE: To investigate the role of the interpeduncular angle (IPA) as a new indicator of intracranial hypotension in fetuses with open spinal dysraphism (SD). METHODS: Two groups of fetuses undergoing magnetic resonance imaging (MRI) examination were identified. The study group included fetuses with open SD (n = 21), while the control group included fetuses with a normal brain and spine (n = 43). Two observers retrospectively evaluated axial T2-weighted images of the brain and the IPA was identified and measured. Other features of the Chiari II malformation were also evaluated in the study group and correlated with the IPA. RESULTS: The average value (±SD) of the IPA for the study and control groups was 9.8° ± 18.5° and 60.2° ± 5.9°, respectively. The intergroup analysis of the IPA measurements revealed a statistically significant difference between the groups (p < 0.005). Brainstem slumping or cerebellar tonsillar descent, collapse of the fourth ventricle, and ventriculomegaly also had a significant correlation with a lower IPA (p = 0.001). CONCLUSION: Measuring the IPA may be a useful technique for assessing the degree of intracranial hypotension in fetuses with open SD. This technique can also detect less severe cases of prenatal intracranial hypotension, even before cerebellar tonsillar descent. The lower IPA in fetuses with open SD further supports the theory that cerebrospinal fluid leakage, and not traction, is the underlying cause of Chiari II malformation.

Aom2 S: A new web-based application for DNA/RNA tandem mass spectrometry data interpretation.

RATIONALE: The Analysis of Oligonucleotide Modifications from Mass Spectra (Aom2 S) was created to support the analysis of oligonucleotide mass spectra. This application complements the existing software tools by providing a comprehensive analysis of oligonucleotide fragments from high-resolution tandem mass spectrometry (HR-MS/MS) data in a flexible and user-friendly manner, directly accessible through a web browser without any need for installation. METHODS: MS measurements of aminoC6-DNA and inosine-RNA were performed using an LTQ Orbitrap FT-MS instrument. The obtained data were analyzed by our newly developed open-source package Aom2 S accessible from the ms.epfl.ch web page or directly at https://mstools.epfl.ch/am2s/ to demonstrate the various functionalities of this tool, notably the possibility to identify different product ions from a nucleotide sequence with any fixed/variable modification by matching theoretical isotopic patterns to any experimental mass spectra with similarity scores ranking. RESULTS: A detailed description of the Aom2 S tool with its user-friendly interface is exemplified using HR-MS/MS data of modified DNA and RNA oligonucleotides. Explanations of analysis parameters and tool workflow, as well as multiple options for viewing and exporting the results, are provided. Product ion assignment and modification localization can be achieved in seconds, and results can be exported as tables, matched mass spectra, and fragmentation maps. CONCLUSIONS: A new open source tool (Aom2 S) for the analysis of HR-MS/MS data for modified DNA and RNA oligonucleotides is described. Aom2 S is fast, highly flexible, and versatile, allowing automatic precursor and product ion assignment in a comprehensive manner, including internal fragments and variable modification localization, with clear graphical representation of the results.

Comparison between covert sound-production task (sound-imagery) vs. motor-imagery for onset detection in real-life online self-paced BCIs.

BACKGROUND: Even though the BCI field has quickly grown in the last few years, it is still mainly investigated as a research area. Increased practicality and usability are required to move BCIs to the real-world. Self-paced (SP) systems would reduce the problem but there is still the big challenge of what is known as the 'onset detection problem'. METHODS: Our previous studies showed how a new sound-imagery (SI) task, high-tone covert sound production, is very effective for onset detection scenarios and we expect there are several advantages over most common asynchronous approaches used thus far, i.e., motor-imagery (MI): 1) Intuitiveness; 2) benefits to people with motor disabilities and, especially, those with lesions on cortical motor areas; and 3) no significant overlap with other common, spontaneous cognitive states, making it easier to use in daily-life situations. The approach was compared with MI tasks in online real-life scenarios, i.e., during activities such as watching videos and reading text. In our scenario, when a new message prompt from a messenger program appeared on the screen, participants watching a video (or reading text, browsing images) were asked to open the message by executing the SI or MI tasks, respectively, for each experimental condition. RESULTS: The results showed the SI task performed statistically significantly better than the MI approach: 84.04% (SI) vs 66.79 (MI) True-False positive rate for the sliding image scenario, 80.84% vs 61.07% for watching video. The classification performance difference between SI and MI was found not to be significant in the text-reading scenario. Furthermore, the onset response speed showed SI (4.08 s) being significantly faster than MI (5.46 s). In terms of basic usability, 75% of subjects found SI easier to use. CONCLUSIONS: Our novel SI task outperforms typical MI for SP onset detection BCIs, therefore it would be more easily used in daily-life situations. This could be a significant step forward for the BCI field which has so far been mainly restricted to research-oriented indoor laboratory settings.

Elucidating the Structural Basis of the Intracellular pH Sensing Mechanism of TASK-2 K2P Channels.

Two-pore domain potassium (K2P) channels maintain the cell's background conductance by stabilizing the resting membrane potential. They assemble as dimers possessing four transmembrane helices in each subunit. K2P channels were crystallized in "up" and "down" states. The movements of the pore-lining transmembrane TM4 helix produce the aperture or closure of side fenestrations that connect the lipid membrane with the central cavity. When the TM4 helix is in the up-state, the fenestrations are closed, while they are open in the down-state. It is thought that the fenestration states are related to the activity of K2P channels and the opening of the channels preferentially occurs from the up-state. TASK-2, a member of the TALK subfamily of K2P channels, is opened by intracellular alkalization leading the deprotonation of the K245 residue at the end of the TM4 helix. This charge neutralization of K245 could be sensitive or coupled to the fenestration state. Here, we describe the relationship between the states of the intramembrane fenestrations and K245 residue in TASK-2 channel. By using molecular modeling and simulations, we show that the protonated state of K245 (K245+) favors the open fenestration state and, symmetrically, that the open fenestration state favors the protonated state of the lysine residue. We show that the channel can be completely blocked by Prozac, which is known to induce fenestration opening in TREK-2. K245 protonation and fenestration aperture have an additive effect on the conductance of the channel. The opening of the fenestrations with K245+ increases the entrance of lipids into the selectivity filter, blocking the channel. At the same time, the protonation of K245 introduces electrostatic potential energy barriers to ion entrance. We computed the free energy profiles of ion penetration into the channel in different fenestration and K245 protonation states, to show that the effects of the two transformations are summed up, leading to maximum channel blocking. Estimated rates of ion transport are in qualitative agreement with experimental results and support the hypothesis that the most important barrier for ion transport under K245+ and open fenestration conditions is the entrance of the ions into the channel.

A novel Kir7.1 splice variant expressed in various mouse tissues shares organisational and functional properties with human Leber amaurosis-causing mutations of this K+ channel.

Kir7.1 is an inwardly rectifying K+ channel present in epithelia where it shares membrane localization with the Na+/K+-pump. In the present communication we report the presence of a novel splice variant of Kir7.1 in mouse tissues including kidney, lung, choroid plexus and retinal pigment epithelium (RPE). The variant named mKir7.1-SV2 lacks most of the C-terminus domain but is predicted to have the two transmembrane domains and permeation pathway unaffected. Similarly truncated predicted proteins, Kir7.1-R166X and Kir7.1-Q219X, would arise from mutations associated with Leber Congenital Amaurosis, a rare recessive hereditary retinal disease that results in vision loss at early age. We found that mKir7.1-SV2 and the pathological variants do not produce any channel activity when expressed alone in HEK-293 cells due to their scarce presence in the plasma membrane. Simultaneous expression with the full length Kir7.1 however leads to a reduction in activity of the wild-type channel that might be due to partial proteasome degradation of WT-mutant channel heteromers.

Kir7.1 inwardly rectifying K+ channel is expressed in ciliary body non pigment epithelial cells and might contribute to intraocular pressure regulation.

Inwardly rectifying K+ channel Kir7.1 is expressed in epithelia where it shares membrane localisation with the Na+/K+-pump. The ciliary body epithelium (CBE) of the eye is a determinant of intraocular pressure (IOP) through NaCl-driven fluid secretion of aqueous humour. In the present study we explored the presence Kir7.1 in this epithelium in the mouse and its possible functional role in the generation of IOP. Use heterozygous animals for total Kir7.1 knockout expressing ß-galactosidase under the control of Kir7.1 promoter, identified the expression of Kir7.1 in non-pigmented epithelial cells of CBE. Using conditional, floxed knockout Kir7.1 mice as negative controls, we found Kir7.1 at the basolateral membrane of the same CBE cell layer. This was confirmed using a knockin mouse expressing the Kir7.1 protein tagged with a haemagglutinin epitope. Measurements using the conditional knockout mouse show only a minor effect of Kir7.1 inactivation on steady-state IOP. Transient increases in IOP in response to general anaesthetics, or to water injection, are absent or markedly curtailed in Kir7.1-deficient mice. These results suggest a role for Kir7.1 in IOP regulation through a possible modulation of aqueous humour production by the CBE non-pigmented epithelial cells. The location of Kir7.1 in the CBE, together with the effect of its removal on dynamic changes in IOP, point to a possible role of the channel as a leak pathway preventing cellular overload of K+ during the secretion process. Kir7.1 could be used as a potential therapeutic target in pathological conditions leading to elevated intraocular pressure.

Structure/Activity Analysis of TASK-3 Channel Antagonists Based on a 5,6,7,8 tetrahydropyrido[4,3-d]pyrimidine.

TASK-3 potassium (K+) channels are highly expressed in the central nervous system, regulating the membrane potential of excitable cells. TASK-3 is involved in neurotransmitter action and has been identified as an oncogenic K+ channel. For this reason, the understanding of the action mechanism of pharmacological modulators of these channels is essential to obtain new therapeutic strategies. In this study we describe the binding mode of the potent antagonist PK-THPP into the TASK-3 channel. PK-THPP blocks TASK-1, the closest relative channel of TASK-3, with almost nine-times less potency. Our results confirm that the binding is influenced by the fenestrations state of TASK-3 channels and occurs when they are open. The binding is mainly governed by hydrophobic contacts between the blocker and the residues of the binding site. These interactions occur not only for PK-THPP, but also for the antagonist series based on 5,6,7,8 tetrahydropyrido[4,3-d]pyrimidine scaffold (THPP series). However, the marked difference in the potency of THPP series compounds such as 20b, 21, 22 and 23 (PK-THPP) respect to compounds such as 17b, inhibiting TASK-3 channels in the micromolar range is due to the presence of a hydrogen bond acceptor group that can establish interactions with the threonines of the selectivity filter.

Correction to: The Relative Contribution of High-Gamma Linguistic Processing Stages of Word Production, and Motor Imagery of Articulation in Class Separability of Covert Speech Tasks in EEG Data.

The author regrets that the acknowledgment was left out from the original publication. The acknowledgement is written below.