C-terminal phosphorylation of NaV1.5 impairs FGF13-dependent regulation of channel inactivation

International audienceVoltage-gated Na(+) (NaV) channels are key regulators of myocardial excitability, and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na(+) current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts

A Kir6.2 mutation causing severe functional effects in vitro produces neonatal diabetes without the expected neurological complications

AIMS/HYPOTHESIS: Heterozygous activating mutations in the pancreatic ATP-sensitive K+ channel cause permanent neonatal diabetes mellitus (PNDM). This results from a decrease in the ability of ATP to close the channel, which thereby suppresses insulin secretion. PNDM mutations that cause a severe reduction in ATP inhibition may produce additional symptoms such as developmental delay and epilepsy. We identified a heterozygous mutation (L164P) in the pore-forming (Kir6.2) subunit of the channel in three unrelated patients and examined its functional effects. METHODS: The patients (currently aged 2, 8 and 20 years) developed diabetes shortly after birth. The two younger patients attempted transfer to sulfonylurea therapy but were unsuccessful (up to 1.1 mg kg(-1) day(-1)). They remain insulin dependent. None of the patients displayed neurological symptoms. Functional properties of wild-type and mutant channels were examined by electrophysiology in Xenopus oocytes. RESULTS: Heterozygous (het) and homozygous L164P K(ATP) channels showed a marked reduction in channel inhibition by ATP. Consistent with its predicted location within the pore, L164P enhanced the channel open state, which explains the reduction in ATP sensitivity. HetL164P currents exhibited greatly increased whole-cell currents that were unaffected by sulfonylureas. This explains the inability of sulfonylureas to ameliorate the diabetes of affected patients. CONCLUSIONS/INTERPRETATION: Our results provide the first demonstration that mutations such as L164P, which produce a severe reduction in ATP sensitivity, do not inevitably cause developmental delay or neurological problems. However, the neonatal diabetes of these patients is unresponsive to sulfonylurea therapy. Functional analysis of PNDM mutations can predict the sulfonylurea response

Consensus statements on the information to deliver after a febrile seizure

Febrile seizures (FS) are usually self-limiting and cause no morbidity. Nevertheless they represent very traumatic events for families. There is a need to identify key messages that reassure carers and help to prevent inappropriate, anxiety-driven behaviors associated with “fever phobia.” No recommendations have been proposed to date regarding the content of such messages. Using a Delphi process, we have established a consensus regarding the information to be shared with families following a FS. Twenty physicians (child neurologists and pediatricians) from five European countries participated in a three-step Delphi process between May 2018 and October 2019. In the first step, each expert was asked to give 10 to 15 free statements about FS. In the second and third steps, statements were scored and selected according to the expert ranking of importance. A list of key messages for families has emerged from this process, which offer reassurance about FS based on epidemiology, underlying mechanisms, and the emergency management of FS should they recur. Interestingly, there was a high level of agreement between child neurologists and general pediatricians. Conclusion: We propose key messages to be communicated with families in the post-FS clinic setting.What is Known:• Febrile seizures (FS) are traumatic events for families.• No guidelines exist on what information to share with parents following a FS.What is New:• A Delphi process involving child neurologists and pediatricians provides consensual statement about information to deliver after a febrile seizure.• We propose key messages to be communicated with families in the post-FS clinic setting

Cortisol in hair measured in young adults - a biomarker of major life stressors?

<p>Abstract</p> <p>Background</p> <p>Stress as a cause of illness has been firmly established. In public health and stress research a retrospective biomarker of extended stress would be an indispensible aid. The objective of this pilot study was to investigate whether concentrations of cortisol in hair correlate with perceived stress, experiences of serious life events, and perceived health in young adults.</p> <p>Methods</p> <p>Hair samples were cut from the posterior vertex area of (n = 99) university students who also answered a questionnaire covering experiences of serious life events, perceived Stress Scale and perceived health during the last three months. Cortisol was measured using a competitive radioimmunoassay in methanol extracts of hair samples frozen in liquid nitrogen and mechanically pulverised.</p> <p>Results</p> <p>Mean cortisol levels were significantly related to serious life events (p = 0.045), weakly negatively correlated to perceived stress (p = 0.025, r = -0.061) but nor affected by sex, coloured/permed hair, intake of pharmaceuticals or self-reported health. In a multiple regression model, only the indicator of serious life events had an independent (p = 0.041) explanation of increased levels of cortisol in hair. Out of four outliers with extremely high cortisol levels two could be contacted, both reported serious psychological problems.</p> <p>Conclusions</p> <p>These findings suggest that measurement of cortisol in hair could serve as a retrospective biomarker of increased cortisol production reflecting exposure to major life stressors and possibly extended psychological illness with important implications for research, clinical practice and public health. Experience of serious life events seems to be more important in raising cortisol levels in hair than perceived stress.</p

Pineoblastoma segregates into molecular sub-groups with distinct clinico-pathologic features: a Rare Brain Tumor Consortium registry study.

Pineoblastomas (PBs) are rare, aggressive pediatric brain tumors of the pineal gland with modest overall survival despite intensive therapy. We sought to define the clinical and molecular spectra of PB to inform new treatment approaches for this orphan cancer. Tumor, blood, and clinical data from 91 patients with PB or supratentorial primitive neuroectodermal tumor (sPNETs/CNS-PNETs), and 2 pineal parenchymal tumors of intermediate differentiation (PPTIDs) were collected from 29 centres in the Rare Brain Tumor Consortium. We used global DNA methylation profiling to define a core group of PB from 72/93 cases, which were delineated into five molecular sub-groups. Copy number, whole exome and targeted sequencing, and miRNA expression analyses were used to evaluate the clinico-pathologic significance of each sub-group. Tumors designated as group 1 and 2 almost exclusively exhibited deleterious homozygous loss-of-function alterations in miRNA biogenesis genes (DICER1, DROSHA, and DGCR8) in 62 and 100% of group 1 and 2 tumors, respectively. Recurrent alterations of the oncogenic MYC-miR-17/92-RB1 pathway were observed in the RB and MYC sub-group, respectively, characterized by RB1 loss with gain of miR-17/92, and recurrent gain or amplification of MYC. PB sub-groups exhibited distinct clinical features: group 1-3 arose in older children (median ages 5.2-14.0 years) and had intermediate to excellent survival (5-year OS of 68.0-100%), while Group RB and MYC PB patients were much younger (median age 1.3-1.4 years) with dismal survival (5-year OS 37.5% and 28.6%, respectively). We identified age < 3 years at diagnosis, metastatic disease, omission of upfront radiation, and chr 16q loss as significant negative prognostic factors across all PBs. Our findings demonstrate that PB exhibits substantial molecular heterogeneity with sub-group-associated clinical phenotypes and survival. In addition to revealing novel biology and therapeutics, molecular sub-grouping of PB can be exploited to reduce treatment intensity for patients with favorable biology tumors

Affinity for phosphatidylinositol 4,5-bisphosphate determines muscarinic agonist sensitivity of Kv7 K+ channels

Kv7 K+-channel subunits differ in their apparent affinity for PIP2 and are differentially expressed in nerve, muscle, and epithelia in accord with their physiological roles in those tissues. To investigate how PIP2 affinity affects the response to physiological stimuli such as receptor stimulation, we exposed homomeric and heteromeric Kv7.2, 7.3, and 7.4 channels to a range of concentrations of the muscarinic receptor agonist oxotremorine-M (oxo-M) in a heterologous expression system. Activation of M1 receptors by oxo-M leads to PIP2 depletion through Gq and phospholipase C (PLC). Chinese hamster ovary cells were transiently transfected with Kv7 subunits and M1 receptors and studied under perforated-patch voltage clamp. For Kv7.2/7.3 heteromers, the EC50 for current suppression was 0.44 ± 0.08 µM, and the maximal inhibition (Inhibmax) was 74 ± 3% (n = 5–7). When tonic PIP2 abundance was increased by overexpression of PIP 5-kinase, the EC50 was shifted threefold to the right (1.2 ± 0.1 µM), but without a significant change in Inhibmax (73 ± 4%, n = 5). To investigate the muscarinic sensitivity of Kv7.3 homomers, we used the A315T pore mutant (Kv7.3T) that increases whole-cell currents by 30-fold without any change in apparent PIP2 affinity. Kv7.3T currents had a slightly right-shifted EC50 as compared with Kv7.2/7.3 heteromers (1.0 ± 0.8 µM) and a strongly reduced Inhibmax (39 ± 3%). In contrast, the dose–response curve of homomeric Kv7.4 channels was shifted considerably to the left (66 ± 8 nM), and Inhibmax was slightly increased (81 ± 6%, n = 3–4). We then studied several Kv7.2 mutants with altered apparent affinities for PIP2 by coexpressing them with Kv7.3T subunits to boost current amplitudes. For the lower affinity (Kv7.2 (R463Q)/Kv7.3T) or higher affinity (Kv7.2 (R463E)/Kv7.3T) channels, the EC50 and Inhibmax were similar to Kv7.4 or Kv7.3T homomers (0.12 ± 0.08 µM and 79 ± 6% [n = 3–4] and 0.58 ± 0.07 µM and 27 ± 3% [n = 3–4], respectively). The very low-affinity Kv7.2 (R452E, R459E, and R461E) triple mutant was also coexpressed with Kv7.3T. The resulting heteromer displayed a very low EC50 for inhibition (32 ± 8 nM) and a slightly increased Inhibmax (83 ± 3%, n = 3–4). We then constructed a cellular model that incorporates PLC activation by oxo-M, PIP2 hydrolysis, PIP2 binding to Kv7-channel subunits, and K+ current through Kv7 tetramers. We were able to fully reproduce our data and extract a consistent set of PIP2 affinities

Pharmacological targeting of apelin impairs glioblastoma growth

Glioblastoma are highly aggressive brain tumours that are associated with an extremely poor prognosis. Within these tumours, a subpopulation of highly plastic self-renewing cancer cells exist that retain the ability to expand ex vivo as tumourspheres, induce tumour growth in mice, and have been implicated in radio- and chemo-resistance. Although their identity and fate are regulated by external cues emanating from endothelial cells, the nature of such angiocrine signals remains unknown. Here, we deployed a mass spectrometry proteomic approach to characterise the factors released by brain endothelial cells. We report the identification of the vasoactive peptide apelin as a central regulator for endothelial-mediated maintenance of glioblastoma patient-derived cells with stem-like properties (GSCs). Genetic and pharmacological targeting of apelin cognate receptor APLNR abrogates apelin- and endothelial-mediated expansion of GSCs and suppresses tumour initiation and growth. Functionally, selective competitive antagonists of APLNR were shown to be safe and effective in lengthening the survival of intracranially xenografted mice. Therefore, the APLN/APLNR signalling nexus may operate as a paracrine signal that sustains tumour cell expansion and progression, suggesting that apelin is a druggable factor in glioblastoma.WT107715/Z/15/

Voltage-Dependent Gating in a “Voltage Sensor-Less” Ion Channel

An unusual mechanism of ion channel regulation generates voltage-dependent gating in the absence of a canonical voltage-sensing domain

The pore structure and gating mechanism of K2P channels

K2P potassium channels are important regulators of cellular excitability. This study reveals that in contrast to most other K+ channels the primary gating mechanism in the K2P channel TREK-1 does not involve opening and closure of the cytoplasmic bundle crossing, but takes place close to or within the selectivity filter