KCNT2-Related Disorders: Phenotypes, Functional, and Pharmacological Properties.

OBJECTIVE: Pathogenic variants in KCNT2 are rare causes of developmental epileptic encephalopathy (DEE). We herein describe the phenotypic and genetic features of patients with KCNT2-related DEE, and the in vitro functional and pharmacological properties of KCNT2 channels carrying 14 novel or previously untested variants. METHODS: Twenty-five patients harboring KCNT2 variants were investigated: 12 were identified through an international collaborative network, 13 were retrieved from the literature. Clinical data were collected and included in a standardized phenotyping sheet. Novel variants were detected using exome sequencing and classified using ACMG criteria. Functional and pharmacological studies were performed by whole-cell electrophysiology in HEK-293 and SH-SY5Y cells. RESULTS: The phenotypic spectrum encompassed: (a) intellectual disability/developmental delay (21/22 individuals with available information), ranging from mild to severe/profound; (b) epilepsy (15/25); (c) neurological impairment, with altered muscle tone (14/22); (d) dysmorphisms (13/20). Nineteen pathogenic KCNT2 variants were found (9 new, 10 reported previously): 16 missense, 1 in-frame deletion of a single amino acid, 1 nonsense, and 1 frameshift. Among tested variants, 8 showed gain-of-function (GoF), and 6 loss-of-function (LoF) features when expressed heterologously in vitro. Quinidine and fluoxetine blocked all GoF variants, whereas loxapine and riluzole activated some LoF variants while blocking others. INTERPRETATION: We expanded the phenotypic and genotypic spectrum of KCNT2-related disorders, highlighting novel genotype-phenotype associations. Pathogenic KCNT2 variants cause GoF or LoF in vitro phenotypes, and each shows a unique pharmacological profile, suggesting the need for in vitro functional and pharmacological investigation to enable targeted therapies based on the molecular phenotype. ANN NEUROL 2023;94:332-349.

Calcium cytotoxicity sensitizes prostate cancer cells to standard-of-care treatments for locally advanced tumors.

Therapy resistance is a major roadblock in oncology. Exacerbation of molecular dysfunctions typical of cancer cells have proven effective in twisting oncogenic mechanisms to lethal conditions, thus offering new therapeutic avenues for cancer treatment. Here, we demonstrate that selective agonists of Transient Receptor Potential cation channel subfamily M member 8 (TRPM8), a cation channel characteristic of the prostate epithelium frequently overexpressed in advanced stage III/IV prostate cancers (PCa), sensitize therapy refractory models of PCa to radio, chemo or hormonal treatment. Overall, our study demonstrates that pharmacological-induced Ca2+ cytotoxicity is an actionable strategy to sensitize cancer cells to standard therapies.

Effects of natural and synthetic isothiocyanate-based H2S-releasers against chemotherapy-induced neuropathic pain: Role of Kv7 potassium channels.

Hydrogen sulfide (H2S) is a crucial signaling molecule involved in several physiological and pathological processes. Nonetheless, the role of this gasotransmitter in the pathogenesis and treatment of neuropathic pain is controversial. The aim of the present study was to investigate the pain relieving profile of a series of slow releasing H2S donors (the natural allyl-isothiocyanate and the synthetics phenyl- and carboxyphenyl-isothiocyanate) in animal models of neuropathic pain induced by paclitaxel or oxaliplatin, anticancer drugs characterized by a dose-limiting neurotoxicity. The potential contribution of Kv7 potassium channels modulation was also studied. Mice were treated with paclitaxel (2.0 mg kg-1) i.p. on days 1, 3, 5 and 7; oxaliplatin (2.4 mg kg-1) was administered i.p. on days 1-2, 5-9, 12-14. Behavioral tests were performed on day 15. In both models, single subcutaneous administrations of H2S donors (1.33, 4.43, 13.31 µmol kg-1) reduced the hypersensitivity to cold non-noxious stimuli (allodynia-related measurement). The prototypical H2S donor NaHS was also effective. Activity was maintained after i.c.v. administrations. On the contrary, the S-lacking molecule allyl-isocyanate did not increase pain threshold; the H2S-binding molecule hemoglobin abolished the pain-relieving effects of isothiocyanates and NaHS. The anti-neuropathic properties of H2S donors were reverted by the Kv7 potassium channel blocker XE991. Currents carried by Kv7.2 homomers and Kv7.2/Kv7.3 heteromers expressed in CHO cells were potentiated by H2S donors. Sistemically- or centrally-administered isothiocyanates reduced chemotherapy-induced neuropathic pain by releasing H2S. Activation of Kv7 channels largely mediate the anti-neuropathic effect.

ß-Adrenergic response is counteracted by extremely-low-frequency pulsed electromagnetic fields in beating cardiomyocytes.

Proper ß-adrenergic signaling is indispensable for modulating heart frequency. Studies on extremely-low-frequency pulsed electromagnetic field (ELF-PEMF) effects in the heart beat function are contradictory and no definitive conclusions were obtained so far. To investigate the interplay between ELF-PEMF exposure and ß-adrenergic signaling, cultures of primary murine neonatal cardiomyocytes and of sinoatrial node were exposed to ELF-PEMF and short and long-term effects were evaluated. The ELF-PEMF generated a variable magnetic induction field of 0-6mT at a frequency of 75Hz. Exposure to 3mT ELF-PEMF induced a decrease of contraction rate, Ca(2+) transients, contraction force, and energy consumption both under basal conditions and after ß-adrenergic stimulation in neonatal cardiomyocytes. ELF-PEMF exposure inhibited ß-adrenergic response in sinoatrial node (SAN) region. ELF-PEMF specifically modulated ß2 adrenergic receptor response and the exposure did not modify the increase of contraction rate after adenylate cyclase stimulation by forskolin. In HEK293T cells transfected with ß1 or ß2 adrenergic receptors, ELF-PEMF exposure induced a rapid and selective internalization of ß2 adrenergic receptor. The ß-adrenergic signaling, was reduced trough Gi protein by ELF-PEMF exposure since the phosphorylation level of phospholamban and the PI3K pathway were impaired after isoproterenol stimulation in neonatal cardiomyocytes. Long term effects of ELF-PEMF exposure were assessed in cultures of isolated cardiomyocytes. ELF-PEMF counteracts cell size increase, the generation of binucleated of cardiomyocytes and prevents the up-regulation of hypertrophic markers after ß-adrenergic stimulation, indicating an inhibition of cell growth and maturation. These data show that short and long term exposure to ELF-PEMF induces a reduction of cardiac ß-adrenergic response at molecular, functional and adaptative levels.

Characterization of two de novoKCNT1 mutations in children with malignant migrating partial seizures in infancy.

The KCNT1 gene encodes for subunits contributing to the Na(+)-activated K(+) current (KNa), expressed in many cell types. Mutations in KCNT1 have been found in patients affected with a wide spectrum of early-onset epilepsies, including Malignant Migrating Partial Seizures in Infancy (MMPSI), a severe early-onset epileptic encephalopathy characterized by pharmacoresistant focal seizures migrating from one brain region or hemisphere to another and neurodevelopment arrest or regression, resulting in profound disability. In the present study we report identification by whole exome sequencing (WES) of two de novo, heterozygous KCNT1 mutations (G288S and, not previously reported, M516V) in two unrelated MMPSI probands. Functional studies in a heterologous expression system revealed that channels formed by mutant KCNT1 subunits carried larger currents when compared to wild-type KCNT1 channels, both as homo- and heteromers with these last. Both mutations induced a marked leftward shift in homomeric channel activation gating. Interestingly, the KCNT1 blockers quinidine (3-1000µM) and bepridil (0.03-10µM) inhibited both wild-type and mutant KCNT1 currents in a concentration-dependent manner, with mutant channels showing higher sensitivity to blockade. This latter result suggests two genotype-tailored pharmacological strategies to specifically counteract the dysfunction of KCNT1 activating mutations in MMPSI patients.

A new Italian FHM2 family: clinical aspects and functional analysis of the disease-associated mutation.

OBJECTIVE: To describe a new FHM kindred, and to analyse the functional consequences of the disease-associated ATP1A2 p.G301R mutation in human cellular models grown at 37°C. PATIENTS AND METHODS: Seven patients were clinically evaluated and gave informed consent for molecular analysis. Extra-pyramidal rigidity of the limbs was present in four subjects and in three of them tongue apraxia was also observed. ATP1A2 and CACNA1A were analysed by direct sequencing. Functional consequences of the mutation were investigated by cell viability assays, Western blots, and immunocytochemistry. Three-dimensional models of the human Na(+)/K(+)-ATPase α2 subunit were generated by homology modelling using SWISS-MODEL. FINDINGS: Analysis of ATP1A2 showed a heterozygous mutation, c.901G>A predicting the replacement of arginine for glycine at residue 301 (p.G301R). Functional analysis suggested that the mutation completely abolished Na(+)/K(+)-ATPase function. CONCLUSIONS: The phenotypic spectrum of our FHM2 family includes some peculiar features. Functional data confirm that Na(+)/K(+)-ATPase haploinsufficiency caused by the ATP1A2 p.G301R mutation is responsible for FHM in the described family.

Cardiotoxic effects of antihistamines: from basics to clinics (...and back).

Drug-induced arrhythmias, particularly those caused by a prolonged QT interval, have become a critical safety issue for compound selection during development by pharmaceutical companies and for health care regulators. The last two decades have witnessed enormous progress in the definition of the clinical conditions that facilitate the occurrence of such serious adverse effects, of its molecular basis, and in the preclinical strategies aimed at early identification of the cardiotoxic liability of compounds undergoing investigation or already used in the clinic. Moreover, despite the fact that acquired factors play an obvious role in drug-induced arrhythmias, it has become evident that the disease is often manifested upon the interaction of strong environmental stressors with specific genetic determinants of the affected individuals; in that sense, few examples can illustrate the existing interaction between acquired and genetic factors in disease manifestation better than drug-induced arrhythmogenesis. Progress in this field has been mainly driven by a strong interaction among various disciplines, including medicinal chemistry, pharmacology, electrophysiology, molecular genetics, and clinical cardiology; such an interdisciplinary approach has often generated unexpected discoveries of great clinical value, allowing clinicians to drive drug selection toward compounds of proven efficacy and safety. Historically, studies on antihistamines have paved the way for much of our current understanding of the mechanisms and problems associated with QT prolongation and drug-induced arrhythmogenesis; therefore, in this perspective, we will attempt to summarize how basic research studies have helped the interpretation of clinically relevant phenomena (from basics to clinics...) and how this information has prompted new emphasis in preclinical studies aimed at predicting the cardiotoxic potential of compounds (...and back). The current availability of several strategies provided with great predictive potential, together with an increased awareness of physicians, pharmaceutical industries, and health care regulators to this potentially serious cardiovascular side effect, has significantly decreased the risk associated with drug-induced arrhythmias caused by drugs newly introduced into the market; nevertheless, given the large number of cases of QT prolongation still occurring during treatment with a wide variety of congeners, it seems appropriate to review the issue of the cardiotoxic actions of antihistamines, as a better comprehension of the underlying mechanisms and risk factors is likely to contribute to the improvement of the risk/benefit ratio for pharmacological treatment in several therapeutic areas.

Human neoplastic mesothelial cells express voltage-gated sodium channels involved in cell motility.

Given the pivotal role of ion channels in neoplastic transformation, the aim of the present study has been to assess possible differences in the expression patterns of voltage-gated monovalent cationic (Na(+) and K(+)) currents between normal and neoplastic mesothelial cells (NM, MPM, respectively), and to evaluate the role of specific ion channels in mesothelioma cells proliferation, apoptosis, and motility. To achieve this aim, membrane currents expressed in NM and MPM cells derived from surgically-removed human specimens were investigated by means of patch-clamp electrophysiology. NM cells were found to express three main classes of K(+) currents, which were defined as K(IR), maxiK(Ca), and K(V) currents on the basis of their biophysical and pharmacological properties. Each of these K(+) currents was absent in MPM cells; by contrast, MPM cells revealed the novel appearance of tetrodotoxin (TTX)-sensitive voltage-gated Na(+) currents undetected in normal mesothelial cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR analysis of MPM cells transcripts showed significant expression of the mRNAs encoding for Na(V)1.2, and Na(V)1.6, and Na(V)1.7 (and less so for Na(V)1.3, Na(V)1.4, and Na(V)1.5) main voltage-gated sodium channel (VGSC) alpha-subunit(s). Interestingly, blockade of VGSCs with TTX decreased mesothelioma cell migration in in vitro motility assays; on the other hand, TTX failed to interfere with cell viability, proliferation, and apoptosis progression triggered by UV exposure. In summary, the results of the present study suggest that VGSCs expression in MPM cells may favor the increased motility of the neoplastic cells, a phenotypic feature often associated with the malignant phenotype.

Decreased subunit stability as a novel mechanism for potassium current impairment by a KCNQ2 C terminus mutation causing benign familial neonatal convulsions.

KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current (I(KM)), a widespread regulator of neuronal excitability. Mutations in KCNQ2- or KCNQ3-encoding genes cause benign familiar neonatal convulsions (BFNCs), a rare autosomal-dominant idiopathic epilepsy of the newborn. In the present study, we have investigated, by means of electrophysiological, biochemical, and immunocytochemical techniques in transiently transfected cells, the consequences prompted by a BFNC-causing 1-bp deletion (2043deltaT) in the KCNQ2 gene; this frameshift mutation caused the substitution of the last 163 amino acids of the KCNQ2 C terminus and the extension of the subunit by additional 56 residues. The 2043deltaT mutation abolished voltage-gated K+ currents produced upon homomeric expression of KCNQ2 subunits, dramatically reduced the steady-state cellular levels of KCNQ2 subunits, and prevented their delivery to the plasma membrane. Metabolic labeling experiments revealed that mutant KCNQ2 subunits underwent faster degradation; 10-h treatment with the proteasomal inhibitor MG132 (20 microm) at least partially reversed such enhanced degradation. Co-expression with KCNQ3 subunits reduced the degradation rate of mutant KCNQ2 subunits and led to their expression on the plasma membrane. Finally, co-expression of KCNQ2 2043deltaT together with KCNQ3 subunits generated functional voltage-gated K+ currents having pharmacological and biophysical properties of heteromeric channels. Collectively, the present results suggest that mutation-induced reduced stability of KCNQ2 subunits may cause epilepsy in neonates.

Lysinuric protein intolerance: identification and functional analysis of mutations of the SLC7A7 gene.

Lysinuric protein intolerance (LPI) is an inherited hyperdibasic aminoaciduria caused by defective cationic amino acid (CAA) transport at the basolateral membrane of epithelial cells in the intestine and kidney. LPI is relatively common in Finland and a few clusters of patients are known in Italy and Japan. The SLC7A7 gene, mutated in LPI patients, encodes the y+LAT-1 protein which is the light subunit of a heterodimeric CAA transporter. We performed the mutation analysis in seven probands from five unrelated LPI families and identified five novel SLC7A7 mutations (p.M50K, p.T188I, p.R333M, p.Y457X, and c.499+?_629-?). By expression studies in X. laevis oocytes or patient's renal tubular cells, the functional analysis of altogether eight SLC7A7 mutations is here reported. Noteworthy, the p.R333M mutation, caused by a G to T transversion of the last nucleotide at 3' end of exon 7, disrupts a functional splicing motif generating misspliced transcripts. Three of the novel mutations were found in patients originating from Greece and Pakistan thus increasing the list of ethnic backgrounds where LPI mutant alleles are present. This reinforces the view that the rarity of LPI outside Finland might be ascribed to misdiagnosis of this disease.