The L1624Q Variant in SCN1A Causes Familial Epilepsy Through a Mixed Gain and Loss of Channel Function

Variants of the SCN1A gene encoding the neuronal voltage-gated sodium channel NaV1.1 cause over 85% of all cases of Dravet syndrome, a severe and often pharmacoresistent epileptic encephalopathy with mostly infantile onset. But with the increased availability of genetic testing for patients with epilepsy, variants in SCN1A have now also been described in a range of other epilepsy phenotypes. The vast majority of these epilepsy-associated variants are de novo, and most are either nonsense variants that truncate the channel or missense variants that are presumed to cause loss of channel function. However, biophysical analysis has revealed a significant subset of missense mutations that result in increased excitability, further complicating approaches to precision pharmacotherapy for patients with SCN1A variants and epilepsy. We describe clinical and biophysical data of a familial SCN1A variant encoding the NaV1.1 L1624Q mutant. This substitution is located on the extracellular linker between S3 and S4 of Domain IV of NaV1.1 and is a rare case of a familial SCN1A variant causing an autosomal dominant frontal lobe epilepsy. We expressed wild-type (WT) and L1642Q channels in CHO cells. Using patch-clamp to characterize channel properties at several temperatures, we show that the L1624Q variant increases persistent current, accelerates fast inactivation onset and decreases current density. While SCN1A-associated epilepsy is typically considered a loss-of-function disease, our results put L1624Q into a growing set of mixed gain and loss-of-function variants in SCN1A responsible for epilepsy.

Membrane pools of phosphatidylinositol-4-phosphate regulate KCNQ1/KCNE1 membrane expression

Plasma membrane phosphatidylinositol 4-phosphate (PI4P) is a precursor of PI(4,5)P2, an important regulator of a large number of ion channels. Although the role of the phospholipid PI(4,5)P2 in stabilizing ion channel function is well established, little is known about the role of phospholipids in channel membrane localization and specifically the role of PI4P in channel function and localization. The phosphatidylinositol 4-kinases (PI4Ks) synthesize PI4P. Our data show that inhibition of PI4K and prolonged decrease of levels of plasma membrane PI4P lead to a decrease in the KCNQ1/KCNE1 channel membrane localization and function. In addition, we show that mutations linked to Long QT syndrome that affect channel interactions with phospholipids lead to a decrease in membrane expression. We show that expression of a LQT1-associated C-terminal deletion mutant abolishes PI4Kinase-mediated decrease in membrane expression and rescues membrane expression for phospholipid-targeting mutations. Our results indicate a novel role for PI4P on ion channel regulation. Our data suggest that decreased membrane PI4P availability to the channel, either due to inhibition of PI4K or as consequence of mutations, dramatically inhibits KCNQ1/KCNE1 channel membrane localization and current. Our results may have implications to regulation of other PI4P binding channels.

A new side-effect of sufentanil: increased monocyte-endothelial adhesion

Background Opioids have been identified by the World Health Organization to be ‘indispensable for the relief of pain and suffering’. Side-effects, such as nausea, vomiting, postoperative delirium, and effects on breathing, of opioids have been well investigated; however, the influence of opioids on monocyte-endothelial adherence has never been reported. Therefore, we explored the effects of representative opioids, fentanyl, sufentanil, and remifentanil, on monocyte-endothelial adherence and the underlying mechanisms. Methods We built a cell adhesion model with U937 monocytes and human umbilical vein endothelial cells (HUVECs). Two kinds of connexin43 (Cx43) channel inhibitors, 18-α-GA and Gap 27, were used to alter Cx43 channel function in U937 monocytes and HUVECs, respectively, to determine the effects of Cx43 channels on U937-HUVEC adhesion. Subsequently, the effects of fentanyl, sufentanil and remifentanil on Cx43 channel function and U937-HUVEC adhesion were explored. Results When fentanyl, sufentanil and remifentanil acted on monocytes or endothelial cells, their effects on monocyte-endothelial adherence differed. When acting on U937 monocytes, sufentanil significantly increased U937-HUVEC adhesion which was associated with reduced release of ATP from Cx43 channels, while fentanyl and remifentanil did not have these influences. Although sufentanil could also inhibit Cx43 channel function in HUVECs, it had no effect on ATP release from HUVECs or U937-HUVECs adhesion. Conclusions We demonstrated that sufentanil application increases monocyte-endothelial adherence which was associated with reduced release of ATP from Cx43 channels in monocytes. This side-effect of sufentanil should be considered seriously by clinicians.

The molecular evolution of function in the CFTR chloride channel

The ATP-binding cassette (ABC) transporter superfamily includes many proteins of clinical relevance, with genes expressed in all domains of life. Although most members use the energy of ATP binding and hydrolysis to accomplish the active import or export of various substrates across membranes, the cystic fibrosis transmembrane conductance regulator (CFTR) is the only known animal ABC transporter that functions primarily as an ion channel. Defects in CFTR, which is closely related to ABCC subfamily members that bear function as bona fide transporters, underlie the lethal genetic disease cystic fibrosis. This article seeks to integrate structural, functional, and genomic data to begin to answer the critical question of how the function of CFTR evolved to exhibit regulated channel activity. We highlight several examples wherein preexisting features in ABCC transporters were functionally leveraged as is, or altered by molecular evolution, to ultimately support channel function. This includes features that may underlie (1) construction of an anionic channel pore from an anionic substrate transport pathway, (2) establishment and tuning of phosphoregulation, and (3) optimization of channel function by specialized ligand–channel interactions. We also discuss how divergence and conservation may help elucidate the pharmacology of important CFTR modulators.

Replacing traditional sections with Teams-based groupwork: Remote learning and beyond

The sudden shift to online teaching and learning brought about by the Covid-19 pandemic provided an opportunity to consider alternatives to entrenched teaching practices. Making use of the private channel function in Microsoft Teams, I replaced traditional sections in an introductory linguistics course with asynchronous groupwork. This enabled students to form learning communities that facilitated peer learning and support in spite of remote learning, while unexpectedly connecting students with instructors in more personalized ways than typically witnessed in traditional sections. The medium allowed the teaching team to provide tailored feedback on each group’s work, as well as point out errors that were common across groups. I reflect on some of the problems encountered and consider how these might be addressed in the future.

Efficient Suppression of Endogenous CFTR Nonsense Mutations Using Anticodon Engineered Transfer RNAs

Nonsense mutations or premature termination codons (PTCs) comprise ~11% of all genetic lesions, which result in over 7,000 distinct genetic diseases. Due to their outsized impact on human health, considerable effort has been made to find therapies for nonsense-associated diseases. Suppressor tRNAs have long been identified as a possible therapeutic for nonsense-associated diseases, however their ability to inhibit nonsense-mediated mRNA decay (NMD) and support significant protein translation from endogenous transcripts has not been determined in mammalian cells. Here we investigated the ability of anticodon edited (ACE)-tRNAs to suppress cystic fibrosis (CF) causing PTCs in the cystic fibrosis transmembrane regulator (CFTR) gene in gene-edited immortalized human bronchial epithelial (16HBEge) cells. Delivery of ACE-tRNAs to 16HBEge cells harboring three common CF mutations G542X-, R1162X- and W1282X-CFTR PTCs significantly inhibited NMD and rescued endogenous mRNA expression. Furthermore, delivery of our highly active leucine encoding ACE-tRNA resulted in rescue of W1282X-CFTR channel function to levels that significantly exceed the necessary CFTR channel function for therapeutic relevance. This study establishes the ACE-tRNA approach as a potential stand-alone therapeutic for nonsense-associated diseases due to its ability to rescue both mRNA and full-length protein expression from PTC containing endogenous genes.