Biology subjects

Nahorski, M.

Publications and source records attributed to Nahorski, M..

3 recordsLinked to original sources

bioRxiv2025

Painless Nav1.7 mutations reveal function-critical residuesin the outer vestibule and N-terminus

Chronic pain is a common condition, placing a large cost on society and a heavy burden on the individual. Nav1.7 has emerged as a non-redundant and axiomatically important part of human pain pathways. Biallelic loss-of-function (LOF) mutations in SCN9A, encoding voltage-gated sodium channel Nav1.7, cause the Mendelian disorder Congenital Insensitivity to Pain (CIP). Studying novel missense mutations in this channel has the potential to uncover unrealised functions of critical residues that could enable new strategies for analgesia. Here we describe detailed functional studies of six SCN9A missense variants found in individuals with typical SCN9A-CIP: c.224T>C/p.Leu75Pro, c.1025C>T/p.Thr342Met, c.2686C>T/p.Arg896Trp, c.2732G>A/p.Arg911His, c.5059G>C/p.Ala1687Pro, and c.5173G>C/p.Gly1725Arg. While all variants were extremely rare and predicted to alter function, current tools cannot specify the extent or direction of change in function, nor provide information about the mechanism(s) of pathogenicity. Stable cell lines were generated for each variant, using the isogenic Jump-In T-REx HEK293 system. We investigated how each variant caused molecular dysfunction using multiple approaches: sequence conservation analysis and 3D modelling to examine the structural importance of the affected residues; automated whole-cell patch clamp electrophysiology to assess channel biophysics; and finally, immunocytochemistry along with cell-surface biotinylation to detect any alterations in subcellular localisation. Our studies revealed that all mutations reached the cell membrane but caused a complete LOF through small localised structural changes in the extracellular vestibule, ion selectivity filter and N-terminus that abolished sodium conductance. Functional analysis remains the most reliable method of classifying SCN9A missense variants and can help to pinpoint important subdomains. Online SummaryVoltage-gated sodium channel Nav1.7 is a key modulator of nociceptor excitability and associated with congenital painlessness. Functional analysis and in silico modelling reveal critical roles for residues in the extracellular vestibule, ion selectivity filter and unexpectedly, the N-terminus.

molecular biology↗

bioRxiv2021

EROS-mediated control of NOX2 and P2X7 biosynthesis

EROS (Essential for Reactive Oxygen Species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease (CGD), but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase (OST) machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions and P2X7 is almost absent in EROS deficient mouse and human primary cells. Accordingly, lack of EROS results in markedly abnormal P2X7 signalling, inflammasome activation and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation and possibly gene therapy.

immunology↗

bioRxiv2018

Human labor pain is influenced by the voltage-gated potassium channel KV6.4 subunit

AO_SCPLOWBSTRACTC_SCPLOWWe sought genetic effects on labour pain by studying healthy women who did not request analgesia during their first delivery. Extensive sensory and psychometric testing were normal in these women, except for significantly higher cuff-pressure pain. We found an excess of heterozygotes carrying the rare allele of SNP rs140124801 in KCNG4. The rare variant KV6.4-Met419 exerts a dominant negative effect and cannot modulate the voltage-dependence of KV2.1 inactivation because it fails to traffic to the plasma membrane. In vivo, we observed Kcng4 (KV6.4) expression in 40% of retrograde labelled mouse uterine sensory neurones, all of which expressed KV2.1, and over 90% expressed nociceptor genes Trpv1 and Scn10a. In neurones overexpressing KV6.4-Met419, the voltage-dependence of inactivation for KV2.1 is more depolarised compared to neurones overexpressing KV6.4. Finally, KV6.4-Met419 overexpressing neurones have a higher action potential threshold. We conclude KV6.4 can influence human labour pain by modulating the excitability of uterine nociceptors.

neuroscience↗