Drissi, I. | Search bioRxiv

bioRxiv2025

A pain-reducing Kv6.4 variant spares other Kv6 channels, offering a target for uterine pain

Uterine pain conditions such as dysmenorrhea and endometriosis are highly prevalent, poorly managed, and associated with long-term impacts on womens health. The identification of a rare KCNG4 variant (rs140124801; p.Val419Met) previously linked to reduced labor pain suggests Kv6.4 (encoded by KCNG4) may play a role in visceral nociception and offer a new target for non-opioid uterine pain relief. We analyzed UK Biobank data to evaluate clinical phenotypes associated with rare single nucleotide polymorphisms (SNPs) in the conserved TVGYG selectivity filter motif of the Kv6 family wherein p.Val419Met is located. Functional consequences of these variants were assessed using immunofluorescence in SHSY5Y cells to examine membrane trafficking, co-immunoprecipitation to investigate interactions between Kv6 subunits and Kv2.1, and single-cell RNA sequencing to determine expression patterns in mouse sensory neurons. Our genetic analysis identified 5,816 individuals heterozygous and 28 homozygous for the p.Val419Met variant, as well as 292 heterozygous carriers of the p.Thr418Met variant, all located in the Kv6.4 subunit. Neither variant was associated with increased risk for general, neurological, or pain-related disorders, even in homozygous p.Val419Met carriers, supporting a favorable safety profile. Kv6.4Val419Met has a dominant negative effect on wild type Kv6.4. However, this effect is specific to Kv6.4 as, in SHSY5Y cells, co-expression of Kv6.4Val419Met along with Kv6.1, Kv6.2 or Kv6.3, showed no effects on the efficient membrane localization of Kv6.1-3. In contrast, Kv6.4Thr418Met does not interfere with Kv6.4 trafficking or its heteromerization with Kv2.1. Additionally, In SHSY5Y cells, the equivalent p.Val419Met substitution, when introduced into Kv6.1, Kv6.2 and Kv6.3, disrupts their membrane localization, noting that these variants have never been reported. Co-immunoprecipitation shows that Kv6.4 does not interact with other Kv6 subunits and transcriptomic analysis shows that Kv6.4 is expressed in a distinct subset of mouse lumbar dorsal root ganglion neurons innervating pelvic organs. Our findings show that the Kv6.4Val419Met variant selectively disrupts Kv6.4 function without affecting other family members and is not linked to adverse phenotypes. This finding supports Kv6.4 as a highly selective and functionally distinct Kv6 subunit with no widespread deleterious effects on other Kv6 subunits, making it an attractive candidate for therapeutic targeting for uterine pain.

genetics↗

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↗