Samantha C. Salvage

Despite significant advances in early detection and therapeutic opportunities, cancer remains a critical global health challenge. The greatest barrier to reducing cancer‐associated mortality is the persistent problem of therapeutic resistance and metastasis. Standard treatment approaches, including surgery combined with chemotherapy and radiation, are effective for many patients with localised disease. However, a substantial proportion of patients will experience recurrence and therapy resistance, leading to treatment failure and poor outcomes. This adverse prognosis for patients highlights the critical need to identify new therapeutic targets capable of improving long‐term survival. The ion transportome comprises ion channels, solute transporters and pumps that regulate controlled movement of sodium (Na ⁺ ), potassium (K ⁺ ), calcium (Ca ²⁺ ), chloride (Cl ⁻ ) and hydrogen (H ⁺ ) across cell membranes. Ion transport is central to many cellular processes, including cell growth, volume regulation, proliferation and migration. In many solid tumours, the finely tuned ionic balance becomes dysregulated, with aberrant expression or activity of membrane transport proteins being closely linked to many of the hallmarks of cancer. Ion channel modulation using pharmacological agents designed to treat channelopathies has demonstrated promise in suppressing cancer progression in preclinical models. Thus, targeting ion‐driven vulnerabilities within solid tumours may complement existing therapies to overcome resistance and improve patient outcomes. The dynamic nature of ion transport mechanisms means that addressing critical research questions regarding their suitability as pharmacological targets is challenging, highlighting the need to develop innovative approaches to study these processes in oncology. image

KV7 (KCNQ) channels generate slowly activating, non-inactivating K+ currents that stabilize the membrane potential and limit cellular excitability [1–3]. Within the cardiovascular and visceral syst...

Bioelectrical signalling is fundamental for regulating biological processes in all forms of life. Ion channels and transporters generate and propagate electrical currents by selectively allowing ions to flow across membranes in response to voltage changes. Although recent breakthroughs in structural determination methods, such as cryogenic electron microscopy, have provided novel insights into the structure–function relationships of ion channels and scaffolding proteins, their precise roles in bioelectrical signal generation and propagation within and across different cells and tissues remain unresolved. This article examines the biochemical and ultrastructural features of the three most studied modes of bioelectrical conduction in human tissues—electrotonic, saltatory and ephaptic conduction—and how biophysical constraints set by membranes and proteins give rise to bioelectricity. Notably, ion channel clustering and scaffolding proteins that define intermembrane distances are common key features among all forms of bioelectrical signalling. Techniques like cryogenic electron tomography offer promising avenues for exploring ion channels and their regulatory protein interactions in situ. The central question is: ‘How does the spatial organization of ions, molecules and tissues give rise to bioelectricity?’ These insights may inform novel therapeutic approaches for various diseases, while also potentially offering new perspectives on life, evolution and consciousness.

Background and Purpose: Peptide toxins isolated from animal venom are potent and selective modulators of ion channels, and promising therapeutic leads. Due to intricate disulphide bridge patterns, they are often challenging to produce in standard laboratory settings, which limits engineering approaches to manipulate their structure-function properties. Given the low cost, wide accessibility, and versatility of recombinant expression systems for protein production, we set out to establish a straightforward high-yield strategy across a broad panel of peptide toxins from snakes, spiders and scorpions. Experimental Approach: 13 toxin DNA sequences were genetically fused to the C-terminus of either bivalent or monovalent human IgG1 antibody fragment crystallisable (Fc) domain sequences and expressed recombinantly from mammalian Expi293F cells. Affinity-purified proteins were evaluated by SDS-PAGE and size-exclusion chromatography (SEC). Function was assessed by Ca ²⁺ flux assays on CN21 cells, or whole-cell electrophysiology on human embryonic kidney (HEK293T) cells, Chinese hamster ovary (CHO) cells, or dorsal root ganglion (DRG) neurons. Immunocytochemistry using HEK293T cells and mouse DRG neurons assessed Fc-toxin fusion binding. Key Results: Monovalent Fc-toxin fusions consistently yielded 1-6 mg of pure, non-proteolytically cleaved protein from 20-70 ml cultures for several toxin types, including three-finger toxins from snakes, inhibitory cystine knot (ICK) toxins from spiders, and α-toxins from scorpions, substantially surpassing the performance of unfused toxins or bivalent Fc-toxin fusions which gave low or no yield. Snake toxins targeting nicotinic acetylcholine receptors retained high single digit nanomolar inhibitory potency. Spider and scorpion toxins targeting the voltage-gated Na ⁺ channel Na _v 1.7 retained pharmacological function and selectivity across a panel of five Na _v subtypes, albeit with reduced potencies that did not exceed ∼70 nM. Conclusions and Implications: We present a strategy for straightforward robust production of pure, monodisperse, and functional animal venom-derived toxins. This lowers the barrier to toxin production in a standard laboratory setting for follow-on engineering purposes.

Voltage-gated sodium channels (Navs) are critical for membrane potential depolarisation in cells, with especially important roles in neuronal and cardiomyocyte membranes. Their malfunction results in a range of disorders, and they are the target of many widely used drugs. A rapid yet accurate functional assay is therefore desirable both to probe for novel active compounds and to better understand the many different Nav isoforms. Here, we use fluorescence to monitor Nav function: cells expressing either the cardiac Nav 1.5 or pain-associated Nav 1.7 were loaded with fluorescent membrane potential sensitive dye and then stimulated with veratridine. Cells expressing Nav 1.5 show a concentration-dependent slow rise and then a plateau in fluorescence. In contrast, cells expressing Nav 1.7 show a more rapid rise and then unexpected oscillatory behavior. Inhibition by flecainide and mexiletine demonstrates that these oscillations are Nav-dependent. Thus, we show that this fluorescent membrane potential dye can provide useful functional data and that we can readily distinguish between these two Nav isoforms because of the behavior of cells expressing them when activated by veratridine. We consider these distinct behaviors may be due to different interactions of veratridine with the different Nav isoforms, although more studies are needed to understand the mechanism underlying the oscillations.

Voltage-gated sodium (Na _V ) channel α-subunits are modulated by associated β-subunits that affect their localization, trafficking and gating behaviour. The β-subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules and the interactions between their extracellular Ig-domains may modify channel clustering. The full-length β3-subunit can form cis trimers on the plasma membrane. The atomic resolution structure of a deglycosylated trimeric β3-subunit Ig-domain has been solved by X-ray crystallography. However, it is not clear whether this particular trimeric Ig-domain structure is plausible for cell-expressed, glycosylated β3-subunits. Here we use glycan profiling to confirm an extensive and heterogeneous pattern of β3-subunit glycosylation, with the majority of glycans being bi- and tri-antennary structures with one or two terminal sialic acids. Two tryptic peptides of the β3 Ig-domain are predicted to contain potential N-linked glycosylation sites. When the isolated, glycosylated full-length β3-subunit was trypsin-digested and analysed by LC-MS/MS, only one of these peptides - containing an N-linked glycosylation site at residue N95 and located close to the trimer interface - was identified in its unmodified form, suggesting that residue N95 is under-glycosylated. All-atom molecular dynamics simulations of the glycosylated, membrane-bound full-length β3 trimer confirmed that glycans can be accommodated with the Ig-domain trimer and indeed, may contribute to protein-membrane and inter-protomer interactions within the full-length, membrane-embedded trimer. Further biochemical studies are warranted to explore the interactions between oligomeric β-subunits with corresponding α-subunit sodium channels.

Voltage-gated sodium channel α-subunits (NaV1.1–1.9) initiate and propagate action potentials in neurons and myocytes. The NaV β-subunits (β1–4) have been shown to modulate α-subunit properties. Homo-oligomerization of β-subunits on neighboring or opposing plasma membranes has been suggested to facilitate cis or trans interactions, respectively. The interactions between several NaV channel isoforms and β-subunits have been determined using cryogenic electron microscopy (cryo-EM). Interestingly, the NaV cryo-EM structures reveal the presence of N-linked glycosylation sites. However, only the first glycan moieties are typically resolved at each site due to the flexibility of mature glycan trees. Thus, existing cryo-EM structures may risk de-emphasizing the structural implications of glycans on the NaV channels. Herein, molecular modeling and all-atom molecular dynamics simulations were applied to investigate the conformational landscape of N-linked glycans on NaV channel surfaces. The simulations revealed that negatively charged sialic acid residues of two glycan sites may interact with voltage-sensing domains. Notably, two NaV1.5 isoform-specific glycans extensively cover the α-subunit region that, in other NaV channel α-subunit isoforms, corresponds to the binding site for the β1- (and likely β3-) subunit immunoglobulin (Ig) domain. NaV1.8 contains a unique N-linked glycosylation site that likely prevents its interaction with the β2 and β4-subunit Ig-domain. These isoform-specific glycans may have evolved to facilitate specific functional interactions, for example, by redirecting β-subunit Ig-domains outward to permit cis or trans supraclustering within specialized cellular compartments such as the cardiomyocyte perinexal space. Further experimental work is necessary to validate these predictions.

Highlights Three N-linked glycosylation sites conserved among all Nav channels Glycan modelling and molecular dynamics simulations highlight 3D landscape Unique Nav1.5 N-linked glycans may prevent binding to Ig-domains of β1 and β3 Unique Nav1.8 N-linked glycan may prevent binding to Ig-domains of β2 and β4 N-linked glycans likely contribute to supra-molecular clustering of Nav channels Voltage-gated sodium channel α-subunits (Nav1.1-1.9) initiate and propagate action potentials in neurons and myocytes. The Nav β-subunits (β1-4) have been shown to modulate α-subunit properties. Homo-oligomerization of β-subunits on neighboring or opposing plasma membranes has been suggested to facilitate cis or trans interactions, respectively. The interactions between several Nav channel isoforms and β-subunits have been determined using cryogenic electron microscopy (cryo-EM). Interestingly, the Nav cryo-EM structures reveal the presence of N-linked glycosylation sites. However, only the first glycan moieties are typically resolved at each site due to the flexibility of mature glycan trees. Thus, existing cryo-EM structures may risk de-emphasizing the structural implications of glycans on the Nav channels. Herein, molecular modelling and all-atom molecular dynamics simulations were applied to investigate the conformational landscape of N-linked glycans on Nav channel surfaces. The simulations revealed that negatively-charged sialic acid residues of two glycan sites may interact with voltage-sensing domains. Notably, two Nav1.5 isoform-specific glycans extensively cover the α-subunit region that, in other Nav channel α-subunit isoforms, corresponds to the binding site for the β1-(and likely β3-) subunit immunoglobulin (Ig) domain. Nav1.8 contains a unique N-linked glycosylation site that likely prevents its interaction with the β2 and β4-subunit Ig domain. These isoform-specific glycans may have evolved to facilitate specific functional interactions, for example by redirecting β-subunit Ig-domains outwards to permit cis or trans supra-clustering within specialized cellular compartments such as the cardiomyocyte perinexal space. Further experimental work is necessary to validate these predictions.