Rebecca Lewis

Ion channels selectively transfer ions across cell membranes, yet the precise basis of selectivity remains unresolved. Acid-Sensing Ion Channels (ASICs), part of the sodium-selective ENaC/DEG family, are implicated in processes such as pain, fear, memory, and cancer. Like other sodium channels, ASICs permit partially hydrated cations to permeate their pore, with solvent deuteration influencing conductance and ion permeability. Here, we investigate ion selectivity in ASIC1 and the associated kinetic isotope effects that arise from ion solvation. We combined molecular dynamics, density functional theory, and open quantum systems simulations to study sodium, potassium, and lithium in H\textsubscript{2}O and D\textsubscript{2}O. Analyses focused on the conserved selectivity filter, comprising the GAS belt and HG motif of the re-entrant loop. Results reveal that binding energies, dehydration costs, and conformational changes jointly shape selectivity. Ion escape rates correlate with ionic mass and solvent shell radius, partially explaining experimental observations. Crucially, coherence lifetimes fall within the sub-femtosecond regime, ruling out quantum coherence and tunnelling as drivers of high selectivity. These findings clarify the biophysical principles of ASIC1 function and provide insights for therapeutic strategies targeting ASIC-related disorders, including chronic pain and cancer.

Cultured chondrocytes have potential for cartilage regeneration to treat degenerative diseases. However, when explanted chondrocytes are cultured in monolayer, they are known to dedifferentiate over time, adopting a more fibroblastic phenotype. This greatly impacts both research and potential clinical applications; cell-based cartilage repair therapies require significant in vitro expansion to obtain sufficient chondrocyte numbers for reimplantation, as chondrocytes adopt a more fibroblastic phenotype causing up to 70% of patients to develop fibrocartilaginous fill. We used dielectrophoresis (DEP) to observe changes in the electrophysiological properties of primary bovine chondrocytes over time. Using a multi-conductivity approach, we demonstrate that monitoring the cytoplasmic conductivity is a reliable method of observing cell changes over 100 days in culture. Results show statistically significant changes in both membrane capacitance (p = 0.0039) and cytoplasm conductivity (p = < 0.0001) when tested at multiple conductivities. Analysis of cytoplasmic vs. medium conductivity allowed simple tracking of chondrocyte membrane potential, which exhibited transitions between three stable values of V_m which correspond to patch-clamp-derived literature values as they transition from chondrocytes (− 13 to − 18 mV) to proliferating fibroblasts (− 32 to − 43 mV) and ultimately to non-proliferating fibroblasts (− 55 to − 71 mV), transitions occurring around days 40 and 80 respectively.

The analysis of cell electrophysiology for pathogenic samples at BSL3 can be problematic. It is virtually impossible to isolate infected from uninfected without a label, for example green fluorescent protein, which can potentially alter the cell electrical properties. Furthermore, the measurement of highly pathogenic organisms often requires equipment dedicated only for use with these organisms due to safety considerations. To address this, we have used dielectrophoresis to study the electrical properties of the human THP‐1 cell line and monocyte‐derived macrophages before and after infection with non‐labelled Mycobacterium tuberculosis. Infection with these highly pathogenic bacilli resulted in changes including a raised surface conductance (associated with reduced zeta potential) and increased capacitance, suggesting an increase in surface roughness. We have also investigated the effect of fixation on THP‐1 cells as a means to enable study on fixed samples in BSL1 or 2 laboratories, which suggests that the properties of these cells are largely unaffected by the fixation process. This advance results in a novel technique enabling the isolation of infected and non‐infected cells in a sample without labelling.

Musculoskeletal disorders represent one of the main causes of disability worldwide, and their prevalence is predicted to increase in the coming decades. Stem cell therapy may be a promising option for the treatment of some of the musculoskeletal diseases. Although significant progress has been made in musculoskeletal stem cell research, osteoarthritis, the most-common musculoskeletal disorder, still lacks curative treatment. To fine-tune stem-cell-based therapy, it is necessary to focus on the underlying biological mechanisms. Ion channels and the bioelectric signals they generate control the proliferation, differentiation, and migration of musculoskeletal progenitor cells. Calcium- and voltage-activated potassium (K_Ca) channels are key players in cell physiology in cells of the musculoskeletal system. This review article focused on the big conductance (BK) K_Ca channels. The regulatory function of BK channels requires interactions with diverse sets of proteins that have different functions in tissue-resident stem cells. In this narrative review article, we discuss the main ion channels of musculoskeletal stem cells, with a focus on calcium-dependent potassium channels, especially on the large conductance BK channel. We review their expression and function in progenitor cell proliferation, differentiation, and migration and highlight gaps in current knowledge on their involvement in musculoskeletal diseases.

Electrical conductivity is of great significance to cardiac tissue engineering and permits the use of electrical stimulation in mimicking cardiac pacing. The development of biomaterials for tissue engineering can incorporate physical properties that are uncommon to standard cell culture and can facilitate improved cardiomyocyte function. In this review, the PICOT question asks, “How has the application of external electrical stimulation in conductive scaffolds for tissue engineering affected cardiomyocyte behavior in in vitro cell culture?” The Preferred Reporting Items for Systematic Reviews and Meta‐Analysis guidelines, with predetermined inclusion and quality appraisal criteria, were used to assess publications from PubMed, Web of Science, and Scopus. Results revealed carbon nanotubes to be the most common conductive agent in biomaterials and rodent‐sourced cell types as the most common cardiomyocytes used. To assess cardiomyocytes, immunofluorescence was used most often, utilizing proteins, such as connexin 43, cardiac α‐actinin, and cardiac troponins. It was determined that the modal average stimulation protocol comprised 1–3 V square biphasic 50‐ms pulses at 1 Hz, applied toward the end of cell culture. The addition of electrical stimulation to in vitro culture has exemplified it as a powerful tool for cardiac tissue engineering and brings researchers closer to creating optimal artificial cardiac tissue constructs.

There is a significant need to understand the complexity and heterogeneity of articular cartilage to develop more effective therapeutic strategies for diseases such as osteoarthritis. Here, we show that carbon nanotubes (CNTs) are excellent candidates as a material for synthetic scaffolds to support the growth of chondrocytes—the cells that produce and maintain cartilage. Chondrocyte morphology, proliferation, and alignment were investigated as nanoscale CNT networks were applied to macroscopically textured polydimethylsiloxane (PDMS) scaffolds. The application of CNTs to the surface of PDMS‐based scaffolds resulted in an up to 10‐fold increase in cell adherence and 240% increase in proliferation, which is attributable to increased nanoscale roughness and hydrophilicity. The introduction of macroscale features to PDMS induced alignment of chondrocytes, successfully mimicking the cell behavior observed in the superficial layer of cartilage. Raman spectroscopy was used as a noninvasive, label‐free method to monitor extracellular matrix production and chondrocyte phenotype. Chondrocytes on these scaffolds successfully produced collagen, glycosaminoglycan, and aggrecan. This study demonstrates that introducing physical features at different length scales allows for a high level of control over tissue scaffold design and, thus, cell behavior. Ultimately, these textured scaffolds can serve as platforms to improve the understanding of osteoarthritis and for early‐stage therapeutic testing.

The Epithelial Sodium Channel/Degenerin (ENaC/DEG) family is a superfamily of sodium-selective channels that play diverse and important physiological roles in a wide variety of animal species. Despite their differences, they share a high homology in the pore region in which the ion discrimination takes place. Although ion selectivity has been studied for decades, the mechanisms underlying this selectivity for trimeric channels, and particularly for the ENaC/DEG family, are still poorly understood. This systematic review follows PRISMA guidelines and aims to determine the main components that govern ion selectivity in the ENaC/DEG family. In total, 27 papers from three online databases were included according to specific exclusion and inclusion criteria. It was found that the G/SxS selectivity filter (glycine/serine, non-conserved residue, serine) and other well conserved residues play a crucial role in ion selectivity. Depending on the ion type, residues with different properties are involved in ion permeability. For lithium against sodium, aromatic residues upstream of the selectivity filter seem to be important, whereas for sodium against potassium, negatively charged residues downstream of the selectivity filter seem to be important. This review provides new perspectives for further studies to unravel the mechanisms of ion selectivity.

Diseases such as osteoarthritis (OA) are commonly characterized at the molecular scale by gene expression and subsequent protein production; likewise, the effects of pharmaceutical interventions are typically characterized by the effects of molecular interactions. However, these phenomena are usually preceded by numerous precursor steps, many of which involve significant ion influx or efflux. As a consequence, rapid assessment of cell electrophysiology could play a significant role in unravelling the mechanisms underlying drug interactions and progression of diseases, such as OA. In this study, we used dielectrophoresis (DEP), a technique that allows rapid, label-free determination of the dielectric parameters to assess the role of potassium ions on the dielectric characteristics of chondrocytes, and to investigate the electrophysiological differences between healthy chondrocytes and those from an in vitro arthritic disease model. Our results showed that DEP was able to detect a significant decrease in membrane conductance (6191 ± 738 vs. 8571 ± 1010 S/m²), membrane capacitance (10.3 ± 1.47 vs. 14.5 ± 0.01 mF/m²), and whole cell capacitance (5.4 ± 0.7 vs. 7.5 ± 0.3 pF) following inhibition of potassium channels using 10 mM tetraethyl ammonium, compared to untreated healthy chondrocytes. Moreover, cells from the OA model had a different response to DEP force in comparison to healthy cells; this was seen in terms of both a decreased membrane conductivity (782 S/m² vs. 1139 S/m²) and a higher whole cell capacitance (9.58 ± 3.4 vs. 3.7 ± 1.3 pF). The results show that DEP offers a high throughput method, capable of detecting changes in membrane electrophysiological properties and differences between disease states.

Cardiovascular disease is currently the top global cause of death, however, research into new therapies is in decline. Tissue engineering is a solution to this crisis and in combination with the use of carbon nanotubes (CNTs), which have drawn recent attention as a biomaterial, could facilitate the development of more dynamic and complex in vitro models. CNTs’ electrical conductivity and dimensional similarity to cardiac extracellular proteins provide a unique opportunity to deliver scaffolds with stimuli that mimic the native cardiac microenvironment in vitro more effectively. This systematic review aims to evaluate the use and efficacy of CNTs for cardiac tissue scaffolds and was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Three databases were searched: PubMed, Scopus, and Web of Science. Papers resulting from these searches were then subjected to analysis against pre-determined inclusion and quality appraisal criteria. From 249 results, 27 manuscripts met the criteria and were included in this review. Neonatal rat cardiomyocytes were most commonly used in the experiments, with multi-walled CNTs being most common in tissue scaffolds. Immunofluorescence was the experimental technique most frequently used, which was employed for the staining of cardiac-specific proteins relating to contractile and electrophysiological function.

Chondrogenic progenitor cells (CPCs) may be used as an alternative source of cells with potentially superior chondrogenic potential compared to mesenchymal stem cells (MSCs), and could be exploited for future regenerative therapies targeting articular cartilage in degenerative diseases such as osteoarthritis (OA). In this study, we hypothesised that CPCs derived from OA cartilage may be characterised by a distinct channelome. First, a global transcriptomic analysis using Affymetrix microarrays was performed. We studied the profiles of those ion channels and transporter families that may be relevant to chondroprogenitor cell physiology. Following validation of the microarray data with quantitative reverse transcription‐polymerase chain reaction, we examined the role of calcium‐dependent potassium channels in CPCs and observed functional large‐conductance calcium‐activated potassium (BK) channels involved in the maintenance of the chondroprogenitor phenotype. In line with our very recent results, we found that the KCNMA1 gene was upregulated in CPCs and observed currents that could be attributed to the BK channel. The BK channel inhibitor paxilline significantly inhibited proliferation, increased the expression of the osteogenic transcription factor RUNX2, enhanced the migration parameters, and completely abolished spontaneous Ca²⁺ events in CPCs. Through characterisation of their channelome we demonstrate that CPCs are a distinct cell population but are highly similar to MSCs in many respects. This study adds key mechanistic data to the in‐depth characterisation of CPCs and their phenotype in the context of cartilage regeneration.