Investigating Electrophysiological Changes in Blood Cells Using Dielectrophoresis

"The electrophysiological variation of RBCs and platelets is intriguing, as both are anucleate and lack traditional cellular apparatus; their behaviour is driven by the ion channels within the membrane. Assessment and characterisation of the electrophysiological rhythms of red blood cells (RBCs), and the observation of similar rhythms in platelets, is potentially of importance due to the temporal incidence of many diseases associated with these cell types. RBCs and platelets were examined using various methods including dielectrophoresis, flow cytometry, and zeta potential to assess for rhythmic properties.This work demonstrates that casein kinase is involved in RBC electrophysiological rhythms. Inhibition of casein kinase in RBCs altered the observed rhythm, revealing its involvement in both RBC electrophysiological rhythms and temperature compensation. The possible roles of intrinsic and extrinsic factors in RBC electrophysiological rhythms were examined. Levels of haemoglobin, ionic abundance, and passive electrophysiology were found to show time of day variation. Furthermore, zeta potential was used as a novel method to examine rhythmic electrophysiology.This thesis provides the first evidence of the presence of electrophysiological rhythms in platelets. Platelet electrophysiology was assessed in: platelet rich plasma immediately after venepuncture, stored platelet rich plasma, and stored resuspended platelets. Each condition exhibited a rhythm of differing period, suggesting the platelet entrainment mechanism involves at least three components: an intrinsic platelet component, entraining plasma component, and endogenous circulation component. Furthermore, activated platelets showed altered rhythms in each condition, suggesting the presence of an additional clock, functioning on platelet activation. The electrophysiological rhythm of a platelet precursor cell line was examined (in the assessment of the origin of platelet rhythms) and was found to be different to that of platelets.

Dielectrophoresis as a Tool to Reveal the Potential Role of Ion Channels and Early Electrophysiological Changes in Osteoarthritis

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

The platelet electrome: evidence for a role in regulation of function and surface interaction

Introduction. Platelets protect the body from injury through formation of blood clots, changing from a normal, quiescent state to become “activated” in response to external stimuli such as chemical cues, shear stress and temperature. This causes changes in shape, increased adhesion, and alteration of the electrical properties such as membrane potential Vm and zeta potential ζ. These phenomena have been regarded as largely unconnected; for example, changes in ζ have been attributed solely to alteration of surface lipid concentration. However, recent reports suggest that cells can alter ζ electrostatically by alteration of Vm in red blood cells. We hypothesised that if platelets also modulate ζ via Vm, this may provide an alternative mechanism to alter cell-cell interaction.Methods. We investigated platelets stored at different temperatures (4°C, 22°C, 37°C) for 24h, which is known to alter platelet behaviour and electrical properties, and compared these with analyses of freshly-harvested platelets. These four conditions exhibited unique sets of electrical properties (Vm, ζ, membrane conductance Geff and cytoplasm conductivity σcyto) as well as surface exposure of the adhesion molecule P-Selectin. These were analysed to identify correlations between electrical parameters and platelet activation state.Results. Many parameters exhibit pairwise correlation across all four conditions, in particular between ζ and Geff, and also between Vm and σcyto. Furthermore, when the electrical behaviour of platelets stored at 4°C (known to activate the cells) was removed from the analysis, additional relationships were observed among the remaining conditions, including those connecting ζ and Vm to the amount of P-selectin binding.Conclusion. Results suggest that Vm may mechanistically alter the availability of cationic molecules at the cell surface, a process never reported before, with implications for our wider understanding of cell-molecule and cell-cell interaction

Vm-related extracellular potentials observed in red blood cells

Even in nonexcitable cells, the membrane potential Vm is fundamental to cell function, with roles from ion channel regulation, development, to cancer metastasis. Vm arises from transmembrane ion concentration gradients; standard models assume homogeneous extracellular and intracellular ion concentrations, and that Vm only exists across the cell membrane and has no significance beyond it. Using red blood cells, we show that this is incorrect, or at least incomplete; Vm is detectable in the extracellular ion concentration beyond the cell surface, and that modulating Vm produces quantifiable and consistent changes in extracellular potential. Evidence strongly suggests this is due to capacitive coupling between Vm and the electrical double layer, rather than molecular transporters. We show that modulating Vm changing the extracellular ion composition mimics the behaviour of voltage-activated ion channel in non-excitable channels. We also observe Vm-synchronised circadian rhythms in extracellular potential, with significant implications for cell-cell interactions and cardiovascular disease