Biophysical study of the effect of lithium isotopes from cells to ion channels, towards uncovering quantum phenomena in neuroscience

Abstract

The effects of lithium (Li) isotopes and their impact on biological processes have recently gained increased attention due to the significance of Li as a pharmacological agent and the potential that Li isotopic effects in neuroscience contexts may constitute a new example of quantum effects in biology. Previous studies have shown that the two Li isotopes, which differ in mass and nuclear spin, have unusually different effects in vivo and in vitro and, although some molecular targets for Li isotope fractionation have been proposed, the experimental evidence of Li isotope differentiation is lacking. The goal of this study was to elucidate the molecular and cellular targets at which Li isotopes exert differential effects on neuronal function, in order to support and clarify previously observed Li isotope effects in animal studies. The first research objective was to study fluxes of Li+, sodium (Na+) and calcium (Ca2+) ions in the mitochondrial sodium/calcium/lithium exchanger (NCLX), the only transporter known with recognized specificity for Li+. We studied the effect of Li+ isotopes on Ca2+ efflux from heart mitochondria in comparison to natural Li+ and Na+ using Ca2+-induced fluorescence and investigated a possible Li isotope fractionation in mitochondria using inductively coupled plasma mass spectrometry (ICP-MS). Our fluorescence data indicate that Ca2+ efflux increases with higher concentrations of either Li+ or Na+. We found that the simultaneous presence of Li+ and Na+ increases Ca2+ efflux compared to Ca2+ efflux caused by the same concentration of Li+ alone. However, no differentiation in the Ca2+ efflux between the two Li+ isotopes was observed, either for Li+ alone or in mixtures of Li+ and Na+. Our ICP-MS data demonstrate that there is selectivity between Na+ and Li+ (greater Na+ than Li+ uptake) and, most interestingly, between the Li+ isotopes (greater 6Li+ than 7Li+ uptake) by the inner mitochondrial membrane. In summary, we observed no Li+ isotope differentiation for Ca2+ efflux in mitochondria via NCLX but found a Li+ isotope fractionation during Li+ uptake by mitochondria with NCLX active or blocked. Our results suggest that the transport of Li+ via NCLX is not the main pathway for Li+ isotope fractionation and that this differentiation does not affect Ca2+ efflux in mitochondria. Therefore, explaining the puzzling effects of Li+ isotopes observed in other contexts will require further investigation to identify the molecular targets for Li+ isotope differentiation. A second research objective focused on calcium phosphate (CaP) clusters, which are relevant to mitochondrial calcium buffering and have been proposed to form stable units such as Posner clusters with potential quantum properties. As a foundational step, we developed and validated an imaging protocol to resolve amorphous CaP clusters using atomic force microscopy (AFM). This allowed us to visualize and characterize CaP cluster formation. While the isotope-specific experiments were not completed at this stage, the proposed project path is to investigate this further with nano-infrared (IR) spectroscopy. This methodological groundwork establishes a platform for future studies aimed at probing whether Li or its isotopes influence CaP assembly or stability. The third research objective was to investigate how Na+ and K+ ion channels are impacted by Li. While Na+ channels serve as the primary pathway for Li+ to enter the cell, it remains unclear whether Li isotopes exert differential effects on these channels. One of the research aims was to study Li isotope effects on Na+ channels, particularly in SH-SY5Y human neuroblastoma cells and human induced pluripotent stem cell (iPSC)-derived neurons using the whole-cell patch-clamp. We were able to show that there are significant differences between the two neuronal cell models in terms of Na+ current as well as differences between Na+, Li+ and Li+ isotopes in some of the current parameters. Although the found difference between Li+ and Na+ and between Li+ isotopes is significant in some cases, it is not drastic and opposite, suggesting other mechanisms to be responsible for impressing behavioural and tissue experimental results. Finally, we used the KcsA potassium (K+) channel, reconstituted into artificial lipid bilayers, as a structurally defined model to investigate whether Li or its isotopes can permeate K+-selective channels. This followed protocol optimization using gramicidin, a simpler monovalent cation channel. KcsA displayed typical single-channel behaviour in K+ and Li+ buffers, with no significant differences in conductance or gating kinetics. While sporadic signal instability was observed in 6Li recordings, the limited number of successful measurements precluded definitive conclusions. Overall, these results indicate that simple passive permeation through K+ channels is unlikely to be the primary source of observed Li+ isotope effects in more complex systems. In summary, this work presents novel experimental studies on the role of Li isotope effects across several biological systems, including mitochondria, neuronal cells and ion channels. Additionally, we provided experimental evidence for the existence of the small calcium phosphate clusters. Our findings suggest that while some degree of Li isotope fractionation occurs at the mitochondrial membrane, particularly in uptake processes, direct isotope differentiation at the level of ion channel permeation or CaP cluster formation is either very minimal or not possible to detect with the technique used. This thesis lays foundational groundwork for the emerging field of Li isotope biology, testing key hypotheses and experimentally probing some of the most plausible molecular targets. In doing so, it not only opens new avenues for isotope-based mechanistic studies but also yields several interesting and novel results along the way. These results highlight the complexity of tracing Li isotope effects in biology and point to the need for further studies on multicomponent systems or indirect mechanisms involving mitochondrial bioenergetics, ROS production, protein-lipid interactions, or spin-dependent processes.