Physiological oxygen levels determine ion channel activities: characterising the role of physoxia in neuronal function
Jennifer Cale1, Sébastien Serres1, Tracy D. Farr2 and Joern R. Steinert1
1School of Life Sciences, University of Nottingham, NG7 2UH, UK
2Institute for Neuroscience and Cardiovascular Research, University of Edinburgh, Queen’s Medical Research Institute, EH16 4TJ
Introduction and aims:
Vascular dementia caused by vascular dysfunction is the second most common type of dementia after Alzheimer’s disease and it is associated with hypoxia; oxygen supply drops below physiological levels of about 5kPa to less than ~2kPa. Mechanistic studies are often carried out under hyperoxic (21kPa) conditions. We sought to characterise neurophysiological regulation at different oxygenation concentrations to addresses this gap by examining ion channel behavior under physioxia (5kPa) to reflect physiological environments. We cultured differentiated mouse-derived neuroblastoma cells for 4 days under physiological or hyperxoxic (21kPa) conditions as well as for 1 day under hypoxic O2 (1kPa) conditions.
Methods:
Mouse neuroblastoma cells (N2a) were cultured in DMEM+GlutaMAX media supplemented with 10% FBS and differentiated by a reduced-serum media supplemented with 1% FBS and 20μM of retinoic acid (RA) for 7-10 days. Cells were cultured under 5% CO2 at 37ºC and subjected to reduced serum and RA for 7-10 days to differentiate into neurons prior to any experimentation[GU1] . For 5kPa and 1kPa O2 levels, cells were cultured in a chamber set to physioxic or hypoxic O2 for four and one day(s), respectively, prior to data collection.
Electrophysiology: Whole-cell currents and action potentials were recorded using a MultiClamp 700B/HEKA EPC10 amplifier for data acquisition (n=10 cells per condition) and analysis was performed with Clampex 11.2 (Molecular Devices)/Patchmaster (HEKA) software. Whole-cell currents were recorded in HEPES buffered standard solution at a perfusion rate of 4ml/min and ∼35ºC in the respective 1kPa, 5kPa or 21kPa O2 environments.
Statistical Analysis: Whole-cell current amplitudes were analysed with a two-way ANOVA and half-activation voltages, and action potential amplitudes were compared using Student’s t-tests (p<0.05 was considered significant). Data is presented as mean ± SEM.
Results and Conclusions:
Physioxia significantly increased TEA-sensitive K⁺ currents (amplitudes at +50mV: 21kPa: 205.9±39.6pA; 5kPa: 240.6±67.3pA, p<0.001) and shifted half-activation voltages to hyperpolarised values (V1/2 K+: 21kPa: 13.8±2.0mV; 5kPa: 3.6±3.1mV, p=0.01). Voltage-gated Na+ currents were enhanced under physioxia (amplitude at 0mV: 21kPa: -93.2±23.9pA; 5kPa: -143.6±23.0pA, p=0.001) without displaying a changed voltage activation curve (V1/2 Na+: 21kPa: -14.3±1.9mV; 5kPa: -12.6±2.3mV, p=0.59). Preliminary data indicate that K+ currents are strongly diminished under hypoxic conditions. Action potential amplitudes were affected by ambient O2 levels resulting in lower values at 5kPa following 300pA current injection at a holding potential of -60mV (21kPa: 22±2mV; 5kPa: 17±0.4mV, p<0.05) in physioxia, indicating that cells in physiological oxygen levels possess different ion channel regulation and electrophysiological phenotypes than under hyperoxic conditions.
These findings demonstrate that physiological oxygen levels critically shape neuronal ion channel function and excitability, underscoring the need to consider physioxia in neurophysiological studies and vascular dementia research. Understanding oxygen-dependent ion channel regulation may inform therapeutic strategies for vascular dementia and other hypoxia-related neurological disorders
[GU1]I am not sure I understand why this is important?