Outward potassium current in neurons of aestivated land snail Achatina fulica.

Aestivation and hibernation represent distinct forms of animal quiescence, characterized by physiological changes, including ion composition. Intracellular ion flows play a pivotal role in eliciting alterations in membrane potential and facilitating cellular communication, while outward K+ currents aid in the restitution and upkeep of the resting membrane potential. This study explores the relationship between inward and outward currents during aestivation in Achatina fulica snails. Specimens were collected near MSUBIT University in Shenzhen and divided into two groups. The first group was kept on a lattice diet, while the second one consisted of aestivating individuals, that were deprived of food and water until a cork-like structure sealed their shells. Recording of current from isolated neurons were conducted using the single-electrode voltage clamp mode with an AxoPatch 200B amplifier. Electrophysiological recordings on pedal ganglia neurons revealed significant differences in the inactivation processes of the Ia and Ikdr components. Alterations in the Ikdr component may inhibit pacemaker activity in pedal ganglion neurons, potentially contributing to locomotion cessation in aestivated animals. The KS current remains unaffected during aestivation. Changes in slow K+ current components could disrupt the resting membrane potential, possibly leading to cell depolarization and influx of Ca2+ and Na+ ions, impacting cell homeostasis. Thus, maintaining the constancy of outward K+ current is essential for cell stability.

Different fluorescent labels report distinct components of spHCN channel voltage sensor movement.

We used voltage clamp fluorometry to probe the movement of the S4 helix in the voltage-sensing domain of the sea urchin HCN channel (spHCN) expressed in Xenopus oocytes. We obtained markedly different fluorescence responses with either ALEXA-488 or MTS-TAMRA covalently linked to N-terminal Cys332 of the S4 helix. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly, consistent with it reporting the initial inward movement of S4, as previously described. In contrast, MTS-TAMRA fluorescence increased more slowly and its early phase correlated with that of channel opening. Additionally, a slow fluorescence component that tracked the development of the mode shift, or channel hysteresis, could be resolved with both labels. We quantitated this component as an increased deactivation tail current delay with concomitantly longer activation periods and found it to depend strongly on the presence of K+ ions in the pore. Using collisional quenching experiments and structural predictions, we established that ALEXA-488 was more exposed to solvent than MTS-TAMRA. We propose that components of S4 movement during channel activation can be kinetically resolved using different fluorescent probes to reveal distinct biophysical properties. Our findings underscore the need to apply caution when interpreting voltage clamp fluorometry data and demonstrate the potential utility of different labels to interrogate distinct biophysical properties of voltage-gated membrane proteins.

Identification of Distinct, Quantitative Pattern Classes from Emergent Tissue-Scale hiPSC Bioelectric Properties.

Bioelectric signals possess the ability to robustly control and manipulate patterning during embryogenesis and tissue-level regeneration. Endogenous local and global electric fields function as a spatial 'pre-pattern', controlling cell fates and tissue-scale anatomical boundaries; however, the mechanisms facilitating these robust multiscale outcomes are poorly characterized. Computational modeling addresses the need to predict in vitro patterning behavior and further elucidate the roles of cellular bioelectric signaling components in patterning outcomes. Here, we modified a previously designed image pattern recognition algorithm to distinguish unique spatial features of simulated non-excitable bioelectric patterns under distinct cell culture conditions. This algorithm was applied to comparisons between simulated patterns and experimental microscopy images of membrane potential (Vmem) across cultured human iPSC colonies. Furthermore, we extended the prediction to a novel co-culture condition in which cell sub-populations possessing different ionic fluxes were simulated; the defining spatial features were recapitulated in vitro with genetically modified colonies. These results collectively inform strategies for modeling multiscale spatial characteristics that emerge in multicellular systems, characterizing the molecular contributions to heterogeneity of membrane potential in non-excitable cells, and enabling downstream engineered bioelectrical tissue design.

Sex differences in membrane properties and cellular excitability of dopamine D1 receptor-expressing neurons within the shell of the nucleus accumbens of pre- and mid-adolescent mice.

BACKGROUND: The transition from childhood to adulthood, or adolescence, a developmental stage, is characterized by psychosocial and biological changes. The nucleus accumbens (NAc), a striatal brain region composed of the core (NAcC) and shell (NAcSh), has been linked to risk-taking behavior and implicated in reward seeking and evaluation. Most neurons in the NAc are medium spiny neurons (MSNs) that express dopamine D1 receptors (D1R +) and/or dopamine D2 receptors (D2R +). Changes in dopaminergic and glutamatergic systems occur during adolescence and converge in the NAc. While there are previous investigations into sex differences in membrane excitability and synaptic glutamate transmission in both subdivisions of the NAc, to our knowledge, none have specified NAcSh D1R + MSNs from mice during pre- and mid-adolescence. METHODS: Sagittal brain slices containing the NAc were prepared from B6.Cg-Tg(Drd1a-tdTomato)6Calak/J mice of both sexes from postnatal days 21-25 and 35-47, representing pre- and mid-adolescence, respectively. Whole-cell electrophysiology recordings were collected from NAcSh D1R + MSNs in the form of membrane-voltage responses to current injections, to assess membrane properties and action potential waveform characteristics, and spontaneous excitatory postsynaptic currents (sEPSCs) to assess glutamatergic synaptic activity. RESULTS: Relative to pre-adolescent males, pre-adolescent female NAcSh D1R + MSNs exhibited a less hyperpolarized resting membrane potential, increased input resistance, and smaller action potential afterhyperpolarization amplitudes. During mid-adolescence, decreased input resistance and a shorter action potential duration in females were the only sex differences observed. CONCLUSIONS: Taken together, our results indicate that NAcSh D1R + MSNs in mice exhibit sex differences in membrane properties and AP waveform during pre-adolescence that are overall indicative of increased cellular excitability in females and are suggestive of possible sex differences in glycine receptors, inwardly-rectifying potassium channels, and large conductance voltage-gated potassium channels. These differences do not appear to persist into mid-adolescence, when sex was observed to affect input resistance oppositely to that of pre-adolescence and AP waveform in a manner suggestive of differences in voltage-gated potassium channels.

Adolescence marks a period of substantial changes in both the mind and body, where alterations in the brain's structure can influence behavior. One change in behavior exhibited by many adolescents is an increased tendency to take risks, particularly in males. While taking risks can result in positive outcomes, like learning new skills, it can also lead to reckless behaviors that may result in negative outcomes. The nucleus accumbens, a brain region tied to risk-taking and reward perception, is not well-studied during the transition from childhood to adulthood, particularly in terms of sex differences. To fill this gap in understanding, this study examined a specific type of brain cell in the nucleus accumbens of pre- and mid-adolescent male and female mice. We measured the electrical properties of these cells and assessed how they responded to manipulation of their electrical state. We also measured how much and how often excitatory electrical information is sent to these cells from other brain regions. Our results suggest that in pre-adolescent females, these brain cells are more excited by manipulations of their electrical state and that these brain cells in mid-adolescent males may take longer to communicate information to other brain regions than in similarly aged females. Understanding these intricacies of brain cell communication sheds light on potential sex-specific vulnerabilities during the transition from childhood to adulthood.

Development of covalent chemogenetic K2P channel activators.

K2P potassium channels regulate excitability by affecting cellular resting membrane potential in the brain, cardiovascular system, immune cells, and sensory organs. Despite their important roles in anesthesia, arrhythmia, pain, hypertension, sleep, and migraine, the ability to control K2P function remains limited. Here, we describe a chemogenetic strategy termed CATKLAMP (covalent activation of TREK family K+ channels to clamp membrane potential) that leverages the discovery of a K2P modulator pocket site that reacts with electrophile-bearing derivatives of a TREK subfamily small-molecule activator, ML335, to activate the channel irreversibly. We show that CATKLAMP can be used to probe fundamental aspects of K2P function, as a switch to silence neuronal firing, and is applicable to all TREK subfamily members. Together, our findings exemplify a means to alter K2P channel activity that should facilitate molecular and systems level studies of K2P function and enable the search for new K2P modulators.

Inhibition and Mechanism of Citral on Bacillus cereus Vegetative Cells, Spores, and Biofilms.

Bacillus cereus causes food poisoning by producing toxins that cause diarrhea and vomiting and, in severe cases, endocarditis, meningitis, and other diseases. It also tends to form biofilms and spores that lead to contamination of the food production environment. Citral is a potent natural antibacterial agent, but its antibacterial activity against B. cereus has not been extensively studied. In this study, we first determined the minimum inhibitory concentrations and minimum bactericidal concentrations, growth curves, killing effect in different media, membrane potential, intracellular adenosine triphosphate (ATP), reactive oxygen species levels, and morphology of vegetative cells, followed by germination rate, morphology, germination state of spores, and finally biofilm clearance effect. The results showed that the minimum inhibitory concentrations and minimum bactericidal concentrations of citral against bacteria ranged from 100 to 800 µg/mL. The lag phase of bacteria was effectively prolonged by citral, and the growth rate of bacteria was slowed down. Bacteria in Luria-Bertani broth were reduced to below the detection limit by citral at 800 µg/mL within 0.5 h. Bacteria in rice were reduced to 3 log CFU/g by citral at 4000 µg/mL within 0.5 h. After treatment with citral, intracellular ATP concentration was reduced, membrane potential was altered, intracellular reactive oxygen species concentration was increased, and normal cell morphology was altered. After treatment with citral at 400 µg/mL, spore germination rate was reduced to 16.71%, spore morphology was affected, and spore germination state was altered. It also had a good effect on biofilm removal. The present study showed that citral had good bacteriostatic activity against B. cereus vegetative cells and its spores and also had a good clearance effect on its biofilm. Citral has the potential to be used as a bacteriostatic substance for the control of B. cereus in food industry production.

Mathematical modeling of intracellular osmolarity and cell volume stabilization: The Donnan effect and ion transport.

The presence of impermeant molecules within a cell can lead to an increase in cell volume through the influx of water driven by osmosis. This phenomenon is known as the Donnan (or Gibbs-Donnan) effect. Animal cells actively transport ions to counteract the Donnan effect and regulate their volume, actively pumping Na+ out and K+ into their cytosol using the Na+/K+ ATPase (NKA) pump. The pump-leak equations (PLEs) are a system of algebraic-differential equations to model the membrane potential, ion (Na+, K+, and Cl-), and water flux across the cell membrane, which provide insight into how the combination of passive ions fluxes and active transport contribute to stabilizing cell volume. Our broad objective is to provide analytical insight into the PLEs through three lines of investigation: (1) we show that the provision of impermeant extracellular molecules can stabilize the volume of a passive cell; (2) we demonstrate that the mathematical form of the NKA pump is not as important as the stoichiometry for cell stabilization; and (3) we investigate the interaction between the NKA pump and cation-chloride co-transporters (CCCs) on cell stabilization, showing that NCC can destabilize a cell while NKCC and KCC can stabilize it. We incorporate extracellular impermeant molecules, NKA pump, and CCCs into the PLEs and derive the exact formula for the steady states in terms of all the parameters. This analytical expression enables us to easily explore the effect of each of the system parameters on the existence and stability of the steady states.

A versatile functional interaction between electrically silent KV subunits and KV7 potassium channels.

Voltage-gated K+ (KV) channels govern K+ ion flux across cell membranes in response to changes in membrane potential. They are formed by the assembly of four subunits, typically from the same family. Electrically silent KV channels (KVS), however, are unable to conduct currents on their own. It has been assumed that these KVS must obligatorily assemble with subunits from the KV2 family into heterotetrameric channels, thereby giving rise to currents distinct from those of homomeric KV2 channels. Herein, we show that KVS subunits indeed also modulate the activity, biophysical properties and surface expression of recombinant KV7 isoforms in a subunit-specific manner. Employing co-immunoprecipitation, and proximity labelling, we unveil the spatial coexistence of KVS and KV7 within a single protein complex. Electrophysiological experiments further indicate functional interaction and probably heterotetramer formation. Finally, single-cell transcriptomic analyses identify native cell types in which this KVS and KV7 interaction may occur. Our findings demonstrate that KV cross-family interaction is much more versatile than previously thought-possibly serving nature to shape potassium conductance to the needs of individual cell types.

From the Microbiome to the Electrome: Implications for the Microbiota-Gut-Brain Axis.

The gut microbiome plays a fundamental role in metabolism, as well as the immune and nervous systems. Microbial imbalance (dysbiosis) can contribute to subsequent physical and mental pathologies. As such, interest has been growing in the microbiota-gut-brain brain axis and the bioelectrical communication that could exist between bacterial and nervous cells. The aim of this study was to investigate the bioelectrical profile (electrome) of two bacterial species characteristic of the gut microbiome: a Proteobacteria Gram-negative bacillus Escherichia coli (E. coli), and a Firmicutes Gram-positive coccus Enterococcus faecalis (E. faecalis). We analyzed both bacterial strains to (i) validate the fluorescent probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol, DiBAC4(3), as a reliable reporter of the changes in membrane potential (Vmem) for both bacteria; (ii) assess the evolution of the bioelectric profile throughout the growth of both strains; (iii) investigate the effects of two neural-type stimuli on Vmem changes: the excitatory neurotransmitter glutamate (Glu) and the inhibitory neurotransmitter γ-aminobutyric acid (GABA); (iv) examine the impact of the bioelectrical changes induced by neurotransmitters on bacterial growth, viability, and cultivability using absorbance, live/dead fluorescent probes, and viable counts, respectively. Our findings reveal distinct bioelectrical profiles characteristic of each bacterial species and growth phase. Importantly, neural-type stimuli induce Vmem changes without affecting bacterial growth, viability, or cultivability, suggesting a specific bioelectrical response in bacterial cells to neurotransmitter cues. These results contribute to understanding the bacterial response to external stimuli, with potential implications for modulating bacterial bioelectricity as a novel therapeutic target.

Calculating transmembrane voltage on the electric pulse-affected cancerous cell membrane: using molecular dynamics and finite element simulations.

CONTEXT: Electroporation is a technique that creates electrically generated pores in the cell membrane by modifying transmembrane potential. In this work, the finite element method (FEM) was used to examine the induced transmembrane voltage (ITV) of a spherical-shaped MCF-7 cell, allowing researchers to determine the stationary ITV. A greater ITV than the critical value causes permeabilization of the membrane. Furthermore, the present study shows how a specific surface conductivity can act as a stand-in for the thin layer that constitutes a cell membrane as the barrier between extracellular and intracellular environments. Additionally, the distribution of ITV on the cell membrane and its maximum value were experimentally evaluated for a range of applied electric fields. Consequently, the entire cell surface area was electroporated 66% and 68% for molecular dynamics (MD) simulations and FEM, respectively, when the external electric field of 1500 V/cm was applied to the cell suspension using the previously indicated numerical methods. Furthermore, the lipid bilayers' molecular structure was changed, which led to the development of hydrophilic holes with a radius of 1.33 nm. Applying MD and FEM yielded threshold values for transmembrane voltage of 700 and 739 mV, respectively. METHOD: Using MD simulations of palmitoyloleoyl-phosphatidylcholine (POPC), pores in cell membranes exposed to external electric fields were numerically investigated. The dependence on the electric field was estimated and developed, and the amount of the electroporated cell surface area matches the applied external electric field. To investigate more, a mathematical model based on an adaptive neuro-fuzzy inference system (ANFIS) is employed to predict the percent cell viability of cancerous cells after applying four pulses during electroporation. For MD simulations, ArgusLab, VMD, and GROMACS software packages were used. Moreover, for FEM analysis, COMSOL software package was used. Also, it is worth mentioning that for mathematical model, MATLAB software is used.

Use of FRET-Sensor 'Mermaid' to Detect Subtle Changes in Membrane Potential of Primary Mouse PASMCs.

Subtle changes in the membrane potential of pulmonary arterial smooth muscle cells (PASMCs) are pivotal for controlling pulmonary vascular tone, e.g., for initiating Hypoxic Pulmonary Vasoconstriction, a vital mechanism of the pulmonary circulation. In our study, we evaluated the ability of the fluorescence resonance energy transfer (FRET)-based voltage-sensor Mermaid to detect such subtle changes in membrane potential. Mouse PASMCs were isolated and transduced with Mermaid-encoding lentiviral vectors before the acceptor/donor emission ratio was assessed via live cell FRET-imaging. Mermaid's sensitivity was tested by applying specific potassium chloride (KCl) concentrations. These KCl concentrations were previously validated by patch clamp recordings to induce depolarization with predefined amplitudes that physiologically occur in PASMCs. Mermaid's emission ratio dose-dependently increased upon depolarization with KCl. However, Mermaid formed unspecific intracellular aggregates, which limited the usefulness of this voltage sensor. When analyzing the membrane rim only to circumvent these unspecific signals, Mermaid was not suitable to resolve subtle changes in the membrane potential of ≤10 mV. In summary, we found Mermaid to be a suitable alternative for reliably detecting qualitative membrane voltage changes of more than 10 mV in primary mouse PASMCs. However, one should be aware of the limitations associated with this voltage sensor.

Structural basis for hyperpolarization-dependent opening of human HCN1 channel.

Hyperpolarization and cyclic nucleotide (HCN) activated ion channels are critical for the automaticity of action potentials in pacemaking and rhythmic electrical circuits in the human body. Unlike most voltage-gated ion channels, the HCN and related plant ion channels activate upon membrane hyperpolarization. Although functional studies have identified residues in the interface between the voltage-sensing and pore domain as crucial for inverted electromechanical coupling, the structural mechanisms for this unusual voltage-dependence remain unclear. Here, we present cryo-electron microscopy structures of human HCN1 corresponding to Closed, Open, and a putative Intermediate state. Our structures reveal that the downward motion of the gating charges past the charge transfer center is accompanied by concomitant unwinding of the inner end of the S4 and S5 helices, disrupting the tight gating interface observed in the Closed state structure. This helix-coil transition at the intracellular gating interface accompanies a concerted iris-like dilation of the pore helices and underlies the reversed voltage dependence of HCN channels.

Hippocampal-dependent navigation in head-fixed mice using a floating real-world environment.

Head-fixation of mice enables high-resolution monitoring of neuronal activity coupled with precise control of environmental stimuli. Virtual reality can be used to emulate the visual experience of movement during head fixation, but a low inertia floating real-world environment (mobile homecage, MHC) has the potential to engage more sensory modalities and provide a richer experimental environment for complex behavioral tasks. However, it is not known whether mice react to this adapted environment in a similar manner to real environments, or whether the MHC can be used to implement validated, maze-based behavioral tasks. Here, we show that hippocampal place cell representations are intact in the MHC and that the system allows relatively long (20 min) whole-cell patch clamp recordings from dorsal CA1 pyramidal neurons, revealing sub-threshold membrane potential dynamics. Furthermore, mice learn the location of a liquid reward within an adapted T-maze guided by 2-dimensional spatial navigation cues and relearn the location when spatial contingencies are reversed. Bilateral infusions of scopolamine show that this learning is hippocampus-dependent and requires intact cholinergic signalling. Therefore, we characterize the MHC system as an experimental tool to study sub-threshold membrane potential dynamics that underpin complex navigation behaviors.

Membrane depolarization mediates both the inhibition of neural activity and cell-type-differences in response to high-frequency stimulation.

Neuromodulation using high frequency (>1 kHz) electric stimulation (HFS) enables preferential activation or inhibition of individual neural types, offering the possibility of more effective treatments across a broad spectrum of neurological diseases. To improve effectiveness, it is important to better understand the mechanisms governing activation and inhibition with HFS so that selectivity can be optimized. In this study, we measure the membrane potential (Vm) and spiking responses of ON and OFF α-sustained retinal ganglion cells (RGCs) to a wide range of stimulus frequencies (100-2500 Hz) and amplitudes (10-100 µA). Our findings indicate that HFS induces shifts in Vm, with both the strength and polarity of the shifts dependent on the stimulus conditions. Spiking responses in each cell directly correlate with the shifts in Vm, where strong depolarization leads to spiking suppression. Comparisons between the two cell types reveal that ON cells are more depolarized by a given amplitude of HFS than OFF cells-this sensitivity difference enables the selective targeting. Computational modeling indicates that ion-channel dynamics largely account for the shifts in Vm, suggesting that a better understanding of the differences in ion-channel properties across cell types may improve the selectivity and ultimately, enhance HFS-based neurostimulation strategies.

Considering Joule heating in coupled electroporation and electrodeformation modeling of glioblastoma cells.

Cells exposed to a pulsed electric field undergo electroporation(EP) and electrodeformation(ED) under electric field stress, and a coupled model of EP and ED of glioblastoma(GBM) taking into account Joule heating is proposed. The model geometry is extracted from real cell boundaries, and the effects of Joule heating-induced temperature rise on the EP and ED processes are considered. The results show that the temperature rise will increase the cell's local conductivity, leading to a decrease in the transmembrane potential(TMP). The temperature rise also causes a decrease in the dynamic Young's modulus of the cell membrane, making the cell less resistant to deformation. In addition, GBM cells are more susceptible to EP in the middle portion of the cell and ED in the three tentacle portions under pulsed electric fields, and the cells undergo significant positional shifts. The ED of the nucleus is similar to spherical cells, but the degree of ED is smaller.

Elevated Membrane Potential as a Tetracycline Resistance Mechanism in Escherichia coli.

The metabolic environment is responsible for antibiotic resistance, which highlights the way in which the antibiotic resistance mechanism works. Here, GC-MS-based metabolomics with iTRAQ-based proteomics was used to characterize a metabolic state in tetracycline-resistant Escherichia coli K12 (E. coli-RTET) compared with tetracycline-sensitive E. coli K12. The repressed pyruvate cycle against the elevation of the proton motive force (PMF) and ATP constructed the most characteristic feature as a consequence of tetracycline resistance. To understand the role of the elevated PMF in tetracycline resistance, PMF inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the pH gradient were used to investigate how the elevation influences bacterial viability and intracellular antibiotic concentration. A strong synergy was detected between CCCP and tetracycline to the viability, which was consistent with increasing intracellular drug and decreasing external pH. Furthermore, E. coli-RTET and E. coli-RGEN with high and low PMF concentrations were susceptible to gentamicin and tetracycline, respectively. The elevated PMF in E. coli-RTET was attributed to the activation of other metabolic pathways, except for the pyruvate cycle, including a malate-oxaloacetate-phosphoenolpyruvate-pyruvate-malate cycle. These results not only revealed a PMF-dependent mechanism for tetracycline resistance but also provided a solution to tetracycline-resistant pathogens by aminoglycosides and aminoglycoside-resistant bacteria by tetracyclines.

Neural network aided extended Kalman filtering for inverse imaging of cardiac transmembrane potential.

Objective.The aim of this study is to address the limitations in reconstructing the electrical activity of the heart from the body surface electrocardiogram, which is an ill-posed inverse problem. Current methods often assume values commonly used in the literature in the absence ofa prioriknowledge, leading to errors in the model. Furthermore, most methods ignore the dynamic activation process inherent in cardiomyocytes during the cardiac cycle.Approach.To overcome these limitations, we propose an extended Kalman filter (EKF)-based neural network approach to dynamically reconstruct cardiac transmembrane potential (TMP). Specifically, a recurrent neural network is used to establish the state estimation equation of the EKF, while a convolutional neural network is used as the measurement equation. The Jacobi matrix of the network undergoes a correction feedback process to obtain the Kalman gain.Main results.After repeated iterations, the final estimated state vector, i.e. the reconstructed image of the TMP, is obtained. The results from both the final simulation and real experiments demonstrate the robustness and accurate quantification of the model.Significance.This study presents a new approach to cardiac TMP reconstruction that offers higher accuracy and robustness compared to traditional methods. The use of neural networks and EKFs allows dynamic modelling that takes into account the activation processes inherent in cardiomyocytes and does not requirea prioriknowledge of inputs such as forward transition matrices.

Resting membrane potential and intracellular [Na+] at rest, during fatigue and during recovery in rat soleus muscle fibres in situ.

Large trans-sarcolemmal ionic shifts occur with fatiguing exercise or stimulation of isolated muscles. However, it is unknown how resting membrane potential (EM) and intracellular sodium concentration ([Na+]i) change with repeated contractions in living mammals. We investigated (i) whether [Na+]i (peak, kinetics) can reveal changes of Na+-K+ pump activity during brief or fatiguing stimulation and (ii) how resting EM and [Na+]i change during fatigue and recovery of rat soleus muscle in situ. Muscles of anaesthetised rats were stimulated with brief (10 s) or repeated tetani (60 Hz for 200 ms, every 2 s, for 30 s or 300 s) with isometric force measured. Double-barrelled ion-sensitive microelectrodes were used to quantify resting EM and [Na+]i. Post-stimulation data were fitted using polynomials and back-extrapolated to time zero recovery. Mean pre-stimulation resting EM (layer 2-7 fibres) was -71 mV (surface fibres were more depolarised), and [Na+]i was 14 mM. With deeper fibres, 10 s stimulation (2-150 Hz) increased [Na+]i to 38-46 mM whilst simultaneously causing hyperpolarisations (7.3 mV for 2-90 Hz). Fatiguing stimulation for 30 s or 300 s led to end-stimulation resting EM of -61 to -53 mV, which recovered rapidly (T1/2, 8-22 s). Mean end-stimulation [Na+]i increased to 86-101 mM with both fatigue protocols and the [Na+]i recovery time-course (T1/2, 21-35 s) showed no difference between protocols. These combined findings suggest that brief stimulation hyperpolarises the resting EM, likely via maximum Na+-induced stimulation of the Na+-K+ pump. Repeated tetani caused massive depolarisation and elevations of [Na+]i that together lower force, although they likely interact with other factors to cause fatigue. [Na+]i recovery kinetics provided no evidence of impaired Na+-K+ pump activity with fatigue. KEY POINTS: It is uncertain how resting membrane potential, intracellular sodium concentration ([Na+]i), and sodium-potassium (Na+-K+) pump activity change during repeated muscle contractions in living mammals. For rat soleus muscle fibres in situ, brief tetanic stimulation for 10 s led to raised [Na+]i, anticipated to evoke maximal Na+-induced stimulation of the Na+-K+ pump causing an immediate hyperpolarisation of the sarcolemma. More prolonged stimulation with repeated tetanic contractions causes massive elevations of [Na+]i, which together with large depolarisations (via K+ disturbances) likely reduce force production. These effects occurred without impairment of Na+-K+ pump function. Together these findings suggest that rapid activation of the Na+-K+ pump occurs with brief stimulation to maintain excitability, whereas more prolonged stimulation causes rundown of the trans-sarcolemmal K+ gradient (hence depolarisation) and Na+ gradient, which in combination can impair contraction to contribute to fatigue in living mammals.