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The membrane potential is a key aspect of cellular function and cell-to-cell signaling, and thereby ultimately body functions. This article describes the basic principles regarding electrical and chemical properties of ions and membranes, how these forces combine to determine electrochemical gradients that, when combined with membrane permeability give rise to membrane potentials. The article quantifies these principles using a range of simple equations such as Ohm’s Law, the Nernst equation, and the Goldman–Hodgkin–Katz (GHK) equation, and illustrates how to calculate the number of ions flowing in or out of a cell during typical membrane potential changes. The role of active and passive transporters in generating membrane potentials is briefly described, with a focus on the important role of a channel’s selectivity filter and gating mechanism. The final section discusses ways to experimentally measure membrane potentials and some caveats to be aware of when using these approaches. The challenges associated with an accurate measurement of membrane potential is illustrated by the range of absolute values reported for the resting membrane potential in hippocampal pyramidal neurons, and it may be a little misleading to consider a membrane as ever at ‘rest.’ Changes in membrane potential are a major cellular unit of organ and body function. It is hoped this article provides a simple and clear outline of the membrane potential and its underlying principles and mechanisms to assist students and researchers in cell biology.

GABAergic inhibition in neurons plays a critical role in determining the output of neural circuits. Neurons in avian nucleus magnocellularis (NM) use several tonotopic-region-dependent specializations to relay the timing information of sound in the auditory nerve to higher auditory nuclei. Previously, we showed that feedforward GABAergic inhibition in NM has a different dependence on the level of auditory nerve activity, with the low-frequency region having a low-threshold and linear relationship, while the high-frequency region has a high-threshold and step-like relationship. However, it remains unclear how the GABAergic synapses are tonotopically regulated and interact with other specializations of NM neurons. In this study, we examined GABAergic transmission in the NM of chickens of both sexes and explored its contributions to the temporal coding of sound at each tonotopic region. We found that the number and size of unitary GABAergic currents and their reversal potential were finely tuned at each tonotopic region in the NM. At the lower-frequency region, unitary GABAergic currents were larger in number but smaller in size. In addition, their reversal potential was close to the resting potential of neurons, which enabled reliable inhibition despite the smaller potassium conductance. At the higher-frequency region, on the other hand, unitary GABAergic currents were fewer, larger, and highly depolarizing, which enabled powerful inhibition via activating the large potassium conductance. Thus, we propose that GABAergic synapses are coordinated with the characteristics of excitatory synapses and postsynaptic neurons, ensuring the temporal coding for wide frequency and intensity ranges.SIGNIFICANCE STATEMENTWe found in avian cochlear nucleus that the number and size of unitary GABAergic inputs differed among tonotopic regions and correlated to respective excitatory inputs; it was larger in number but smaller in size for neurons tuned to lower-frequency sound. Furthermore, GABAergic reversal potential also differed among the regions in accordance with the size of Kv1 current; it was less depolarized in the lower-frequency neurons with smaller Kv1 current. These differentiations of GABAergic transmission maximized the effects of inhibition at each tonotopic region, ensuring precise and reliable temporal coding across frequencies and intensities. Our results emphasize the importance of optimizing characteristics of GABAergic transmission within individual neurons for proper neural circuit function.

High sodium (HS) intake inhibited epithelial Na+ channel (ENaC) in the aldosterone-sensitive distal nephron and Na+-Cl- cotransporter (NCC) by suppressing basolateral Kir4.1/Kir5.1 in the distal convoluted tubule (DCT), thereby increasing renal Na+ excretion but not affecting K+ excretion. The aim of the present study was to explore whether deletion of Kir5.1 compromises the inhibitory effect of HS on NCC expression/activity and renal K+ excretion. Patch-clamp experiments demonstrated that HS failed to inhibit DCT basolateral K+ channels and did not depolarize K+ current reversal potential of the DCT in Kir5.1 knockout (KO) mice. Moreover, deletion of Kir5.1 not only increased the expression of Kir4.1, phospho-NCC, and total NCC but also abolished the inhibitory effect of HS on the expression of Kir4.1, phospho-NCC, and total NCC and thiazide-induced natriuresis. Also, low sodium-induced stimulation of NCC expression/activity and basolateral K+ channels in the DCT were absent in Kir5.1 KO mice. Deletion of Kir5.1 decreased ENaC currents in the late DCT, and HS further inhibited ENaC activity in Kir5.1 KO mice. Finally, measurement of the basal renal K+ excretion rate with the modified renal clearance method demonstrated that long-term HS inhibited the renal K+ excretion rate and steadily increased plasma K+ levels in Kir5.1 KO mice but not in wild-type mice. We conclude that Kir5.1 plays an important role in mediating the effect of HS intake on basolateral K+ channels in the DCT and NCC activity/expression. Kir5.1 is involved in maintaining renal ability of K+ excretion during HS intake. NEW & NOTEWORTHY Kir5.1 plays an important role in mediating the effect of high sodium intake on basolateral K+ channels in the distal convoluted tubule and Na+-Cl- cotransporter activity/expression.

The availability of high-resolution structures of ion channels opens the doors to reliable computations of electrophysiological properties, such as the dependence of ionic currents and selectivities on applied voltage. We develop two theoretical approaches for calculating these properties from molecular dynamics simulations at a single voltage, or even in the absence of voltage, combined with the electrodiffusion model in which ion motion in the channel is represented as one-dimensional diffusion in the potential of mean force exerted by other components of the system and the applied electric field. No knowledge of diffusivity or ion densities at other voltages is needed. Instead, in one approach, one-sided ion fluxes and density profiles are used to determine the free energy profile. In the other approach, committor probabilities for ions transported at the selected voltage are used for this purpose. Both approaches have been validated in an example of a simple ion channel formed by trichotoxin. The potentials of mean force calculated by way of the proposed approaches and obtained from traditional methods are in excellent agreement. Furthermore, the current-voltage dependence agrees very well with results obtained by way of computationally more demanding methods. We also have readily calculated the reversal potential, a computationally challenging electrophysiological property. The key assumptions of the electrodiffusion model, such as the independence of crossing events or the insensitivity of the potential of mean force to applied voltage, have been found to be satisfied. We also show that the voltage changes linearly in the hydrophobic core of the membrane and is constant elsewhere.

Quantifying the passage of the large peptide protamine (Ptm) across CymA, a passive channel for cyclodextrin uptake, is in the focus of this study. Using a reporter‐pair‐based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation through CymA is the rate‐limiting factor. Furthermore, we reconstituted single CymA channels into planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage‐dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1–2 events per second and long dwell times, in agreement with the liposome study. Applied‐field and steered molecular dynamics simulations added an atomistic view of the permeation events. It can be concluded that a concentration gradient of 1 μm Ptm leads to a translocation rate of about one molecule per second and per channel., Surprisingly, large peptides (Protamine, 5.1 kDa) can permeate through bacterial outer membrane channels. The use of fluorescence, electrophysiology, and all‐atom modeling allows to quantify the flux. This approach can be transferred to related problems.

Voltage-gated Cav1 and Cav2 Ca2+ channels are comprised of a pore-forming α1 subunit (Cav1.1-1.4, Cav2.1-2.3) and auxiliary β (β1-4) and α2δ (α2δ-1-4) subunits. The properties of these channels vary with distinct combinations of Cav subunits and alternative splicing of the encoding transcripts. Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Cav subunits and splice variants. Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Cav1.4 channels typical of those in human retina: Cav1.4 splice variants with or without exon 47 (Cav1.4+ex47 and Cav1.4Δex47, respectively), and the auxiliary subunits, β2X13 and α2δ-4. We find that IT caused both Cav1.4 splice variants to activate at significantly more negative voltages and with slower deactivation kinetics than the corresponding WT channels. These effects of the IT mutation, along with unexpected alterations in ion selectivity, were generally larger in channels lacking exon 47. The weaker ion selectivity caused by IT led to hyperpolarizing shifts in the reversal potential and large outward currents that were evident in channels containing the auxiliary subunits β2X13 and α2δ-4 but not in those with β2A and α2δ-1. We conclude that the IT mutation stabilizes channel opening and alters ion selectivity of Cav1.4 in a manner that is strengthened by exclusion of exon 47 and inclusion of β2X13 and α2δ-4. Our results reveal complex actions of IT in modifying the properties of Cav1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors.

The currents of α7 nicotinic acetylcholine receptors activated by acetylcholine (ACh) are brief. The channel has high permeability to calcium relative to monovalent cations and shows inward rectification. It has been previously noted that in the presence of positive allosteric modulators (PAMs), currents through the channels of α7 receptors differ from normal α7 currents both in sensitivity to specific channel blockers and their current-voltage (I-V) relationships, no longer showing inward rectification. Linear I-V functions are often associated with channels lacking calcium permeability, so we measured the I-V functions of α7 receptors activated by ACh when PAMs were bound to the allosteric binding site in the transmembrane domain. Currents were recorded in chloride-free Ringer’s solution with low or high concentrations of extracellular calcium to determine the magnitude of the reversal potential shift in the two conditions as well as the I-V relationships. ACh-evoked currents potentiated by the allosteric agonist–PAMs (ago-PAMs) (3aR,4S,9bS)-4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (GAT107) and 3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propenamide (B-973B) showed reduced inward rectification and calcium-dependent reversal potential shifts decreased by 80%, and 50%, respectively, compared with currents activated by ACh alone, indicative of reduced calcium permeability. Currents potentiated by 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide were also linear and showed no calcium-dependent reversal potential shifts. The ago-PAMs GAT-107 and B-973B stimulated increases in intracellular calcium in stably transfected HEK293 cells. However, these calcium signals were delayed relative to channel activation produced by these agents and were insensitive to the channel blocker mecamylamine. Our results indicate that, although allosterically activated α7 nicotinic ACh receptor may affect intracellular calcium levels, such effects are not likely due to large channel-dependent calcium influx. SIGNIFICANCE STATEMENT: Positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptor can increase channel activation by two or more orders of magnitude, raising the concern that, due to the relatively high calcium permeability of α7 receptors activated by acetylcholine alone, such efficacious PAMs may have cytotoxic side effects. We show that PAMs alter the ion conduction pathway and, in general, reduce the calcium permeability of the channels. This supports the hypothesis that α7 effects on intracellular calcium may be independent of channel-mediated calcium influx.

Background: Prenatal adverse effects of radiofrequency electromagnetic fields (RF-EMF) exposure on nervous system are an issue of major concern.Objective: Thus, in this study we evaluated the membrane current flow properties of Purkinje neurons after maternal exposure to 900 MHz pulsed RF-EMF.Material and Methods: In this experimental study, during all days of pregnancy, rats in the EMF-exposed group were exposed to 900 MHz pulsed-EMF radiation for 6 h per day. The effects of RF-EMF exposure on the electrophysiological properties of the Purkinje cerebellum neurons from male pups were evaluated by whole-cell patch clamp recordings in current and voltage clamp modes. In voltage-clamp experiments, the holding potential was -60 mV, and a depolarizing voltage step (1000 ms duration) was applied from -60 to +50 mV in 10 mV increments at 2s intervals. Results: The exposure group demonstrated reduced spontaneous firing associated with upward and rightward shift in I/V curve compared to the control rats. Moreover, the peak amplitude of the current for the exposure pups also revealed a significant decrement. The reversal potential was +40 mV and +20 mV for the control and RF-EMF groups, respectively and showed significant differences between the two groups. Conclusion: The decrease in ion’s conductance could be attributed to the observed decrease in the voltage onset of the inward current, peak amplitude and voltage shift.

Serotonin (5-HT) has an important role in the pathophysiology of the mood disorders like major depression and anxiety disorders in central nervous system. On the one hand, dorsal raphe nucleus (DRN) neurons send serotonergic projections to almost all brain regions. On the other hand, they affect themselves through 5-HT1A autoreceptors. Many electrophysiological studies have investigated the ionic mechanism of the 5-HTs effect on the DRN neurons of the rat. However, there is no study characterizing the current that mediates the 5-HTs effect on mouse DRN neurons. In the present electrophysiological study, the whole-cell patch-clamp technique was used in the neurons of the DRN from one-month-old Balb/c mice to investigate the effect of 5-HT on the DRN neurons of mice and its ionic mechanism of action. The application of 5-HT resulted in a 14.3 ± 3.1 mV hyperpolarization (n = 9, P < 0.01) of resting membrane potential and 25.7 ± 3.5 pA outward current (n = 7) in the DRN neurons. The reversal potential (E5-HT) of the current induced by 5-HT was close to the potassium equilibrium potential (EK). This current had an inward rectification feature and was blocked by quinine pretreatment (n = 5, P < 0.05). In conclusion, 5-HT inhibits the DRN neurons of mice by inducing a current that is carried by potassium ions through G-protein-coupled inward rectifier potassium channels.

TOKs are outwardly rectifying K+ channels in fungi with two pore-loops and eight transmembrane spans. Here, we describe the TOKs from four pathogens that cause the majority of life-threatening fungal infections in humans. These TOKs pass large currents only in the outward direction like the canonical isolate from Saccharomyces cerevisiae (ScTOK), and distinct from other K+ channels. ScTOK, AfTOK1 (Aspergillus fumigatus) and H99TOK (Cryptococcus neoformans grubii) are K+-selective and pass current above the K+ reversal potential. CaTOK (Candida albicans) and CnTOK (Cryptococcus neoformans neoformans) pass both K+ and Na+ and conduct above a reversal potential reflecting the mixed permeability of their selectivity filter. Mutations in CaTOK and ScTOK at sites homologous to those that open the internal gates in classical K+ channels are shown to produce inward TOK currents. A favored model for outward rectification is proposed whereby the reversal potential determines ion occupancy, and thus conductivity, of the selectivity filter gate that is coupled to an imperfectly restrictive internal gate, permitting the filter to sample ion concentrations on both sides of the membrane.

Key points Cation-chloride cotransporters (CCCs) play a critical role in controlling the efficacy and polarity of GABAA receptor (GABAA R)-mediated transmission in the brain, yet their expression and function in GABAergic interneurons has been overlooked. We compared the polarity of GABA signalling and the function of CCCs in mouse hippocampal pyramidal neurons and parvalbumin-expressing interneurons. Under resting conditions, GABAA R activation was mostly depolarizing and yet inhibitory in both cell types. KCC2 blockade further depolarized the reversal potential of GABAA R-mediated currents often above action potential threshold. However, during repetitive GABAA R activation, the postsynaptic response declined independently of the ion flux direction or KCC2 function, suggesting intracellular chloride build-up is not responsible for this form of plasticity. Our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal pyramidal neurons and parvalbumin interneurons. Abstract Transmembrane chloride gradients govern the efficacy and polarity of GABA signalling in neurons and are usually maintained by the activity of cation-chloride cotransporters, such as KCC2 and NKCC1. Whereas their role is well established in cortical principal neurons, it remains poorly documented in GABAergic interneurons. We used complementary electrophysiological approaches to compare the effects of GABAA receptor (GABAA R) activation in adult mouse hippocampal parvalbumin interneurons (PV-INs) and pyramidal cells (PCs). Loose cell-attached, tight-seal and gramicidin-perforated patch recordings all show GABAA R-mediated transmission is slightly depolarizing and yet inhibitory in both PV-INs and PCs. Focal GABA uncaging in whole-cell recordings reveal that KCC2 and NKCC1 are functional in both PV-INs and PCs but differentially contribute to transmembrane chloride gradients in their soma and dendrites. Blocking KCC2 function depolarizes the reversal potential of GABAA R-mediated currents in PV-INs and PCs, often beyond firing threshold, showing KCC2 is essential to maintain the inhibitory effect of GABAA Rs. Finally, we show that repetitive 10 Hz activation of GABAA Rs in both PV-INs and PCs leads to a progressive decline of the postsynaptic response independently of the ion flux direction or KCC2 function. This suggests intraneuronal chloride build-up may not predominantly contribute to activity-dependent plasticity of GABAergic synapses in this frequency range. Altogether our data demonstrate similar mechanisms of chloride regulation in mouse hippocampal PV-INs and PCs and suggest KCC2 downregulation in the pathology may affect the valence of GABA signalling in both cell types.

Long-term memory of complex olfactory learning is expressed by wide spread enhancement in excitatory and inhibitory synaptic transmission onto piriform cortex pyramidal neurons. A particularly interesting modification in synaptic inhibition is the hyperpolarization of the reversal potential of the fast post synaptic inhibitory potential (fIPSP). Here we study the mechanism underlying the maintenance of such a shift in the fIPSP. Blocking of the neuronal specific K+-Cl− co-transporter (KCC2) in neurons of trained rats significantly depolarized the averaged fIPSP reversal potential of the spontaneous miniature inhibitory post synaptic currents (mIPSCs), to the averaged pre-training level. A similar effect was obtained by blocking PKC, which was previously shown to upregulate KCC2. Accordingly, the level of PKC-dependent phosphorylation of KCC2, at the serine 940 site, was significantly increased after learning. In contrast, blocking two other key second messenger systems CaMKII and PKA, which have no phosphorylation sites on KCC2, had no effect on the fIPSP reversal potential. Importantly, the PKC inhibitor also reduced the averaged amplitude of the spontaneous miniature excitatory synaptic currents (mEPSCs) in neurons of trained rats only, to the pre-training level. We conclude that learning-induced hyper-polarization of the fIPSP reversal potential is mediated by PKC-dependent increase of KCC2 phosphorylation.

In this paper, based on geometric singular perturbation analysis of a quasi-one dimensional Poisson-Nernst-Planck model for ionic flows, we study the problem of zero current condition for ionic flows through membrane channels with a simple profile of permanent charges. For ionic mixtures of multiple ion species, under equal diffusion constant condition, Eisenberg, et al [{\em Nonlinearity {\bf 28} (2015), 103-128}] derived a system of two equations for determining the reversal potential and an equation for the reversal permanent charge. The equal diffusion constant condition is significantly degenerate from physical points of view. For unequal diffusion coefficients, the analysis becomes extremely challenging. This work will focus only on two ion species, one positively charged (cation) and one negatively charged (anion), with two arbitrary diffusion coefficients. Dependence of reversal potential on channel geometry and diffusion coefficients has been investigated experimentally, numerically, and analytically in simple setups, in many works. In this paper, we identify two governing equations for the zero current, which enable one to mathematically analyze how the reversal potential depends on the channel structure and diffusion coefficients. In particular, we are able to show, with a number of concrete results, that the possible different diffusion constants indeed make significant differences. The inclusion of channel structures is also far beyond the situation where the Goldman-Hodgkin-Katz (GHK) equation might be applicable. A comparison of our result with the GHK equation is provided. The dual problem of reversal permanent charges is briefly discussed too., Comment: 29 pages, 5 figures

Auditory transduction is mediated by chordotonal (Cho) neurons in Drosophila larvae, but the molecular identity of the mechanotransduction (MET) channel is elusive. Here, we established a whole-cell recording system of Cho neurons and showed that two transient receptor potential vanilloid (TRPV) channels, Nanchung (NAN) and Inactive (IAV), are essential for MET currents in Cho neurons. NAN and IAV form active ion channels when expressed simultaneously in S2 cells. Point mutations in the pore region of NAN-IAV change the reversal potential of the MET currents. Particularly, residues 857 through 990 in the IAV carboxyl terminus regulate the kinetics of MET currents in Cho neurons. In addition, TRPN channel NompC contributes to the adaptation of auditory transduction currents independent of its ion-conduction function. These results indicate that NAN-IAV, rather than NompC, functions as essential pore-forming subunits of the native auditory transduction channel in Drosophila and provide insights into the gating mechanism of MET currents in Cho neurons.

Acid-sensing ion channels (ASICs) are activated by extracellular acidification. Because ASIC currents are transient, these channels appear to be ideal sensors for detecting the onset of rapid pH changes. ASICs are involved in neuronal death after ischemic stroke, and in the sensation of inflammatory pain. Ischemia and inflammation are associated with a slowly developing, long-lasting acidification. Recent studies indicate however that ASICs are unable to induce an electrical signaling activity under standard experimental conditions if pH changes are slow. In situations associated with slow and sustained pH drops such as high neuronal signaling activity and ischemia, the extracellular K+ concentration increases, and the Ca2+ concentration decreases. We hypothesized that the concomitant changes in H+, K+, and Ca2+ concentrations may allow a long-lasting ASIC-dependent induction of action potential (AP) signaling. We show that for acidification from pH7.4 to pH7.0 or 6.8 on cultured cortical neurons, the number of action potentials and the firing time increased strongly if the acidification was accompanied by a change to higher K+ and lower Ca2+ concentrations. Under these conditions, APs were also induced in neurons from ASIC1a–/– mice, in which a pH of ≤ 5.0 would be required to activate ASICs, indicating that ASIC activation was not required for the AP induction. Comparison between neurons of different ASIC genotypes indicated that the ASICs modulate the AP induction under such changed ionic conditions. Voltage-clamp measurements of the Na+ and K+ currents in cultured cortical neurons showed that the lowering of the pH inhibited Na+ and K+ currents. In contrast, the lowering of the Ca2+ together with the increase in the K+ concentration led to a hyperpolarizing shift of the activation voltage dependence of voltage-gated Na+ channels. We conclude that the ionic changes observed during high neuronal activity mediate a sustained AP induction caused by the potentiation of Na+ currents, a membrane depolarization due to the changed K+ reversal potential, the activation of ASICs, and possibly effects on other ion channels. Our study describes therefore conditions under which slow pH changes induce neuronal signaling by a mechanism involving ASICs.