Intensified expression of calcium and sodium voltage-operated channels in oocytes ofXenopus following the injection of total RNA from the rat brain

Expression of voltage-gated calcium and sodium ionic channels was found with the help of electrophysiological methods in the membrane of oocytes of theXenopus laevis frog following the injection of total RNA from the brain of 15-to-20-day-old rats. The amplitudes of currents through these channels were much higher than those induced by poly(A)⁺-mRNA from the same source. Barium currents induced by both RNA preparations were insensitive to Bay K 8644 (10 µM) and nitrendipine (50 µM), but were blocked by addition of 100 µM Cd²⁺ to extracellular solution; in both cases -conotoxin GVIA (1 µM) suppressed currents through the expressed calcium channels. The conclusion is that processes responsible for the expression of alien ionic channels in oocyte membrane become more intensive following the injection of total RNA separated in a sucrose gradient than those following the injection of poly(A)⁺-mRNA. The results suggest that the effectiveness of the expression is positively affected by some factors contained in the preparations of total RNA. The question of the nature of these factors remains open.Neirofiziologiya/Neurophysiology, Vol. 25, No. 6, pp. 433–437, November–December, 1993.     

Ionic components of electric current at rat corneal wounds

Background

Endogenous electric fields and currents occur naturally at wounds and are a strong signal guiding cell migration into the wound to promote healing. Many cells involved in wound healing respond to small physiological electric fields in vitro. It has long been assumed that wound electric fields are produced by passive ion leakage from damaged tissue. Could these fields be actively maintained and regulated as an active wound response? What are the molecular, ionic and cellular mechanisms underlying the wound electric currents?

Methodology/Principal Findings

Using rat cornea wounds as a model, we measured the dynamic timecourses of individual ion fluxes with ion-selective probes. We also examined chloride channel expression before and after wounding. After wounding, Ca²⁺ efflux increased steadily whereas K⁺ showed an initial large efflux which rapidly decreased. Surprisingly, Na⁺ flux at wounds was inward. A most significant observation was a persistent large influx of Cl⁻, which had a time course similar to the net wound electric currents we have measured previously. Fixation of the tissues abolished ion fluxes. Pharmacological agents which stimulate ion transport significantly increased flux of Cl⁻, Na⁺ and K⁺. Injury to the cornea caused significant changes in distribution and expression of Cl⁻ channel CLC2.

Conclusions/Significance

These data suggest that the outward electric currents occurring naturally at corneal wounds are carried mainly by a large influx of chloride ions, and in part by effluxes of calcium and potassium ions. Ca²⁺ and Cl⁻ fluxes appear to be mainly actively regulated, while K⁺ flux appears to be largely due to leakage. The dynamic changes of electric currents and specific ion fluxes after wounding suggest that electrical signaling is an active response to injury and offers potential novel approaches to modulate wound healing, for example eye-drops targeting ion transport to aid in the challenging management of non-healing corneal ulcers.     

Newborn neuroblasts feel the field in the adult brain

A paper in this issue of EMBO reports shows that endogenous electric currents exist in the adult mouse brain and that they may guide neuroblast migration. These findings and their implications are discussed here. EMBO reports (2013) 14 2, 184–190 doi:10.1038/embor.2012.215 In the adult mammalian brain, new neurons are continuously generated from a small supply of neural stem cells in two regions—the dentate gyrus of the hippocampus and the subventricular zone (SVZ)—in a manner that modulates numerous learning and memory processes. In the latter region, immature neuroblasts must traverse millimetres of cortical tissue to reach their final destination in the olfactory bulb [1]. How do these cells navigate along this route, termed the ‘rostral migratory stream'' (RMS)? Our understanding is that RMS migration is guided by molecular and cellular mechanisms including chemoattractive and chemorepulsive factor gradients—some established with the aid of cerebrospinal fluid flow [1, 2]. A new study published in this issue of EMBO reports raises the intriguing possibility that an endogenous electric field lying along the RMS might also be important in guiding directional cell migration [3].In 1974, the development of the vibrating probe technique enabled highly sensitive measurements of small endogenous currents within living tissues to be taken [4]. The presence of electric currents and their associated direct current fields has since been established in a variety of adult and developing tissues. As one example, polarized ionic transport through Na⁺/K⁺-ATPases can establish a large potential difference (40–200 mV mm⁻¹) from the apical to basolateral surface of an epithelial cell layer, aided by the high ionic resistance of tight junctions. Furthermore, injury to such epithelial sheets triggers an electrical current that guides epithelial migration during subsequent wound closure [5].Although electrophysiological investigations of neurons and their synaptic connections within the brain have been conducted for over a century, comparatively less is known about whether long distance, macroscopic electric fields are generated as a natural consequence of cellular membrane depolarization or currents. The work by Zhao and colleagues published in this issue presents, for the first time to our knowledge, evidence that endogenous electric currents exist along the RMS and that neuroblasts might migrate in the direction of the associated electric field, a process generally known as galvanotaxis.By using the vibrating probe method, an endogenous electric potential gradient of 3.3 mV mm⁻¹ was measured along the RMS; a separate determination based on current and resistance measurements arrived at a slightly smaller value of 2 mV mm⁻¹. As in the case of transepithelial potentials, this field might be generated by the spatial organization of Na⁺/K⁺-ATPases in the SVZ and olfactory bulb. Specifically, the authors found that epithelia lining the lateral ventricular wall, at the beginning of the RMS, had a high concentration of ATPases on the basal side, which might thus pump excess Na⁺ ions into the brain. On the other end of the RMS, in the olfactory bulb, they found Na⁺/K⁺-ATPases primarily on the apical side of the epithelia, which might pump Na⁺ ions outward from the olfactory bulb and thus create an ionic sink. The authors suggest that a resulting flow of Na⁺ cations from the SVZ to the olfactory bulb is responsible for the low level direct current electrical field along the length of the RMS, which is supported by their finding that inhibition of the ATPase by ouabain significantly reduced the field strength.In vitro and in vivo time-lapse data from this study and previous work [6] demonstrated that electric fields direct neural stem cell migration. Cao and colleagues found that high field strengths (>10 mV mm⁻¹) promoted clear and sustained directional migration towards the cathode. At lower strengths, closer to those measured between the SVZ and olfactory bulb (approximately 3.5 mV mm⁻¹), the in vitro migratory bias was slight yet statistically significant. Additionally, time-lapse analysis of labelled neuroblasts in live explant slices showed that migration towards the olfactory bulb was strongly enhanced by exogenous fields as low as 10 mV mm⁻¹—approximately three times the endogenous potential. Furthermore, reversing the field direction with a high exogenous potential (50 mV mm⁻¹) caused cells to steer off course and in the direction of the imposed field. Finally, by using pharmacological inhibition as well as RNA interference knockdown, Cao and colleagues implicate the P2Y1 purinergic receptor, which is expressed specifically in migrating neuroblasts, as a mediator of the galvanotaxis.…endogenous electric currents exist along the RMS and [that] neuroblasts might migrate in the direction of the associated electric fieldThe field of stem cell biology is increasingly recognizing the importance of not only biochemical but also biophysical regulatory cues, and this work provides further support for investigating the role of electrostatics in controlling cellular function. Naturally, it also raises several interesting and open questions. It is clear in this study that neuroblasts migrate in response to strong imposed electrical fields in vitro and in vivo, and weaker fields in vitro, although future work is necessary to establish definitively that the low electrical field measured in vivo (approximately 2–3.5 mV mm⁻¹) is sufficient to influence directional cell migration within the RMS.In addition, these results raise interesting questions about possible relationships between electrostatic and biochemical cues in regulating neuroblast migration. Such migration depends on gradients of the chemorepulsive factor Slit along the RMS [2], as well as the cell adhesion molecule PSA-NCAM, which enables cells to migrate as chains within the rodent RMS [7]. Investigating the relative importance of galvanotaxis compared with chemotaxis in guiding neuroblasts might benefit from inducible genetic manipulation of neural stem cells and their progeny in situ to establish further underlying molecular mechanisms. In addition, future work might address whether electric fields have any role in regulating neuroblasts that do not undergo RMS migration within the human brain [8].Although the phenomenon of galvanotaxis in weak direct current fields is well established across cell types, and Cao and colleagues suggest a role for the P2Y1 receptor, in general the cellular and molecular mechanisms that underlie this process are not well understood. Many cells—including these SVZ neuroblasts—respond robustly to electrical fields of 10 mV mm⁻¹ or more, yet these fields correspond to small potential differences (roughly 0.1 mV) across the dimensions of a cell [5]. One hypothesis is that small direct current fields drive ionic flow of free cations—namely Na⁺—the hydration shell of which drags concordantly along charged membrane-associated proteins towards the anode (Fig 1). The result might be a cell surface gradient of key receptors that in turn direct migration. Another theory is that small potential differences may differentially bias voltage-gated ion channels, although the activation voltages for such gated channels typically range from 50–100 mV [5]. A third explanation is that electrical fields can generate forces on negatively charged cell surface adhesion molecules (such as integrins), leading to differences in cell–extracellular matrix interactions and migratory properties across the length of the cell [9]. Finally, the lipid phosphatase PTEN—a repressor of phosphatidylinositide 3-kinase signalling—mediates an electric field response during wound healing [10], and a structurally related phosphatase (Ci-VSP) that regulates the activity of phosphoinositide-sensitive ion channels was discovered to be voltage sensitive [11]. Given the established importance of PTEN in neural stem cells and glioblastomas, these factors might also offer potential mechanisms.Open in a separate windowFigure 1There are numerous hypothesized mechanisms by which cells might sense an electrical field. A weak electrical field could impose a force on negatively charged cell surface receptors, or alternatively the electric force imposed on positive ions (Na⁺) could result in the flow of their associated hydration shell, which exerts a drag force on cell surface membranes. The resulting asymmetrical redistribution of cell surface receptors, such as the ones involved in sensing chemokines or motogens, could affect cell migration. Alternatively, the electric field could conceivably trigger voltage-gated ion channels or exert forces on adhesion receptors, such as integrins, which result in asymmetrical binding to extracellular matrix (ECM) proteins. Finally, phosphatases, such as Ci-VSP or PTEN, mediate cellular responses to electric fields.In summary, Cao and colleagues establish that electrical currents and field gradients exist along the RMS, provide further support for the observation that adult neuroblasts migrate directionally within electrical fields and provide evidence that an endogenous potential gradient along the RMS might help guide these immature neurons to the olfactory bulb. This study thus lays stimulating groundwork for future investigations to explore the roles of biophysical cues in guiding the fate and flow of stem cells and their progeny in the nervous system.     

Sodium Channelopathies: From Molecular Physiology towards Medical Genetics

Voltage-gated sodium channels are heteromeric transmembrane proteins involved in the conduction of sodium ion currents in response to membrane depolarization. In humans, nine homologous genes, SCN1A–11A, which encode different isoforms of the voltage-gated sodium channel family, are known. Sodium channel isoforms exhibit different kinetic properties that determine different types of neurons. Mutations in different channels are described in patients with various congenital disorders, from epilepsy to congenital insensitivity to pain. This review presents an analysis of the current literature on the properties of different isoforms of voltage-gated sodium channels and associated diseases.     

Modeling and simulation of ion channels and action potentials in taste receptor cells

Based on patch clamp data on the ionic currents of rat taste receptor cells, a mathematical model of mammalian taste receptor cells was constructed to simulate the action potentials of taste receptor cells and their corresponding ionic components, including voltage-gated Na⁺ currents and outward delayed rectifier K⁺ currents. Our simulations reproduced the action potentials of taste receptor cells in response to electrical stimuli or sour tastants. The kinetics of ion channels and their roles in action potentials of taste receptor cells were also analyzed. Our prototype model of single taste receptor cell and simulation results presented in this paper provide the basis for the further study of taste information processing in the gustatory system.     

Single-microelectrode voltage clamp measurements of pancreatic β-cell membrane ionic currents in situ

A conventional patch clamp amplifier was used to test the feasibility of measuring whole-cell ionic currents under voltage clamp conditions from -cells in intact mouse islets of Langerhans perifused with bicarbonate Krebs buffer at 37°C. Cells impaled with a high resistance microelectrode (ca. 0.150 G) were identified as -cells by the characteristic burst pattern of electrical activity induced by 11 mm glucose. Voltage-dependent outward K⁺ currents were enhanced by glucose both in the presence and absence of physiological bicarbonate buffer and also by bicarbonate regardless of the presence or absence of glucose. For comparison with the usual patch clamp protocol, similar measurements were made from single rat -cells at room temperature; glucose did not enhance the outward currents in these cells. Voltage-dependent inward currents were recorded in the presence of tetraethylammonium (TEA), an effective blocker of the K⁺ channels known to be present in the -cell membrane. Inward currents exhibited a fast component with activation-inactivation kinetics and a delayed component with a rather slow inactivation; inward currents were dependent on Ca²⁺ in the extracellular solution. These results suggest the presence of either two types of voltage-gated Ca²⁺ channels or a single type with fast and slow inactivation. We conclude that it is feasible to use a single intracellular microelectrode to measure voltage-gated membrane currents in the -cell within the intact islet at 37°C, under conditions that support normal glucose-induced insulin secretion and that glucose enhances an as yet unidentified voltage-dependent outward K⁺ current.The authors are pleased to thank Dr. M.X. Li for performing some of the experiments in Fig. 2, Dr. A. Sherman for many illuminating discussions, and Drs. J. Rinzel and N. Sheppard for support. D.M. was supported in part by a National Institutes of Health training grant to The Johns Hopkins University, Department of Biomedical Engineering. Thanks are also given to B. Chidakel for electronic design and construction.     

Veratridine blocks voltage-gated potassium current in human T lymphocytes and in mouse neuroblastoma cells

(i) Effects of veratridine on ionic conductances of human peripheral blood T lymphocytes have been investigated using the whole-cell patch-clamp technique, (ii) Veratridine reduces the net outward current evoked by membrane depolarizations. The reduction originates from block of a 4-aminopyridine-sensitive, voltage-gated K⁺ current, (iii) Human T lymphocytes do not appear to express voltage-gated Na⁺ channels, since inward currents are observed neither in control nor in veratridine- and bretylium-exposed lymphocytes. (iv) The effect of veratridine consists of an increase in the rate of decay of the voltage-gated K⁺ current and a reduction of the peak current amplitude. Both effects depend on veratridine concentration. Halfmaximum block occurs at 97 m and the time constant of decay is reduced by 50% at 54 m of veratridine. (v) Possible mechanisms of veratridine action are discussed. The increased rate of K⁺ current decay is most likely due to open channel block. The decrease of current amplitude may involve an additional mechanism. (vi) In cultured mouse neuroblastoma N1E-115 cells, veratridine blocks a component of voltage-gated K⁺ current, in addition to its effect on voltage-gated Na⁺ current. This result shows that the novel effect of veratridine is not confined to lymphocytes.We thank Jacobien Künzel of the Wilhelmina Hospital for Children, Utrecht, for providing the blood samples and Aart de Groot for technical assistance. The research was supported by a fellowship of the Royal Netherlands Academy of Arts and Sciences to M. Oortgiesen.     

The Human Red Cell Voltage-regulated Cation Channel. The Interplay with the Chloride Conductance,the Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> Channel and the Ca<Superscript>2+</Superscript> Pump

Electrocytes from the electric organ of Electrophorus electricus exhibited sodium action potentials that have been proposed to be repolarized by leak currents and not by outward voltage-gated potassium currents. However, patch-clamp recordings have suggested that electrocytes may contain a very low density of voltage-gated K⁺ channels. We report here the cloning of a K⁺ channel from an eel electric organ cDNA library, which, when expressed in mammalian tissue culture cells, displayed delayed-rectifier K⁺ channel characteristics. The amino-acid sequence of the eel K⁺ channel had the highest identity to Kv1.1 potassium channels. However, different important functional regions of eel Kv1.1 had higher amino-acid identity to other Kv1 members, for example, the eel Kv1.1 S4-S5 region was identical to Kv1.5 and Kv1.6. Northern blot analysis indicated that eel Kv1.1 mRNA was expressed at appreciable levels in the electric organ but it was not detected in eel brain, muscle, or cardiac tissue. Because electrocytes do not express robust outward voltage-gated potassium currents we speculate that eel Kv1.1 channels are chronically inhibited in the electric organ and may be functionally recruited by an unknown mechanism.     

VOCCs and TREK-1 ion channel expression in human tenocytes

Mechanosensitive and voltage-gated ion channels are known to perform important roles in mechanotransduction in a number of connective tissues, including bone and muscle. It is hypothesized that voltage-gated and mechanosensitive ion channels also may play a key role in some or all initial responses of human tenocytes to mechanical stimulation. However, to date there has been no direct investigation of ion channel expression by human tenocytes. Human tenocytes were cultured from patellar tendon samples harvested from five patients undergoing routine total knee replacement surgery (mean age: 66 yr; range: 63-73 yr). RT-PCR, Western blotting, and whole cell electrophysiological studies were performed to investigate the expression of different classes of ion channels within tenocytes. Human tenocytes expressed mRNA and protein encoding voltage-operated calcium channel (VOCC) subunits (Ca alpha(1A), Ca alpha(1C), Ca alpha(1D), Ca alpha(2)delta(1)) and the mechanosensitive tandem pore domain potassium channel (2PK(+)) TREK-1. They exhibit whole cell currents consistent with the functional expression of these channels. In addition, other ionic currents were detected within tenocytes consistent with the expression of a diverse array of other ion channels. VOCCs and TREK channels have been implicated in mechanotransduction signaling pathways in numerous connective tissue cell types. These mechanisms may be present in human tenocytes. In addition, human tenocytes may express other channel currents. Ion channels may represent potential targets for the pharmacological management of chronic tendinopathies.     

Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes

The ability of azobenzene trimethylammonium bromide (azoTAB) to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav), calcium (ICav), and potassium (IKv) currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na⁺) and calcium (Ca²⁺) currents and potentiation of net potassium (K⁺) currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential.     

Taurine- and FMRFamide-dependent modulation of receptor- and voltage-gated currents by cerebrolysine in molluscan neurons

The action of cerebrolysine, a biogenic stimulator, on the receptor- and voltage-gated ionic currents was studied in identifiedHelix pomatia neurons. Cerebrolysine reversibly suppressed the acetylcholine (ACh)- and glutamate (GLU)-induced chloride currents in some neurons (LP11, B4, E12) with a latency of 9±3 sec, while not affecting these currents in other neurons. The suppressing effect of cerebrolysine on the voltage-gated sodium and calcium currents was also selective. There were fast and slow phases, with latencies of 52±8 sec and 5±1 min, respectively, in the cerebrolysine effect on the voltage-gated sodium current. The effect of cerebrolysine on the sodium current during the fast suppression phase could be simulated with FMRFamide (10^–5 M), while those exerted on the ACh- and GLU-induced currents could be simulated with taurine (10^–6 M). The effects of cerebrolysine and the above substances were non-additive. These facts allow us to suggest that both taurine and FMRFamide (or its fragment) are involved in the mechanism of posttraumatic and postsurgical curative effects of cerebrolysine.Neirofiziologiya/Neurophysiology, Vol. 26, No. 3, pp. 190–196, May–June, 1994.     

Trigeminal-Rostral Ventromedial Medulla circuitry is involved in orofacial hyperalgesia contralateral to tissue injury

Background

Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. ?? and ??ENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear.

Results

Here we show that deleting ?? and ??ENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro.

Conclusion

Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.     

Basis for allosteric open-state stabilization of voltage-gated potassium channels by intracellular cations

The open state of voltage-gated potassium (Kv) channels is associated with an increased stability relative to the pre-open closed states and is reflected by a slowing of OFF gating currents after channel opening. The basis for this stabilization is usually assigned to intrinsic structural features of the open pore. We have studied the gating currents of Kv1.2 channels and found that the stabilization of the open state is instead conferred largely by the presence of cations occupying the inner cavity of the channel. Large impermeant intracellular cations such as N-methyl-d-glucamine (NMG⁺) and tetraethylammonium cause severe slowing of channel closure and gating currents, whereas the smaller cation, Cs⁺, displays a more moderate effect on voltage sensor return. A nonconducting mutant also displays significant open state stabilization in the presence of intracellular K⁺, suggesting that K⁺ ions in the intracellular cavity also slow pore closure. A mutation in the S6 segment used previously to enlarge the inner cavity (Kv1.2-I402C) relieves the slowing of OFF gating currents in the presence of the large NMG⁺ ion, suggesting that the interaction site for stabilizing ions resides within the inner cavity and creates an energetic barrier to pore closure. The physiological significance of ionic occupation of the inner cavity is underscored by the threefold slowing of ionic current deactivation in the wild-type channel compared with Kv1.2-I402C. The data suggest that internal ions, including physiological concentrations of K⁺, allosterically regulate the deactivation kinetics of the Kv1.2 channel by impairing pore closure and limiting the return of voltage sensors. This may represent a primary mechanism by which Kv channel deactivation kinetics is linked to ion permeation and reveals a novel role for channel inner cavity residues to indirectly regulate voltage sensor dynamics.     

Intramembrane Charge Movement Associated with Endogenous K+ Channel Activity in HEK-293 Cells

1. The use of molecular biology in combination with electrophysiology in the HEK-293 cell line has given fascinating insights into neuronal ion channel function. Nevertheless, to fully understand the properties of channels exogenously expressed in these cells, a detailed evaluation of endogenous channels is indispensable. 2. Previous studies have shown the expression of endogenous voltage-gated K+, Ca2+, and Cl- channels and this predicts that changes in membrane potential will cause intramembrane charge movement, though this gating charge translocation remain undefined. Here, we confirm this prediction by performing patch-clamp experiments to record ionic and gating currents. Our data show that HEK-293 cells express at least two types of K+-selective endogenous channels which sustain the majority of the ionic current, and exclude a significant contribution from Ca2+ and Cl- channels to the whole-cell current. 3. Gating currents were unambiguously resolved after ionic current blockade enabling this first report of intramembrane charge movement in HEK-293 cells arising entirely from endogenous K+ channel activity, and providing valuable information concerning the activation mechanism of voltage-gated K+ channels in these cells.     

Using mutagenesis to study potassium channel mechanisms

The voltage-activated K⁺ channels are members of an ion channel family that includes the voltage-activated Na⁺ and Ca²⁺ channels. These ion channels mediate the transmembrane ionic currents that are responsible for the electrical signals produced by cells. The recent cloning of numerous voltage-activated K⁺ channels has made it possible to combine molecular-genetic and biophysical methods to study K⁺ channel mechanisms. These mutagenesis-function studies are beginning to provide new information about the architecture of K⁺ channel proteins and how they form a voltage-gated, K⁺-selective pore.     

Concentration-dependent modulations of potassium and calcium currents of rat osteoblastic cells by arachidonic acid

We show that the voltage-gated K⁺ and Ca²⁺ currents of rat osteoblastic cells are strongly modulated by arachidonic acid (AA), and that these modulations are very sensitive to the AA concentration. At 2 or 3 μm, AA reduces the amplitude and accelerates the inactivation of the K⁺ current activated by depolarization; at higher concentrations (≥5 μm), AA still blocks this K⁺ current, but also induces a very large noninactivating K⁺ current. At 2 or 3 μm, AA enhances the T-type Ca²⁺ current, close to its threshold of activation, whereas at 10 μm, it blocks that current. AA (1–10 μm) also blocks the dihydropyridine-sensitive L-type Ca²⁺ current. Thus, the effect of AA on Ca²⁺ entry through voltage-gated Ca²⁺ channels can change qualitatively with the AA concentration: at 2 or 3 μm, AA will favor Ca²⁺ entry through T channels, both by lowering the voltage-gated K⁺ conductance and by increasing the T current, whereas at 10 μm, AA will prevent Ca²⁺ entry through voltage-gated Ca²⁺ channels, both by inducing a K⁺ conductance and by blocking Ca²⁺ channels.     

Current Transients Associated with BK Channels in Human Glioma Cells

We have previously demonstrated the expression of BK channels in human glioma cells. There was a curious feature to the whole-cell currents of glioma cells seen during whole-cell patch-clamp: large, outward current transients accompanied repolarization of the cell membrane following an activating voltage step. This transient current, I _transient, activated and inactivated rapidly (1 ms). The I-V relationship of I _transient had features that were inconsistent with simple ionic current through open ion channels: (i) I _transient amplitude peaked with a –80 mV voltage change and was invariant over a 200 mV range, and (ii) I _transient remained large and outward at –140 mV. We provide evidence for a direct relationship of I _transient to glioma BK currents. They had an identical time course of activation, identical pharmacology, identical voltage-dependence, and small, random variations in the amplitude of the steady-state BK current and I _transient seen over time were often perfectly in phase. Substituting intracellular K⁺ with Cs⁺, Li⁺, or Na ⁺ ions reversibly reduced I _transient and BK currents. I _transient was not observed in recordings of other BK currents (hbr5 expressed in HEK cells and BK currents in rat neurons), suggesting I _transient is unique to BK currents in human glioma cells. We conclude that I _transient is generated by a mechanism related to the deactivation, and level of prior activation, of glioma BK channels. To account for these findings we propose that K⁺ ions are trapped within glioma BK channels during deactivation and are forced to exit to the extracellular side in a manner independent of membrane potential.     

Simulations of voltage clamping poorly space-clamped voltage-dependent conductances in a uniform cylindrical neurite

Significant error is made by using a point voltage clamp to measure active ionic current properties in poorly space-clamped cells. This can even occur when there are no obvious signs of poor spatial control. We evaluated this error for experiments that employ an isochronal I(V) approach to analyzing clamp currents. Simulated voltage clamp experiments were run on a model neuron having a uniform distribution of a single voltage-gated inactivating ionic current channel along an elongate, but electrotonically compact, process. Isochronal Boltzmann I(V) and kinetic parameter values obtained by fitting the Hodgkin-Huxley equations to the clamp currents were compared with the values originally set in the model. Good fits were obtained for both inward and outward currents for moderate channel densities. Most parameter errors increased with conductance density. The activation rate parameters were more sensitive to poor space clamp than the I(V) parameters. Large errors can occur despite normal-looking clamp curves.     

G protein activation inhibits gating charge movement in rat sympathetic neurons

G protein-coupled receptors (GPCRs) control neuronal functions via ion channel modulation. For voltage-gated ion channels, gating charge movement precedes and underlies channel opening. Therefore, we sought to investigate the effects of G protein activation on gating charge movement. Nonlinear capacitive currents were recorded using the whole cell patch-clamp technique in cultured rat sympathetic neurons. Our results show that gating charge movement depends on voltage with average Boltzmann parameters: maximum charge per unit of linear capacitance (Q_max) = 6.1 ± 0.6 nC/µF, midpoint (V_h) = –29.2 ± 0.5 mV, and measure of steepness (k) = 8.4 ± 0.4 mV. Intracellular dialysis with GTPS produces a nonreversible 34% decrease in Q_max, a 10 mV shift in V_h, and a 63% increase in k with respect to the control. Norepinephrine induces a 7 mV shift in V_h and 40% increase in k. Overexpression of G protein ₁₄ subunits produces a 13% decrease in Q_max, a 9 mV shift in V_h, and a 28% increase in k. We correlate charge movement modulation with the modulated behavior of voltage-gated channels. Concurrently, G protein activation by transmitters and GTPS also inhibit both Na⁺ and N-type Ca²⁺ channels. These results reveal an inhibition of gating charge movement by G protein activation that parallels the inhibition of both Na⁺ and N-type Ca²⁺ currents. We propose that gating charge movement decrement may precede or accompany some forms of GPCR-mediated channel current inhibition or downregulation. This may be a common step in the GPCR-mediated inhibition of distinct populations of voltage-gated ion channels. ion channel modulation; G protein-coupled receptors; charge movement     

BK channels mediate cholinergic inhibition of high frequency cochlear hair cells

Background

Outer hair cells are the specialized sensory cells that empower the mammalian hearing organ, the cochlea, with its remarkable sensitivity and frequency selectivity. Sound-evoked receptor potentials in outer hair cells are shaped by both voltage-gated K⁺ channels that control the membrane potential and also ligand-gated K⁺ channels involved in the cholinergic efferent modulation of the membrane potential. The objectives of this study were to investigate the tonotopic contribution of BK channels to voltage- and ligand-gated currents in mature outer hair cells from the rat cochlea.

Methodology/Principal

Findings In this work we used patch clamp electrophysiology and immunofluorescence in tonotopically defined segments of the rat cochlea to determine the contribution of BK channels to voltage- and ligand-gated currents in outer hair cells. Although voltage and ligand-gated currents have been investigated previously in hair cells from the rat cochlea, little is known about their tonotopic distribution or potential contribution to efferent inhibition. We found that apical (low frequency) outer hair cells had no BK channel immunoreactivity and little or no BK current. In marked contrast, basal (high frequency) outer hair cells had abundant BK channel immunoreactivity and BK currents contributed significantly to both voltage-gated and ACh-evoked K⁺ currents.

Conclusions/Significance

Our findings suggest that basal (high frequency) outer hair cells may employ an alternative mechanism of efferent inhibition mediated by BK channels instead of SK2 channels. Thus, efferent synapses may use different mechanisms of action both developmentally and tonotopically to support high frequency audition. High frequency audition has required various functional specializations of the mammalian cochlea, and as shown in our work, may include the utilization of BK channels at efferent synapses. This mechanism of efferent inhibition may be related to the unique acetylcholine receptors that have evolved in mammalian hair cells compared to those of other vertebrates.