Voltage Dependence of ATP Secretion in Mammalian Taste Cells

Mammalian type II taste cells release the afferent neurotransmitter adenosine triphosphate (ATP) through ATP-permeable ion channels, most likely to be connexin (Cx) and/or pannexin hemichannels. Here, we show that ion channels responsible for voltage-gated (VG) outward currents in type II cells are ATP permeable and demonstrate a strong correlation between the magnitude of the VG current and the intensity of ATP release. These findings suggest that slowly deactivating ion channels transporting the VG outward currents can also mediate ATP secretion in type II cells. In line with this inference, we studied a dependence of ATP secretion on membrane voltage with a cellular ATP sensor using different pulse protocols. These were designed on the basis of predictions of a model of voltage-dependent transient ATP efflux. Consistently with curves that were simulated for ATP release mediated by ATP-permeable channels deactivating slowly, the bell-like and Langmuir isotherm–like potential dependencies were characteristic of ATP secretion obtained for prolonged and short electrical stimulations of taste cells, respectively. These observations strongly support the idea that ATP is primarily released via slowly deactivating channels. Depolarizing voltage pulses produced negligible Ca²⁺ transients in the cytoplasm of cells releasing ATP, suggesting that ATP secretion is mainly governed by membrane voltage under our recording conditions. With the proviso that natural connexons and pannexons are kinetically similar to exogenously expressed hemichannels, our findings suggest that VG ATP release in type II cells is primarily mediated by Cx hemichannels.     

Voltage-gated Ca2+ channels in type III taste cells

In the mammalian taste bud, the heterogeneous cell population includes three morphologically distinct types of cells, type I to type III, which are also different in their electrophysiological features. Particularly, voltage-gated (VG) Ca²⁺ channels are functional solely in taste cells of the type III. These channels were studied here with external Ba²⁺ ions as current carriers. It was specifically shown that VG Ba²⁺ currents were almost completely blockable with nifedipine as well as with ionic blockers, such as Cd²⁺, Ni²⁺, and Co²⁺. Kinetic properties of VG Ba²⁺ currents in type III cells and their sensitivity to the blockers indicated that these currents were largely mediated by VG Ca²⁺ channels of the L-type. The expression of genes, which encode pore-forming α1-subunits of Ca²⁺ channels, was analyzed using methods of molecular biology. Among four genes encoding L-type Ca²⁺ channel α1-subunits (Ca _ν 1.1-Ca _ν 1.4), the expression of Ca _ν 1.2 was demonstrated in taste cells.     

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.     

Channel activity of recombinant pannexin 1

Mammalian taste cells of the type II release ATP, an afferent neurotransmitter, by employing unselective ATP-permeable ion channels. The molecular identity of these channels is not known with confidence, although evidence implicates certain channel proteins from the connexin and pannexin families as most likely candidates. Here we carried out the comparative analysis of biophysical features and pharmacological profiles of unselective channels operative in type II cells and recombinant pannexin 1 (Panx1), which was cloned from the taste tissue and heterologously expressed in eukaryotic cells of several lines, including HEK-293, CHO, and neuroblastoma SK-N-SH. Integral currents mediated by Panx1 hemichannels were recorded to elucidate their kinetics characteristics, such as activation and deactivation, voltage dependence, and sensitivity to a variety of blockers, including carbenoxolone, DIDS, and NPPB. It was shown that the heterologous expression of Panx1 in cells of each type induced specific conductance, which exhibited outward rectification and was effectively blockable with carbenoxolone and anionic channel blockers DIDS and NPPB. Panx1 activity was studied at the single channel level as well. As was found, transfection of HEK-293 cells with the plasmid harboring cDNA encoding Panx1 gave rise to single channel current-like events in excised patches that were inhibited by 20 μM carbenoxolone, the relatively specific blocker of Panx1. These carbenoxolone-sensitive channels were peculiar in that single-channel current versus membrane voltage was not linear but exhibited outward rectification. In addition, the open-channel probability strongly increased with membrane voltage. Taken together, the data obtained here and earlier demonstrate clearly that by their biophysical and pharmacological features, ATP-permeable channels operative in type II cells are rather distinct from recombinant Panx1 hemichannels, thus arguing against Panx1 as the main conduit of ATP release in taste cells.     

A heterogeneous electrophysiological profile of bone marrow-derived mast cells

Electrophysiological properties of mouse bone marrow-derived mast cells (BMMC) were studied under the whole-cell clamp configuration. About one third of the cells were quiescent, but others expressed either inward or outward currents. Inwardly rectifying (IR) currents were predominant in 14% of the cells, and outwardly rectifying (OR) currents in 24%. The rest (22%) of the cells exhibited both inward and outward currents. The IR currents were eliminated by 1 mm Ba²⁺, and were partially inhibited by 100 μm quinidine. The reversal potential was dependent on extracellular K⁺, thereby indicating that K⁺ mediated the IR currents. The negative conductance region was seen at potentials positive to E _K. The OR currents did not apparently depend on the extracellular K⁺ concentration, but were reduced by lowering the extracellular Cl^? concentration. The OR currents were partially blocked by 1 mm Ba²⁺, and were further blocked by a Cl^? channel blocker, 4,4′-diisothiocyano-2, 2′-stilbenedisulfonate (DIDS). In addition, the reversal potential of the OR currents was positively shifted by decreasing the ratio of external and internal Cl^? concentrations, suggesting that Cl^? was a major ion carrier. In cells exhibiting IR currents, the membrane potential varied among cells and tended to depolarize by elevating the external K⁺ concentration. In cells with OR currents, the resting potential was hyperpolarized in association with an increase in conductance. These results suggest that BMMC have a heterogeneous electrophysiological profile that may underlie a variety of ion channels expressed in different phenotypes of mast cells. Activities of both the inwardly rectifying K⁺ channel and the outwardly rectifying Cl^? channel seem to contribute to the regulation of the membrane potential.     

Human Adipose Cells Have Voltage-dependent Potassium Currents

The whole-cell patch-clamp method was used to study the membrane electrical properties of human adipocyte cells obtained by differentiating from precursors of human abdominal and mammary tissues. All differentiated cells exhibited outward currents with sigmoidal activation kinetics. The outward currents showed activation thresholds between –20 to –30 mV and slow inactivation. The ionic channels underlying the macroscopic current were highly selective for K⁺. Their selectivity was for typical K⁺ channels with relative permeabilities of K⁺>NH ₄ ⁺ >Cs⁺>Na⁺. No evidence of any other type of voltage-gated channel was found. The potassium currents (I _KV) were blocked reversibly by tetraethylammonium and barium. The IC ₅₀ value and Hill coefficient of tetraethylammonium inhibition of I _KV were 0.56 mM and 1.17 respectively. These results demonstrate that human adipose cells have voltage-dependent potassium currents.     

Electrical excitability of taste cells. Mechanisms and possible physiological significance

Neuronal, muscle and some endocrine cells are electrically excitable. While in muscle and endocrine cells AP stimulates and synchronizes intracellular processes, neurons employ action potentials (APs) to govern discontinuous synapses located distantly. Meanwhile, such axonless sensory cells as photoreceptors and hair cells exemplify afferent output, which is not driven by APs; instead, gradual receptor potentials elicited by sensory stimuli control the release of afferent neurotransmitter glutamate. Mammalian taste cells of the type II and type III are electrically excitable and respond to stimulation by firing APs. Since taste cells also have no axons, physiological significance of the electrical excitability for taste transduction and encoding sensory information is unclear. Perhaps, AP facilitates transmitter release, ATP in type II cells and 5-HT in type III cells, although via different mechanisms. The ATP release is mediated by connexin hemichannels, does not require a Ca²⁺ trigger, and largely gated by membrane voltage. 5-HT secretion is driven by intracellular Ca²⁺ and involves VG Ca²⁺ channels. Here, we discuss ionic mechanisms of excitability of taste cells and speculate on a likely role of APs in mediating their afferent output.     

Identification of TRPM5 Ion Channels in Type-II Taste Cells of Mice

TRPM5 are ion channels belonging to the TRP family, which demonstrate a nonselective permeability for monovalent cations and are activated by an increase in the intracellular calcium level. TRPM5 are present in taste receptor cells of type II responsible for reception of bitter, sweet, and umami taste sensations. Knockout of the trpm5 gene in mice results in a nearly complete loss of sensitivity to taste stimuli of the above-mentioned modalities (taste blindness). The physiological activity of TRPM5 in taste receptive cells has practically not been studied. Using a patch-clamp technique, we carried out a comparative analysis of the properties of recombinant TRPM5 and Ca²⁺-activated membrane channels in type-II taste cells in mice. Dialysis of the studied cells with a high-Ca²⁺ solution and application of a calcium ionophore, ionomycin, caused activation of outward-rectification ion channels permeable for Na⁺, Cs⁺, and K⁺ in CHO-strain cells with exogenous TRPM5. These channels were blocked by 100 μM triphenylphosphine oxide (TPPO). Calcium-activated channels in type-II taste cells also possessed analogous properties. Application of the calcium ionophore ionomycin or a stepwise increase in the intracellular Ca²⁺ level using photolysis (uncaging) caused activation of channels nonselective with respect to Na⁺ and Cs⁺ and impermeable for N-methyl-D-glucamine (NMDG⁺). These channels had the current–voltage characteristics of outward rectification and a high thermosensitivity (Q₁₀ = 6.7 ± 0.5); they could be blocked by TPPO. It should be emphasized that TRPM5 were specific with respect to type-II cells. An increase in the intracellular calcium level induced the appearance of Cl– current in type-I cells and did not influence the basic current in type-III cells.     

Voltage-gated inward currents of morphologically identified cells of the frog taste disc

We used the patch clamp technique to record from taste cells in vertical slices of the bullfrog (Rana catesbeiana) taste disc. Cell types were identified by staining with Lucifer yellow in a pipette after recording their electrophysiological properties. Cells could be divided into the following three groups: type Ib (wing) cells with sheet-like apical processes, type II (rod) cells with single thick rod-like apical processes and type III (rod) cells with thin rod-like apical processes. No dye-coupling was seen either between cells of the same type or between cells of different types. We focused on the voltage-gated inward currents of the three types of cells. Type Ib and type II cells exhibited tetrodotoxin (TTX)-sensitive voltage-gated Na+ currents. Surprisingly, type III cells showed TTX-resistant voltage-gated Na+ currents and exhibited a lack of TTX-sensitive Na+ currents. TTX-resistant voltage-gated Na+ currents in taste cells are reported for the first time here. The time constant for the inactivating portion of the voltage-gated inward Na+ currents of type III cells was much larger than that of type Ib and type II cells. Therefore, slow inactivation of inward Na+ currents characterizes type III cells. Amplitudes of the maximum peak inward currents of type III cells were smaller than those of type Ib and type II cells. However, the density (pA/pF) of the maximum peak inward currents of type III cells was much higher than that of type Ib cells and close to that of type II cells. No evidence of the presence of voltage-gated Ca2+ channels in frog taste cells has been presented up to now. In this study, voltage-gated Ba2+ currents were observed in type III cells but not in type Ib and type II cells when the bath solution was a standard Ba2+ solution containing 25 mM Ba2+. Voltage-gated Ba2+ currents were blocked by addition of 2 mM CoCl2 to the standard Ba2+ solution, suggesting that type III cells possess the voltage-gated Ca2+ channels and they do classical (calcium-influx) synaptic transmission. It appears that type III cells are taste receptor cells.     

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.     

Regulation of tonoplast k channels by voltage in the range of physiological electric potentials

The presence of tonoplast ion channels regulated by voltage in the physiological range of transtonoplast electric potential was studied in isolated vacuoles from Acer pseudoplatanus cultured cells. In symmetrical KCl or K-gluconate depolarizing pulses induced instantaneously developing, decaying outward currents, while in symmetrical tetramethylammonium chloride these currents were absent. The outward currents were reduced if the depolarizations were applied from a holding potential of +30 millivolts and increased upon depolarizations from a holding potential of −30 millivolts and even more from a holding potential of −50 millivolts. These results indicate that the outward currents are due to K⁺ movement through channels which are open around 0 millivolt and close at positive potentials. These K⁺ channels, regulated in the range of the physiological electric potentials reported for the vacuoles in situ, are likely the same K⁺ channels activated by hyperpolarizations which we have previously described (R Colombo, R Cerana, P Lado, A Peres [1988] J Membr Biol 103: 227-236).     

Potassium currents in human myogenic cells from healthy and congenital myotonic dystrophy foetuses

The whole-cell patch clamp technique was used to record potassium currents in in vitro differentiating myoblasts isolated from healthy and myotonic dystrophy type 1 (DM1) foetuses carrying 2000 CTG repeats. The fusion of the DM1 myoblasts was reduced in comparison to that of the control cells. The dystrophic muscle cells expressed less voltage-activated K⁺ (delayed rectifier and non-inactivating delayed rectifier) and inward rectifier channels than the age-matched control cells. However, the resting membrane potential was not significantly different between the control and the DM1 cells. After four days in a differentiation medium, the dystrophic cells expressed the fast-inactivating transient outward K⁺ channels, which were not observed in healthy cells. We suggest that the low level of potassium currents measured in differentiated DM1 cells could be related to their impaired fusion.     

Volume-activated chloride channels in neuroblastoma cells are blocked by the antiestrogen toremifene

Summary The presence of volume-activated chloride channels has been examined in neuroblastoma C1300 cells using the whole-cell configuration of the patch-clamp technique. Chloride channels could not be detected under isotonic conditions. However, hypotonic challenge induced slowly developed inward and outward anionic currents that exhibited outward rectification and inactivation at the most depolarizing potentials, features that were similar to the currents described in other cell preparations where volume-activated Cl^– channels have been associated with the expression of P-glycoprotein. This hypotonicity-activated Cl^– currents could be reversibly blocked by extracellular exposure to toremifene, a novel synthetic antioestrogen. The fact that toremifene and its analog tamoxifen, have been shown to block P-glycoprotein-associated chloride channels and to reverse P-glycoprotein associated multidrug resistance in a number of cell lines suggest that P-glycoprotein could be involved in the generation of hypotomic-induced chloride conductance in neuroblastoma cells.     

Zebrafish heart as a model for human cardiac electrophysiology

The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart.     

Moderate Hypoxia Influences Potassium Outward Currents in Adipose-Derived Stem Cells

Moderate hypoxic preconditioning of adipose-derived stem cells (ASCs) enhances properties such as proliferation and secretion of growth factors, representing a valuable strategy to increase the efficiency of cell-based therapies. In a wide variety of cells potassium (K⁺) channels are key elements involved in the cellular responses to hypoxia, suggesting that ASCs cultured under low oxygen conditions may display altered electrophysiological properties. Here, the effects of moderate hypoxic culture on proliferation, whole-cell currents, and ion channel expression were investigated using human ASCs cultured at 5% and 20% oxygen. Although cell proliferation was greatly enhanced, the dose-dependent growth inhibition by the K⁺ channel blocker tetraethylammonium (TEA) was not significantly affected by hypoxia. Under both normoxic and hypoxic conditions, ASCs displayed outward K⁺ currents composed by Ca²⁺-activated, delayed rectifier, and transient components. Hypoxic culture reduced the slope of the current-voltage curves and caused a negative shift in the voltage activation threshold of the whole-cell currents. However, the TEA-mediated shift of voltage activation threshold was not affected by hypoxia. Semiquantitative real-time RT-PCR revealed that expression of genes encoding for various ion channels subunits related to oxygen sensing and proliferation remained unchanged after hypoxic culture. In conclusion, outward currents are influenced by moderate hypoxia in ASCs through a mechanism that is not likely the result of modulation of TEA-sensitive K⁺ channels.     

Cell permeabilization and inhibition of voltage-gated Ca(2+) and Na(+) channel currents by nanosecond pulsed electric field

Previous studies have found that nanosecond pulsed electric field (nsPEF) exposure causes long-term permeabilization of the cell plasma membrane. In this study, we utilized the whole-cell patch-clamp method to study the nsPEF effect on currents of voltage-gated (VG) Ca(2+) and Na(+) channels (I(Ca) and I(Na)) in cultured GH3 and NG108 cells. We found that a single 300 or 600 ns pulse at or above 1.5-2 kV/cm caused prolonged inhibition of I(Ca) and I(Na). Concurrently, nsPEF increased a non-inactivating "leak" current (I(leak)), presumably due to the formation of nanoelectropores or larger pores in the plasma membrane. The nsPEF effects were similar in cells that were exposed intact and subsequently brought into the whole-cell recording configuration, and in cells that were first brought into the whole-cell configuration and then exposed. Although both I(leak) and the inhibition of VG currents were enhanced at higher E-field levels, these two nsPEF effects showed relatively weak correlation with each other. In some cells, I(leak) increased 10-fold or more while VG currents remained unchanged. At longer time intervals after exposure (5-15 min), I(Ca) and I(Na) could remain inhibited although I(leak) had largely recovered. The causal relation of nsPEF inhibitory effects on VG currents and permeabilization of the plasma membrane is discussed.     

Proton-activated K+-channels of frog taste receptor cells

It is conventionally accepted that sour transduction does not require a receptor mechanism and is based on a direct interaction of acid stimuli with apical ion channels. At the same time, it has been shown that a number of neuronal cells express H(+)-gated cation channels. We studied the effect of acid stimuli on ion currents recorded from frog Rana temporaria taste receptor cells and found that a substantial subpopulation of them exhibited K+ currents activated by extracellular protons. To our knowledge, this is the first demonstration of H(+)-gated K+ channels in cells of any type including taste receptor cells. These channels are presumably involved in sour transduction and/or contribute to intercellular communications between discoid cells.     

Voltage-sensitive chloride ion channels in Anopheles gambiae Sua-1B cells

In this study, we performed electrophysiological analysis of Anopheles gambiae Sua-1B cells having “neuron-like” morphologies using the patch clamp method. The recorded cells (n = 79) had processes resembling axons/dendrites, with 63 % unipolar, 22 % bipolar, and 15 % multipolar. While no inward currents were observed following step depolarizations (holding potential = ?80 mV), a slowly activating outward current was observed in 96 % of the cells, especially at depolarized potentials. The amplitude of the current was attenuated nearly 70 % by reducing extracellular Cl^? ion concentration, or by incubating with 100 μM DIDS, a known voltage-sensitive chloride channel blocker, suggesting that the current was mediated by chloride ions. No qualitative difference was found between recordings made with Cs⁺ ions in the intracellular pipette solution (inhibits K⁺ currents) and those made with normal physiological solution, indicating a deficiency of potassium channels. Additionally, recordings made with Ca²⁺-free extracellular bath solution eliminated the slowly activating outward current. A subset of cells (n = 3) lacked this current, but had outward currents with voltage-dependent properties similar to those of volume-regulated chloride channels. Taken together, our results suggest that the voltage-sensitive currents observed in the majority of Sua-1B cells are mediated primarily by chloride channels of the calcium-dependent subtype.     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

Properties of the charibdotoxin-sensitive component of Ca2+-dependent K+ current in smooth muscle cells of the guinea pigTaenia Coli

Using the voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea pigtaenia coli. In our study, we identified and studied a charibdotoxin-sensitive component of Ca²⁺-dependent K⁺ current carried through the channels of high conductance (in most publications called “big conductance,”I _BK(Ca)). This component was completely blocked by 100 nM charibdotoxin and by tetraethylammonium in concentrations as low as 1 mM.I _BK(Ca) demonstrated fast kinetics of inactivation, which nearly coincided with that of Ca²⁺ current. In addition to the dependence on Ca²⁺ concentration, this current also showed voltage-dependent properties: with a rise in the level of depolarization its amplitude increased. In many cells, depolarizing shifts in the membrane potential evoke spontaneous outward currents. Such currents probably represent the secondary effect of cyclic Ca²⁺ release from the caffeine-sensitive intracellular stores that result in short-term activation of charibdotoxin-sensitive Ca²⁺-dependent K⁺ channels.