Distribution and characterization of functional amiloride-sensitive sodium channels in rat tongue

The role of amiloride-sensitive Na+ channels (ASSCs) in the transduction of salty taste stimuli in rat fungiform taste buds has been well established. Evidence for the involvement of ASSCs in salt transduction in circumvallate and foliate taste buds is, at best, contradictory. In an attempt to resolve this apparent controversy, we have begun to look for functional ASSCs in taste buds isolated from fungiform, foliate, and circumvallate papillae of male Sprague-Dawley rats. By use of a combination of whole-cell and nystatin-perforated patch-clamp recording, cells within the taste bud that exhibited voltage-dependent currents, reflective of taste receptor cells (TRCs), were subsequently tested for amiloride sensitivity. TRCs were held at - 70 mV, and steady-state current and input resistance were monitored during superfusion of Na(+)-free saline and salines containing amiloride (0.1 microM to 1 mM). Greater than 90% of all TRCs from each of the papillae responded to Na+ replacement with a decrease in current and an increase in input resistance, reflective of a reduction in electrogenic Na+ movement into the cell. ASSCs were found in two thirds of fungiform and in one third of foliate TRCs, whereas none of the circumvallate TRCs was amiloride sensitive. These findings indicate that the mechanism for Na+ influx differs among taste bud types. All amiloride-sensitive currents had apparent inhibition constants in the submicromolar range. These results agree with afferent nerve recordings and raise the possibility that the extensive labeling of the ASSC protein and mRNA in the circumvallate papillae may reflect a pool of nonfunctional channels or a pool of channels that lacks sensitivity to amiloride.     

Distribution of amiloride-sensitive sodium channels in the oral cavity of the hamster

The distribution of amiloride-sensitive sodium channels (ASSCs) in taste buds isolated from the oral cavity of hamsters was assessed by patch clamp recording. In contrast to the case for rats, taste cells from the fungiform, foliate and vallate papillae and from the soft palate all contain functional ASSCs. The differential distribution of ASSCs between the hamster and the rat may be important for understanding the physiology underlying the differing behavioral responses of these species to sodium salts.      

Characterization of sodium transport in gustatory epithelia from the hamster and rat

The transduction of sodium salts occurs through a variety of mechanisms, including sodium influx through amiloride-sensitive sodium channels, anion-dependent sodium movement through intercellular junctions and unidentified amiloride-insensitive mechanisms. Characterizations of sodium transport in lingual epithelium mounted in Ussing chambers have focused almost exclusively on epithelia containing only fungiform taste buds. In the present study we have investigated sodium transport by measuring NaCl-induced short-circuit current from lingual epithelia containing fungiform, foliate, vallate and palatine taste buds in the hamster and the rat. All areas show measurable sodium transport, yet significant differences were noted between the epithelia from the rat and the hamster and among the different epithelia within a single species in terms of current density, transepithelial resistance and mucosal amiloride sensitivity. In general, epithelia from the anterior tongue were of a lower resistance and transported sodium more effectively than from the posterior tongue. Moreover, fungiform- and vallate-containing epithelia in the rat had a greater current density than did the corresponding tissues in the hamster. Amiloride sensitivity also differed between the rat and the hamster. In the hamster all gustatory areas showed some amiloride sensitivity, while in the rat the vallate-containing epithelia were devoid of amiloride- sensitive sodium transport. The results are consistent with the interpretation that all chemosensitive areas may participate in the detection of salts but the degree of salt transport and the mechanism of transport is variable among different lingual epithelia and different species.      

Insulin activates epithelial sodium channel (ENaC) via phosphoinositide 3-kinase in mammalian taste receptor cells

Diabetes is a profound disease that results in a severe lack of regulation of systemic salt and water balance. From our earlier work on the endocrine regulation of salt taste at the level of the epithelial sodium channel (ENaC), we have begun to investigate the ability of insulin to alter ENaC function with patch-clamp recording on isolated mouse taste receptor cells (TRCs). In fungiform and vallate TRCs that exhibit functional ENaC currents (e.g., amiloride-sensitive Na(+) influx), insulin (5-20 nM) caused a significant increase in Na(+) influx at -80 mV (EC(50) = 7.53 nM). The insulin-enhanced currents were inhibited by amiloride (30 μM). Similarly, in ratiometric Na(+) imaging using SBFI, insulin treatment (20 nM) enhanced Na(+) movement in TRCs, consistent with its action in electrophysiological assays. The ability of insulin to regulate ENaC function is dependent on the enzyme phosphoinositide 3-kinase since treatment with the inhibitor LY294002 (10 μM) abolished insulin-induced changes in ENaC. To test the role of insulin in the regulation of salt taste, we have characterized behavioral responses to NaCl using a mouse model of acute hyperinsulinemia. Insulin-treated mice show significant avoidance of NaCl at lower concentrations than the control group. Interestingly, these differences between groups were abolished when amiloride (100 μM) was added into NaCl solutions, suggesting that insulin was regulating ENaC. Our results are consistent with a role for insulin in maintaining functional expression of ENaC in mouse TRCs.     

Characteristics of an amiloride-sensitive sodium entry pathway in cultured rodent glial and neuroblastoma cells

We have studied the induction of an amiloride-sensitive sodium influx into C6 glioma, NIE, and NB2A neuroblastoma cell lines. In late log phase, cells grown continuously in the presence of 10% fetal calf serum showed Na⁺ influxes of approximately 25–30 nmol/mg protein min; < 5% of this flux was inhibited by amiloride. Removal of serum for 24 h caused a decrease in the total Na⁺ influx to 15–20 nmol/mg protein/min. Upon readdition of serum to the incubation medium, there was an increase in total Na⁺ influx, depending on the cell type, of 20–400% within 2 min. This increment in Na⁺ influx represented an increase in amiloride-sensitive Na⁺ transport with an apparent K′, of 0.4 mM. By adding serum back at various times after serum deprivation, it was determined that 4 h was required to observe a detectable increase in the amiloride-sensitive Na⁺ flux. Thus, serum removal results in the induction of the amiloride transport system which, however, remains latent until the reintroduction of serum to the medium. Addition of 5 μg/ml of cycloheximide blocked the increase in Na⁺ transport, indicating that de novo protein synthesis mediated this serum deprivation–induced increase in Na⁺ transport. Moreover, inhibition of de novo lipid synthesis by 0.1 mM fenfluramine also blocked the induction of this transport activity, suggesting that a coordinated synthesis of lipid and protein is required for the expression of this sodium transport site. We have also found that this serum stimulated Na⁺ influx did not saturate with Na⁺ concentration, up to 140 mM. Also, among commonly used inhibitors of passive Na⁺ entry into epithelial tissues, only amiloride was capable of inhibiting this transport system in these neural cell lines.     

Detection of NaCl and KCl in TRPV1 knockout mice

Both amiloride-sensitive and -insensitive mechanisms contribute to NaCl taste transduction. The amiloride-sensitive mechanism relies on the epithelial Na(+) channel ENaC, which is widely expressed on the apical membrane of fungiform taste cells. The amiloride-insensitive mechanism, which predominates in circumvallate and foliate taste buds, was recently reported to involve a variant of the nonselective cation channel TRPV1. We performed 2-bottle preference and threshold experiments with TRPV1 knockout mice and wild-type (C57BL/6J) controls to test for NaCl preference and detection thresholds in the presence and absence of amiloride. Surprisingly, TRPV1 knockout mice not only detected NaCl in the presence of amiloride but they preferred NaCl over water at concentrations avoided by the wild-type mice. NaCl detection thresholds were between 2 and 3 mM for both genotypes. Amiloride increased the detection thresholds of wild-type mice but not knockout mice. The knockout mice also preferred 100 mM KCl compared with wild-type controls, suggesting that TRPV1 receptors may mediate a general aversive response to salts. Analyses of consumption data also revealed that TRPV1 knockout mice ingested more of the NaCl, with and without amiloride, and KCl solutions than the wild-type mice. However, comparisons of preference ratios and consumption volumes indicated that both wild-type and TRPV1 knockout mice avoided citric acid in quite a similar manner, suggesting that TRPV1 receptors do not mediate the detection of citric acid. These data, taken together, suggest that additional mechanisms must contribute to the amiloride-insensitive NaCl response.     

Membrane potential and sodium flux in neuroblastoma X glioma hybrid cells: Effects of amiloride and serum

The Na⁺ uptake into neuroblastoma x glioma hybrid cells was measured in Hepes-buffered EMEM containing 10% calf serum and 5 mM ouabain in the presence and absence of amiloride (1.0 mM). Amiloride was found to markedly inhibit net Na⁺ influx (by approximately 50%). Examination of the effect of amiloride on net Na⁺ influx in the absence of calf serum revealed that a significant amiloride-sensitive Na⁺ influx remains even under serum-deprived conditions, although the degree of amiloride inhibition (35%) is substantially lower than that found in the presence of serum. The amiloride-insensitive portion of Na⁺ influx was found to be independent of serum effects. Estimation of resting membrane potential was made by measurement of the steady state distribution of the lipophilic cation, TPP⁺, in the presence and absence of amiloride. A large, immediate increase in TPP⁺ uptake, indicative of a membrane hyperpolarization, was seen upon addition of amiloride. Determination of the effect of amiloride on resting membrane potential of serum-deprived cells showed that cells are hyperpolarized to a greater extent in the presence than in the absense of amiloride, and that serum exerts a depolarizing effect on the cells. Thus, serum-stimulation of Na⁺ influx results in a depolarization of resting membrane potential, while amiloride inhibition of Na⁺ influx causes a hyperpolarization. These data strongly suggest that NG108-15 cells possess an electrogenic Na⁺ influx pathway that is sensitive to amiloride inhibition and enhanced by serum.     

Stimulation of Transepithelial Na<Superscript>+</Superscript> Current by Extracellular Gd<Superscript>3+</Superscript> in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Alveolar Epithelium

In the present study we investigated the effect of extracellular gadolinium on amiloride-sensitive Na⁺ current across Xenopus alveolar epithelium by Ussing chamber experiments and studied its direct effect on epithelial Na⁺ channels with the patch-clamp method. As observed in various epithelia, the short-circuit current (I _sc) and the amiloride-sensitive Na⁺ current (I _ami) across Xenopus alveolar epithelium was downregulated by high apical Na⁺ concentrations. Apical application of gadolinium (Gd³⁺) increased I _sc in a dose-dependent manner (EC ₅₀ = 23.5 µM). The effect of Gd³⁺ was sensitive to amiloride, which indicated the amiloride-sensitive transcellular Na⁺ transport to be upregulated. Benz-imidazolyl-guanidin (BIG) and p-hydroxy-mercuribenzonic-acid (PHMB) probably release apical Na⁺ channels from Na⁺-dependent autoregulating mechanisms. BIG did not stimulate transepithelial Na⁺ currents across Xenopus lung epithelium but, interestingly, it prevented the stimulating effect of Gd³⁺ on transepithelial Na⁺ transport. PHMB increased I _sc and this stimulation was similar to the effect of Gd³⁺. Co-application of PHMB and Gd³⁺ had no additive effects on I _sc. In cell-attached patches on Xenopus oocytes extracellular Gd³⁺ increased the open probability (NP _o) of Xenopus epithelial sodium channels (ENaC) from 0.72 to 1.79 and decreased the single-channel conductance from 5.5 to 4.6 pS. Our data indicate that Xenopus alveolar epithelium exhibits Na⁺-dependent non-hormonal control of transepithelial Na⁺ transport and that the earth metal gadolinium interferes with these mechanisms. The patch-clamp experiments indicate that Gd³⁺ directly modulates the activity of ENaCs.     

Molecular and functional identification of sodium channels in K562 cells

Modulations of ion channel activity underlie rapid changes in membrane transport of cations in various nonexcitable cells. Previously, in smooth muscle cells, macrophages, lymphocytes, carcinoma and leukemia cell lines, non-voltage-gated sodium (NVGS) channels have been found. The activity of NVGS channels was shown to be critically dependent on the organization of actin cytoskeleton. The molecular identity of NVGS channels remains unclear. The present work is focused on molecular and functional identification of NVGS channels in human myeloid leukemia K562 cells. Degenerin/epithelial Na⁺ channels (DEG/ENaC) can be considered as possible molecular correlates. By using RT-PCR, expression of ??-, ??-, and ??-hENaC subunits in the K562 cells was detected. Various modes of the patch-clamp method were used to examine functional properties of sodium channels??specifically, to test the effect of amiloride on single channel and integral currents. The biophysical characteristics of the NVSG channels were close to those of ENaC; the channels have unitary conductance of 12 pS (145 mM Na⁺) and were impermeable to divalent cations (Ca²⁺ and Mg²⁺). We found that amiloride did not inhibit NVGS channels. Importantly, no amiloride-blockable sodium current was detected in the plasma membrane of K562 cells. Taken together, our observations suggest that amiloride-insensitive sodium channels in the K562 cells belong to the ENaC family.     

Na+/H+ exchange in ehrlich ascites tumor cells: Activation by cytoplasmic acidification and by treatment with cupric sulphate

Summary Exposure of Ehrlich cells to isotonic Na⁺-propionate medium induces a rapid cell swelling. This treatment is likely to impose an acid load on the cells. Cell swelling is absent in K⁺-propionate medium but may be induced by the ionophore nigericin, which mediates K⁺/H⁺ exchange. Cell swelling in Na⁺-propionate medium is blocked by amiloride, but an alternative pathway is introduced by addition of the ionophore monensin, which mediates Na⁺/H⁺ exchange. Consequently, swelling of Ehrlich cells in Na⁺-propionate medium is due to the operation of an amiloride-sensitive, Na⁺-specific mechanism. It is concluded that this mechanism is a Na⁺/H⁺ exchange system, activated by cytoplasmic acidification. We have previously demonstrated that the heavy metal salt CuSO₄ in micromolar concentrations inhibits regulatory volume decrease (RVD) of Ehrlich cells following hypotonic swelling. The present work shows that CuSO₄ inhibits RVD as a result of a net uptake of sodium, of which the major part is sensitive to amiloride. Measurements of intracellular pH show that CuSO₄ causes significant cytoplasmic alkalinization, which is abolished by amiloride. Concomitantly, CuSO₄ causes an amiloride-sensitive net proton efflux from the cells. The combined results confirm that a Na⁺/H⁺ exchange system exists in Ehrlich cells and demonstrate that the heavy metal salt CuSO₄ activates this Na⁺/H⁺ exchange system.     

Na+ transport by human placental brush border membranes: Are there several mechanisms?

Na⁺ transport was evaluated in brush border membrane vesicles isolated from the human placental villous tissue. Na⁺ uptake was assayed by the rapid filtration technique in the presence and the absence of an uphill pH gradient. Amiloride strongly decreased Na⁺ uptake whether a pH gradient was present or not. In pH gradient conditions (pH 7.5 in and 9.0 out), 1 mM amiloride decreased the 10 mM Na⁺ uptake by 84%. In the absence of pH gradient (pH 7.5 in and out), Na⁺ uptake was lower but still sensitive to amiloride. The Lineweaver-Burk plot of Na⁺ uptake consistently showed a single kinetics. Increasing the pH gradient decreased Km values of the amiloride-sensitive Na⁺ uptake, leaving the Vmax unchanged. In the absence of a pH gradient, the amiloride sensitive Na⁺ transport was maximal at pH 7.5. Here again, a single kinetics was observed, and pH influenced exclusively the Km of Na⁺. Since ethylisopropylamiloride, the specific Na/H exchanger inhibitor mimicked the effects of amiloride, decreasing by 98% the 10 mM Na⁺ uptake, whereas benzamil, the Na⁺ channel blocker, had no effect, it was concluded that the amiloride sensitive Na⁺ uptake was predominantly or exclusively due to a Na⁺-H⁺ exchanger activity. K⁺ in trans-position significantly decreased the amiloride sensitive uptake. In contrast, the presence of the cation in cis-position had no effect. The amiloride resistant Na⁺ transport was neither influenced by pH, nor saturable. Incubation of the placental tissue with 100 μM or 1 mM dibutyryl cAMP, 0.1 or 1 μM phorbol myristate acetate, 10⁻⁷ M insulin, 10⁻¹⁰ M angiotensin II, or 10⁻⁸ M human parathyroid hormone (PTH) did not influence Na⁺ transport by subsequently prepared brush border membranes. Finally, we failed to demonstrate any Na⁺-H⁺ exchange activity in the basal plasma membrane. These results indicate that (1) in the absence of co-substrates such as phosphate and aminoacids, the Na⁺-H⁺ exchange is probably the unique mechanism of Na⁺ transport by the placental brush border membrane, (2) the placental isoform of the exchanger is not regulated by PTH, angiotensin, nor insulin and, therefore, is different from the isoform present in the renal brush border membrane, and (3) there is no exchanger activity in the basal plasma membrane. © 1996 Wiley-Liss, Inc.     

Extracellular Allosteric Regulatory Subdomain within the γ Subunit of the Epithelial Na+ Channel

The activity of the epithelial Na⁺ channel (ENaC) is modulated by Na⁺ self-inhibition, a down-regulation of the open probability of ENaC by extracellular Na⁺. A His residue within the extracellular domain of γENaC (γHis²³⁹) was found to have a critical role in Na⁺ self-inhibition. We investigated the functional roles of residues in the vicinity of this His by mutagenesis and analyses of Na⁺ self-inhibition responses in Xenopus oocytes. Significant changes in the speed and magnitude of Na⁺ self-inhibition were observed in 16 of the 47 mutants analyzed. These 16 mutants were distributed within a 22-residue tract. We further characterized this scanned region by examining the accessibility of introduced Cys residues to the sulfhydryl reagent MTSET. External MTSET irreversibly increased or decreased currents in 13 of 47 mutants. The distribution patterns of the residues where substitutions significantly altered Na⁺ self-inhibition or/and conferred sensitivity to MTSET were consistent with the existence of two helices within this region. In addition, single channel recordings of the γH239F mutant showed that, in the absence of Na⁺ self-inhibition and with an increased open probability, ENaCs still undergo transitions between open and closed states. We conclude that γHis²³⁹ functions within an extracellular allosteric regulatory subdomain of the γ subunit that has an important role in conferring the response of the channel to external Na⁺.     

Na+ channels and amiloride-induced noise in the mammalian colon epithelium

(1) The effect of the Na⁺-channel blocker, amiloride, on the short-circuit current carried by Na⁺ was studied with fluctuation analysis, in rabbit descending colon epithelium. (2) In the presence of mucosal amiloride, the power spectrum of the Na⁺-current noise showed a Lorentzian component. When the Na⁺ current was reduced by increasing the blocker concentrations, the Lorentzian plateau decreased and corner frequency increased. Microscopic short-circuit current and current-noise data are evidence for a two-state mechanism of the blocker interaction with the Na⁺ channel. (3) On- and off-rate constants for the blocker-receptor reaction, single-channel currents and Na⁺-channel density were calculated at room temperature and at 37°C. Also, the activation energy for the amiloride-receptor reaction was estimated. The microscopic parameters obtained for the Na⁺ channel in the colon were similar to those found for Na⁺ channels in other tight epithelia.     

Ion transport across the skin of a larval salamander

The transport characteristics of the skin of neotenic Ambystoma tigrinum were investigated using ion substitution and circuit analysis. When bathed with sodium Ringer solution on both sides, a transepithelial potential of up to 50 mV (inside positive) and a short-circuit current (I_sc) of up to 10 μA/cm² were observed. When amiloride was added or Na⁺ was replaced by tetramethylammonium in the apical solution, I_sc was decreased from 3.7 ± 0.4 to 1.5 ± 0.2 μA/cm² (n = 10). When K⁺ replaced Na⁺, there was a smaller change in I_sc from 5.8 ± 0.6 to 3.7 ± 0.5 μA/cm² (n = 10). Although barium had no effect when added to 100 K Ringer on normal skin, it inhibited I_sc on skins taken from K⁺-loaded animals. Nystatin caused substantial increases in I_sc with either Na⁺ or K⁺ as the dominant cation in the apical solution. Current voltage analysis using amiloride was used to estimate the resistances and electromotive forces (EMF) associated with ion transport. The EMF for ion transport was partially dependent on K⁺ in the basolateral solution and it was similar to that observed in other epithelia. The resistance of the transport pathway was high, consistent with the low I_sc. These results suggest that there is an amiloride-sensitive Na⁺ channel in parallel with a small K⁺ conductance in the apical membrane of this preparation.     

Development of Na+ transport in the chicken colon

To evaluate the developmental changes in colonic Na⁺ transport, Na, K-ATPase activity and the sensitivity of the short-circuit current to amiloride were investigated. The amiloride-sensitive short-circuit current which represents the electrogenic, amiloride-sensitive Na⁺ transport through Na⁺ channels, was not present in chicken embryos but rose significantly after hatching in chicks which were kept on a low-salt diet. Amiloride-sensitive short-circuit current increased gradually but the plateau was not reached during the first 15 days of life. Drinking of 0.9% NaCl totally inhibited the induction of amiloride-sensitive Na⁺ transport. Na⁺, K⁺-ATPase activity increased during development but was not influenced by changes in salt intake. Na⁺ transport in chicken colon therefore undergoes profound developmental changes. The increase of Na⁺ transport refleets not only the adaptation of colonocytes to low salt intake but also the maturation of Na⁺ absorption in colon. The possible role of aldosterone in the adaptation to low-salt intake is discussed.Abbreviations LS low-salt - HS high-salt - I _sc short-circuit current     

Na+ channel activity in cultured renal (A6) epithelium: Regulation by solution osmolarity

Summary Solution osmolarity is known to affect Na⁺ transport rates across tight epithelia but this variable has been relatively ignored in studies of cultured renal epithelia. Using electrophysiological methods to study A6 epithelial monolayers, we observed a marked effect of solution tonicity on amiloride-sensitive Na⁺ currents (I _sc).I _sc for tissues bathed in symmetrical hyposmotic (170 mOsm), isosmotic (200 mOsm), and hyperosmotic (230 or 290 mOsm) NaCl Ringer's solutions averaged 25±2, 9±2, 3±0.4, and 0.6±0.5 A/cm², respectively. Similar results were obtained following changes in the serosal tonicity; mucosal changes did not significantly affectI _sc. The changes inI _sc were slow and reached steady-state within 30 min. Current fluctuation analysis measurements indicated that single-channel currents and Na⁺ channel blocker kinetics were similar for isosmotic and hyposmotic conditions. However, the number of conducting Na⁺ channels was approximately threefold higher for tissues bathed in hyposmotic solutions. No channel activity was detected during hyperosmotic conditions. The results suggest that Na⁺ channels in A6 epithelia are highly sensitive to relatively small changes in serosal solution tonicity. Consequently, osmotic effects may partly account for the large variability in Na⁺ transport rates for A6 epithelia reported in the literature.     

Effects of amiloride on the mechanical,electrical and biochemical aspects of ischemia-reperfusion injury

Although many causal factors have been proposed for the ischemia-reperfusion injury, the exact mechanisms for interdependent derangements of mechanical, electrical and metabolic events remains unclear. For this purpose, the Langendorff-perfused rat hearts were subjected to regional brief ischemia followed by reperfusion to study the protective effects of amiloride, an inhibitor of Na⁺–H⁺ exchange. Amiloride (0.1 mM) attenuated the rise in tissue Na⁺ and Ca²⁺, both duration and incidence of arrhythmias (p<0.05 vs. control), sarcolemmal injury (assessed by Na–K ATPase) and lipid peroxidation (assessed by malonedialdehyde formation) during reperfusion. Treatment of hearts with monensin, a sodium inophore, reversed the protective effects of amiloride. Reduction in transsarcolemmal Na⁺ and pH gradients during ischemia exhibited protective effects similar to those seen with amiloride. These results suggest that cardiac dysfunction, sarcolemmal injury and triggered arrhythmias during ischemia-reperfusion are due to the occurrence of intracellular Ca²⁺ overload caused by the activation of Na⁺–H⁺ exchange and Na⁺–Ca²⁺ exchange systems in the myocardium.     

Membrane currents in taste cells of the rat fungiform papilla. Evidence for two types of Ca currents and inhibition of K currents by saccharin

Taste buds were isolated from the fungiform papilla of the rat tongue and the receptor cells (TRCs) were patch clamped. Seals were obtained on the basolateral membrane of 281 TRCs, protruding from the intact taste buds or isolated by micro-dissection. In whole-cell configuration 72% of the cells had a TTX blockable transient Na inward current (mean peak amplitude 0.74 nA). All cells had outward K currents. Their activation was slower than for the Na current and a slow inactivation was also noticeable. The K currents were blocked by tetraethylammonium, Ba, and 4-aminopyridine, and were absent when the pipette contained Cs instead of K. With 100 mM Ba or 100 mM Ca in the bath, two types of inward current were observed. An L-type Ca current (ICaL) activated at -20 mV had a mean peak amplitude of 440 pA and inactivated very slowly. At 3 mM Ca the activation threshold of ICaL was near -40 mV. A transient T-type current (ICaT) activated at -50 mV had an average peak amplitude of 53 pA and inactivated with a time constant of 36 ms at -30 mV. ICaL was blocked more efficiently by Cd and D600 than ICaT. ICaT was blocked by 0.2 mM Ni and half blocked by 200 microM amiloride. In whole-cell voltage clamp, Na-saccharin caused (in 34% of 55 cells tested) a decrease in outward K currents by 21%, which may be expected to depolarize the TRCs. Also, Na-saccharin caused some taste cells to fire action potentials (on-cell, 7 out of 24 cells; whole-cell, 2 out of 38 cells responding to saccharin) of amplitudes sufficient to activate ICaL. Thus the action potentials will cause Ca inflow, which may trigger release of transmitter.     

The effects of amiloride on the labellar taste receptor cells of the fleshfly Boettcherisca peregrina

Amiloride is known to inhibit the taste response of vertebrates to salt by blocking the amiloride-sensitive sodium channel. In this study, we investigated electrophysiologically the effect of amiloride on the taste response of the fleshfly Boettcherisca peregrina. When 0.5 mM amiloride was included in taste solutions, the response of the salt receptor cell (salt response) to sodium chloride (NaCl) was not depressed but those of the sugar receptor cell (sugar responses) to sucrose, glucose, fructose, l-valine (l-Val) and l-phenylalanine (l-Phe) were strongly depressed. An inhibitory effect of amiloride on the concentration-response relationship for both sucrose and l-Phe was clearly revealed, but not at high concentrations of sucrose. After pretreatment of a chemosensory seta with 0.15 mM amiloride for 10 min, the salt response to NaCl was not affected. On the other hand, the sugar responses to sucrose, fructose, l-Val and l-Phe were depressed just after amiloride pretreatment. The sugar response to adenosine 5’-diphosphate (ADP) mixed with 0.5 mM amiloride was not depressed, but the response to ADP alone was depressed after amiloride pretreatment. It was therefore observed that amiloride depressed the responses to all stimulants that react with each of the receptor sites of the sugar receptor cell.