A quinine-activated cationic conductance in vertebrate taste receptor cells

The chincona alkaloid quinine is known to be a bitter tasting substance for various vertebrates. We examined the effects of quinine on isolated taste receptor cells from the bullfrog (Rana catesbeiana). Membrane currents were recorded by whole-cell recording, while quinine hydrochloride was applied extracellularly from a puffer pipette. At the resting potential (-77 +/- 9 mV, mean +/- SD, n = 49 cells), taste cells generated inward currents in response to quinine stimulation (> 1 mM), indicating a depolarizing response in the taste cells. Two types of current responses were observed; a newly found quinine-activated cationic conductance and a previously reported blocking effect of quinine on K+ conductances. The cationic current was isolated from the K+ current by using a Cs(+)-containing patch pipette. The relative permeabilities (Pion) of the quinine-activated cationic conductance were: PNa/PK/PCs = 1:0.5:0.42. The quinine dose-response relation was described by the Hill equation with the K1/2 of 3.6 mM and Hill coefficient of 5.3. When extracellular [Ca2+] (1.8 mM) was reduced to nominally free, the conductance was enhanced by about sixfold. This property is consistent with observations on quinine responses recorded from the gustatory nerve, in vivo. The quinine-induced cationic current was decreased with an application of 8-bromo-cAMP. We conclude that the bitter substance quinine activates a cation channel in taste receptor cells and this channel plays an important role in bitter taste transduction.     

Pancuronium inactivates alamethicin-induced conductance in artificial membranes.

The effect of pancuronium on alamethicin-induced currents was studied in negatively charged lipid bilayer membranes. Pancuronium induces inactivation of the alamethicin-induced current. Inactivation is only observed if this compound is added to the compartment containing alamethicin. Moreover, the process of inactivation is reduced or abolished if pancuronium is added to the alamethicin-free side of the membrane. The time needed to recover from inactivation is greatly reduced if the aqueous solution in the alamethicin-free compartment is stirred. These data suggest that pancuronium permeates through the membrane when the alamethicin-induced conductance is "turned on," binds to the other membrane surface, and changes the surface potential.     

Pressure effects on alamethicin conductance in bilayer membranes.

We report here the first observations of the effects of elevated hydrostatic pressure on the kinetics of bilayer membrane conductance induced by the pore-forming antibiotic, alamethicin. Bacterial phosphatidylethanolamine-squalene bilayer membranes were formed by the apposition of lipid monolayers in a vessel capable of sustaining hydrostatic pressures in the range, 0.1-100 MPa (1-1,000 atm). Principal observations were (a) the lifetimes of discrete conductance states were lengthened with increasing pressure, (b) both the onset and decay of alamethicin conductance accompanying application and removal of supra-threshold voltage pulses were slowed with increasing pressure, (c) the onset of alamethicin conductance at elevated pressure became distinctly sigmoidal, suggesting an electrically silent intermediate state of channel assembly, (d) the magnitudes of the discrete conductance levels observed did not change with pressure, and, (e) the voltage threshold for the onset of alamethicin conductance was not altered by pressure. Apparent activation volumes for both the formation and decay of conducting states were positive and of comparable magnitude, namely, approximately 100 A3/event. Observation d indicates that channel geometry and the kinetics of ion transport through open channels were not affected by pressure in the range employed. The remaining observations indicate that, while the relative positions of free-energy minima characterizing individual conducting states at a given voltage were not modified by pressure, the heights of intervening potential maxima were increased by its application.     

Human epithelial cystic fibrosis transmembrane conductance regulator without exon 5 maintains partial chloride channel function in intracellular membranes.

The cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR) is a splice variant of the epithelial CFTR, with lacks 30 amino acids encoded by exon 5 in the first intracellular loop. For examination of the role of exon 5 in CFTR channel function, a CFTR deletion mutant, in which exon 5 was removed from the human epithelial CFTR, was constructed. The wild type and delta exon5 CFTR were expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (approximately 170 kDa) was immunoprecipitated from cells transfected with wild type CFTR cDNA, whereas cells transfected with delta exon5 CFTR express only a core-glycosylated from (approximately 140 kDa). The Western blot test performed on subcellular membrane fractions showed that delta exon5 CFTR was located in the intracellular membranes. Neither incubation at lower temperature (26 degrees C) nor stimulation of 293 HEK cells with forskolin or CPT-cAMP caused improvement in glycosylation and processing of delta exon5 CFTR proteins, indicating that the human epithelial CFTR lacking exon5 did not process properly in 293 HEK cells. On incorporation of intracellular membrane vesicles containing the delta exon5 CFTR proteins into the lipid bilayer membrane, functional phosphorylation- and ATP-dependent chloride channels were identified. CFTR channels with an 8-pS full-conductance state were observed in 14% of the experiments. The channel had an average open probability (Po) of 0.098 +/- 0.022, significantly less than that of the wild type CFTR (Po = 0.318 +/- 0.028). More frequently, the delta exon5 CFTR formed chloride channels with lower conductance states of approximately 2-3 and approximately 4-6 pS. These subconductance states were also observed with wild type CFTR but to a much lesser extent. Average Po for the 2-3-pS subconductance state, estimated from the area under the curve on an amplitude histogram, was 0.461 +/- 0.194 for delta exon5 CFTR and 0.332 +/- 0.142 for wild type (p = 0.073). The data obtained indicate that deleting 30 amino acids from the first intracellular loop of CFTR affects both processing and function of the CFTR chloride channel.     

A synthetic peptide based on a glycine-gated chloride channel induces a novel chloride conductance in isolated epithelial cells

CK(4)-M2GlyR, an aqueous soluble peptide derived from the transmembrane M2 segment of the glycine-gated Cl(-) channel found in postsynaptic membranes of the central nervous system, has previously been shown to increase transepithelial Cl(-) and fluid secretion of epithelial monolayers. The goal of this study was to determine whether CK(4)-M2GlyR exerts these effects via formation of a novel chloride conductance pathway, modulation of endogenous chloride channel activity, or a combination of these effects. Ionic currents were recorded from isolated epithelial cells before and after treatment with the peptide using the whole-cell configuration of the patch-clamp technique. CK(4)-M2GlyR increased whole-cell Cl(-) currents in all epithelial cell lines that were studied, including: Madin-Darby canine kidney cells, a human colonic epithelial cell line (T84), and airway epithelial cells derived from a human cystic fibrosis patient (IB3-1). No evidence was found for modulation of endogenous Cl(-) channels by CK(4)-M2GlyR based on both the electrophysiological properties of the observed currents and the pharmacological profile of the CK(4)-M2GlyR-induced current. These results suggest that CK(4)-M2GlyR increases Cl(-) permeability in epithelial cells directly, by forming a distinct conduction pathway in cell membranes.     

Zinc localization in taste bud membranes

Zinc was measured by flame aspiration atomic absorption spectrophotometry in homogenates and in enriched fractions and subfractions from bovine taste bud membranes and from surrounding control tissues that contained no taste buds. Zinc was found in significantly higher concentrations in tissues containing taste buds and increased in concentration as biochemical and electron microscopic purity increased. The role of zinc in taste bud membranes could relate to its role in membrane stabilization or to its activity in alkaline phosphatase, a zinc-dependent enzyme whose specific activity increased in taste bud membranes in the same manner as did zinc concentration.     

Voltage-induced conductance in human erythrocyte membranes

Isotonic suspensions of erythrocytes were exposed to intense electric fields for a duration in microseconds. Time-dependent increase in the conductivity of the suspension was observed under fields greater than a threshold of about 1.5 kV/cm. The threshold was independent of the ionic strength of the medium, and changed little with temperature or with the rise time of the applied field. Under fields greater than 3 kV/cm, the time course of the conductivity increase consisted of a rapid (approx. 1 μs) and a slow (approx. 100 μs) phases. The increase is attributed primarily to large membrane conductance induced by the applied field. The membrane conductance is in the order of $\text{10 Ω}{}^{\mathrm{?1}}\text{/}\text{cm}{}^2$ in the rapid phase and $\text{10}{}^2\text{Ω}{}^{\mathrm{?1}}\text{/}\text{cm}{}^2$ in the slow phase. Comparison with previous results indicates that this induced membrane conductance corresponds to the formation of aqueous pores in the cell membrane. After the applied field was removed, the conductivity of the suspension returned nearly to its initial value, indicating that the induced membrane conductance is strongly dependent on the membrane potential. The conductivity then increased again in the time range of 10 s. This is attributed to the diffusional efflux of intracellular ions through the voltage-induced pores. From the rate of the efflux, number of the pores/cell is estimated to be in the order of 10². Final stage of the conductivity change was a slow decrease, corresponding to the colloid osmotic swelling of the perforated cells.     

A three state model for alamethicin conductance in bilayer membranes

Alamethicin is an antibiotic which produces voltage gated channels in lipid bilayer membranes. Recently completed studies of the pressure dependence of alamethicin conductance have shown that its onset following application of a suprathreshold voltage step at a pressure of 100 MPa (1000 atm) is markedly slowed relative to that observed at ambient pressure. Furthermore, the time course of the onset of conductance becomes distinctly sigmoidal at elevated pressure, a condition which is not evident at atmospheric pressure. The decay of alamethicin conductance upon removal of suprathreshold applied voltage is also slowed by application of hydrostatic pressure, but it follows a single exponential time course at all pressures. In addition, kinetic parameters characterizing the onset and decay of conductance show distinctly different pressure dependences. These observations cannot be explained by a two state model in which alamethicin moves reversibly between nonconducting and conducting states. Therefore we re-examine critically a hypothesis made by previous workers, namely that alamethicin, in monomeric or aggregate form, moves upon application of suprathreshold voltage first from a nonconducting surface state to a nonconducting preassembly or precursor state, and then finally into a conducting state. Parameters of this three state model are related to a geometric factor which measures the degree of sigmoidal conductance response and which can be evaluated directly from experimental data. An alternative aggregation-type analysis, equivalent to that applied by Hodgkin & Huxley to the potassium conductance in squid axon, is also considered in the context of this same geometric factor. The possibility of distinguishing between these analyses on the basis of experimental data is discussed.     

Proton conductance of cell membranes

Several workers have suggested that cell membranes have a high proton conductance. Our interest in this concept arose from the possibility that the nutrient (submucosal-facing) membrane of the gastric mucosa may have a high proton or hydroxyl ion conductance which would play a role in the regulation of the acid-base balance of the cell. We found that wide changes in the H⁺ concentration of the fluid bathing the nutrient side of the in vitro frog gastric mucosa did not result in significant changes in p.d. However, a maintained change of the H⁺ concentration of the bathing fluid would be expected to produce only a temporary change in p.d. Since a diffusion barrier is present on the nutrient side the temporary change in p.d. might be masked. An analysis of this possibility was made on the basis of a conceptual model and as a result of the analysis it is concluded that the proton (and/or OH^?) conductance of the nutrient membrane of the frog gastric mucosa is not a significant fraction of its total conductance. The present status of the proton conductance hypothesis with respect to striated muscle and to the secretory membrane of the gastric mucosa is reviewed.     

Channels in epithelial cell membranes and junctions.

Epithelia may be classified as "tight" or "leaky," depending on whether there is a significant pathway for transepithelial ion permeation via the junctions and bypassing the cells. The resistance of this paracellular channel may depend partly on structures visible in the electron microscope, partly on wall charge. Permeability determinations in the leaky junctions of gallbladder epithelium, using many different organic cations, suggest that the critical barriers barriers to ion permeation are 5--8 A in radius and bind cations by up to four strongly proton-accepting oxygens. The apical cell membrane of tight epithelia contains a Na+-selective channel that is blocked by amiloride and Ca2+, subject to negative feedback control by the Na+ pump in the basolateral membrane, and somehow promoted by aldosterone. To determine the permeabilities of these two channels (the junctional channel of leaky epithelia, and the Na+ channel of tight epithelia) to water and nonelectrolytes remains a major unsolved problem.     

Space charge-limited conductance in lipid bilayer membranes

Summary A mathematical treatment is given for the flux of ions of one charge sign across lipid bilayer membranes. This treatment is a generalization of a previous analysis of the membrane conductance by D. Walz, E. Bamberg and P. Läuger which was restricted to systems with negligible space charge in the membrane. The present theory includes space charge effects, and it is no longer assumed that the electric field strength in the membrane is constant. It is found that the ohmic membrane conductivity ₀ is reduced by space charges; if only ions of one charge sign are soluble in the membrane, ₀ approaches a limiting value for increasing concentration of the permeable ion in the aqueous solution. The theory also predicts the range in which the constant field approximation is valid. It is found that space charge effects become predominant when the mean concentration of the permeable ion in the membrane exceeds 5×10^–5 m. The currentvoltage characteristic of the membrane remains practically linear even in the presence of a high space charge. It is therefore concluded that the experimentally observed nonlinearity is caused mainly by the distortion of the potential energy profile of an ion due to image forces.     

Step conductance increases in bilayer membranes induced by antibody-antigen-complement action.

A sharp rise in the electrical conductance of lipid bilayer membranes was observed following the addition of antigen (bovine serum), antibody (rabbit anti-bovine serum), and complement to the neighboring aqueous phases. At low concentrations, step increases in the conductivity occurred which are consistent with the appearance of about 2.2 nm holes in the membrane. Probably attack or lysis of the lipid bilayer by complement is responsible.     

Hypoosmotic stimuli activate a chloride conductance in rat taste cells

The oral cavity is subjected to a wide range of osmotic conditions, yet little is known about how solution osmolarity affects performance of the gustatory system. In order to elucidate the mechanism by which hypoosmotic stimuli affect the peripheral taste system, I have attempted to characterize the effects of hypoosmotic stimuli on individual rat taste receptor cells (TRCs) using whole-cell patch clamp recording. Currents elicited in response to voltage ramps (-90 to +60 mV) were recorded in control saline and in solutions varying only in osmolarity (-30, -60 and -90 mOsm). In roughly two-thirds of cells, hypoosmotic solutions (230 mOsm) caused a 15% increase in cell capacitance and activated a reversible conductance that exhibited marked adaptation in the continued presence of the stimulus. Similar responses could be elicited in taste cells from taste buds in the foliate and vallate papillae, the soft palate, the nasopharynx and the epiglottis. Ion substitution experiments were consistent with the interpretation that the predominant ion carried through these apparent volume- or stretch-activated channels was Cl(-) under normal conditions. Reversal potentials for the hypoosmotic-induced current closely matched those predicted by the Goldman-Hodgkin-Katz constant field equation for a Cl(-) conductance. The relative permeability sequence of the hypoosmotic-activated current in TRCs was thiocyanate(-) > or = l(-) > or = Br(-) > Cl(-) > or = F(-) > or = isethionate(-) > gluconate(-). Pharmacological experiments revealed that this Cl(-) conductance was inhibited by 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid and 5-nitro-3-(3-phenyl-propylamino)benzoic acid (EC(50) = 1.3 and 4.6 microM, respectively), but not by CdCl(2) (300 microM) nor GdCl(3) (200 microM). I hypothesize that this hypoosmotic-activated Cl(-) conductance, which is similar to the well-characterized swelling-activated Cl(-) current, may contribute to volume regulation and could represent the transduction mechanism by which the presence of hypoosmotic stimuli, including water, may be signaled in taste receptor cells.     

Biochemical studies of taste sensation. XI. Isolation, characterization and taste ligand binding activity of plasma membranes from catfish taste tissue

Variants of creatine kinase-MM (variant of ATP:creatine N-phosphotransferase, EC 2.7.3.2), present in human heart and skeletal muscle, have been purified to homogeneity using DEAE-Sepharose column chromatography and column chromatofocusing techniques. Creatine kinase-MM I-IV were present in both heart and skeletal muscle, while MM-V was found only in heart. The number, ratio and elution profile of the variants during chromatofocusing remained identical even when they were purified in the presence of proteinase inhibitors. MM-I-V, on chromatofocusing, were eluted at pH 8.3, 7.9, 7.6, 7.2 and 6.8, respectively. Isoelectric focusing revealed the pI of MM-I-V to be 7.2, 6.9, 6.7, 6.4 and 6.2. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed a doublet pattern for creatine kinase-MM variants III-V. However, polyacrylamide gel electrophoresis without SDS indicated homogeneity because each variant showed a single band. The doublet pattern observed in the presence of SDS may reflect the presence of two subunits of slightly different mass.