MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons

Gamma-aminobutyric acid(GABA)ergic neurons,the most abundant inhibitory neurons in the human brain,have been found to be reduced in many neurological disorders,including Alzheimer's disease and Alzheimer's disease-related dementia.Our previous study identified the upregulation of microRNA-502-3p(miR-502-3p)and downregulation of GABA type A receptor subunitα-1 in Alzheimer's disease synapses.This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function.In vitro studies were perfo rmed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs.In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunitα-1 mRNA.Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunitα-1 gene and suppresses the luciferase activity.Furthermore,quantitative reve rse transcription-polymerase chain reaction,miRNA in situ hybridization,immunoblotting,and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunitα-1 level,while suppression of miR-502-3p increased the level of GABA type A receptor subunitα-1 protein.Notably,as a result of the overexpression of miR-502-3p,cell viability was found to be reduced,and the population of necrotic cells was found to be increased.The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney(HEK)recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function,suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function.Additionally,the levels of proteins associated with Alzheimer s disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression.The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p.We propose that micro-RNA,in particular miR-502-3p,could be a potential therapeutic to rget to modulate GABAergic synapse function in neurological disorders,including Alzheimer's disease and Alzheimer's diseaserelated dementia.

Action Mechanism Research of Lanthanons to Slow Vacuolar Ion Channels in Raphanus Satirus L.(Xinlimei) Radish by Patch-Clamp

We used whole-vacuolar patch-clamp recording mode to study the action mechanism of La3+ to Slow Vacuolar (SV) channels for the first time. We recorded SV channel currents of Xinlimei (Raphanus satirus L.) vacuolars. The minimum activation potentials of voltage-dependent SV channels tied in 25+/-5 mV. The increase in cytoplasmic Ca2+ led to enhancement of SV-type currents. It was found that the threshold potential of activation shifted towards more depolarized values whenever cytoplasmic Ca2+ was increased. When 10(-10) mol/L free La3+ was added to the bath, SV-type current was suppressed by 60 similar to 75%. These data showed La3+ reduced ion permeabilities of Xinlimei root vacuolar membrane.

Study of Oligonucleotide Fixation on Bilayer Lipid Membranes by Patch-clamp Pipette Support

One kind of novel BLMs was fabricated by patch-clamp pipette technology characterized in considerably sensitive to changes of electrochemical parameters.Detectiye currents and voltage presented linear relationship when BLMs was formed and it could be confirmed by Gramicidin method.Ion current was increased by dihexyl (C_ (12)) modified ssDNA fixed on the BLMs and also indicated linear relationship to ssDNA's concentration due to the interaction of (C_ 12)-ssDNA and BLMs.Further more,the regression equations were different from BLMs fixed with ssDNA probe and a blank control BLM in the same experimental conditions.The ssDNA probe was successfully fixed on patch-clamp pipette supported-BLMs.Based on our studies,a biosensor with reactive element of patch-clamp pipette-supported BLMs has been established.

L-and T-type Ca^(2+)channels dichotomously contribute to retinal ganglion cell injury in experimental glaucoma

Retinal ganglion cell apoptotic death is the main pathological characteristic of glaucoma,which is the leading cause of irreversible blindness.Disruption of Ca^(2+)homeostasis plays an important role in glaucoma.Voltage-gated Ca^(2+)channel blockers have been shown to improve vision in patients with glaucoma.However,whether and how voltage-gated Ca^(2+)channels are involved in retinal ganglion cell apoptotic death are largely unknown.In this study,we found that total Ca^(2+)current densities in retinal ganglion cells were reduced in a rat model of chronic ocular hypertension experimental glaucoma,as determined by whole-cell patch-clamp electrophysiological recordings.Further analysis showed that L-type Ca^(2+)currents were downregulated while T-type Ca^(2+)currents were upregulated at the later stage of glaucoma.Western blot assay and immunofluorescence experiments confirmed that expression of the Ca_(V)1.2 subunit of L-type Ca^(2+)channels was reduced and expression of the Ca_(V)3.3 subunit of T-type Ca^(2+)channels was increased in retinas of the chronic ocular hypertension model.Soluble tumor necrosis factor-α,an important inflammatory factor,inhibited the L-type Ca^(2+)current of isolated retinal ganglion cells from control rats and enhanced the T-type Ca^(2+)current.These changes were blocked by the tumor necrosis factor-αinhibitor XPro1595,indicating that both types of Ca^(2+)currents may be mediated by soluble tumor necrosis factor-α.The intracellular mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and nuclear factor kappa-B signaling pathway mediate the effects of tumor necrosis factor-α.TUNEL assays revealed that mibefradil,a T-type calcium channel blocker,reduced the number of apoptotic retinal ganglion cells in the rat model of chronic ocular hypertension.These results suggest that T-type Ca^(2+)channels are involved in disrupted Ca^(2+)homeostasis and apoptosis of retinal ganglion cells in glaucoma,and application of T-type Ca^(2+)channel blockers,especially a specific CaV3.3 blocker,may be a potential strategy for the treatment of glaucoma.

Two outward potassium current types are expressed during the neural differentiation of neural stem cells

The electrophysiological properties of potassium ion channels are regarded as a basic index for determining the functional differentiation of neural stem cells. In this study, neural stem cells from the hippocampus of newborn rats were induced to differentiate with neurotrophic growth factor, and the electrophysiological properties of the voltage-gated potassium ion channels were observed. Immunofluorescence staining showed that the rapidly proliferating neural stem cells formed spheres in vitro that expressed high levels of nestin. The differentiated neurons were shown to express neuron-specific enolase. Flow cytometric analysis revealed that the neural stem cells were actively dividing and the percentage of cells in the S ＋ G2/M phase was high. However, the ratio of cells in the S ＋ G2/M phase decreased obviously as differentiation proceeded. Whole-cell patch-clamp re- cordings revealed apparent changes in potassium ion currents as the neurons differentiated. The potassium ion currents consisted of one transient outward potassium ion current and one delayed rectifier potassium ion current, which were blocked by 4-aminopyridine and tetraethylammonium, respectively. The experimental findings indicate that neural stem cells from newborn rat hippo- campus could be cultured and induced to differentiate into functional neurons under defined condi- tions in vitro. The differentiated neurons expressed two types of outward potassium ion cur＇ents similar to those of mature neurons in vivo.

Inhibitory Effects of Neferine on Na_v1.5 Channels Expressed in HEK293 Cells

Neferine, a bisbenzylisoquinoline alkaloid in Lotus Plumule, was proved to have a wide range of biological activities. In the present study, using whole-cell patch-clamp technique, we investigated the effects of neferine on Nav1.5 channels that are stably expressed in HEK 293 cells. We found that neferine potently and reversibly inhibited Nav1.5 currents in a concentration dependent manner with a half-maximal inhibition（IC50） being 26.15 μmol/L. The inhibitory effects of neferine on Nav1.5 currents were weaker than those of quinidine at the same concentration. The steady-state inactivation curve was significantly shifted towards hyperpolarizing direction in the presence of 30 μmol/L neferine, while the voltage-dependent activation was unaltered. Neferine prolonged the time to peak of activation, increased the inactivation time constants of Nav1.5 currents and markedly slowed the recovery from inactivation. The inhibitory effect of neferine could be potentiated in a frequency-dependent manner. These results suggested that neferine can block Nav1.5 channels under the open state and inactivating state and it is an open channel blocker of Nav1.5 channels.

Effects of antigliomatin from the scorpion venom of Buthus martensii Karsch on chloride channels on C6 glioma cells

Using whole-cell patch-clamp recordings, the effects of antigliomatin were observed on chloride channels on C6 glioma cells cultured in vitro. Antigliomatin was extracted from the venom of the scorpion Buthus martensii Karsch. Chloride channels are closed under normal osmotic pressure. When osmotic pressure was reduced to 120, 110 and 100 mV, the cell volume enlarged, chloride channels opened, and the chloride channel current increased. Three minutes after antigliomatin treatment, the chloride channel current decreased in a dose-dependent manner. These results show that antigliomatin extracted from the venom of the scorpion Buthus martensii Karsch diminishes chloride channel currents on C6 glioma cells.

Effects of simvastatin on ion channel currents in ventricular myocytes from rabbit with acute myocardial infarction

Objective To investigate the effects of simvastatin on membrane ionic currents in left ventricular myocytes after acute myocardial infarction (AMI,so as to explore the ionic mechanism of statin treatment for antiarrhythmia.Methods Fourty-five New Zeland rabbits were randomly divided into three groups:AMI group,simvastatin intervention group （statin group） and sham-operated control group （CON）.Rabbits were infarcted by ligation of the left anterior descending coronary artery after administration of oral simvastatin 5 mg·kg-1·d-1 （Statin group） or placebo （AMI group）for 3 days.Twenty-four hours later,single ventricular myocytes were isolated enzymatically from the epicardial zone of the infracted region.Whole cell patch clamp technique was used to record membrane ionic currents,including sodium current (INa),L-type calcium current (ICa-L) and transient outward potassium current (Ito).Results①There was no significant difference in serum cholesterol concentration among three groups.②The peak INa current density （at-30 mV） was significantly decreased in AMI group （-23.26±5.18） compared with CON （-42.78±5.48,P【0.05）,while it was significantly increased in Statin group （-39.23±5.45） compared with AMI group （P【0.01）;The peak ICa-L current density （at 0 mV） was significantly decreased in AMI group （-3.23±0.91） compared with CON （-4.56±1.01,P【0.05）,while it was significantly increased in Statin group （- 4.18±0.95） compared with AMI group （P【0.05）;The Ito current density（at +60 mV） was significantly decreased in AMI group（10.41±1.93）compared with CON （17.41±3.13,P【0.01）,while it was significantly increased in Statin group（16.21±2.42）compared with AMI group （P【0.01）.Conclusions AMI induces significant down-regulation of INa,ICa-L and Ito.Pretreatment with simvastatin could attenuate this change without lowering the serum cholesterol level,suggesting that simvastatin reverse this electrical remodeling,thus contributing to the ionic mechanism of statin treatment for antiarrhythmia.

Induced neuronal differentiation of neural stem cells by a combination of cytokines One-step versus two-step methods

BACKGROUND： A combination of basic fibroblast growth factor （bFGF）, platelet-derived growth factor （PDGF）, human heregulin-beta-1, beta-mercaptoethanol retinoic acid and forskolin has been reported to induce the differentiation of rat bone marrow stromal cells into myelinating Schwann-like cells. OBJECTIVE： To investigate the inducing effects of a combination of bFGF, PDGF, human heregulin-beta-1, beta-mercaptoethanol retinoic acid and forskolin on neural stem cell differentiation by one- and two-step methods. DESIGN, TIME AND SETTING： A cytobiology experiment was performed at the Department of Histology and Embryology, Medical School of Nantong University, and Jiangsu Province Key Laboratory of Neuroregeneration, China, between August 2005 and January 2007. MATERIALS： A total of 30 healthy Sprague Dawley rat embryos at gestational days 14-16 were selected, bFGF, PDGF, human heregulin-beta-t, beta-mercaptoethanol, retinoic acid, and forskolin were purchased from Sigma, USA. METHODS： Passage 3 rat neural stem cells were cultured by a one-step method in serum-free medium plus 10 ng/m/bFGF, 5 ng/mL PDGF, 200 ng/mL heregulin-beta-1,35 ng/mL all-trans retinoic acid, and 5 pmol/L forskolin or by a two-step method in serum-free medium plus 35 ng/m/ all-trans retinoic acid for 72 hours, followed by serum-free medium plus 10 ng/mL bFGF, 5 ng/mL PDGF, 200 ng/mL heregulin-beta-t and 5 μmol/L forskolin. The control condition consisted of 10% fetal bovine serum alone or 20 ng/mL bFGF alone. MAIN OUTCOME MEASURES： Differentiated cells were identified by immunocytochemical staining for microtubule associate protein-2 （MAP2） and St 00 protein. Geometric parameters and sodium ion currents of the differentiated cells were measured by image analysis and whole-cell patch-clamp techniques, respectively. RESULTS： Compared with the two-step culture method, neuronal-like cells exhibited longer processes and a similar appearance to mature neurons using the one-step method. The percentage of MAP2 positive cells induced by the one-step method was significantly greater than the serum-alone group （P 〈 0.05）. Furthermore, the MAP2 positive cells induced by the one-step method had greater surface areas, cell body perimeters, and longer process than cells induced by serum-alone and bFGF-alone （P 〈 0.05）. There were no significant differences in these parameters between the one-step and two-step methods （P 〉 0.05）. In addition, 80% of the induced neuronal-like cells from the one-step method and 20% from the two-step method displayed inwardly-evoked currents. CONCLUSION： The combination of bFGF, PDGF, human heregulin-beta-t, beta-mercaptoethanol retinoic acid and forskolin successfully induced neuronal differentiation from neural stem cells, with the one-step induction being more effective than the two-step method.

Roles of the ocular pressure, pressure-sensitive ion channel, and elasticity in pressure-induced retinal diseases

The intraocular pressure inside the human eye maintains 10–21 mmHg above the atmospheric pressure.Elevation of intraocular pressure is highly correlated with the retinopathy in glaucoma,and changes in the exterior pressure during mountain hiking,air traveling,and diving may also induce vision decline and retinopathy.The pathophysiological mechanism of these pressure-induced retinal disorders has not been completely clear.Retinal neurons express pressure-sensitive channels intrinsically sensitive to pressure and membrane stretch,such as the transient receptor potential channel(TRP)family permeable to Ca^2+and Na^+and the two-pore domain K channel family.Recent data have shown that pressure excites the primate retinal bipolar cell by opening TRP vanilloid 4 to mediate transient depolarizing currents,and TRP vanilloid 4 agonists enhance the membrane excitability of primate retinal ganglion cells.The eyeball wall is constructed primarily by the sclera and cornea of low elasticity,and the flow rate of the aqueous humor and intraocular pressure both fluctuate,but the mathematical relationship between the ocular elasticity,aqueous humor volume,and intraocular pressure has not been established.This review will briefly review recent literature on the pressure-related retinal pathophysiology in glaucoma and other pressure-induced retinal disorders,the elasticity of ocular tissues,and pressure-sensitive cation channels in retinal neurons.Emerging data support the global volume and the elasticity and thickness of the sclera and cornea as variables to affect the intraocular pressure level like the volume of the aqueous humor.Recent results also suggest some potential routes for TRPs to mediate retinal ganglion cell dysfunction:TRP opening upon intraocular pressure elevation and membrane stretch,enhancing glutamate release from bipolar cells,increasing intracellular Na^+,Ca^2+concentration in retinal ganglion cells and extracellular glutamate concentration,inactivating voltage-gated Na^+channels,and causing excitotoxicity and dysfunction of retinal ganglion cells.Further studies on these routes likely identify novel targets and therapeutic strategies for the treatment of pressure-induced retinal disorders.

2-aminoethoxydiphenyl borate or lanthanum potentiates transient receptor potential-like channels in rat CA1 hippocampal neurons

Expression of transient receptor potential （TRP） channels is widespread with transcripts distributed throughout the brain. All TRP channel subunits are activated following phospholipase C activation and form cation-selective ion channels. Previous studies examining the existence of TRP channels in hippocampal CA1 pyramidal neurons were based on cultured neurons. Therefore, their relevance for living tissue remains unclear. In the present study, patch-clamp recordings were conducted from CA1 pyramidal neurons in hippocampal slices from 7-day-old rats. Whole-cell currents were obtained from CA1 hippocampal neurons with potentiation effects of 2-aminoethoxydiphenyl borate and lanthanum, revealing that recorded experimental currents were characteristic TRP-like channel currents. Identification of rat hippocampal mRNA transcripts of TRPC4, TRPC5, TRPV1, TRPV2, and TRPV3 channels further verified the expression of characteristic TRP-like channels on rat CA1 hippocampal neurons.

Effect of Shenmai Injection on L-type Calcium Current of Diaphragmatic Muscle in Rats

In this study, whole cell patch clamp recording technique was employed to investigate the effect of Shenmai Injection (SMI) on L-type calcium current ofdiaphragmatic muscle in rats. The result showed that when the diaphragmatic muscle cell was held at —80 mV and depolarized to +60 mV, 10 μl/ml, 50 μl/ml and 100 μl/ml SMI enhanced the inner peak L-type calcium current from -(6.8±0.7) pA/pF (n=7) to -(7.3±0.8) pA/pF (P>0.05, n=7), -(8.6±1.0) pA/pF (P<0.05, n=7) and -(9.4±1.2) pA/pF (P<0.05, n=7), respectively. The rates of L-type calcium current were increased by (7.34±2.37) %, (25.72±5.94)% , and (38.16±7.33)% , respectively. However, it had no significant effect on maximal activation potential and reversal potential. Our results suggested that SMI could activate the calcium channel of the diaphragmatic fibers of the rats, increase the influx of Ca 2+, and enhance the contractility of diaphragmatic muscles.

Potassium channel in peripheral blood lymphocytes of turbot (Scophthalmus maximus)

In order to provide pertinent evidence of ion channel with immune response in the fish, whole cell patch-clamp technique was employed for potassium ion channel study in turbot （Scophthalmus maximus）. Lymphocytes were isolated by Percoll density gradient centrifugation from peripheral blood samples, and electrophysiological characters of the channel were analyzed. In the recorded cells, activated voltage of the channels was -42.5±3.7 mV and the average peak current was 313.12±28.2 pA. The channel was identified as voltage dependent, the current was outward and it could be inhibited by 10 mmol/dma TEA or 5 mmol/dm^3 4-AP, a specific potassium channel inhibitor, identifying the existence of potassium channel in peripheral lymphocytes of the turbot.

Effect of Radiofrequency Catheter Ablation on Calcium Channel of Ventricular Myocytes

Using whole-cell patch-clamp technique,the effect of radiofrequency catheter ablation(RFCA)on calcium current(Ica)of guinea pig ventricular myocytes was examined.The radiofrequency energy delivered was 20W×10 s.RFCA decreased Ica apparently with the affected area reaching up to 1.2 cm from the ablation focus.In the meanwhile,the pathological lesion size resulted from RFCA was merely 0.41±0.11 cm.These findings indicate that RFCA,apart from causing tissue necrosis by heat,can affect myocyte membrane currents in a large area.This may explain why RFCA has a very high success rate with a small pathological lesion.

CHARACTERISTICS OF ACTIONPOTENTIAL AND THEIR UNDERLYING OUTWARD CURRENTS IN MAMMALIAN TASTE RECEPTOR CELLS

Many rat taste receptor cells conduct action potentials(APs).APs had a mean threshold of -35 mV(n=95 cells)and a spike height of 52mV above threshold in current clamp(hold= -80mV).Aps could be classified into two significantly different (P<0.001) groups-fast,with short half-time durations and large outward currents (mean1.3 ms and 2.7nA),and slow,with long duration and small outward currents(mean9.2ms and 0. 29nA).AP upstrokes were conducted by TTX-sensitive sodium currents whereas the downstroke by TEA-blockable outward currents. Voltage dependent analysis of outward current separated transient and sustained components.The transient component was specifically blocked by 4-AP(1mmol/L).A calcium-dependent outward component was also revealed modulating voltage and external calcium concentration.The fast recovery phase of the AP appears related the sustained outward current whereas the after hyperpolarization(AHP) was blocked by 4AP suggesting a significant contribution of the transient component.Forskolin (FSK),which elevates cAMP,reversibly blocked the majority of the sustained current without influencing the transient. FSK greatly exaggerated the AHP without changing the spike height or duration. These data suggest that several components of the outward current contribute specifically to the gustatory AP and that the AP may be modulated by cyclic nucleotides.

Anandamide Depresses Glycinergic and GABAergic Inhibitory Transmissions in Adult Rat Substantia Gelatinosa Neurons

Cannabinoid CB1 receptors have been found in the superficial dorsal horn of the spinal cord, particularly the substantia gelatinosa (SG), which is thought to play a pivotal role in modulating nociceptive transmission. Although cannabinoids are known to inhibit excitatory transmission in SG neurons, their effects on inhibitory transmission have not yet been examined fully. In order to know further about a role of cannabinoids in regulating nociceptive transmission, we examined the effects of cannabinoids on inhibitory transmissions in adult rat SG neurons using whole-cell voltage-clamp recordings. Anandamide (10 μM) superfused for 2 min reduced glycinergic and GABAergic electrically-evoked inhibitory postsynaptic current (IPSC) amplitudes;these actions persisted for more than 6 min after washout. Similar actions were produced by cannabinoid-receptor agonist WIN55,212-2 (5 μM) and 2-arachidonoyl glycerol (20 μM). The evoked IPSC amplitudes reduced by anandamide recovered to the control level following superfusion of CB1-receptor antagonist SR141716A (5 μM). A ratio of the second to first evoked IPSC amplitude in paired-pulse experiments was increased by anandamide (10 μM). The frequencies of glycinergic and GABAergic spontaneous IPSCs were reduced by anandamide (10 μM) without a change in their amplitudes. It is concluded that cannabinoids depress inhibitory transmissions in adult rat SG neurons by activating CB1 receptors in nerve terminals. This action could contribute to the modulation of nociceptive transmission by cannabinoids.

Conductance and Capacity of Plain Lipid Membranes under Conditions of Variable Gravity

Biological cell membranes are complex structures containing mainly lipids and proteins. Functional aspects of such membranes are usually attributed to membrane integral proteins. However, it is well established that parameters of the lipid matrix are modifying the function of proteins. Additionally, electrical capacity and conductance of the plain lipid matrix of membranes are contributing directly to cellular functions as there is, for example, the propagation of action potentials. Accordingly the dependence of these parameters on changes of gravity might be important in the field of life sciences under space conditions. In this study consequently we have performed experiments in parabolic flight campaigns utilizing the patch-clamp technology to investigate conductance and capacity of plain lipid vesicle membranes under conditions of changing gravity. Both capacity and conductance were found to be gravity dependent. The changes in capacity could be contributed to changes in membrane geometry. Significant permeability in plain lipid membranes could be only observed at high potentials, where spontaneous current fluctuations occurred. The probability of these fluctuations was gravity dependent.

Protein Kinase C Enhances the Swelling-Induced Chloride Current in Human Atrial Myocytes

Swelling-activated chloride currents（ICl.swell） are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C（PKC） in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate（PDBu） enhanced ICl.swellin a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.

Electrophysiology of Sodium Receptors in Taste Cells

Sodium intake is important to maintain proper osmolarity and volume of extracellular fluid in vertebrates. The ability to find sources of sodium ions for managing electrolyte homeostasis relies on the activity of the taste system to sense salt. Several studies have been performed to understand the mechanisms underlying Na+ reception in taste cells, the peripheral detectors for food chemicals. It is now generally accepted that Na+ interacts with specific ion channels in taste cell membrane, called sodium receptors. As ion channels, these proteins mediate transmembrane ion fluxes (that is, electrical currents) during their operation. Thus, a lot of information on the functional properties of sodium receptors has been obtained by using electrophysiological techniques. Here, I review our current knowledge on the biophysical and physiological features of these receptors obtained by applying the patch-clamp recording techniques to single taste cells.