An optimized recording method to characterize biophysical and pharmacological properties of acid-sensing ion channel

Objective To re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a （hASIC1a） current in HEK293 cells with a modified perfusion methods. Methods With cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASICla currents evoked by low pH external solution. Results Using cell floating method, the amplitude of hASICla currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is （21±5） ms and （270±25） ms, respectively. Inactivation time constants are （496±23） ms and （2284±120） ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC 1 a [IC50 is （3.4± 1.1 ） μmol/L and （2.4± 0.9） μmol/L, respectively]. Both recording methods have similar pH activation ECs0 （6.6±0.6, 6.6±0.7, respectively）. Conclusion ASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC la current was re-confirmed and the biophysical and pharmacological properties of hASIC la channel in HEK293 cells was precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.

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.

Axonal bleb recording

Patch-clamp recording requires direct accessibility of the cell membrane to patch pipettes and allows the investigation of ion channel properties and functions in specific cellular compartments.The cell body and relatively thick dendrites are the most accessible compartments of a neuron,due to their large diameters and therefore great membrane surface areas.However,axons are normally inaccessible to patch pipettes because of their thin structure;thus studies of axon physiology have long been hampered by the lack of axon recording methods.Recently,a new method of patchclamp recording has been developed,enabling direct and tight-seal recording from cortical axons.These recordings are performed at the enlarged structure（axonal bleb） formed at the cut end of an axon after slicing procedures.This method has facilitated studies of the mechanisms underlying the generation and propagation of the main output signal,the action potential,and led to the finding that cortical neurons communicate not only in action potential-mediated digital mode but also in membrane potential-dependent analog mode.

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.

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.

Study of transmembrane La^(3+) movement in rat ventricular myocytes by the patch-clamp technique

We have studied transmembrane La3+ movement in rat ventricular myocytes for the first time by using the whole-cell patch-clamp recording mode. La3+ (0.01-5.0 mmol/L) could not bring out inward currents through the L-type calcium channel in rat ventricular myocytes, while it could enter the cells by the same way carried by 1μmo1/L ionomycin. When the outward Na+ concentration gradient is formed, La3+ can enter the cells via Na-Ca exchange, and the exchange currents increase with the increase of external La3+ concentrations. But compared with Na-Ca exchange currents in the same concentration, the former is only 14%-38% of the latter. The patch-clamp experiment indicates that La3+ normally can not enter ventricular myocytes through L-type calcium channel, but it can enter the cells via Na-Ca exchange.

Effects of BaCl_2 on slow vacuolar ion channels on radish by patch-clamp

The effects of BaCl_2 on slow vacuolar (SV) currents of radish are studied byusing the whole-vacuolar patch-clamp recording mode. The Ca^(2+)-dependent SV channel can beactivated by cytosolic Ca^(2+). When 1 mmol/L BaCI_2 is added into pipette solution, SV currents aresuppressed remarkably. Then adding BaCI_2 of different concentrations into the bath solution, SVcurrents reflect different effects. The results show that BaCl_2 with a lower concentration (< 3mmol/L) promotes the channel currents and the currents are saturated when BaCl_2 concentrations arebetween 1 μmol/L and 1 mmol/L, but BaCl_2 with higher concentration (≥ 3 mmol/L) inhibits SVcurrents.

Research at channel level on the effect of LaCl3 on Radish (Raphanus satirus L.) vacuolar membrane

We recorded slow vacuolar (SV-type) channel currents of Radish vacuoles successfully for the first time by using the whole-vacuolar patch-clamp recording mode. SV-type currents would increase and threshold potentials of activation would shift towards more negative values with the increase of concentrations of cytosolic Ca2+. When 2.5 mmol/ L LaCl3 and 4 mmol/L EGTA were added to bath solutions, SV-type currents were suppressed remarkably. Then adding LaCl3 with different concentrations to pipette solutions, we found that LaCl3 with higher concentrations (】4 × 10-7mol/L) had a strong inhibitory effect on SV-type currents, while LaCl3 with lower concentrations (≤4 × 10-7mol/L) promoted channel currents. This promoting effect provides an impor-tant basis at channel level for researching further the effects of rare earth on physiological activities of plants and the production-increase effects of rare earth fertilizers on crops.

Pathological Networks Involving Dysmorphic Neurons in Type ⅡFocal Cortical Dysplasia

Focal cortical dysplasia(FCD)is one of the most common causes of drug-resistant epilepsy.Dysmorphic neurons are the major histopathological feature of typeⅡFCD,but their role in seizure genesis in FCD is unclear.Here we performed whole-cell patch-clamp recording and morphological reconstruction of cortical principal neurons in postsurgical brain tissue from drug-resistant epilepsy patients.Quantitative analyses revealed distinct morphological and electrophysiological characteristics of the upper layer dysmorphic neurons in typeⅡFCD,including an enlarged soma,aberrant dendritic arbors,increased current injection for rheobase action potential firing,and reduced action potential firing frequency.Intriguingly,the upper layer dysmorphic neurons received decreased glutamatergic and increased GABAergic synaptic inputs that were coupled with upregulation of the Na^(+)-K^(+)-Cl^(−)cotransporter.In addition,we found a depolarizing shift of the GABA reversal potential in the CamKⅡ-cre::PTENflox/flox mouse model of drug-resistant epilepsy,suggesting that enhanced GABAergic inputs might depolarize dysmorphic neurons.Thus,imbalance of synaptic excitation and inhibition of dysmorphic neurons may contribute to seizure genesis in typeⅡFCD.

Spontaneous firing properties of rat medial vestibular nucleus neurons in brain slices by infrared visual patch clamp technique

Domestic application of infrared patch clamp techniques on brain slices is limited.The key of the tech-nique is to prepare high-quality brain slices.The present paper describes the preparation procedure of brainstem slices and the spontaneous firing properties of rat medial vestibular nucleus(MVN)neurons.By infrared differ-ential interference contrast technique,neurons of rat MVN were visualized directly at the depth of 50–100 mm underneath the surface of slices.Firing activities of MVN neurons were recorded by the whole-cell patch clamp technique in artificial cerebrospinal fluid(ACSF)and low Ca^(2+)-high Mg^(2+) fluid.The firing mode was more irregular and depressive in low Ca^(2+)-high Mg2+fluid than in ACSF.According to the averaged waveform of action potentials,cells were classified as the neurons with mono-phasic after-hyperpolarization potential(AHP),and the neurons with biphasic AHP.The resting membrane potential(RMP),input resistance(Rin)and membrane capacitance(Cm)of neurons were recorded and com-pared between groups.With infrared videomicroscopy,patch clamp recordings could be made under direct obser-vation in freshly prepared brainstem slices.The discharge activities of MVN neurons were spontaneous and the fir-ing mode was modulated by extracellular calcium concen-tration.The basic membrane properties of two types of neurons were not significantly different,while the differ-ences in waveform might play a role in the segregation between tonic and kinetic cells.