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The ion channels located on the cell fine structures play an important role in the physiological functions of cell membrane. However, it is impossible to achieve precise positioning on the nanometer scale cellular microstructures by conventional patch-clamp technique, due to the 200 nm resolution limit of optical microscope. To solve this problem, we have established a high-resolution patch-clamp technique, which combined commercial scanning ion conductance microscopy (SICM) and patch-clamp recording through a nanopipette probe, based on SICM feedback control. MDCK cells were used as observation object to test the capability of the technique. Firstly, a feedback controlled SICM nanopipette (approximately 150 MOmega) non-contactly scanned over a selected area of living MDCK cells monolayer to obtain high-resolution topographic images of microvilli and tight-junction microstructures on the MDCK cells monolayer. Secondly, the same nanopipette was non-contactly moved and precisely positioned over the microvilli or tight-junction microstructure under SICM feedback control. Finally, the SICM feedback control was switched off, the nanopipette slowly contacted with the cell membrane to get a patch-clamp giga-ohm sealing in the cell-attached patch-clamp configuration, and then performed ion channel recording as a normal patch-clamp electrode. The ion channel recordings showed that ion channels of microvilli microstructure opened at pipette holding potential of -100, -60, -40, 0, +40, +60, +100 mV (n=11). However, the opening of ion channels of tight-junction microstructure was not detected at pipette holding potential of -100, -40, 0, +40, +100 mV (n=9). These results suggest that our high-resolution patch-clamp technique can achieve accurate nanopipette positioning and nanometer scale high-resolution patch-clamp recording, which may provide a powerful tool to study the spatial distribution and functions of ion channel in the nanometer scale microstructures of living biological samples.

A modified protocol for quick and economic treatment of cultured human umbilical vein endothelial cells has been established, which result in high viability of the cells to allow better performance in patch-clamp studies. The electrophysiological properties of Ca (2+)-activated K(+) (KCa) channel of the cultured cells were investigated with a cell-attached configuration. With this modified treatment method, the cultured cells appear fusiform in shape under microscope, and KCa channel currents could be detected readily, suggesting their eligibility for patch-clamp studies.

A USB bus based data interface technology for full auto Patch-Clamp system is discussed in the article. The main controller is CY2131QC (Cypress) and the logic controller is EPM3256A (Altera). Optocouplers are used to get rid of the noise from the interface. It makes the installation of the Patch-Clamp system easier by using the USB bus, and is suitable for the new generation of the Patch-Clamp system with a high speed of 1M bytes/s.

The patch-clamp technique, a dominant technique in cellular electrophysiology, is always being regarded as the gold standard for ion channel research. Application of the patch-clamp technique can demonstrate the existences of ion channels and provide valuable information for ion channels, including their electrophysiological properties, molecular structures and the mechanism of drug action. Genomics and proteomics research has showed that the development of drugs for ion channel target would be very promising in future. In recent years, numerous improvements have been achieved, which will facilitate the application of patch clamp technique in drug high-throughput screening. These techniques will break through the bottleneck in the development of drugs aiming at ion channels. New advancements in patch-clamp technique and application in drug high-throughput screening are reviewed in the paper.

Aim and Methods: Using blind whole-cell recording techniques on rat hippocampal slices, the function and mechanisms of several leak subtraction methods of Axon patch clamp system (Axopatch amplifier and pClamp software) were analyzed. That how to select and use scaled P/N leak subtraction, patch clamp amplifier leak subtraction and Clampfit leak subtraction were emphasized in our present study., Results: The noise induced by scaled P/N leak subtraction of Clampex soft ware was smaller than that of P/N leak subtraction. Axon patch clamp amplifier leak subtraction could subtract the leak current produced by single depolarizing pulse but not the leak current produced by a series of different step depolarizing pulses. Due to its assumption that leak current would be produced if only potential difference arises across membrane, Clampfit leak subtraction was not suitable to subtracting the steady-state leak current while recording voltage-gated channel currents., Conclusion: P/N- and scaled P/N leak subtraction, but not Clampfit leak subtraction, cad be used to subtract steady-state leak current while recording voltage-gated channel currents.

In order to overcome the effects of the anti-aliasing filter and the colored background noise, an adaptive algorithm is proposed to estimate the parameters of ion channel kinetics and the background noise, and whereafter the ion channel signal could be restored from the strong noisy patch-clamp recordings. The algorithm cross-couples the recursive expectation-maximization algorithm, which estimates optimally the parameters of hidden Markov model, and the recursive extended least square algorithm, which estimates optimally the characteristics of the background noise. Simulation suggests that this cross-coupling algorithm convergences consistently, and is very robust to the inexact conformation number.

Aim: To investigate membrane electro-physiological features in vestibular ganglion neuronal population using a voltage-sensitive dye and optical recording technique., Methods: Dissociated and cultured mouse vestibular ganglion neurons were stained with an absorption voltage-sensitive dye, RH 155, and were imaged in 16 x 16 elements Photodiode arrays (PDA) optical recording system., Results: When the cells were depolarized during perfusion with 150 mmol/L potassium solution, optical absorption of the dye that bound to the external surface of neuron membranes increased. The relative ratio (delta I/I) of optical absorption change was 0.23% +/- 0.08% (x +/- s, n = 28). These optical responses were wavelength dependent. Under our experimental conditions, photo toxicity and pharmacological effects of the dye were either absent or insignificant., Conclusion: Our results suggest that optical recording provides a new, practical and less toxic method to simultaneously monitor changes in membrane potential from several cultured vestibular ganglion cells.

Aim and Methods: By using blind whole-cell recording techniques, the dynamic properties of voltage-gated Ca2+ channel of CA1 pyramidal neuron in rat hippocampal slices were investigated., Results: The voltage-gated Ca2+ channel of CA1 pyramidal neuron in rat hippocampal slices has following features: (1) The threshold potential of activation is relatively lower. The mean value is -49.3 +/- 8.6 mV with range from -65 to -30 mV (n = 23). (2) Current decay is Ca(2+) -dependent and its time constant (tau) is longer with a wide range from 100 to 700 ms (n = 12). (3) The steady-state inactivation is voltage-dependent and the half inactivation potential (V1/2) is -55.4 +/- 9.7 mV, slope factor (k) is 5.3 +/- 0.9 (n = 10). (4) The reversal potential (E(rev)) of Ca2+ channel is 55 +/- 13 mV (n = 10) when extracellular Ca2+ concentration is 2.5 mmol/L. (5) Tail current, which is voltage-independent, primarily has only one component. In addition, Ca2+ channel is insensitive to verapamil and nifedipine., Conclusion: N-type Ca2+ channel is strongly suggested in CA1 pyramidal neuron in rat hippocampal slices.

The single ion channel signal is stochastic ionic current on the order of 1 pA recorded by patch clamp. Because the weakness of the signal, the background noise always dominates in the recordings, the threshold detector traditionally used in patch clamp to denoise and restore the channel signal can't work satisfactorily. This problem was analyzed mathematically, and a signal restoring and parameters estimating scheme called HMM algorithm was studied. The algorithm has been validated by simulation and the results suggest it performs effectively in the situation of low signal to noise ratio where the threshold detector fails completely.

The blind patch clamp whole-cell recording technique for CA1 pyramidal neurons in hippocampal slices was carefully introduced, the key procedures and the points for attention were emphasized. Using the "blind" method, we analyzed the properties of synaptic activity, the current features of Ca2+ channels and glutamate, GABA receptors. Our results provide a scientific basis for study of dynamic properties of ion channels on hippocampal neurons and the effects of drugs on synaptic activity by using the blind whole-cell recording technique.

Chemical communication for neurons and endocrine cells depends on their secretion activity. Development of variety of biophysical techniques have greatly promoted the understanding of the mechanism of cellular secretion. Here we will introduce three recently developed biophysical techniques which have been frequently used to monitor secretion activity in real-time.

Action of ion channel on membrane is the key events of messenger transduction for excitable cells, which can be detected by the patch clamp technique. The developments of patch clamp technique have brought a revolution of life science research. The theory, work modes, single channel and whole cell recording techniques in single cells are described here in detail.

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of serotonin (5-hydroxytryptamine, 5-HT) on taurine (Tau)-activated current (ITau) was investigated in neurons dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results are as follows: (1) Tau acted on strychnine-sensitive glycine (Gly) receptors and elicited inward currents at a holding potential (VH) of -40 mV in SDCN neurons; (2)in a range of 0.01 to 100 mumol/L, 5-HT enhanced ITau in a concentration-dependent manner; (3) both the reversal potential of Tau and the binding affinity of Tau to Gly receptor were not affected by 5-HT; (4) when the neurons were loaded with 3 mumol/L chelerythrine, application of 1 mumol/L 5-HT failed to enhance ITau. The above results thus indicate that intracellular PKC pathway is at least partially involved in the enhancement of ITau by 5-HT. In accordance with our previous study, it is suggested that 5-HT may play an important antinociceptive function in SDCN through enhanced Gly and Tau inhibitory effects.