Bayesian restoration of single-channel patch clamp recordings.

The technique of patch clamp recording makes possible the measurement of current flowing through a single ion channel in a cell membrane. Examination of such recordings suggests that the current is quantal in nature, alternating in a seemingly random manner between "on" and "off," but the recordings are corrupted by noise from a variety of sources. In this paper we propose and illustrate methods for restoring the underlying quantal signal from such noisy measurements. The methods use a Markov chain prior distribution for the underlying quantal process and base the restoration on the resulting posterior distribution.     

Micromolded PDMS planar electrode allows patch clamp electrical recordings from cells

The patch clamp method measures membrane currents at very high resolution when a high-resistance 'gigaseal' is established between the glass microelectrode and the cell membrane (Pflugers Arch. 391 (1981) 85; Neuron 8 (1992) 605). Here we describe the first use of the silicone elastomer, poly(dimethylsiloxane) (PDMS), for patch clamp electrodes. PDMS is an attractive material for patch clamp recordings. It has low dielectric loss and can be micromolded (Annu. Rev. Mat. Sci. 28 (1998) 153) into a shape that mimics the tip of the glass micropipette. Also, the surface chemistry of PDMS may be altered to mimic the hydrophilic nature of glass (J. Appl. Polym. Sci. 14 (1970) 2499; Annu. Rev. Mat. Sci. 28 (1998) 153), thereby allowing a high-resistance seal to a cell membrane. We present a planar electrode geometry consisting of a PDMS partition with a small aperture sealed between electrode and bath chambers. We demonstrate that a planar PDMS patch electrode, after oxidation of the elastomeric surface, permits patch clamp recording on Xenopus oocytes. Our results indicate the potential for high-throughput patch clamp recording with a planar array of PDMS electrodes.     

Blind patch clamp recordings in embryonic and adult mammalian brain slices

To obtain electrophysiological recordings in brain slices, sophisticated and expensive pieces of equipment can be used. However, costly microscope equipment with infrared differential interference contrast optics is not always necessary or even desirable. For instance, obtaining a randomized unbiased sample in a given preparation would be better accomplished if cells were not directly visualized before recording. In addition, some preparations require thick slices, and direct visualization is not possible. Here we describe a protocol for the 'blind patch clamp method' that we developed several years ago to perform electrophysiological recordings in mammalian brain slices using a standard patch clamp amplifier, dissecting microscope and recording chamber. Overall, it takes approximately 3-4 h to set up this procedure.     

Scanning electron microscopy and gramicidin patch clamp recordings of microvillous receptor neurons dissociated from the rat vomeronasal organ

Vomeronasal organs from female rats were dissociated and isolated microvillous receptor neurons were studied. The isolated receptor neurons kept the typical bipolar shape which they have in situ as observed by scanning electron microscopy. We applied the perforated patch-clamp technique using the cation-selective ionophore gramicidin on freshly isolated and well differentiated receptor neurons. The mean resting potential was -58+/-14 mV (n=39). The contribution of the sodium pump current to the resting potential was demonstrated by lowering the K+ concentration in the bath or by application of 100 microM dihydro-ouabain. The input resistance was in the range of 1-6 GOmega and depolarizing current pulses of a few pA were sufficient to trigger overshooting action potentials. In voltage clamp conditions a fast transient sodium inward current and a sustained outward potassium current were activated by membrane depolarization. These observations indicate that freshly isolated vomeronasal receptor neurons of rats can be recorded, using gramicidin, with little modification of the intracellular content. Their electrophysiological properties are very similar to those observed in situ. Four out of eight female vomeronasal receptor cells were depolarized by diluted rat male urine.      

Resolution of patch capacitance recordings and of fusion pore conductances in small vesicles

We investigated the noise levels in cell-attached patch capacitance recordings with a lock-in amplifier. The capacitance noise level decreases with increasing sine wave frequency up to 20-40 kHz. With a 20-mV rms sine wave the rms noise level above 8 kHz is <50 aF. With increasing sine wave amplitudes a further reduction down to 14 aF could be achieved. Capacitance measurements with a lock-in amplifier may also be used to measure the conductance of fusion pores connecting the vesicular lumen to the extracellular space. It is estimated that at noise levels of 14 aF fusion pore conductances between 20 pS and 700 pS may be resolved in vesicles with 380-aF capacitance by using a 50-kHz sine wave. This corresponds to vesicles with a approximately 110-nm diameter. It is suggested that with low-noise techniques fusion pores may be detectable in vesicles approaching the size of large synaptic vesicles.     

New fin-line devices for radiofrequency exposure of small biological samples in vitro allowing whole-cell patch clamp recordings

The development and analysis of three waveguides for the exposure of small biological in vitro samples to mobile communication signals at 900 MHz (GSM, Global System for Mobile Communications), 1.8 GHz (GSM), and 2 GHz (UMTS, Universal Mobile Telecommunications System) is presented. The waveguides were based on a fin‐line concept and the chamber containing the samples bathed in extracellular solution was placed onto two fins with a slot in between, where the exposure field concentrates. Measures were taken to allow for patch clamp recordings during radiofrequency (RF) exposure. The necessary power for the achievement of the maximum desired specific absorption rate (SAR) of 20 W/kg (average over the mass of the solution) was approximately P_in = 50 mW, P_in = 19 mW, and P_in = 18 mW for the 900 MHz, 1800 MHz, and 2 GHz devices, respectively. At 20 W/kg, a slight RF‐induced temperature elevation in the solution of no more than 0.3 °C was detected, while no thermal offsets due to the electromagnetic exposure could be detected at the lower SAR settings (2, 0.2, and 0.02 W/kg). A deviation of 10% from the intended solution volume yielded a calculated SAR deviation of 8% from the desired value. A maximum ±10% variation in the local SAR could occur when the position of the patch clamp electrode was altered within the area where the cells to be investigated were located. Bioelectromagnetics 32:102–112, 2011. © 2010 Wiley‐Liss, Inc.     

Patch clamp recordings from membranes which contain gap junction channels.

The septal membranes of the median and lateral giant axons of earthworm, which contain gap junctions, were exposed by cutting one segment of the cord. Patch recordings were obtained from the exposed cytoplasmic side of the septum. Seal resistances ranged from 2 to 15 G omega. The patch could be excised (detached) or left attached to the whole cell. Two types of channels were observed. One type was blocked by tetraethylammonium (TEA) or Cs+ and had a unitary conductance of 30-40 pS. It appears to be a K+ channel. The other channel type had a unitary conductance of 90-110 pS and was unaffected by TEA+ or Cs+. In the detached configuration the channel was shown to conduct Cs+, K+, Na+, TMA+, Cl- and TEA+ even in the presence of 2 mM Zn2+, 1 mM Ni2+, 1 mM Co2+, and 4 mM 4-aminopyridine. The conductance ratios relative to K+ were 1.0 for Cs+, 0.84 for Na+, 0.64 for TMA+, 0.52 for Cl- and 0.2 for TEA+. The channel appears to be voltage insensitive whether monitored in detached or attached recording mode. Both H+ and Ca2+ reduce the probability of opening. Thus, the 100 pS channel has many of the properties expected of a gap junction channel.     

Channels in the mitochondrial outer membrane: Evidence from patch clamp studies

Patch clamp techniques were applied to outer mitochondrial membranes of giant mitochondria from mice kept on a cuprizone diet or to vesicles produced by fusing membranes derived from the outer membrane ofNeurospora mitochondria. In the negative range of potentials the conductances decreased with increases in the magnitude of voltage, suggesting the closing of channels. Experiments in which mitochondria were treated with the polyanion polymethacrylate maleate styrene (1:2:3) or succinic anhydride suggest that the channels correspond to VDAC. Although sometimes conductance also decreased with increasing potential over a narrow range of positive potentials, more commonly the conductances increased. Although this phenomenon may represent a detachment of the patch, the changes in conductance are reversible, suggesting that they correspond to the formation or the opening of channels.     

Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.     

Estimating the number of channels in patch recordings

The estimation of the number of channels in a patch was assumed to be equivalent to the estimation of the binomial parameter n. Seven estimators were evaluated, using data sets simulated for a range of parameters appropriate for single channel recording experiments. No single estimator was best for all parameters; a combination of estimators is a possible option to avoid the biases of individual estimators. All estimators were highly accurate in estimating n in the case that n = 1. For n ≤ 4 the simplest estimator, the maximum number of simultaneously open channels, was the best, For larger values of n the best estimators were Bayesian.     

Introduction: patch clamp and single-cell voltage clamp techniques and selected data

Molecular and Cellular Biochemistry -     

Achieving maximal speed of solution exchange for patch clamp experiments

Background

Resolving the kinetics of agonist binding events separately from the subsequent channel gating processes requires the ability of applying and removing the agonist before channel gating occurs. No reported system has yet achieved pulses shorter than 100 µs, necessary to study nicotinic ACh receptor or AMPA receptor activation.

Methodology/Principal Findings

Solution exchange systems deliver short agonist pulses by moving a sharp interface between a control and an experimental solution across a channel preparation. We achieved shorter pulses by means of an exchange system that combines a faster flow velocity, narrower partition between the two streams, and increased velocity and bandwidth of the movement of the interface. The measured response of the entire system was fed back to optimize the voltage signal applied to the piezoelectric actuator overcoming the spurious oscillations arising from the mechanical resonances when a high bandwidth driving function was applied. Optimization was accomplished by analyzing the transfer function of the solution exchange system. When driven by optimized command pulses the enhanced system provided pulses lasting 26 ± 1 µs and exchanging 93 ± 1% of the solution, as measured in the open tip of a patch pipette.

Conclusions/Significance

Pulses of this duration open the experimental study of the molecular events that occur between the agonist binding and the opening of the channel.     

Calcium-dependent potassium channel inParamecium studied under patch clamp

Summary We have studied a class of Ca _i ²⁺ -dependent K channels in inside-out excised membrane patches fromParamecium under patch clamp. Single channels had a conductance of 72 ±9.0 pS in a solution containing 100mM K⁺. The channels were selective for K⁺ over Rb⁺ with the permeability ratio of 1 0.56. and over Na⁺, Cs⁺ or NH ₄ ⁺ with a ratio 1<0.1. The channel activity was dependent on Ca _i ²⁺ , which was applied to the cytoplasmic side; the Ca _i ²⁺ concentration for the half maximal activation was 2 m. The Hill coefficient for the Ca _i ²⁺ dependence of the channel activity was 2.58, indicating that more than two Ca _i ²⁺ bindings are necessary for full activation. Unlike most Ca _i ²⁺ -dependent K channels in other organisms, the channels inParamecium were slightly more active upon hyperpolarization than upon depolarization. The voltage dependence was fitted to a Boltzmann curve with 41.2 mV pere-fold change in channel activity. While a high Ca _i ²⁺ concentration activated the channels, it also irreversibly reduced the channel activity over time. The decay of channel activity occurred faster at higher Ca _i ²⁺ concentrations. Quaternary ammonium ions suppressed ion passage through the channel; more highly alkylated quaternary ammonium ions were more efficient in blocking. Ba _i ²⁺ and Ca _i ²⁺ were relatively ineffective in blockage. It was concluded that these Ca _i ²⁺ -dependent K channels inParamecium are different from the previously described Ca _i ²⁺ -dependent K channels, and are perhaps of a novel class.     

Analyzing stochastic events in multi-channel patch clamp data

In an effort to understand the gating properties of ionic channels in biological membranes, an efficient method was developed to estimate the kinetic constants from opening-closing events of the channels. Our method is suitable to single channel patch-clamp recordings that contain several ionic channels functioning simultaneously. It is different from the maximum likelihood method previous developed by Horn and Lange, in that our method is a continuum approach and makes uses of analytic expressions of the probability density functions of the event times. Combinatorial analysis was necessary to correctly include more typical multi-channel recordings. This yields computationally quicker results than the method of Horn and Lange, which uses a discretized time series. Model-dependent portions of the code are minimal and easily modified. To illustrate the goodness of our method, we have generated the open-close processes of the patch-clamp records on a digital computer using the exponential random number generators. For multi-channel patches, we have introduced a few plausible approximations to make our algorithm more efficient. The soundness of the our approximations were tested with such measures as the fraction of the open state at time t, p_open(t), and frequencies of the number of openings per run. A copy of the computer code implementing this algorithm can be obtained from the authors.This work is supported by NIH R01 HL33905-01     

Axon公司膜片钳系统的漏减分析

目的和方法：采用大鼠海马脑片盲法膜片钳的全细胞记录技术,研究美国Axon公司生产的膜片钳系统（Axopatch放大器和pClamp软件）中几种漏减功能的意义和作用机制,重点对定标P／N漏减（Scaled P/N leak subtraction)、膜片钳放大器漏减以及Clampfit处理软件漏减功能的选择与使用进行分析与比较。结果：Clampex采样软件中的定标P／N漏减功能比P／N漏减功能的噪声要小;放大器漏减功能可漏减单一去极化电压幅度所诱发的漏电流,但不能同时对不同电压幅度系列去极化所产生的稳态漏电流进行追踪漏减;Clampfit漏减功能由于其设定只要膜两侧存在电位差就有漏电流产生,因而不适合在记录电压门控性离子通道电流时对稳态漏电流进行漏减。结论：在研究电压门控性离子通道的性质时,可采用P／N漏减功能或定标P／N漏减功能对稳态漏电流进行漏减,而Clampfit漏减功能是不合适的。     

Evidence for cooperativity between nicotinic acetylcholine receptors in patch clamp records

It is often assumed that ion channels in cell membrane patches gate independently. However, in the present study nicotinic receptor patch clamp data obtained in cell-attached mode from embryonic chick myotubes suggest that the distribution of steady-state probabilities for conductance multiples arising from concurrent channel openings may not be binomial. In patches where up to four active channels were observed, the probabilities of two or more concurrent openings were greater than expected, suggesting positive cooperativity. For the case of two active channels, we extended the analysis by assuming that 1) individual receptors (not necessarily identical) could be modeled by a five-state (three closed and two open) continuous-time Markov process with equal agonist binding affinity at two recognition sites, and 2) cooperativity between channels could occur through instantaneous changes in specific transition rates in one channel following a change in conductance state of the neighboring channel. This allowed calculation of open and closed sojourn time density functions for either channel conditional on the neighboring channel being open or closed. Simulation studies of two channel systems, with channels being either independent or cooperative, nonidentical or identical, supported the discriminatory power of the optimization algorithm. The experimental results suggested that individual acetylcholine receptors were kinetically identical and that the open state of one channel increased the probability of opening of its neighbor.     

海马脑片盲法膜片钳全细胞记录技术

本文较为详细地介绍了海马脑片盲法膜片钳全细胞记录技术,对其关键步骤和需要注意的问题进行了重点说明,同时对CA1区锥体神经元突触活动的特点,电压门控性Ca^2 通道以及谷氨酸（glutamate,Glu)γ－氨基丁酸（GABA）受体通道电流性质等进行了观察和分析,实验结果为采用海马脑片盲法膜片钳全细胞记录技术研究海马神经元离子通道动力学性质和中枢神经系统药物对突触活动的影响提供了可靠的依据。