A simple technique for transferring excised patches of membrane to different solutions for single channel measurements

A technical problem associated with the patch clamp technique has been the changing of solutions bathing the membrane patch. The simple technique described here solves this problem by means of a movable polythene sleeve placed on the shaft of the patch clamp pipette. The sleeve is initially placed so that the tip of the pipette is exposed. A gigaohm seal is formed using standard techniques. The patch is then excised and the sleeve is slipped down a few mm past the end of the tip of the pipette. When the pipette and sleeve is now removed from the solution, a small drop of solution covering the membrane patch is held in place at the end of the sleeve by surface tension. The pipette is then easily transferred to a different solution without passing the membrane patch through the air-water interface. The sleeve is then simply pulled back up the pipette shaft to expose the membrane patch to the new solution.     

Single channel recordings of reconstituted ion channel proteins: an improved technique

Single channel recording of reconstituted ion channels is possible by patch clamp measurements of giant liposomes formed by dehydration-rehydration of lipid films. This hydration technique consists of carefully controlled dehydration of a suspension of small vesicles followed by rehydration of the residue resulting in formation of large liposomes. Patch pipettes can be attached to the liposome surface, yielding stable, high resistance seals between membranes and glass pipettes. This method allows the study of the properties of reconstituted ion channels from different tissues. The hydration technique was used to characterize the reconstituted K⁺-channel of sarcoplasmic reticulum from rabbit skeletal muscle. In a solution of 100 mM KCl, the sarcoplasmic reticulum K⁺-channel studied displays a conductance _K ⁺ of 145 pS. The single channel conductance in 100 mM Rb⁺ and Na⁺ is _Rb ⁺ = 98 pS and _Na ⁺ = 65 pS respectively. A concentration of 0.5 mM decamethonium causes a flickering channel block. These properties are in good agreement with the ones found in sarcoplasmic reticulum K⁺-channels characterized by other methods. Other ion channels have also been reconstituted and studied by this technique. This improved method is compared with previous approaches and its applicability for the characterization of reconstituted ion channel proteins is discussed.     

基于HMM的低信噪比离子通道信号的恢复及参数估计

细胞膜离子单通道信号是皮安级的随机电流 ,膜片钳技术可以记录这些信号。一般认为它是一阶的、状态有限的 Markov过程。某些种类的离子通道 ,电流信号特别微弱 ,完全淹没在背景噪声中 ,传统的膜片钳技术很难检测到 ,只能运用数学方法恢复和估计。在低采样频率情况下 ,由于混叠效应 ,可认为背景噪声是白色的 ;在高采样频率条件下 (高于奈奎斯特频率 ) ,背景噪声是有色的。本文分别综述了白色背景噪声条件下基于隐式 Markov模型和有色背景噪声条件下基于隐式矢量 Markov模型的低信噪比离子单通道信号的恢复和参数估计 ,主要包括前后向算法、EM算法等     

Single channel Cl− and K+ currents from cells of uterus not treated with enzymes

Single channel currents were recorded from uterine smooth muscle cells of pregnant guinea-pigs. The cells were within small bundles of smooth muscle of low input resistance. Giga-ohm seals were formed between the patch clamp electrodes and the membrane of smooth muscle cells that had not been treated with enzymes. In cell-attached mode, outward current steps were observed that had a conductance of 130–170 pS, a reversal potential close to the potassium reversal potential, and the probability of a channel being in the open state increased e-fold per 8–11 mV depolarization. In the cell-free, inside-out patch mode, currents were recorded that had a large conductance, 420 pS, and smaller conductance levels. When the chloride concentration was changed the reversal potential shifted in a manner consistent with the Nernst equation indicating that the channels were permeable to chloride ions.     

Pentobarbital promotes bursts of gamma-aminobutyric acid-activated single channel currents in cultured mouse central neurons

The extracellular patch clamp method was used to study the influence of pentobarbital (PB) on membrane channels induced by gamma-aminobutyric acid (GABA) in cultured mouse spinal neurons. PB (200 microM) increased the probability of finding GABA-sensitive channels in the open state; it also increased the average frequency at which GABA-induced single channel currents occur, without decreasing the number of data sweeps which showed no single channel activity. These results indicate that PB potentiates GABA responses by promoting burst-like openings of the GABA-sensitive channel.     

Single synaptic channels recorded at glutamate sensitive patches on a crayfish muscle

When a patch clamp pipette filled with 50 mumol/l glutamate was placed on a muscle fiber of the deep abdominal extensor of crayfish, in some locations current pulses were recorded which were identified as synaptic, glutamate-operated ionic channel openings. At a given site all current pulses had approximately the same amplitude. At resting potential and 19 degrees C, their mean amplitudes were 7-8 pA, corresponding to channel conductances of 70-80 pS. The distribution of open times of the channels could be described by the sum of two exponentials with time constants tau 1 of 0.3-0.5 ms for the longer, and tau 2 of 0.03-0.06 ms for the shorter component. Bursts of channel openings interrupted by gaps occurred in about 10% of the events only. The longer time constant tau 1 conforms to the channel open times estimated by noise analysis [11].     

The on-line direct fitting of low signal-noise ratio single ion channel recordings based on hidden Markov models

INTRODUCTION　　 As fundamental elements of the neural system,ion channels are proteinmolecules spanning in the membrane of excitable cells. They form pores thathave asmall number of discrete conformations. In some of the conformations,the pore isopen and…     

Potential role of reduced basolateral potassium (IKCa3.1) channel expression in the pathogenesis of diarrhoea in ulcerative colitis

Diarrhoea in ulcerative colitis (UC) mainly reflects impaired colonic Na(+) and water absorption. Colonocyte membrane potential, an important determinant of electrogenic Na(+) absorption, is reduced in UC. Colonocyte potential is principally determined by basolateral IK (KCa3.1) channel activity. To determine whether reduced Na(+) absorption in UC might be associated with decreased IK channel expression and activity, we used molecular and patch clamp recording techniques to evaluate IK channels in colon from control patients and patients with active UC. In control patients, immunolabelling revealed basolateral IK channels distributed uniformly along the surface-crypt axis, with substantially decreased immunolabelling in patients with active UC, although IK mRNA levels measured by quantitative PCR were similar in both groups. Patch clamp analysis indicated that cell conductance was dominated by basolateral IK channels in control patients, but channel abundance and overall activity were reduced by 53% (p = 0.03) and 61% (p = 0.04), respectively, in patients with active UC. These changes resulted in a 75% (p = 0.003) decrease in the estimated basolateral membrane K(+) conductance in UC patients compared with controls. Levels of IK channel immunolabelling and activity in UC patients in clinical remission were similar to those in control patients. We conclude that a substantial decrease in basolateral IK channel expression and activity in active UC most likely explains the epithelial cell depolarization observed in this disease, and decreases the electrical driving force for electrogenic Na(+) transport, thereby impairing Na(+) absorption (and as a consequence, Cl(-) and water absorption) across the inflamed mucosa.     

A new method of preparing the basal membrane of renal tubules for patch clamp,using beetle malpighian tubules

Access to the basal membrane of beetle Malpighian tubules (Onymacris rugatipennis) was achieved by a new method of stripping tubules of the surrounding connective tissue and basement membrane, in a simple squeezing process. We recorded single channel currents in the cell-attached configuration from basal K⁺ channels. The peeling method described, in this paper allows quick and easy preparation of Malpighian tubules for patch clamp studies on the basal membrane.     

A direct effect of forskolin on sodium channel bursting

A long-lasting component of current through voltage-dependent Na channels is believed to contribute to the plateau phase of the cardiac action potential. Here we report that in cardiac ventricular myocytes forskolin increases the contribution of a very slow component of decay (=36±16 ms,n=13) in ensemble currents in response to step depolarizations to 0 mV. Long-lasting bursts of openings (mean duration of 27±14 ms,n=10) accounted for this behavior. The slow time constant of decay was not altered by forskolin (5–50 M). Rather, an increase in the probability of bursting behavior produced a forskolin concentration-dependent increase in the amplitude of this very slow component. This action of forskolin was not the result of stimulation of adenylyl cyclase because it was not affected when cAMP-dependent phosphorylation was inhibited by the protein kinase inhibitor H-89, and it could not be mimicked by addition of isoproterenol, membrane-permeant cAMP [8-(4-chloro-phenylthio)-cAMP], or the phosphatase inhibitor okadaic acid. In addition, bursting was not augmented by guanosine 5-O-(3-thiotriphosphate) (GTP [S]) either applied to the bath or directly to the intracellular face of the channel in inside-out macropatches. Further-more, 1,9-dideoxy-forskolin, which does not stimulate adenylyl cyclase and 6-(3-dimethylaminopropionyl)-forskolin, a hydrophilic derivative of forskolin, also augmented late channel activity. Comparison of the characteristics of bursts in the presence of forskolin with those occurring in its absence suggested that the increase in the frequency of long-lasting bursts produced by forskolin represents a direct interaction of forskolin with the channel that augments, by up to tenfold, the probability that channels will have delayed inactivation.     

A novel method for direct application of phospholipids to giant excised membrane patches in the study of sodium-calcium exchange and sodium channel currents

Effects of membrane phospholipids on Na⁺-Ca²⁺ exchange and Na⁺ channel currents were studied in giant excised cardiac sarcolemmal patches. Phospholipids were suspended in an inert vehicle of -tocopherol acetate and hexane and were then directly applied to the side of patch electrodes at a short distance from the tip during current recording. Phosphatidylserine strongly stimulated outward Na⁺-Ca²⁺ exchange current and altered the kinetics of cytoplasmic Na⁺- and Ca²⁺-dependent secondary modulation. This effect was partially reversed by phosphatidylcholine. Prolonged treatment with phosphatidylserine eliminated the inactivation transient normally observed upon rapid application of cytoplasmic Na⁺ but left cytoplasmic Ca²⁺ dependence largely intact. In such cases, subsequent chymotrypsin treatment removed cytoplasmic Ca²⁺ dependence, but had no further stimulatory effect, indicating maximum alleviation of inactivation by phosphatidylserine. While these results indicate that phosphatidylserine acts on a cytoplasmic, protease-sensitive regulatory domain of the exchanger, phosphatidylserine also stimulated the exchange current after deregulation by chymotrypsin, indicating an effect on the exchange mechanism itself. As in other myocyte preparations, cardiac Na⁺ currents in giant patches undergo a time-dependent negative shift in the voltage dependence of steady-state inactivation. Loss of phosphatidylserine from the cytoplasmic leaflet (i.e. loss of transbilayer asymmetry of phosphatidylserine distribution) does not appear to be the underlying cause, since phosphatidylserine did not reverse this shift, despite stimulation of Na⁺-Ca²⁺ exchange current in the same patches.     

(R)-roscovitine prolongs the mean open time of unitary N-type calcium channel currents

(R)-roscovitine (Ros) is a cyclin-dependent kinase inhibitor that also has been shown to have direct agonist and antagonist actions on Ca_v2.1 (P/Q-type) and Ca_v 2.2 (N-type) families of voltage-gated calcium channels. These kinase-independent effects represent a novel opportunity to advance our understanding of calcium channel function and calcium-triggered neurotransmitter release. Furthermore, such actions on calcium channels may direct the development of Ros derivatives as new therapeutic agents. We used patch clamp recordings to characterize mechanisms that underlie the agonist effects of Ros on unitary N-type calcium channel gating. We found that N-type channels normally gate with either a short or long mean open time, that Ros significantly prolonged the mean open time of the long gating component and increased the probability of observing channels that gated with a long open time, but had no effect on single channel conductance. Using Monte Carlo simulations of a single channel kinetic model and Ros interactions, we were able to reproduce our experimental results and investigate the model's microscopic dynamics. In particular, our simulations predicted that the longer open times generated by Ros were due to the appearance of a long open state combined with an increased amount of time spent in transitions between open states. Our results suggest a mechanism for agonist effects of Ros at the level of single channels, and provide a mechanistic explanation for previously reported agonist effects on whole cell calcium currents.     

The extracellular patch clamp: A method for resolving currents through individual open channels in biological membranes

The current contributions of individual ionic channels can be measured by electrically isolating a small patch of membrane. To do this, the tip of a small pipette is brought into close contact with an enzymatically cleaned membrane of a hypersensitive amphibian or mammalian muscle fiber. Current flowing through the pipette is measured. If the pipette contains cholinergic agonist at -molar concentrations, square pulse current waveforms can be observed which represent the activation of individual acetylcholine-receptor channels. The square pulses have amplitudes of 1 to 3 pA and durations of 10–100 ms.In order to obtain the necessary resolution, a delicate compromise had to be found between different experimental parameters. Pipettes with 1–3 m internal diameter and a steep final taper had to be used, extensive enzyme treatment was necessary, and conditions had to be found in which channels open at a relatively low frequency.     

A microflow superfusion system for use with excised membrane patches

A new method for rapidly changing the solution superfusing excised membrane patches is described. The main advantage of this method over previous rapid exchange methods is that it is possible to use very small quantities of solution (minimal volume about 100 l). The method consists of inserting the pipette tip, after obtaining an excised patch, into a small tube which can be independently perfused. Air bubbles are used to separate different solutions and to produce rapid step changes of solution. To test this method we examined the rate of change of: (1) the pipette resistance produced in response to changing to a solution of lower conductivity, (2) the amplitude of single K-channel currents in response to changing external K. The results suggest that the solution exchange is essentially a step function and is complete within 10–20 ms. The recording is briefly interrupted during the time taken for the passage of the air bubble past the pipette tip (<250 ms). In general, the passage of the air bubble did not affect the seal resistance. This method will be particularly useful for studies of the interactions of single ionic channels with chemicals which can only be obtained in small quantities.     

Whole-cell and unitary Ca channel currents in mammalian intestinal smooth muscle cells: evidence for the existence of two types of Ca channels

Whole-cell and single Ca channel currents were recorded in smooth muscle cells isolated from guinea-pig taenia coli to examine whether multiple types of Ca channels exist. Two different types of voltage-dependent Ca channels with different conductances and inactivation kinetics were identified from cell-attached patch clamp recordings using 50 mM Ba in the patch pipettes. One type of channel, with a large conductance of 25 pS, had a threshold of activation near –40 mV and the mean current reconstructed by averaging individual current responses inactivated slowly. A second type of channel, with a small conductance of 12 pS, had a similar threshold value to that of the 25 pS channel, but the averaged current inactivated rapidly. The steady state inactivation of the 12 pS channel was complete at a holding potential of –40 mV. We concluded that both channel types represent fast and slow inactivating voltage-dependent Ca channels which have been found in many excitable cells.     

A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system

(1) A preparation is described which allows patch clamp recordings to be made on mammalian central nervous system (CNS) neurones in situ. (2) A vibrating tissue slicer was used to cut thin slices in which individual neurones could be identified visually. Localized cleaning of cell somata with physiological saline freed the cell membrane, allowing the formation of a high resistance seal between the membrane and the patch pipette. (3) The various configurations of the patch clamp technique were used to demonstrate recording of membrane potential, whole cell currents and single channel currents from neurones and isolated patches. (4) The patch clamp technique was used to record from neurones filled with fluorescent dyes. Staining was achieved by filling cells during recording or by previous retrograde labelling. (5) Thin slice cleaning and patch clamp techniques were shown to be applicable to the spinal cord and almost any brain region and to various species. These techniques are also applicable to animals of a wide variety of postnatal ages, from newborn to adult.