A simple hanging-drop patch-clamp technique for studying single channel activities in excised membrane patches

A simple hanging-drop technique has been developed for examining the effects of reagents available in limited quantities such as some proteolytic enzymes and site-directed antibodies on single sodium channel activities in patches excised from hippocampal neuronal membranes. The advantages of the technique are as follows: 1. a very small volume (2–4 l of the buffer solution containing the agents to be tested; 2. recording under static conditions without using continuous perfusion systems; 3. reduced background current noise. The use of the technique for studying modifications of single channel parameters under the influence of agents available in limited amounts is illustrated by modification of single sodium channels by treatment with papain.     

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.     

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.     

Characterization of Ca2+-activated K+ channels in excised patches of human T lymphocytes

Ca²⁺-activated K⁺ [K(Ca)] channels were studied in excised patches of resting and activated human peripheral blood T lymphocytes. The K(Ca) channel had a single-channel conductance of 50±6 pS in symmetrical high-K⁺ solutions in the potential range of –100 to –10 mV and was inwardly rectifying at more depolarized potentials. The channel was sensitive to block by charybdotoxin (10 nM) and insensitive to apamin (3 nM). Half-maximum activation occurred at an internal free Ca²⁺ concentration of 360±110 nM. The concentration-effect curve had a slope factor of 0.83±0.12, suggesting a 11 interaction of Ca²⁺ ions with the channel. Ca²⁺ affects the open time probability of the K(Ca) channels, mainly by modulating the frequency of channel opening. The open probability did not show voltage dependence. The kinetics of the channel could be described assuming one open state and two closed states. The time constant of the exponential describing the open time distribution amounted to 2.8±1.2 ms, whereas the closed time distribution could be described with two exponentials with time constants of 0.2±0.05 ms and 8.0±2.1 ms, respectively. Resting T lymphocytes expressed a low number of channels but the density of channels increased dramatically during chronic phytohaemagglutinin stimulation.     

Giant excised cardiac sarcolemmal membrane patches: sodium and sodium-calcium exchange currents

Summary A tightly-sealed cardiac sarcolemmal patch preparation of large diameter (10–16 m with 5–25 G seals) has been developed to study regulation of selected electrogenic mechanisms. Formation of a large readily accessible membrane surface area is achieved by incubating freshly isolated myocytes in a high KCl/zero calcium solution, which promotes separation of sarcolemma from myofilaments. The formation of large-diameter, high resistance seals is facilitated by depositing a neutral hydrocarbon film on electrode tips. Sodium currents in excised patches are stable for patch life-times of 15–40 min with peak current densities of 120 to 350 A/cm². Outward sodium-calcium exchange current is identified by its specific dependence on external calcium and internal sodium, inhibition by cobalt and dichlorobenzamil, and activation by <0.1M internal free, calcium. Maximum exchange current density is >20 A/cm².Supported by a Grant-in-Aid and an Established Investigatorship of the American Heart Association.     

Calpastatin and nucleotides stabilize cardiac calcium channel activity in excised patches

The activity of single L-type Ca²⁺ channels is rapidly lost (run-down) when contact between the membrane and cytosol is interrupted. We have now achieved the stabilization of cardiac Ca²⁺ channel activity of guinea-pig ventricular myocytes by using either cytosol or defined components added to excised patches. The endogenous protease inhibitor, calpastatin, together with nucleotides, ATP + GTP, was found to prevent rundown as effectively as cardiac cytosolic solution. These results suggest the involvement of proteolysis by calpain in run-down of channel activity and enable the study of cardiac Ca²⁺ channel regulation with free access to both sides of the membrane.     

Single Ca2+-activated K+ channels in excised membrane patches of hamster oocytes

The properties of the Ca²⁺-activated K⁺ channel in unfertilized hamster oocytes were investigated at the single-channel level using inside-out excised membrane patches. The results indicate a new type of Ca²⁺-activated K⁺ channel which has the following characteristics: (1) single-channel conductance of 40–85 pS for outward currents in symmetrical K⁺ (150 mM) solutions, (2) inward currents of smaller conductance (10–50 pS) than outward currents, i.e. the channel is outwardly rectified in symmetrical K⁺ solutions, (3) channel activity dependent on the internal concentration of free Ca⁺ and the membrane potential, (4) modification of the channel activity by internal adenosine 5 diphosphate (0.1 mM) producing a high open probability regardless of membrane potential.     

Imaging excised apical plasma membrane patches of MDCK cells in physiological conditions with atomic force microscopy

 We combined the patch-clamp technique with atomic force microscopy (AFM) to visualize plasma membrane proteins protruding from the extracellular surface of cultured kidney cells (MDCK cells). To achieve molecular resolution, patches were mechanically isolated from whole MDCK cells by applying the patch-clamp technique. The excised inside-out patches were transferred on freshly cleaved mica and imaged with the AFM in air and under physiological conditions (i.e. in fluid). Thus, the resolution could be increased considerably (lateral and vertical resolutions 5 and 0.1 nm, respectively) as compared to experiments on intact cells, where plasma membrane proteins were hardly detectable. The apical plasma membrane surface of the MDCK cells showed multiple protrusions which could be identified as membrane proteins through the use of pronase. These proteins had a density of about 90 per μm2, with heights between 1 and 9 nm, and lateral dimensions of 20–60 nm. Their frequency distribution showed a peak value of 3 nm for the protein height. A simplified assumption – modelling plasma membrane proteins as spherical structures protruding from the lipid bilayer – allowed an estimation of the possible molecular weights of these proteins. They range from 50 kDa to 710 kDa with a peak value of 125 kDa. We conclude that AFM can be used to study the molecular structures of membranes which were isolated with the patch-clamp technique. Individual membrane proteins and protein clusters, and their arrangement and distribution in a native plasma membrane can be visualized under physiological conditions, which is a first step for their identification. Received: 18 December 1996 / Accepted: 5 March 1997     

Kinetic mode switch of rat brain IIA Na channels in Xenopus oocytes excised macropatches

Na currents recorded from inside-out macropatches excised from Xenopus oocytes expressing the subunit of the rat brain Na channel IIA show at least two distinguishable components in their inactivation time course, with time constants differing about tenfold ( _h1 = approx. 150 s and _h2 = approx. 2 ms). In excised patches, the inactivation properties of Na currents changed with time, favoring the faster inactivation kinetics. Analysis of the fast and slow current kinetics shows that only the relative magnitudes of _h1 and _h2 components are altered without significant changes in the time constants of activation or inactivation. In addition, voltage dependence of both activation and steady-state inactivation of Na currents are shifted to more negative potentials in patches with predominantly fast inactivation, although reversal potentials and valences remained unaltered. We conclude that the two inactivation modes discerned in this study are conferred by two states of Na channel the interconversion of which are regulated by an as yet unknown mechanism that seems to involve cytosolic factors.     

Cation channel blocked by extracellular Ca2+ in the apical membrane of the chick embryonic ectoderm

In the chick embryo (20 h incubation, gastrula stage), the apical membrane of the ectodermal cells shows a high density of a non-selective cation channel which is blocked by very low extracellular Ca²⁺ concentrations. Properties of this channel were studied at the single-channel level using the patch-clamp technique in the cell-attached mode.With 1 mmol/l Ca²⁺ in the pipette, only outward current was present and the channel conductance measured at +120 mV was 25.5 pS. In the absence of Ca²⁺, also inward current through the channel was observed. The conductances measured at –50 mV were 49.5 pS with Na⁺ as the charge carrier, 72.5 pS with K⁺, 49.1 pS with Cs⁺, and 18.5 pS with Li⁺. The conductance measured at +80 mV was around 23 pS in all four cases. The reversal potential was similar (around 25 mV) for all four ions, which indicates a poor selectivity of the channel. In the absence of Ca²⁺ and the presence of 1 mmol/l ethylene-bis(oxonitrilo) tetraacetate (EGTA), the kinetics of the channel were characterized by bursts of the order of seconds. During a burst, the channel flickered between one open and one closed level. The open time was constant between –30 mV and –80 mV, while the closed time decreased with hyperpolarization. The open time varied according to the permeant ion (K⁺+=Cs⁺+).Extracellular Ca²⁺ blocked the inward current in a voltage-dependent manner. The K _d values, 1 mol/l at –30 mV and 3.2 mol/l at –80 mV, indicate that Ca²⁺ ions exit the channel toward the intracellular side. A weak voltage dependency of the association rate constant suggests that the Ca²⁺-binding site is close to the outside mouth. Extracellular Ca²⁺ was much less efficient at blocking the outward current (K _d about 1 mmol/l at 80 mV). Tetracaine, but not uraniumdioxide, decreased the opening probability of the channel. The embryonic channel shows similarities with the Ca²⁺-blockable, poorly selective channel described in the epithelium of toad urinary bladder.     

Single K+ channels in membrane evaginations of smooth muscle cells

Attempts have been made to apply the patch-clamp technique to enzymatically dispersed smooth muscle cells of frog and toad stomach. The rate of successful gigaseal formation has been extremely low, but better results can be obtained when patches are taken from membrane evaginations which develop on single cells after mechanical agitation and incubation in Ca²⁺-containing solutions at 25° C. Also ball-shaped single cells formed by the confluence of membrane evaginations were found to be equally useful for patch-clamp studies. Giga-seal formation was obtained in more than 80% of all attempts. Electron micrographs indicate that the myofilaments in membrane evaginations an in ball-shaped cells are separated from the cell membrane. Channel activity in membrane patches of such myoballs or evaginations is similar to the channel activity as found in intact cells. Two types of K⁺ channels (100 and 200 pS) have been observed that can be blocked by tetraethylammonium. Channels with the conductance of 200 pS are activated by intracellular Ca²⁺. The formation of evaginations has also been observed in other cells and may help to apply the patch-clamp technique to cells contaminated with surface coats.     

Digital processing of the current noise evoked by kainate in cerebellar granule cells

Current induced in cultured cerebellar granule cells by the bath application of kainate (500 μM) was measured using the conventional patch-clamp technique. Two different kinds of responses were observed after the agonits perfusion. Some cells exhibited small inward whole-cell currents: 116±40 pA (7 cells) at a clamp potential of−60 mV; in other cells, the agonist induced significantly larger currents: 420±35 pA (6 cells) at a clamp potential of −60 mV. The current flowing in the agonist-activated ionic channels was indirectly estimated by processing the fluctuations of whole-cell current by means of an original parametric method. Mean conductance of the underlying channels was then determined from the single-channel current estimated at different clamp potentials. In the cells exhibiting small inward currents, the mean conductance was equal to 0.5±0.2 pS (7 cells), whereas in the cells with large inward currents it was 3±0.4 pS (6 cells). This result gives a coherent explanation of the different kinds of responses observed at macroscopic level in the whole-cell current and confirms that kainate-activated channels can exhibit different levels of conductance.     

Direct effects of 9-anthracene compounds on cystic fibrosis transmembrane conductance regulator gating

Anthracene-9-carboxylic acid (9-AC) has been reported to show both potentiation and inhibitory effects on guinea-pig cardiac cAMP-activated chloride channels via two different binding sites, and inhibition of Mg²⁺-sensitive protein phosphatases has been proposed for the mechanism of 9-AC potentiation effect. In this study, we examined the effects of 9-AC on wild-type and mutant human cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels expressed in NIH3T3 or CHO cells. 9-AC inhibits whole-cell CFTR current in a voltage-dependent manner, whereas the potentiation effect is not affected by membrane potentials. Anthracene-9-methanol, an electro-neutral 9-AC analog, fails to block CFTR, but shows a nearly identical potentiation effect, corroborating the idea that two chemically distinct sites are responsible, respectively, for potentiation and inhibitory actions of 9-AC. 9-AC also enhances the activity of R-CFTR, a constitutively active CFTR mutant whose R-domain is removed. In excised inside-out patches, 9-AC increases P_o by prolonging the mean burst durations and shortening the interburst durations. We therefore conclude that two different 9-AC binding sites for potentiation and inhibitory effects on CFTR channels are located outside of the R-domain. We also speculate that 9-AC potentiates CFTR activity by directly affecting CFTR gating.     

A cAMP-activated chloride channel in the plasma membrane of cultured human gastric cells (HGT-1)

Hydrochloric acid (HCl) secretion by gastric parietal cells involves an apical Cl^– conductance, the properties of which have not been defined. In the present study, forskolin and histamine [agonists that increase intracellular cyclic adenosine monophosphate (cAMP)], and dibutyryl cAMP, activated channels in previously quiescent cell-attached membrane patches on cultured human gastric cells (HGT-1). In the cell-attached configuration (Cl^–149 mmol/ 1 in bath and pipette), channels exhibited outward rectification, voltage dependence, inward current (–0.7 pA) at zero holding potential and a reversal potential of +24 mV, consistent with the presence of a Cl^– conductive pathway. In excised inside-out patches, channels (i) exhibited degrees of outward rectification and voltage dependence that were comparable to those seen in cell-attached patches, (ii) demonstrated a –21 mV shift of their reversal potential when bath Cl^– was decreased from 149 mmol/l to 53 mmol/l (calculated Cl^–:cation permeability ratio 171), and (iii) were highly sensitive to the Cl^– channel blocker diphenylamine-2-carboxylic acid (DPC, 10^–3 mol/l). This cAMP-activated Cl^– channel bears many similarities to other Cl^– channels within intestinal epithalia, and may represent the apical Cl^– channel operating in HCl-secreting gastric parietal cells.     

Effects of thrombin on single calcium channels in frog ventricular cells

Single calcium channel (Ca channel) currents were measured using the patch-clamp technique in isolated ventricular myocytes of the frog (Rana esculenta). Sodium was used as the charge carrier. After formation of cell-attached patches, the proteolytic enzyme thrombin was added to the bath solution, where it increased the amplitude of the averaged currents more than twofold, by decreasing the number of empty sweeps and reducing the time constant of the slow exponential term of the shut-time histogram. Single channel conductance was not changed by thrombin. If the activation kinetics of the Ca channels are described by the commonly usedC ₁-C ₂-O model, whereC ₁ andC ₂ indicate closed states 1 and 2 respectively andO denotes the open state, thrombin increases the open-state probability in the non-empty sweeps by increasing the rate constant (k ₁) for the transition fromC ₁ toC ₂. It is shown that thrombin acts via an H-7 blockable pathway.     

Effects of intracellular pH and Ca2+ on the activity of stretch-sensitive cation channels in leech neurons

The effects of intracellular pH and calcium on the activity of the leech mechanosensitive cation channels have been studied. These channels exhibited two activity modes denoted as spike-like (SL) and multiconductance (MC). In the absence of mechanical stimulation, acidification of the intracellular side of membrane patches from 7.2 to 6.2 reversibly increased the mean channel open time as well as the opening frequency in the SL mode. Channels in MC mode were activated by a pH_i reduction from 7.2 to 6.2, but were inhibited at pH_i 5.5. Unlike MC mode, SL mode was strongly activated by intracellular Ca²⁺. Fura-2 imaging experiments showed that intracellular calcium was induced to increase by hypotonic cell swelling. The major component of this response did not require extracellular calcium. A component of the swelling-induced calcium response was sensitive to blockers of stretch-sensitive cation channels. The results indicate that the two activity modes of mechanosensitive channels of leech neurons respond differently to changes of intracellular pH and calcium. The sensitivity of the channel to micromolar concentrations of internal free calcium, along with its permeability to this ion, is consistent with a role in the amplification of mechanically induced Ca²⁺ signals in leech neurons.     

Excision-activated chloride channels in cultured postnatal rat hippocampal neurons

Chloride permeable intermediate conductance single channel events activated on patch excision were found in outside-out patches from cultured postnatal hippocampal neurons. A majority of the channels had a conductance of 83 ± 2.1 pS when recorded in a symmetrical TEAC1 solution. Two other populations of channels with conductance values of 62 ± 2.1 pS and 145 ± 1.9 pS were also observed. The reversal potentials for these intermediate conductance Cl⁻ channels coincided with that of the GABA activated channels. The channels characteristically appeared 5–15 min after patch excision, suggesting that these channels may be blocked by some diffusible factors under physiological conditions. Based on the measurements of channel burst durations while the channel was partially blocked, and the channel open times after complete relief from the block, the mechanism of blockade does not appear to be a simple open channel blockade. The high prevalence and its potential regulation by cytosolic factors suggest an important physiological role for these Cl⁻ channels coupling neuronal excitability with cellular metabolism.     

In situ recording from gut pacemaker cells

Interstitial cells of Cajal (ICC) associated with the myenteric plexus of the small intestine are crucial players in gut physiology performing pacemaker functions and directing peristalsis and segmentation. ICC have been studied after chemical isolation and under culture conditions, but concerns that these methods affect the intrinsic properties have hindered progress in our understanding of ICC. To overcome this problem, we have developed a method to obtain electrophysiological recordings from ICC in situ. The critical feature is the ability to make high resistance seals onto cells that are embedded within tissue to obtain patch clamp recordings. Our first results show a prominent presence of a chloride channel, one of the proposed ICC pacemaker channels. The developed method can be applied to auxiliary cells of the enteric nervous system such as glial cells or fibroblasts and will be ideal for the study of cell–cell communication in tissue.     

Single Ca channel currents in mammalian visceral smooth muscle cells

Recordings of single Ca channel currents in mammalian visceral smooth muscle cells were obtained using patch clamp techniques. Smooth muscle cells from guinea-pig taenia coli were prepared by enzymatic dispersion using 0.3% collagenase. The recordings were obtained from cell-attached membrane patches of isolated cells with a pipette filled with isotonic 50 mM Ba²⁺. When the membrane patch was depolarized, brief inward current pulses of unitary size and small amplitude were observed. The amplitude of these single channel currents decreased linearly with increasing depolarization in a voltage range from –20 mV to +50 mV about the resting potential. The slope conductance was estimated to be about 30 pS. The mean current reconstructed by averaging individual current responses showed kinetic behaviour with a rapid activation and a slower inactivation process similar to the macroscopic Ca²⁺ current observed in strips of guinea-pig taenia coli. The present study suggests that the inward current pulses of unitary size induced by voltage-clamp pulses were due to Ba ions passing through a single voltage dependent Ca channel.