Effects of pipette geometry on the time course of solution change in patch clamp experiments.

The time course of change in current through KATP channels in inside-out membrane patches, after step change of permeant ion (K+) concentration, was measured. A simple model of the patch as a membrane disc at the base of a cone with the apex removed, was able to describe the time course of channel activity after step change of [K+]. By measuring pipette geometry and using jumps of [permeant ion], it was then possible to estimate the time course of concentration at the membrane for jumps of any other ion or gating ligand. A simple channel block mechanism was used to simulate experiments with concentration jumps of a blocking ligand. The rate constants for ligand-channel interaction were extracted by least-squares fitting of computed mass action responses to those observed in simulated experiments. The simulations showed that even with diffusion delays of hundreds of milliseconds (as may occur in inside-out patch experiments), ligand association and dissociation rates of up to 1,000 s-1 could be accurately extracted by this approach. The approach should be generally applicable to the analysis of ligand concentration jump experiments on any ion channel whose activity is modulated by intracellular ligand.     

Hindered diffusion in excised membrane patches from retinal rod outer segments.

Excised inside-out membrane patches are useful for studying the cGMP-activated ion channels that generate the electrical response to light in retinal rod cells. We show that strong ionic current across a patch changes the driving force on the current by altering the ionic concentration near the surface membrane, an effect somewhat like that first described by Frankenhaeuser and Hodgkin (1956) in squid axons. The dominant concentration change occurs in the solution adjacent to the cytoplasmic (inner) surface of the membrane, where diffusion is impaired by intracellular material that adheres to the patch during excision. The magnitude and time course of the ionic changes are consistent with the expected volume of this material and with an effective diffusion coefficient about an order of magnitude less than that in free solution. Methods are described for correcting current transients observed in voltage clamp experiments, so that channel gating kinetics can be obtained without contamination by changes in driving force. We suggest that restricted diffusion may occur in patches excised from other types of cells and influence rapid kinetic measurements.     

Activation of nonselective cation channels by physiological cholecystokinin concentrations in mouse pancreatic acinar cells.

The activation of the nonselective cation channels in mouse pancreatic acinar cells has been assessed at low agonist concentrations using patch-clamp whole cell, cell-attached patch, and isolated inside-out patch recordings. Application of acetylcholine (ACh) (25-1,000 nM) and cholecystokinin (CCK) (2-10 pM) evoked oscillatory responses in both cation and chloride currents measured in whole cell experiments. In cell-attached patch experiments we demonstrate CCK and ACh evoked opening of single 25-pS cation channels in the basolateral membrane. Therefore, at least a component of the whole cell cation current is due to activation of cation channels in the basolateral acinar cell membrane. To further investigate the reported sensitivity of the cation channel to intracellular ATP and calcium we used excised inside-out patches. Micromolar Ca2+ concentrations were required for significant channel activation. Application of ATP and ADP to the intracellular surface of the patch blocked channel opening at concentrations between 0.2 and 4 mM. The nonmetabolizable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP, 0.2-2 mM), also effectively blocked channel opening. The subsequent removal of ATP caused a transient increase in channel activity not seen with the removal of ADP or AMP-PNP. Patches isolated into solutions containing 2 mM ATP showed channel activation at micromolar Ca2+ concentrations. Our results show that ATP has two separate effects. The continuous presence of the nucleotide is required for operation of the cation channels and this action seems to depend on ATP hydrolysis. ATP can also close the channel and this effect can be demonstrated in excised inside-out patches when ATP is added to the bath after a period of exposure to an ATP-free solution. This action does not require ATP hydrolysis. Under physiological conditions hormonal stimulation can open the nonselective cation channels and this can be explained by the rise in the intracellular free Ca2+ concentration.     

Patch cramming: monitoring intracellular messengers in intact cells with membrane patches containing detector ion channels.

This paper introduces "patch cramming," a new procedure that utilizes an ion channel gated directly by an intracellular messenger molecule as a probe for detecting changes in the concentration of that molecule in an intact cell. A patch pipette containing the channel in a membrane patch is inserted into a recipient cell where the channel locally "senses" the intracellular messenger. In this study patches containing Ca2(+)-dependent K+ channels were inserted into Helix neurons, where they were activated by Ca2+ influx during trains of action potentials. Channels gated directly by other messengers, including cyclic nucleotides and IP3, have also been identified. Hence, by using detector channels with appropriate specificity, it may be possible to detect local intracellular fluctuations of these molecules.     

Ion fluxes in giant excised cardiac membrane patches detected and quantified with ion-selective microelectrodes

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2-3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10-15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion-coupled transporters in giant patches.     

Developmental regulation of mechanosensitive calcium channels in skeletal muscle from normal and mdx mice.

Single-channel activity was recorded from cell-attached membrane patches on flexor digitorum brevis fibres acutely isolated from normal and mdx mice at different stages of postnatal development. Recordings from cell-attached patches on both normal and mdx fibres were dominated by the activity of mechanosensitive ion channels with a conductance of approximately 17 pS with 110 mM Ba2+ in the patch electrode. In a small fraction of the patches on mdx fibres from young mice, channels showed very high levels of activity. Channel activity recorded from mdx fibres from older mice was higher than in age-matched normal fibres and the level of activity decreased during development. Channel density decreased in normal fibres, whereas it remained relatively constant in mdx fibres, as if channels are down-regulated in normal, but not mdx, fibres during postnatal development. An early step in the dystrophic process may be an alteration of the mechanisms that regulate the expression of functional channels.     

Membrane-pipette interactions underlie delayed voltage activation of mechanosensitive channels in Xenopus oocytes.

To investigate the mechanism for the delayed activation by voltage of the predominant mechanosensitive (MS) channel in Xenopus oocytes, currents were recorded from on-cell and excised patches of membrane with the patch clamp technique and from intact oocytes with the two-electrode voltage clamp technique. MS channels could be activated by stretch in inside-out, on-cell, and outside-out patch configurations, using pipettes formed of either borosilicate or soft glass. In inside-out patches formed with borosilicate glass pipettes, depolarizing voltage steps activated MS channels in a cooperative manner after delays of seconds. This voltage-dependent activation was not observed for outside-out patches. Voltage-dependent activation was also not observed when the borosilicate pipettes were either replaced with soft glass pipettes or coated with soft glass. When depolarizing voltage steps were applied to the whole oocyte with a two-electrode voltage clamp, currents that could be attributed to MS channels were not observed. Yet the same depolarizing steps activated MS channels in on-cell patches formed with borosilicate pipettes on the same oocyte. These observations suggest that the delayed cooperative activation of MS channels by depolarization is not an intrinsic property of the channels, but requires interaction between the membrane and patch pipette.     

Cation channels in the plasma membrane of retinal rod outer segments activated by cGMP

Properties of cGMP-activated cation channels were investigated on isolated patches of the ROS plasma membrane using the "patch clamp" technique. The channels were shown to be characterized by ideal cation selectivity under physiological conditions and are nearly equally permeable for cations of alkaline metals. At the same time they are permeable for some bivalence cations (PNa approximately PCa). Other channel properties are described and their comparative analysis is given. It suggests that cGMP-activated cation channels represent a new type of cation channels.     

Exocytosis of single chromaffin granules in cell-free inside-out membrane patches

In chromaffin cells, exocytosis of single granules and properties of the fusion pore--the first connection between vesicular lumen and extracellular space --can be studied by cell-attached patch amperometry, which couples patch-clamp capacitance measurements with simultaneous amperometric recordings of transmitter release. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.     

High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formation

Microstructured planar substrates have been shown to be suitable for patch clamp recording from both whole cells and isolated patches of membrane, as well as for measurements from planar lipid bilayers. Here, we further explore this technology with respect to high-resolution, low noise single-channel recording. Using solvent-free lipid bilayers from giant unilamellar vesicles obtained by electro-swelling, we recorded channels formed by the peptaibol alamethicin, a well-studied model system for voltage-dependent channels, focusing on the transient dynamics of single-channel formation upon application of a voltage step. With our setup, we were able to distinctly resolve dwell times well below 100 mus and to perform a thorough statistical analysis of alamethicin gating. Our results show good agreement with models that do not rely on the existence of non-conducting preaggregate states. Microstructured apertures in glass substrates appear promising with respect to future experiments on cellular ion channels reconstituted in suspended lipid membranes.     

The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel.

The role of the tyrosine kinase p60c-src on the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel was investigated with the cell-attached and excised patch clamp technique in conjunction with current noise analysis of recordings containing multiple channels per patch. Spectra of CFTR-generated current noise contained a low-frequency and a high-frequency Lorentzian noise component. In the cell-attached mode, the high-frequency Lorentzian was significantly dependent on the membrane potential, while the low-frequency Lorentzian was unaffected. Excision of forskolin-stimulated patches into ATP-containing solution significantly reduced the amplitude of the voltage-dependent high-frequency Lorentzian. Addition of the tyrosine kinase p60c-src to excised, active, CFTR-containing membrane patches increased mean currents by 54%, increased the corner frequency of the low-frequency Lorentzian, and recovered the high-frequency Lorentzian and its characteristics. Treatment with lambda-phosphatase inactivated src-induced currents and changes in gating. When active patches were excised under conditions in which patch-associated tyrosine phosphatases were blocked with sodium vanadate, the high-frequency gating remained relatively unchanged. The results suggest that CFTR's open probability and its voltage-dependent fast gate are dependent on tyrosine phosphorylation, and that membrane-associated tyrosine phosphatases are responsible for inactivation of the fast gate after patch excision.     

Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.     

GABA increases both the conductance and mean open time of recombinant GABAA channels co-expressed with GABARAP

The single channel properties of recombinant gamma-aminobutyric acid type A (GABA(A))alphabetagamma receptors co-expressed with the trafficking protein GABARAP were investigated using membrane patches in the outside-out patch clamp configuration from transiently transfected L929 cells. In control cells expressing alphabetagamma receptors alone, GABA activated single channels whose main conductance was 30 picosiemens (pS) with a subconductance state of 20 pS, and increasing the GABA concentration did not alter their conductance. In contrast, when GABA(A) receptors were co-expressed with GABARAP, the GABA-activated single channels displayed multiple, high conductances (> or =40 pS), and GABA (> or =10 microM) was able to increase their conductance, up to a maximum of 60 pS. The mean open time of GABA-activated channels in control cells expressing alphabetagamma receptors alone was 2.3 +/- 0.1 ms for the main 30-pS channel and shorter for the subconductance state (20 pS, 0.8 +/- 0.1 ms). Similar values were measured for the 30- and 20-pS channels active in patches from cells co-expressing GABARAP. However higher conductance channels (> or =40 pS) remained open longer, irrespective of whether GABA or GABA plus diazepam activated them. Plotting mean open times against mean conductances revealed a linear relationship between these two parameters. Since high GABA concentrations increase both the maximum single channel conductance and mean open time of GABA(A) channels co-expressed with GABARAP, trafficking processes must influence ion channel properties. This suggests that the organization of extrasynaptic GABA(A) receptors may provide a range of distinct inhibitory currents in the brain and, further, provide differential drug responses.     

High-resolution electrophysiology on a chip: Transient dynamics of alamethicin channel formation

Microstructured planar substrates have been shown to be suitable for patch clamp recording from both whole cells and isolated patches of membrane, as well as for measurements from planar lipid bilayers. Here, we further explore this technology with respect to high-resolution, low noise single-channel recording. Using solvent-free lipid bilayers from giant unilamellar vesicles obtained by electro-swelling, we recorded channels formed by the peptaibol alamethicin, a well-studied model system for voltage-dependent channels, focusing on the transient dynamics of single-channel formation upon application of a voltage step. With our setup, we were able to distinctly resolve dwell times well below 100 μs and to perform a thorough statistical analysis of alamethicin gating. Our results show good agreement with models that do not rely on the existence of non-conducting preaggregate states. Microstructured apertures in glass substrates appear promising with respect to future experiments on cellular ion channels reconstituted in suspended lipid membranes.     

Studying properties of neurotransmitter receptors by non-stationary noise analysis of spontaneous postsynaptic currents and agonist-evoked responses in outside-out patches

Chemical synaptic transmission depends on neurotransmitter-gated ion channels concentrated in the postsynaptic membrane of specialized synaptic contacts. The functional characteristics of these neurotransmitter receptor channels are important for determining the properties of synaptic transmission. Whole-cell recording of postsynaptic currents (PSCs) and outside-out patch recording of transmitter-evoked currents are important tools for estimating the single-channel conductance and the number of receptors contributing to the PSC activated by a single transmitter quantum. When single-channel activity cannot be directly resolved, non-stationary noise analysis is a valuable tool for determining these parameters. Peak-scaled non-stationary noise analysis can be used to compensate for quantal variability in synaptic currents. Here, we present detailed protocols for conventional and peak-scaled non-stationary noise analysis of spontaneous PSCs and responses in outside-out patches. In addition, we include examples of computer code for individual functions used in the different stages of non-stationary noise analysis. These analysis procedures require 3-8 h.     

Clustering and coupled gating modulate the activity in KcsA, a potassium channel model

Different patterns of channel activity have been detected by patch clamping excised membrane patches from reconstituted giant liposomes containing purified KcsA, a potassium channel from prokaryotes. The more frequent pattern has a characteristic low channel opening probability and exhibits many other features reported for KcsA reconstituted into planar lipid bilayers, including a moderate voltage dependence, blockade by Na(+), and a strict dependence on acidic pH for channel opening. The predominant gating event in this low channel opening probability pattern corresponds to the positive coupling of two KcsA channels. However, other activity patterns have been detected as well, which are characterized by a high channel opening probability (HOP patterns), positive coupling of mostly five concerted channels, and profound changes in other KcsA features, including a different voltage dependence, channel opening at neutral pH, and lack of Na(+) blockade. The above functional diversity occurs correlatively to the heterogeneous supramolecular assembly of KcsA into clusters. Clustering of KcsA depends on protein concentration and occurs both in detergent solution and more markedly in reconstituted membranes, including giant liposomes, where some of the clusters are large enough (up to micrometer size) to be observed by confocal microscopy. As in the allosteric conformational spread responses observed in receptor clustering (Bray, D. and Duke, T. (2004) Annu. Rev. Biophys. Biomol. Struct. 33, 53-73) our tenet is that physical clustering of KcsA channels is behind the observed multiple coupled gating and diverse functional responses.     

Activation of mechanosensitive currents in traumatized membrane

Mechanosensitive (MS) channels, ones whose open probabilityvaries with membrane tension in patch recordings, are diverse andubiquitous, yet many are remarkably insensitive to mechanical stimuliin situ. Failure to elicit mechanocurrents from cells with abundant MSchannels suggests that, in situ, the channels are protected frommechanical stimuli. To establish what conditions affect MS channelgating, we monitored Lymnaea neuronstretch-activated K (SAK) channels in cell-attached patches afterdiverse treatments. Mechanosensitivity was gauged by rapidity of onsetand extent of channel activation during a step pressure applied to a"naive" patch. The following treatments enhancedmechanosensitivity: actin depolymerization (cytochalasin B),N-ethylmaleimide, an inhibitor ofATPases including myosin, elevated Ca (using A-23187), and osmoticswelling (acutely and after 24 h). Osmotic shrinking decreased mechanosensitivity. A unifying interpretation is that traumatized cortical cytoskeleton cannot prevent transmission of mechanical stimulito plasma membrane channels. Mechanoprotection and capricious mechanosensitivity are impediments to cloning efforts with MS channels.We demonstrate a potpourri of endogenous MS currents fromL-M(TK) fibroblasts;others had reported these cells to be MS current null and hence to besuitable for expressing putative MS channels.     

Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.     

The interpretation of current-clamp recordings in the cell-attached patch-clamp configuration

In these experiments we have investigated the feasibility and accuracy of recording steady-state and dynamic changes in transmembrane potential noninvasively across an intact cell-attached patch using the current-clamp mode of a conventional patch-clamp amplifier. Using an equivalent circuit mimicking simultaneous whole-cell voltage-clamp and cell-attached current-clamp recordings we have defined both mathematically and experimentally the relationship between the membrane patch resistance, the seal resistance, and the fraction of the whole-cell potential recorded across an intact membrane patch. This analysis revealed a steep increase in the accuracy of recording of steady-state membrane potential as the seal/membrane ratio increases from 0. The recording accuracy approaches 100% as the seal/membrane ratio approaches infinity. Membrane potential measurements across intact cell-attached patches in rat basophilic leukemia cells and rat megakaryocytes revealed a surprisingly high degree of accuracy and demonstrated the ability of this noninvasive technique to follow dynamic changes in potential in nonexcitable cells.     

Lipopolysaccharide can activate BK channels of arterial smooth muscle in the absence of iNOS expression

The role of inducible nitric oxide synthase (iNOS) in the acute activation of large-conductance, Ca(2+)-dependent K(+) channels (BK channels) by Escherichia coli endotoxin (lipopolysaccharide, LPS) was studied in murine vascular smooth muscle cells. Confocal laser scanning microscopy and patch clamp recordings were utilised. Within 2 h of donor rat sacrifice, iNOS-like immunoreactivity could be detected in cerebrovascular smooth muscle cells (CVSMCs) enzymatically dispersed from rat cerebral arteries. This staining was absent in cells fixed immediately after donor rat sacrifice. LPS was then applied to the cytoplasmic face of inside-out membrane patches excised from rat CVSMCs within 2-4 h of donor rat sacrifice. It was found that LPS (10-100 microg/ml) rapidly and reversibly increased the open probability of BK channels in these patches. This LPS response was not altered in the presence of the non-isoform specific NOS inhibitor N(omega)-nitro-L-arginine. LPS responses were then compared in aortic smooth muscle (ASMC) BK channels derived from wild-type and iNOS-knockout (iNOS-KO) mice. LPS activated BK channels in inside-out patches of ASMC membrane derived from both wild-type and iNOS-knockout mice. These studies establish that LPS can activate BK channels by a mechanism quite independent of the well-established pathway mediated by iNOS in vascular smooth muscle cells.