Cell-attached Patch-clamp Experiments Research Articles

A detailed analysis of single-channel Nav 1.5 recordings does not reveal any cooperative gating.

Cardiac voltage-gated sodium (Na+ ) channels (Nav 1.5) are crucial for myocardial electrical excitation. Recent studies based on single-channel recordings have suggested that Na+ channels interact functionally and exhibit coupled gating. However, the analysis of such recordings frequently relies on manual interventions, which can lead to bias. Here, we developed an automated pipeline to de-trend and idealize single-channel currents, and assessed possible functional interactions in cell-attached patch clamp experiments in HEK293 cells expressing human Nav 1.5 channels as well as in adult mouse and rabbit ventricular cardiomyocytes. Our pipeline involved de-trending individual sweeps by linear optimization using a library of predefined functions, followed by digital filtering and baseline offset. Subsequently, the processed sweeps were idealized based on the idea that the ensemble average of the idealized current identified by thresholds between current levels reconstructs at best the ensemble average current from the de-trended sweeps. This reconstruction was achieved by non-linear optimization. To ascertain functional interactions, we examined the distribution of the numbers of open channels at every time point during the activation protocol and compared it to the distribution expected for independent channels. We also examined whether the channels tended to synchronize their openings and closings. However, we did not uncover any solid evidence of such interactions in our recordings. Rather, our results indicate that wild-type Nav 1.5 channels are independent entities or exhibit only very weak functional interactions that are probably irrelevant under physiological conditions. Nevertheless, our unbiased analysis will be important for further studies examining whether auxiliary proteins potentiate functional Na+ channel interactions. KEY POINTS: Nav 1.5 channels are critical for cardiac excitation. They are part of macromolecular interacting complexes, and it was previously suggested that two neighbouring channels may functionally interact and exhibit coupled gating. Manual interventions when processing single-channel recordings can lead to bias and inaccurate data interpretation. We developed an automated pipeline to de-trend and idealize single-channel currents and assessed possible functional interactions between Nav 1.5 channels in HEK293 cells and cardiomyocytes during activation protocols using the cell-attached patch clamp technique. In recordings consisting of up to 1000 sweeps from the same patch, our analysis did not reveal any evidence of functional interactions or coupled gating between wild-type Nav 1.5 channels. Our unbiased analysis may be useful in further studies examining how Na+ channel interactions are affected by mutations and auxiliary proteins.

17β-estradiol does not have a direct effect on the function of striatal cholinergic interneurons in adult mice in vitro.

The striatum is an essential component of the basal ganglia that is involved in motor control, action selection and motor learning. The pathophysiological changes of the striatum are present in several neurological and psychiatric disorder including Parkinson's and Huntington's diseases. The striatal cholinergic neurons are the main regulators of striatal microcircuitry. It has been demonstrated that estrogen exerts various effects on neuronal functions in dopaminergic and medium spiny neurons (MSN), however little is known about how the activity of cholinergic interneurons are influenced by estrogens. In this study we examined the acute effect of 17β-estradiol on the function of striatal cholinergic neurons in adult mice in vitro. We also tested the effect of estrus cycle and sex on the spontaneous activity of cholinergic interneurons in the striatum. Our RNAscope experiments showed that ERα, ERβ, and GPER1 receptor mRNAs are expressed in some striatal cholinergic neurons at a very low level. In cell-attached patch clamp experiments, we found that a high dose of 17β-estradiol (100 nM) affected the spontaneous firing rate of these neurons only in old males. Our findings did not demonstrate any acute effect of a low concentration of 17β-estradiol (100 pM) or show any association of estrus cycle or sex with the activity of striatal cholinergic neurons. Although estrogen did not induce changes in the intrinsic properties of neurons, indirect effects via modulation of the synaptic inputs of striatal cholinergic interneurons cannot be excluded.

Cytoskeleton Rearrangements Modulate TRPC6 Channel Activity in Podocytes.

The actin cytoskeleton of podocytes plays a central role in the functioning of the filtration barrier in the kidney. Calcium entry into podocytes via TRPC6 (Transient Receptor Potential Canonical 6) channels leads to actin cytoskeleton rearrangement, thereby affecting the filtration barrier. We hypothesized that there is feedback from the cytoskeleton that modulates the activity of TRPC6 channels. Experiments using scanning ion-conductance microscopy demonstrated a change in migration properties in podocyte cell cultures treated with cytochalasin D, a pharmacological agent that disrupts the actin cytoskeleton. Cell-attached patch-clamp experiments revealed that cytochalasin D increases the activity of TRPC6 channels in CHO (Chinese Hamster Ovary) cells overexpressing the channel and in podocytes from freshly isolated glomeruli. Furthermore, it was previously reported that mutation in ACTN4, which encodes α-actinin-4, causes focal segmental glomerulosclerosis and solidifies the actin network in podocytes. Therefore, we tested whether α-actinin-4 regulates the activity of TRPC6 channels. We found that co-expression of mutant α-actinin-4 K255E with TRPC6 in CHO cells decreases TRPC6 channel activity. Therefore, our data demonstrate a direct interaction between the structure of the actin cytoskeleton and TRPC6 activity.

A Distinct Pool of Nav1.5 Channels at the Lateral Membrane of Murine Ventricular Cardiomyocytes.

Background: In cardiac ventricular muscle cells, the presence of voltage-gated sodium channels Nav1.5 at the lateral membrane depends in part on the interaction between the dystrophin–syntrophin complex and the Nav1.5 C-terminal PDZ-domain-binding sequence Ser-Ile-Val (SIV motif). α1-Syntrophin, a PDZ-domain adaptor protein, mediates the interaction between Nav1.5 and dystrophin at the lateral membrane of cardiac cells. Using the cell-attached patch-clamp approach on cardiomyocytes expressing Nav1.5 in which the SIV motif is deleted (ΔSIV), sodium current (INa) recordings from the lateral membrane revealed a SIV-motif-independent INa. Since immunostaining has suggested that Nav1.5 is expressed in transverse (T-) tubules, this remaining INa might be carried by channels in the T-tubules. Of note, a recent study using heterologous expression systems showed that α1-syntrophin also interacts with the Nav1.5 N-terminus, which may explain the SIV-motif independent INa at the lateral membrane of cardiomyocytes.Aim: To address the role of α1-syntrophin in regulating the INa at the lateral membrane of cardiac cells.Methods and Results: Patch-clamp experiments in cell-attached configuration were performed on the lateral membranes of wild-type, α1-syntrophin knockdown, and ΔSIV ventricular mouse cardiomyocytes. Compared to wild-type, a reduction of the lateral INa was observed in myocytes from α1-syntrophin knockdown hearts. Similar to ΔSIV myocytes, a remaining INa was still recorded. In addition, cell-attached INa recordings from lateral membrane did not differ significantly between non-detubulated and detubulated ΔSIV cardiomyocytes. Lastly, we obtained evidence suggesting that cell-attached patch-clamp experiments on the lateral membrane cannot record currents carried by channels in T-tubules such as calcium channels.Conclusion: Altogether, these results suggest the presence of a sub-pool of sodium channels at the lateral membrane of cardiomyocytes that is independent of α1-syntrophin and the PDZ-binding motif of Nav1.5, located in membrane domains outside of T-tubules. The question of a T-tubular pool of Nav1.5 channels, however, remains open.

Harnessing red cell membrane pathophysiology towards point‐of‐care diagnosis for sickle cell disease

Harnessing red cell membrane pathophysiology towards point‐of‐care diagnosis for sickle cell disease

Hypertension-Linked Mutation of α-Adducin Increases CFTR Surface Expression and Activity in HEK and Cultured Rat Distal Convoluted Tubule Cells

The CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) activity and localization are influenced by the cytoskeleton, in particular by actin and its polymerization state. In this study we investigated whether the expression of the hypertensive mutations of α-adducin (G460W-S586C in humans, F316Y in rats), an actin capping protein, led to a functional modification of CFTR activity and surface expression. The experiments were performed on HEK293 T cells cotransfected with CFTR and the human wild type (WT) or G460W mutated α-adducin. In whole-cell patch-clamp experiments, both the CFTR chloride current and the slope of current activation after forskolin addition were significantly higher in HEK cells overexpressing the G460W adducin. A higher plasma membrane density of active CFTR channels was confirmed by cell-attached patch-clamp experiments, both in HEK cells and in cultured primary DCT cells, isolated from MHS (Milan Hypertensive Strain, a Wistar rat (Rattus norvegicus) hypertensive model carrying the F316Y adducin mutation), compared to MNS (Milan Normotensive Strain) rats. Western blot experiments demonstrated an increase of the plasma membrane CFTR protein expression, with a modification of the channel glycosylation state, in the presence of the mutated adducin. A higher retention of CFTR protein in the plasma membrane was confirmed both by FRAP (Fluorescence Recovery After Photobleaching) and photoactivation experiments. The present data indicate that in HEK cells and in isolated DCT cells the presence of the G460W-S586C hypertensive variant of adducin increases CFTR channel activity, possibly by altering its membrane turnover and inducing a retention of the channel in the plasmamembrane. Since CFTR is known to modulate the activity of many others transport systems, the increased surface expression of the channel could have consequences on the whole network of transport in kidney cells.

Ethanol Stimulates Epithelial Sodium Channels via Reactive Oxygen Species

Disorders of electrolyte and water metabolism with resultant retention of sodium and water are common in alcoholics. However, the mechanisms responsible are largely unknown. In the present study, we found that ethanol stimulates the epithelial Na+ channel (ENaC) in A6 distal nephron cells. The data from cell-attached patch-clamp experiments showed that ethanol significantly increased both ENaC open probability (Po) and the number of active ENaC in the patch (N). The effects were mimicked by acetaldehyde, the first metabolic product of ethanol, as well as by n-propanol, another primary alcohol which can be oxidized to form an aldehyde, but not by iso-propanol, a secondary alcohol which can also be oxidized, but which forms a ketone rather than an aldehyde. Confocal microscopy combined with biotinylation experiments showed that ethanol significantly increased α-ENaC protein in the apical membrane. Primary alcohols and acetaldehyde elevated intracellular reactive oxygen species (ROS). The effects of ethanol on ENaC Po and N were abolished by a superoxide dismutase mimetic compound, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL). Together with our previous finding that ROS stimulate ENaC, these results suggest that ethanol stimulates ENaC by elevating intracellular ROS probably via its metabolic product acetaldehyde. Supported by 2R01DK067110 to HPM, P01 DK061521, R37 DK037963, and P50 AA013757 to DCE

Anionic phospholipids differentially regulate the epithelial sodium channel (ENaC) by interacting with α, β, and γ ENaC subunits

Anionic phospholipids (APs) present a variety of lipids in the cytoplasmic leaflet of the plasma membrane, including phosphatidylinositol (PI), PI-4-phosphate (PI(4)P), phosphatidylserine (PS), PI-4,5-bisphosphate (PI(4,5)P(2)), PI-3,4,5-trisphosphate (PI(3,4,5)P(3)), and phosphatidic acid (PA). We previously showed that PI(4,5)P(2) and PI(3,4,5)P(3) upregulate the renal epithelial sodium channel (ENaC). Further studies from others suggested that PI(4,5)P(2) and PI(3,4,5)P(3) respectively target beta- and gamma-ENaC subunit. To determine whether PI(4,5)P(2) and PI(3,4,5)P(3) selectively bind to beta and gamma subunit, we performed lipid-protein overlay experiments. Surprisingly, the results reveal that most APs, including PI(4)P, PS, PI(4,5)P(2), PI(3,4,5)P(3), and PA, but not PI, non-selectively bind to not only beta and gamma but also alpha subunit. To determine how these APs regulate ENaC, we performed inside-out patch-clamp experiments and found that PS, but not PI or PI(4)P, maintained ENaC activity, that PI(4,5)P(2) and PI(3,4,5)P(3) stimulated ENaC, and that PA, however, inhibited ENaC. These data together suggest that APs differentially regulate ENaC by physically interacting with alpha-, beta-, and gamma-ENaC. Further, the data from cell-attached patch-clamp and confocal microscopy experiments indicate that PA, a product of phospholipase D, may provide one of the pathways for inhibition of ENaC by endothelin receptors.

Mediation of angiotensin II-induced Ca2+signaling by polycystin 2 in glomerular mesangial cells

Ca(+) influx across the plasma membrane is a major component of mesangial cell (MC) response to vasoconstrictors. Polycystin 2 (PC2), the protein product of the gene mutated in type 2 autosomal dominant polycystic kidney disease, has been shown to function as a nonselective cation channel in a variety of cell types. The present study was performed to test the hypothesis that PC2 and its binding partners constitute a Ca(2+)-permeable channel and contribute to ANG II-induced Ca(2+) signaling in MCs. Western blot and immunocytochemistry showed PC2 expression in cultured human MCs. The existence of PC2 in MCs was further confirmed by immunohistochemsitry in rat kidney sections. Coimmunoprecipitation displayed a selective interaction of PC2 with canonical transient receptor potential (TRPC) proteins TRPC1 and TRPC4. Cell-attached patch-clamp experiments revealed that ANG II-induced membrane currents were enhanced by overexpression of pkd2 but significantly inhibited by knock down of pkd2, 30 microM Gd(3+) (a PC2 channel blocker), and dominant-negative pkd2 mutant (pkd2-D511V). Corresponding to the increase in channel currents, ANG II stimulation increased expression of PC2 on the cell surface of MCs and interaction with TRPC1 and TRPC4. Furthermore, ANG II-induced MC contraction was significantly reduced in pkd2-knocked down MCs. These data suggest that PC2 selectively assembles with TRPC1 and TRPC4 to form channel complexes mediating ANG II-induced Ca(2+) responses in MCs.

Indirect Activation of the Epithelial Na+ Channel by Trypsin

We tested the hypothesis that the serine protease trypsin can indirectly activate the epithelial Na(+) channel (ENaC). Experiments were carried out in Xenopus oocytes and examined the effects on the channel formed by all three human ENaC subunits and that formed by Xenopus epsilon and human beta and gamma subunits (epsilonbetagammaENaC). Low levels of trypsin (1-10 ng/ml) were without effects on the oocyte endogenous conductances and were specifically used to test the effects on ENaC. Addition of 1 ng/ml trypsin for 60 min stimulated the amiloride-sensitive human ENaC conductance (g(Na)) by approximately 6-fold. This effect on the g(Na) was [Na(+)]-independent, thereby ruling out an interaction with channel feedback inhibition by Na(+). The indirect nature of this activation was confirmed in cell-attached patch clamp experiments with trypsin added to the outside of the pipette. Trypsin was comparatively ineffective at activating epsilonbetagammaENaC, a channel that exhibited a high spontaneous open probability. These observations, in combination with surface binding experiments, indicated that trypsin indirectly activated membrane-resident channels. Activation by trypsin was also dependent on catalytic activity of this protease but was not accompanied by channel subunit proteolysis. Channel activation was dependent on downstream activation of G-proteins and was blocked by G-protein inhibition by injection of guanyl-5'-yl thiophosphate and by pre-stimulation of phospholipase C. These data indicate a receptor-mediated activation of ENaC by trypsin. This trypsin-activated receptor is distinct from that of protease-activated receptor-2, because the response to trypsin was unaffected by protease-activated receptor-2 overexpression or knockdown.

Nongenomic, endothelium-independent effects of estrogen on human coronary smooth muscle are mediated by type I (neuronal) NOS and PI3-kinase-Akt signaling

Sex steroids exert profound and controversial effects on cardiovascular function. For example, estrogens have been reported to either ameliorate or exacerbate coronary heart disease. Although estrogen dilates coronary arteries from a variety of species, the molecular basis for this acute, nongenomic effect is unclear. Moreover, we know very little of how estrogen affects human coronary artery smooth muscle cells (HCASMC). The purpose of this study was to elucidate nongenomic estrogen signal transduction in HCASMC. We have used tissue (arterial tension studies), cellular (single-channel patch clamp, fluorescence), and molecular (protein expression) techniques to now identify novel targets of estrogen action in HCASMC: type I (neuronal) nitric oxide synthase (nNOS) and phosphatidylinositol 3-kinase (PI3-kinase)Akt. 17beta-Estradiol (E(2)) increased NO-stimulated fluorescence in HCASMC, and cell-attached patch-clamp experiments revealed that stimulation of nNOS leads to increased activity of calcium-activated potassium (BK(Ca)) channels in these cells. Furthermore, overexpression of nNOS protein in HCASMC greatly enhanced BK(Ca) channel activity. Immunoblot studies demonstrated that E(2) enhances Akt phosphorylation in HCASMC and that wortmannin, an inhibitor of PI3-kinase, attenuated E(2)-stimulated channel activity, NO production, Akt phosphorylation, and estrogen-stimulated coronary relaxation. These studies implicate the PI3-kinase/Akt signaling axis as an estrogen transduction component in vascular smooth muscle cells. We conclude, therefore, that estrogen opens BK(Ca) channels in HCASMC by stimulating nNOS via a transduction sequence involving PI3-kinase and Akt. These findings now provide a molecular mechanism that can explain the clinical observation that estrogen enhances coronary blood flow in patients with diseased or damaged coronary arteries.

Regulation of mesangial cell function by PKD2

Ca2+ influx through plasma membrane is a major component of MC response to vasoconstrictors. PKD2, the protein product of the gene mutated in type 2 autosomal dominant polycystic kidney disease, has been shown to function as an non-selective cation channel (NSCC) in renal tubular epithelial cells. The present study was performed to test the hypothesis that PKD2 and its binding partner constitute NSCC in MCs and contribute to contractile function of the cells. Western blotting and immunocytochemistry showed PKD2 expression in cultured human MCs. The existence of PKD2 in MCs was further confirmed by immunohistochemsitry in rat and human kidney sections. Co-immunoprecipitation showed a selective interaction of PKD2 with TRPC1 and TRPC4. Cell-attached patch clamp experiments revealed nonselective cationic currents stimulated by Ang II. The Agonist-induced currents were enhanced by over-expressing PKD2, but attenuated by knocking down PKD2. Corresponding to the increase in channel currents, Ang II stimulation increased expression of PKD2 in the cell surface as well as interaction with TRPC4. Furthermore, Ang II-induced MC contraction was reduced in PKD2 knocked out MCs. These data suggest that PKD2 might selectively assemble with specific isoform of TRPC proteins to form NSCC in MCs and this channel complex contributes to contractile function of MCs. (supported by AHA SDG)

Phenylglycine and Sulfonamide Correctors of Defective ΔF508 and G551D Cystic Fibrosis Transmembrane Conductance Regulator Chloride-Channel Gating

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel cause cystic fibrosis. The delta F508 mutation produces defects in channel gating and cellular processing, whereas the G551D mutation produces primarily a gating defect. To identify correctors of gating, 50,000 diverse small molecules were screened at 2.5 microM (with forskolin, 20 microM) by an iodide uptake assay in epithelial cells coexpressing delta F508-CFTR and a fluorescent halide indicator (yellow fluorescent protein-H148Q/I152L) after delta F508-CFTR rescue by 24-h culture at 27 degrees C. Secondary analysis and testing of >1000 structural analogs yielded two novel classes of correctors of defective delta F508-CFTR gating ("potentiators") with nanomolar potency that were active in human delta F508 and G551D cells. The most potent compound of the phenylglycine class, 2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide, reversibly activated delta F508-CFTR in the presence of forskolin with K(a) approximately 70 nM and also activated the CFTR gating mutants G551D and G1349D with K(a) values of approximately 1100 and 40 nM, respectively. The most potent sulfonamide, 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide, had K(a) approximately 20 nM for activation of delta F508-CFTR. In cell-attached patch-clamp experiments, phenylglycine-01 (PG-01) and sulfonamide-01 (SF-01) increased channel open probability >5-fold by the reduction of interburst closed time. An interesting property of these compounds was their ability to act in synergy with cAMP agonists. Microsome metabolism studies and rat pharmacokinetic analysis suggested significantly more rapid metabolism of PG-01 than SF-03. Phenylglycine and sulfonamide compounds may be useful for monotherapy of cystic fibrosis caused by gating mutants and possibly for a subset of delta F508 subjects with significant delta F508-CFTR plasma-membrane expression.

Syntaxin 1A Regulates ENaC Channel Activity

Na(+) entry across the apical membranes of many absorptive epithelia is determined by the number (N) and open probability (P(o)) of epithelial sodium channels (ENaC). Previous results showed that the H3 domain of syntaxin-1A (S1A) binds to ENaC to reduce N, supporting a role for S1A in the regulation of ENaC trafficking. The aim of this study was to determine whether S1A-induced reductions in ENaC current also result from interactions between cell surface ENaC and S1A that alter ENaC P(o). Injection of a glutathione S-transferase (GST)-H3 S1A fusion protein into ENaC-expressing Xenopus oocytes inhibited whole cell Na(+) current (I(Na)) by 33% within 5 min. This effect was dose-dependent, with a K(i) of 7 ng/microl (approximately 200 nm). In contrast, injection of GST alone or a H3 domain-deleted GST-S1A fusion protein had no effect on I(Na). In cell-attached patch clamp experiments, GST-H3 acutely decreased ENaC P(o) by 30%, whereas GST-S1A Delta H3 was without effect. Further analysis revealed that ENaC mean closed time was significantly prolonged by S1A. Interestingly, GST-H3 had no effect on channel activity of an ENaC pore mutant that constitutively gates open (P(o) approximately equal 1.0), supporting the idea that S1A alters the closed state of ENaC and indicating that the actions of S1A on ENaC trafficking and gating can be separated experimentally. This study indicates that, in addition to a primary effect on ENaC trafficking, S1A interacts with cell surface ENaC to rapidly decrease channel gating. This rapid effect of S1A may modulate Na(+) entry rate during rapid increases in ENaC N.

Protein Kinase C Activates Store-operated Ca2+Channels in Human Glomerular Mesangial Cells

Store-operated Ca(2+) channels (SOC) are expressed in cultured human mesangial cells and activated by epidermal growth factor through a pathway involving protein kinase C (PKC). We used fura-2 fluorescence and patch clamp experiments to determine the role of PKC in mediating the activation of SOC after depletion of internal stores by thapsigargin. The measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) revealed that the thapsigargin-induced Ca(2+) entry pathway was abolished by calphostin C, a protein kinase C inhibitor. The PKC activator, phorbol 12-myristate 13-acetate (PMA), promoted a Ca(2+) influx that was significantly attenuated by calphostin C and La(3+) but not by diltiazem. Neither PMA nor calphostin C altered the thapsigargin-induced initial transient rise in [Ca(2+)](i). In cell-attached patch clamp experiments, the thapsigargin-induced activation of SOC was potentiated by PMA and abolished by both calphostin C and staurosporine. However, SOC was unaffected by thapsigargin when clamping [Ca(2+)](i) with 1,2-bis (o-Aminophenoxy)ethane-N,N,N',N'tetraacetic acid tetra(acetoxymethyl)ester. In the absence of thapsigargin, PMA and phorbol 12, 13-didecanoate evoked a significant increase in NP(O) of SOC, whereas calphostin C did not affect base-line channel activity. In inside-out patches, SOC activity ran down immediately upon excision but was reactivated significantly after adding the catalytic subunit of 0.1 unit/ml of PKC plus 100 microm ATP. Neither ATP alone nor ATP with heat-inactivated PKC rescued a rundown of SOC. Metavanadate, a general protein phosphatase inhibitor, also enhanced SOC activity in inside-out patches. Bath [Ca(2+)] did not significantly affect the channel activity in inside-out patch. These results indicate that the depletion of Ca(2+) stores activates SOC by PKC-mediated phosphorylation of the channel proteins or a membrane-associated complex.

Carbachol activates a K+ channel of very small conductance in the basolateral membrane of rat pancreatic acinar cells.

Secretion of Cl- requires the presence of a K+ conductance to hyperpolarize the cell, and to provide the driving force for Cl- exit via luminal Cl- channels. In the exocrine pancreas Cl- secretion is mediated by an increase in cytosolic Ca2+ ([Ca2+]i). Two types of Ca2+-activated K+ channels could be shown in pancreatic acinar cells of different species. However, there are no data on Ca2+-activated K+ channels in rat pancreatic acini. Here we examine the basolateral K+ conductance of freshly isolated rat pancreatic acinar cells in cell-attached and cell-excised patch-clamp experiments. Addition of carbachol (CCH, 1 micromol/l) to the bath led to the activation of very small conductance K+ channels in cell-attached patches (n=27), producing a noisy macroscopic outward current. The respective outward conductance increased significantly by a factor of 2.1+/-0.1 (n=27). Noise analysis revealed a Lorentzian noise component with a corner frequency (f(c)) of 30.3+/-3.5 Hz (n=19), consistent with channel activity in these patches. The estimated single-channel conductance was 1.5+/-0.4 pS (n=19). In cell-excised patches (inside out) from cells previously stimulated with CCH, channel activity was only observed in the presence of K+ in the bath solution. Under these conditions f(c) was 47.6+/-11.9 Hz (estimated single-channel conductance 1.1+/-0.2 pS, n=20). The current/voltage relationship of the noise showed weak inward rectification and the reversal potential shifted towards E(K+) when Na+ in the bath was replaced by K+. Channel activity in cell-excised patches was slightly reduced by 10 mmol/l Ba2+ (23.6+/-2.1% of the total outward current) and was completely absent when K+ in the bath was replaced by Na+. Reduction of the [Ca2+]i from 1 mmol/l to 1 micromol/l in cell-excised experiments decreased the current by 52.3+/-12.3% (n=5). Expression of K(v)LQT1, one of the possible candidates for a small-conductance K+ channel in rat pancreatic acinar cells, was shown by reverse transcriptase polymerase chain reaction (RT-PCR). In fact, a K(V)LQT-blocker (chromanol 293B) reduced channel activity in seven excised patches. These data suggest that CCH activates very small conductance K+ channels in rat pancreatic acinar cells, most likely via an increase in [Ca2+]i.

Mechanosensitive ion channels in putative aortic baroreceptor neurons.

Cell-attached patch-clamp experiments were performed on dissociated neurons from nodose ganglia of adult rats. Putative aortic baroreceptor neurons were identified by labeling nerve endings in the adventitia of the aortic arch with the carbocyanine dye DiI. Whereas previous experiments demonstrated the presence of mechanosensitive (MS) whole cell currents, these experiments studied single MS ion channels and examined the influence of culture conditions on their expression. Single MS channels were activated by applying negative pressure through the recording pipette. Channel openings became more frequent as the negative pressure was increased, with open probability increasing significantly above 30 mmHg. MS channels had a slope conductance of 114 pS and a reversal potential of approximately 0 mV, consistent with a nonspecific cation conductance. Channels were not affected by antagonists of voltage-gated conductances but were blocked by 20 microM gadolinium, a known blocker of MS ion channels. When nodose neurons were cocultured with aortic endothelial cells, but not aortic smooth muscle cells, the percentage of patches exhibiting MS ion channels increased significantly, suggesting that aortic endothelial cells secrete a diffusible factor that increases channel expression.

A K+ single channel and whole-cell clamp study on the effects of levcromakalim in guinea pig portal vein cells

Single channel cell-attached patch and whole-cell clamp experiments on the mode of action of the K+ channel opener (KCO), levcromakalim, were performed in guinea pig isolated portal vein cells. At +20 mV (135/23 mM K+ in bath/pipette), 10 microM levcromakalim activated K+ channels with a chord conductance of 23.2 pS (K(KCO)), which were sensitive to the blocker of ATP-dependent K+ channels (K(ATP)), glibenclamide. Voltage steps from -80 mV to +20 mV activated 4-aminopyridine-sensitive K+ channels of 6.5 pS with properties of delayed rectifier K+ channels (Kv). In patches which upon a previous voltage step had revealed the existence of Kv, levcromakalim reduced the open-probability of Kv, but it did not concomitantly activate K(KCO). During the course of the experiments, but unrelated to the presence of levcromakalim, large conductance K+ channels (BK(Ca)) appeared which could be inhibited by iberiotoxin, a selective blocker of BK(Ca), and by the membrane-permeant calcium buffer, BAPTA/AM, but not by glibenclamide. Whole-cell current-voltage (i-V) relations were established in response to voltage ramps from +50 mV to -100 mV; on subtraction of control i-V curves from i-V curves obtained in the presence of 10 microM levcromakalim, the KCO-induced K+ current remained which was proportional to voltage. This is not compatible with the upward-bent curvature predicted by the GHK current equation for purely resistive channels at high [K+]i versus low [K+]o. In conclusion, in the guinea pig portal vein cells, no evidence could be established for the hypotheses that KCOs may act via conversion of Kv to K(ATP) (Beech and Bolton 1989; Edwards et al. 1993) or by activation of BK(Ca) (Balwierczak et al. 1995). In these cells, mild inward rectification of the levcromakalim-induced current was observed which underlines their relationship to K(ATP) in other tissues.

Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes.

CD95/Fas/APO-1 mediated apoptosis is an important mechanism in the regulation of the immune response. Here, we show that CD95 receptor triggering activates an outwardly rectifying chloride channel (ORCC) in Jurkat T lymphocytes. Ceramide, a lipid metabolite synthesized upon CD95 receptor triggering, also induces activation of ORCC in cell-attached patch clamp experiments. Activation is mediated by Src-like tyrosine kinases, because it is abolished by the tyrosine kinase inhibitor herbimycin A or by genetic deficiency of p56lck. In vitro incubation of excised patches with purified p56lck results in activation of ORCC, which is partially reversed upon addition of anti-phosphotyrosine antibody. Inhibition of ORCC by four different drugs correlates with a 30-65% inhibition of apoptosis. Intracellular acidification observed upon CD95 triggering is abolished by inhibition of either ORCC or p56lck. The results suggest that tyrosine kinase-mediated activation of ORCC may play a role in CD95-induced cell death in T lymphocytes.

Regulation of CFTR by protein phosphatase 2B and protein kinase C.

The activity of the CFTR Cl- channel is dependent on its phosphorylation status set by kinases and phosphatases. We report here that protein phosphatase 2B (PP2B) and protein kinase C (PKC) are potential regulators of the cystic fibrosis conductance regulator (CFTR). Treating CFTR-expressing 3T3 cells with either of the two specific PP2B blockers cyclosporin A (CsA, 1 microM) or deltamethrin (DM, 30 nM) caused rapid activation of CFTR in cell-attached patches. As determined by noise analysis of multi channel patches, DM- or CsA-activated CFTR displayed gating kinetics comparable to those of forskolin-activated CFTR. After activation of CFTR by blocking PP2B, CFTR still inactivated. CFTR-mediated currents were, on average, 6.1 times larger when cells were stimulated by forskolin during PP2B block compared to stimulation by forskolin alone. This suggests that, in CFTR-expressing 3T3 cells, a phosphorylation site of CFTR is regulated by cellular PKA, PP2B and another phosphatase. However, in the epithelial cell lines Calu-3 and HT-29/B6, CsA and DM had no effect on CFTR activity in both cell-attached patch-clamp and transepithelial experiments. In contrast, when exogenous PP2B was added to patches excised from 3T3 or Calu-3 cells, PKA-activated CFTR currents were quickly inactivated. This indicates that free exogenous PP2B can inactivate CFTR in patches from both cell types. We propose that in order to regulate CFTR in an intact cell, PP2B may require a selective subcellular localization to become active. When excised patches were PKC-phosphorylated, the gating kinetics of CFTR were significantly different from those of PKA-phosphorylated CFTR. Addition of PP2B also inactivated PKC-activated CFTR showing the indiscriminate dephosphorylation of different phosphorylation sites by PP2B.