A patch-clamp study of GABA- and strychnine-sensitive glycine-activated currents in post-natal tissue-cultured hippocampal neurons.

GABA- and strychnine-sensitive glycine-activated currents in post-natal tissue-cultured hippocampal neurons were studied by using patch-clamp techniques. Current-voltage relations for both agonists in symmetrical Cl- solutions showed outward rectification. Strychnine-sensitive glycine-activated currents have not been studied in detail before in post-natal tissue-cultured hippocampal neurons. Partial desensitization of whole-cell currents was observed in symmetrical Cl- solutions during bath application of GABA- and glycine. In F-/Cl- solutions, both agonists gave a PF/Pcl value of about 0.06. The reversal potentials in mixtures of Cl- and SO4(2-) solutions were close to the equilibrium potentials of Cl- in the presence of both GABA and glycine. Single channels in inside-out excised patches with 2 mumol l-1 GABA and 5 mumol l-1 glycine in the pipette showed outward current rectification. The dose-response curves for GABA and glycine gave Kd values of 52 and 61 mumol l-1, respectively, and Hill coefficients close to 2. GABA and glycine binding were competitively blocked by their respective antagonists (bicuculline and strychnine). The similarities between GABA- and glycine-activated currents and the response in a combination of saturating concentrations of both GABA and glycine implied that the two agonists activated comparable numbers of anatomically distinct channels with very similar permeation properties.     

Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.     

Volume-regulated anion channels serve as an auto/paracrine nucleotide release pathway in aortic endothelial cells

Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant Ca(2+) oscillations and NO production. They did not, however, affect Ca(2+) oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a "permeating blocker model," in which ATP binds with an affinity of 1.0 +/- 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low Cl(-) hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.     

Chloride channels in the small intestinal cell line IEC-18

Small intestinal crypt cells play a critical role in modulating Cl- secretion during digestion. The types of Cl- channels mediating Cl- secretion in the small intestine was investigated using the intestinal epithelial cell line, IEC-18, which was derived from rat small intestine crypt cells. In initial radioisotope efflux studies, exposure to forskolin, ionomycin or a decrease in extracellular osmolarity significantly increased 36Cl efflux as compared to control cells. Whole cell patch clamp techniques were subsequently used to examine in more detail the swelling-, Ca2+-, and cAMP-activated Cl- conductance. Decreasing the extracellular osmolarity from 290 to 200 mOsm activated a large outwardly rectifying Cl- current that was voltage-independent and had an anion selectivity of I- > Cl-. Increasing cytosolic Ca2+ by ionomycin activated whole cell Cl- currents, which were also outwardly rectifying but were voltage-dependent. The increase in intracellular Ca2+ levels with ionomycin was confirmed with fura-2 loaded IEC-18 cells. A third type of whole cell Cl- current was observed after increases in intracellular cAMP induced by forskolin. These cAMP-activated Cl- currents have properties consistent with cystic fibrosis transmembrane regulator (CFTR) Cl- channels, as the currents were blocked by glibenclamide or NPPB but insensitive to DIDS. In addition, the current-voltage relationship was linear and had an anion selectivity of Cl- > I-. Confocal immunofluorescence studies and Western blots with two different anti-CFTR antibodies confirmed the expression of CFTR. These results suggest that small intestinal crypt cells express multiple types of Cl- channels, which may all contribute to net Cl- secretion.     

Characterization of anion channels in the plasma membrane of Arabidopsis epidermal root cells and the identification of a citrate-permeable channel induced by phosphate starvation

Organic-acid secretion from higher plant roots into the rhizosphere plays an important role in nutrient acquisition and metal detoxification. In this study we report the electrophysiological characterization of anion channels in Arabidopsis (Arabidopsis thaliana) root epidermal cells and show that anion channels represent a pathway for citrate efflux to the soil solution. Plants were grown in nutrient-replete conditions and the patch clamp technique was applied to protoplasts isolated from the root epidermal cells of the elongation zone and young root hairs. Using SO4(2-) as the dominant anion in the pipette, voltage-dependent whole-cell inward currents were activated at membrane potentials positive of -180 mV exhibiting a maximum peak inward current (I(peak)) at approximately -130 mV. These currents reversed at potentials close to the equilibrium potential for SO4(2-), indicating that the inward currents represented SO4(2-) efflux. Replacing intracellular SO4(2-) with Cl- or NO3(-) resulted in inward currents exhibiting similar properties to the SO4(2-) efflux currents, suggesting that these channels were also permeable to a range of inorganic anions; however when intracellular SO4(2-) was replaced with citrate or malate, no inward currents were ever observed. Outside-out patches were used to characterize a 12.4-picoSiemens channel responsible for these whole-cell currents. Citrate efflux from Arabidopsis roots is induced by phosphate starvation. Thus, we investigated anion channel activity from root epidermal protoplasts isolated from Arabidopsis plants deprived of phosphate for up to 7 d after being grown for 10 d on phosphate-replete media (1.25 mm). In contrast to phosphate-replete plants, protoplasts from phosphate-starved roots exhibited depolarization-activated voltage-dependent citrate and malate efflux currents. Furthermore, phosphate starvation did not regulate inorganic anion efflux, suggesting that citrate efflux is probably mediated by novel anion channel activity, which could have a role in phosphate acquisition.     

Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl- conductance in mammalian heart

The regulation of cardiac Cl- conductance by cAMP-dependent protein kinase (PKA) and cellular phosphatases was studied in isolated guinea pig ventricular myocytes by using wide-tipped, perfused pipettes to record whole-cell currents. Exposure to forskolin (Fsk) or isoproterenol (Iso) elicits a Cl- conductance that results exclusively from PKA-dependent phosphorylation because it can be completely abolished, or its activation fully prevented, by switching to pipette solution containing PKI, a synthetic peptide inhibitor of PKA. The Cl- conductance activated by micromolar concentrations of either agonist reached its steady-state amplitude in 1-2 min and was deactivated promptly and entirely, usually within 2 min, upon washing out the agonist, implying a continuous high level of activity of endogenous protein phosphatases. Accordingly, intracellular application of okadaic acid or microcystin, both potent inhibitors of protein phosphatases 1 and 2A, during exposure to Fsk enhanced the steady-state Cl- conductance and slowed its deactivation after washing out the Fsk. Maximal potentiation of the conductance, by approximately 60%, was obtained with pipette concentrations of approximately 10 microM okadaic acid (or approximately 5 microM microcystin) and did not result from an increase in the apparent affinity for Fsk. In the presence of maximally effective concentrations of okadaic acid and/or microcystin, deactivation of the enhanced Cl- conductance upon washout of agonist was incomplete, with about half of the conductance persisting indefinitely. That residual conductance did not reflect continued action of PKA because it was insensitive to PKI, but was identified as a fraction of the activated Cl- conductance by its biophysical characteristics. The results suggest that complete deactivation of the PKA-regulated cardiac Cl- conductance requires dephosphorylation by a type 1 and/or 2A phosphatase, but that partial deactivation can be accomplished by activity of some other phosphatase(s). These findings are consistent with sequential phosphorylation of a protein, probably the Cl- channel itself, at two different kinds of sites. The resulting phosphoproteins can be distinguished on the basis of their different contributions to whole-cell Cl- conductance.     

A hypertonicity-activated nonselective conductance in single proximal tubule cells isolated from mouse kidney

The whole-cell patch-clamp technique was used to examine nonselective conductances in single proximal tubule cells isolated from mouse kidney. Single cells were isolated in either the presence or absence of a cocktail designed to stimulate cAMP. Patches were obtained with Na+ Ringer in the bath and Cs+ Ringer in the pipette. On initially achieving the whole-cell configuration, whole-cell currents were small. In cAMP-stimulated cells, with 5 mM ATP in the pipette solution, whole-cell currents increased with time. The activated current was linear, slightly cation-selective, did not discriminate between Na+ and K+ and was inhibited by 100 microM gadolinium. These properties are consistent with the activation of a nonselective conductance, designated G(NS). Activation of G(NS) was abolished with pipette AMP-PNP, ATP plus alkaline phosphatase or in the absence of ATP. In unstimulated cells G(NS) was activated by pipette ATP together with PKA. These data support the hypothesis that G(NS) is activated by a PKA-mediated phosphorylation event. G(NS) was also activated by a hypertonic shock. However, G(NS) does not appear to be involved in regulatory volume increase (RVI), as RVI was unaffected in the presence of the G(NS) blocker gadolinium. Instead, the ATP sensitivity of G(NS) suggests that it may be regulated by the metabolic state of the renal proximal tubule cell.     

Prostaglandin E1 activates a chloride current in Jurkat T lymphocytes via cAMP-dependent protein kinase.

Patch-clamp studies have identified a cAMP-dependent Cl- conductance in lymphocytes that is defectively regulated in cystic fibrosis. In this study we used 125I efflux and whole-cell patch-clamp studies to investigate whether prostaglandin E1 (PGE1), an agonist that generates intracellular cAMP in Jurkat T lymphocytes, activates a Cl- conductance. Stimulation of T cells by externally applied PGE1 stimulated 125I efflux and activated a slowly developing membrane current. When external and internal Cl- were about equal, the current reversed at about zero mV, but when external Cl- was lowered from 157 to 7 mM the reversal potential shifted 75 mV in the positive direction, demonstrating that the current carrier was Cl-. In addition, the current was blocked by 10 microM 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB), a potent Cl- channel blocker. A membrane-permeable cAMP analog mimicked the effect of PGE1, whereas intracellular application of a cAMP antagonist Rp-cAMP blocked the effect of PGE1. Addition of purified catalytic subunit of cAMP-dependent protein kinase (PKA) plus ATP to the recording pipette also activated a similar current, whereas internally applied Walsh inhibitor, the synthetic peptide inhibitor of PKA, blocked the PGE1 effect. These results suggest that PGE1, acting through PKA, activates a Cl- current in Jurkat T cells.     

Nonselective cationic currents activated by acetylcholine in swine tracheal smooth muscle cells

Membrane currents in isolated swine tracheal smooth muscle cells were investigated using a pipette solution containing BAPTA-Ca2+ buffer and Cs+ as the major cation. With a pipette solution containing 100 nM free Ca2+, acetylcholine (ACh; 1-100 microM), in a concentration-dependent manner, activated a current without inducing shortening of cells, although neither 1 mM histamine nor 1 microM leukotriene D4 activated the current (n = 7, n is the number of cells). The effect of 100 microM ACh was suppressed by pretreatment with 100 microM atropine (n = 6) or intracellular application of preactivated pertussis toxin at a concentration of 0.1 microg x mL(-1) (n = 8). Genistein (0.1-100 microM), in a concentration-dependent manner, suppressed the activation of the inward current by 100 microM ACh, whereas it did not significantly suppress that of the outward current (n = 6-8). With a pipette solution containing 50 nM free Ca2+, outward current, but not inward current, was activated by 100 microM ACh (n = 10). When the pipette solution had free Ca2+ concentrations greater than 50 nM, the inward current together with the outward current was activated. The ratio between the amplitude of the inward and outward currents was significantly increased as the free Ca2+ concentration in the pipette solution increased. The steady-state activation curve of the ACh-activated current with the 50 nM free Ca2+ pipette solution was fitted by a single Boltzmann distribution (Vh = +69.8 mV, k = -11.9 mV, n = 10). The activation time constant became smaller as the membrane potential was more depolarized (164.3+/-5.9 ms at +40 mV to 92.4+/-6.3 ms at +120 mV, n = 10). The reversal potential was not significantly changed by reducing extracellular Cl- concentration to one-tenth of the control (n = 8), suggesting that the current is a nonselective cationic current. These results suggest that ACh activates an outward nonselective cationic current via pertussis toxin-sensitive G-protein(s) coupled with muscarinic receptors. Involvement of genistein-sensitive tyrosine kinase in the activation process of the current is unlikely.     

ClC-3-independent, PKC-dependent activity of volume-sensitive Cl channel in mouse ventricular cardiomyocytes.

Volume-sensitive outwardly rectifying (VSOR) Cl- channels are activated during osmotic swelling and involved in the subsequent volume regulation in most animal cells. To test the hypothesis that the ClC-3 protein is the molecular entity corresponding to the VSOR Cl- channel in cardiomyocytes, the properties of VSOR Cl- currents in single ventricular myocytes isolated from ClC-3-deficient (Clcn3(-/-)) mice were compared with those of the same currents in ClC-3-expressing wild-type (Clcn3(+/+)) and heterozygous (Clcn3(+/-)) mice. Basal whole-cell currents recorded under isotonic conditions in ClC-3-deficient and -expressing cells were indistinguishable. The biophysical and pharmacological properties of whole-cell VSOR Cl- currents in ClC-3-deficient cells were identical in ClC-3-expressing cells. The VSOR Cl- current density, which is an indicator of the plasmalemmal expression of functional channels, was essentially the same in cells isolated from these 3 types of mice and C57BL/6 mice. Activation of protein kinase C (PKC) by a phorbol ester was found to upregulate VSOR Cl- currents in ClC-3-deficient and -expressing cardiomyocytes. This effect is opposite to the reported downregulatory effect of PKC activators on ClC-3-associated Cl- currents. We thus conclude that functional expression of VSOR Cl- channels in the plasma membrane of mouse cardiomyocytes is independent of the molecular expression of ClC-3.     

Novel Cl- currents elicited by follicle stimulating hormone and acetylcholine in follicle-enclosed Xenopus oocytes

Voltage-clamp techniques were used to study the membrane currents elicited by follicle stimulating hormone (FSH) and acetylcholine (ACh) in follicle-enclosed oocytes of Xenopus laevis (follicles). Both agonists caused complex responses that were more evident when the follicles were in hypotonic Ringer solution (HR; 190.4 mosM). In this medium, currents activated by FSH regularly showed three phases whereas currents activated by ACh displayed three to six phases. At a holding potential of -60 mV, FSH, and ACh responses involved combinations of inward and outward currents. Both FSH and ACh responses included a slow smooth inward component that was associated with an increase in membrane conductance, mainly to Cl- (S(in)). This current was strongly dependent on the osmolarity of the external solution: an increase in osmolarity of the HR solution of 18-20 mosM caused a 50% decrease in S(in). In contrast, a fast and transient Cl- current (F(in)) specifically elicited by ACh was not dependent on osmolarity. Both, F(in) and S(in) currents required the presence of follicular cells, since defolliculation using three different methods abolished all the response to FSH and at least four components of the ACh responses. The membrane channels carrying F(in) and oscillatory Cl- currents elicited by stimulation of ACh or serum receptors, were much more permeable to I- and Br- than Cl-, whereas S(in) channels were equally permeable to these anions. Unlike the oscillatory Cl- currents generated in the oocyte itself, S(in) and F(in) currents in follicle-enclosed oocytes were not abolished by chelation of intracellular Ca2+, either with EGTA or BAPTA, which suggests that intracellular Ca2+ does not play a critical role in the activation of these currents. Our experiments show that S(in) and F(in) currents are quite distinct from the previously characterized oscillatory Cl- responses of oocytes. Moreover, the results strongly suggest that the FSH and ACh receptors, the Cl- channels mediating the F(in) and S(in) currents, together with the necessary elements for their activation, are all located in the follicular cells and not in the oocyte. Many aspects of follicular cell physiology in Xenopus laevis, and other species, are regulated by hormones and neurotransmitters, including FSH and ACh. The follicular Cl- currents described in this paper may play an important role in the follicular cell-oocyte development.     

Volume-regulated anion and organic osmolyte channels in mouse zygotes

Whole-cell currents in mouse zygotes were measured using the patch-clamp technique in whole-cell mode. Upon exposure to hypotonic medium, patch-clamped zygotes increased in volume and developed a large swelling-activated current. The swelling-activated current was blocked by Cl- channel blockers, and the magnitude of the current and reversal potential were dependent on the Cl- gradient. Thus, the swelling-activated current had the properties of a current mediated by anion channels. However, in addition to being permeable to Cl- and I- (with I- having the greater permeability), there was also a significant swelling-activated conductance to aspartate and taurine, indicating that the swelling-activated channels in zygotes conduct not only inorganic anions but organic osmolytes as well. This swelling-activated anion and organic osmolyte pathway likely underlies the ability of zygotes to recover from an increase in volume, and it may function to regulate intracellular amino acid concentrations.     

Involvement of heparan sulfate proteoglycan in sensing hypotonic stress in bovine aortic endothelial cells

Hypotonic stress (HTS) induces various responses in vascular endothelium, but the molecules involved in sensing HTS are not known. To investigate a possible role of heparan sulfate proteoglycan (HSPG) in sensing HTS, we compared the responses of control bovine aortic endothelial cells (BAECs) with those of cells treated with heparinase III, which exclusively degrades HSPG. Tyrosine phosphorylation of 125 kDa FAK induced by HTS (-30%) in control cells was abolished in heparinase III-treated BAECs. The amplitude of the volume-regulated anion channel (VRAC) current, whose activation is regulated by tyrosine kinase, was significantly reduced by the treatment with heparinase III. Also, HTS-induced ATP release through the VRAC pore and the concomitant Ca(2+) transients were significantly reduced in the heparinase III-treated BAECs. In contrast, exogenously applied ATP evoked similar Ca(2+) transients in both control and heparinase III-treated BAECs. The transient formation of actin stress fibers induced by HTS in control cells was absent in heparinase III-treated BAECs. Lysophosphatidic acid (LPA) also induced FAK phosphorylation, actin reorganization and ATP release in control BAECs, but heparinase III did not affect these LPA-induced responses. We conclude from these observations that HSPG is one of the sensory molecules of hypotonic cell swelling in BAECs.     

Role of GTP-binding proteins in the regulation of mammalian cardiac chloride conductance

Beta-Adrenoceptor agonists activate a time- and voltage-independent Cl- conductance in mammalian cardiac myocytes. To characterize the cellular signaling pathways underlying its regulation, wide-tipped pipettes fitted with a pipette perfusion device were used to record whole-cell current and to introduce nucleotides to the interior of guinea pig ventricular myocytes. Replacement of pipette GTP with GDP beta S prevented activation of the Cl- conductance by Iso, suggesting a requirement for G protein turnover. With GTP in the pipette, the effect of Iso could be abolished by the beta-adrenoceptor antagonist propranolol, and mimicked by histamine or forskolin. These actions of Iso and forskolin are mediated exclusively via cAMP-dependent protein kinase (PKA), because (a) maximal activation of the Cl- conductance by forskolin or pipette cAMP occluded the effect of Iso, and (b) switching to pipette solution containing a synthetic peptide inhibitor (PKI) of PKA completely abolished the Cl- conductance activated by Iso and prevented the action of forskolin, but had no further effect. These results argue against basal activation of the Cl- conductance, and make it extremely unlikely that the stimulatory G protein, Gs, has any direct, phosphorylation-independent influence. The muscarinic receptor agonists acetylcholine (ACh) and carbachol diminished, in a reversible manner, Cl- conductance activated by Iso or forskolin, but not that elicited by cAMP. The muscarinic inhibition was abolished by replacing pipette GTP with GDP beta S, or by preincubating cells with pertussis toxin (PTX), and was therefore mediated by an inhibitory G protein, presumably Gi, influencing adenylyl cyclase activity. Nonhydrolyzable GTP analogues (GTP gamma S or GppNHp) applied via the pipette did not themselves activate Cl- conductance, but rendered Cl- current activation by brief exposures to Iso or histamine, but not to forskolin, irreversible. The Cl- conductance persistently activated by Iso was insensitive to propranolol or ACh, but could still be abolished by pipette application of PKI. The data indicate that stimulation of beta-adrenergic or histaminergic receptors in the presence of nonhydrolyzable GTP analogues causes persistent activation of Gs and uncouples it from the receptors. We conclude that autonomic regulation of cardiac Cl- conductance reflects accurately the underlying modulation of adenylyl cyclase activity and, hence, that this system is a suitable mammalian model for in situ studies of the interactions between adenylyl cyclase, Gs, Gi, and forskolin.     

External anions and volume-sensitive anion current in guinea-pig ventricular myocytes

The objective of this study was to determine the effects of anion replacement on volume-sensitive anion current in guinea-pig ventricular myocytes. Myocytes in the conventional whole-cell voltage-clamp configuration were superfused and dialysed with Na(+)-, K(+)-, and Ca(2+)-free solution, and exposed to external 75 mM Cl- solution of one-half normal osmolality. Prolonged exposures to hyposmotic solution promoted the development of outwardly-rectifying currents that were inactivated at high positive potentials and reversed in a Cl(-)-dependent manner (50 mV per decade pipette Cl- concentration). Replacement of external Cl- by iodide and aspartate affected the reversal potential (E(rev)) and slope conductance of the volume-sensitive current. Relative permeabilities calculated from changes in E(rev) were 1.49 +/- 0.09, 1.00, and 0.29 +/- 0.04 for iodide, Cl-, and aspartate, respectively; relative slope conductances between E(rev) and E(rev) + 40 mV were 1.21 +/- 0.09, 1.00, and 0.43 +/- 0.07, respectively. Replacement of Cl- also affected the time dependence of the volume-sensitive current; replacement by iodide reversibly enhanced the decay of outward current at positive potentials, whereas replacement by aspartate reduced it. These results are compared with earlier findings on noncardiac time- and voltage-dependent anion current activated by hyposmotic solution.     

K+ conductance activated during regulatory volume decrease. The channels in Ehrlich cells and their possible molecular counterpart

K+ currents activated by hypotonic cell swelling have been studied in Ehrlich ascites tumour cells by the whole-cell recording mode of the patch-clamp technique. K+ together with Cl- currents developed in the absence of added intracellular Ca2+ and with strong buffering of internal Ca2+ in experiments conducted at 37 degrees C. Manipulation of the extracellular medium with other cations suggests a selectivity sequence of K+ > Rb+ > NH4+ > or = Na+ approximately equals Li+ approximately equals Cs+. The current-voltage relationship of the volume-sensitive K+ current was well fitted with the Goldman-Hodgkin-Katz current equation between -130 and 20 mV at both physiological and high K+ extracellular solutions. The class III antiarrhytmic drug clofilium blocked the volume-sensitive K+ current in a voltage-independent manner. Clofilium was also found to be a strong inhibitor of the regulatory volume decrease (RVD) response of Ehrlich cells. The leukotriene D4 (LTD4) can activate the same current in isotonicity, consistent with a role for this compound in the signalling process of volume regulation. It is suggested that K+ channels activated by cell swelling belong to the so-called background K+ channel group. These are voltage-independent channels which underlie the resting potential of many cells and have recently been identified as belonging to a family of K+ channels with two pore domains in tandem (2P-4TM). Preliminary experiments show the presence of the TASK-2 channel, a member of the 2P-4TM family inhibited by acid extracellular pH, in Ehrlich cells and suggest that it might underlie the swelling-induced K+ current.     

A cyclic GMP-dependent calcium-activated chloride current in smooth-muscle cells from rat mesenteric resistance arteries

We have previously demonstrated the presence of a cyclic GMP (cGMP)-dependent calcium-activated inward current in vascular smooth-muscle cells, and suggested this to be of importance in synchronizing smooth-muscle contraction. Here we demonstrate the characteristics of this current. Using conventional patch-clamp technique, whole-cell currents were evoked in freshly isolated smooth-muscle cells from rat mesenteric resistance arteries by elevation of intracellular calcium with either 10 mM caffeine, 1 microM BAY K8644, 0.4 microM ionomycin, or by high calcium concentration (900 nM) in the pipette solution. The current was found to be a calcium-activated chloride current with an absolute requirement for cyclic GMP (EC50 6.4 microM). The current could be activated by the constitutively active subunit of PKG. Current activation was blocked by the protein kinase G antagonist Rp-8-Br-PET-cGMP or with a peptide inhibitor of PKG, or with the nonhydrolysable ATP analogue AMP-PNP. Under biionic conditions, the anion permeability sequence of the channel was SCN- > Br- > I- > Cl- > acetate > F- > aspartate, but the conductance sequence was I- > Br- > Cl- > acetate > F- > aspartate = SCN-. The current had no voltage or time dependence. It was inhibited by nickel and zinc ions in the micromolar range, but was unaffected by cobalt and had a low sensitivity to inhibition by the chloride channel blockers niflumic acid, DIDS, and IAA-94. The properties of this current in mesenteric artery smooth-muscle cells differed from those of the calcium-activated chloride current in pulmonary myocytes, which was cGMP-independent, exhibited a high sensitivity to inhibition by niflumic acid, was unaffected by zinc ions, and showed outward current rectification as has previously been reported for this current. Under conditions of high calcium in the patch-pipette solution, a current similar to the latter could be identified also in the mesenteric artery smooth-muscle cells. We conclude that smooth-muscle cells from rat mesenteric resistance arteries have a novel cGMP-dependent calcium-activated chloride current, which is activated by intracellular calcium release and which has characteristics distinct from other calcium-activated chloride currents.     

Patch clamp recording of the responses to three bitter stimuli in mouse taste cells.

Although several pathways of bitter taste signal transduction have been proposed in taste cells, these mechanisms have not been elucidated in detail. To investigate the diversity of responses to bitter stimuli, we recorded the electrophysiological responses to quinine, denatonium and naringin using whole-cell patch clamp technique in isolated taste cells of C57BL/6J mice. Ten mM quinine induced depolarizing response under the current clamp mode, and inward current response under the voltage-clamp mode (holding potential -80 mV) using both K+ (with pseudo intracellular solution) and Cs+ (K+ was substituted by Cs+ in the pseudo intracellular solution) pipettes. However, when the K+ pipette was used, the membrane conductance was suppressed and activated in succession. On the other hand, the membrane conductance was only activated when the Cs+ pipette was used. Half to one mM denatonium induced depolarizing response under the current clamp mode, and outward current response under the voltage clamp mode with both pipettes. Using these pipettes, the membrane conductance was activated or suppressed in the individual case. Naringin-induced responses were not detected in these measurements. These electrophysiological recordings suggest that multiple transduction mechanisms are involved in bitter taste perception in mouse taste cells.     

Simultaneous analysis of cell Ca2+ and Ca2(+)-stimulated chloride conductance in colonic epithelial cells (HT-29).

We used perforated patch, whole-cell current recordings and video-based fluorescence ratio imaging to monitor the relation of plasma membrane ionic conductances to intracellular free Ca2+ within individual colonic epithelial cells (HT-29). The Ca2(+)-mediated agonist, neurotensin, activated K+ and Cl- conductances that showed different sensitivities to [Ca2+]i. The Cl- conductance was sensitive to increases or decreases in [Ca2+]i around the resting value of 76 +/- 32 (mean +/- SD) nM (n = 46), whereas activation of the K+ conductance required at least a 10-fold rise in [Ca2+]i. Neurotensin increased [Ca2+]i by stimulating a transient intracellular Ca2+ release, which was followed by a sustained rise in [Ca2+]i due to Ca2+ influx from the bath. The onset of the initial [Ca2+]i transient, monitored at a measurement window over the cell interior, lagged behind the rise in Cl- current during agonist stimulation. This lag was not present when the [Ca2+]i rise was due to Ca2+ entry from the bath, induced either by the agonist or by the Ca2+ ionophore ionomycin. The temporal differences in [Ca2+]i and Cl- current during the agonist-induced [Ca2+]i transient can be explained by a localized Ca2+ release from intracellular stores in the vicinity of the plasma membrane Cl- channel. Chloride currents recover toward basal values more rapidly than [Ca2+]i after the agonist-induced [Ca2+]i transient, and, during a sustained neurotensin-induced [Ca2+]i rise, Cl- currents inactivate. These findings suggest that an inhibitory pathway limits the increase in Cl- conductance that can be evoked by agonist. Because this Cl- current inhibition is not observed during a sustained [Ca2+]i rise induced by ionomycin, the inhibitory pathway may be mediated by another agonist-induced messenger, such as diacylglycerol.