Stretch-activated single K+ channels account for whole-cell currents elicited by swelling

Functionally significant stretch-activated ion channels have been clearly identified in excitable cells. Although single-channel studies suggest their expression in other cell types, their activity in the whole-cell configuration has not been shown. This discrepancy makes their physiological significance doubtful and suggests that their mechanical activation is artifactual. Possible roles for these molecules in nonexcitable cells are acute cell-volume regulation and, in epithelial cells, the complex adjustment of ion fluxes across individual cell membranes when the rate of transepithelial transport changes. We report the results of experiments on isolated epithelial cells expressing in the basolateral membrane stretch-activated K+ channels demonstrable by the cell-attached patch-clamp technique. In these cells, reversible whole-cell currents were elicited by both isosmotic and hyposmotic cell swelling. Cation selectivity and block by inorganic agents were the same for single-channel and whole-cell currents, indicating that the same entity underlies single-channel and whole-cell currents and that the single-channel events are not artifactual. In these cells, when the rate of apical-membrane NaCl entry increases, the cell Na+ content and volume also increase, stimulating the Na+,K+-ATPase at the basolateral membrane, i.e., both Na+ extrusion and K+ uptake increase. We speculate that, under these conditions, the parallel activation of basolateral K+ channels (by the swelling) elevates conductive K+ loss, tending to maintain the cell K+ content constant (“pump-leak parallelism”). This study describes a physiologically relevant stretch-activated channel, at both the single-channel and whole-cell levels, in a nonneural cell type.

A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells

Sustained (noninactivating) outward-rectifying K+ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K+ currents, an inactivating outward-rectifying K+ current was characterized (plant A-type current: IAP). IAP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. IAP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to −100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward IAP was mainly carried by K+ ions. In contrast to the sustained outward-rectifying K+ currents, cytosolic alkaline pH was found to inhibit IAP and extracellular K+ was required for IAP activity. Furthermore, increasing cytosolic free Ca2+ in the physiological range strongly inhibited IAP activity with a half inhibitory concentration of ≈ 94 nM. We present a detailed characterization of an inactivating K+ current in a higher plant cell. Regulation of IAP by diverse factors including membrane potential, cytosolic Ca2+ and pH, and extracellular K+ and Ca2+ implies that the inactivating IAP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.

DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature

Several DEG/ENaC cation channel subunits are expressed in the tongue and in cutaneous sensory neurons, where they are postulated to function as receptors for salt and sour taste and for touch. Because these tissues are exposed to large temperature variations, we examined how temperature affects DEG/ENaC channel function. We found that cold temperature markedly increased the constitutively active Na+ currents generated by epithelial Na+ channels (ENaC). Half-maximal stimulation occurred at 25°C. Cold temperature did not induce current from other DEG/ENaC family members (BNC1, ASIC, and DRASIC). However, when these channels were activated by acid, cold temperature potentiated the currents by slowing the rate of desensitization. Potentiation was abolished by a “Deg” mutation that alters channel gating. Temperature changes in the physiologic range had prominent effects on current in cells heterologously expressing acid-gated DEG/ENaC channels, as well as in dorsal root ganglion sensory neurons. The finding that cold temperature modulates DEG/ENaC channel function may provide a molecular explanation for the widely recognized ability of temperature to modify taste sensation and mechanosensation.

Ion channels gated by heat

All animals need to sense temperature to avoid hostile environments and to regulate their internal homeostasis. A particularly obvious example is that animals need to avoid damagingly hot stimuli. The mechanisms by which temperature is sensed have until recently been mysterious, but in the last couple of years, we have begun to understand how noxious thermal stimuli are detected by sensory neurons. Heat has been found to open a nonselective cation channel in primary sensory neurons, probably by a direct action. In a separate study, an ion channel gated by capsaicin, the active ingredient of chili peppers, was cloned from sensory neurons. This channel (vanilloid receptor subtype 1, VR1) is gated by heat in a manner similar to the native heat-activated channel, and our current best guess is that this channel is the molecular substrate for the detection of painful heat. Both the heat channel and VR1 are modulated in interesting ways. The response of the heat channel is potentiated by phosphorylation by protein kinase C, whereas VR1 is potentiated by externally applied protons. Protein kinase C is known to be activated by a variety of inflammatory mediators, including bradykinin, whereas extracellular acidification is characteristically produced by anoxia and inflammation. Both modulatory pathways are likely, therefore, to have important physiological correlates in terms of the enhanced pain (hyperalgesia) produced by tissue damage and inflammation. Future work should focus on establishing, in molecular terms, how a single ion channel can detect heat and how the detection threshold can be modulated by hyperalgesic stimuli.

A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca2+-permeable channel in the yeast vacuolar membrane

The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltage-dependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (TRP) family. By using a combination of gene deletion and re-expression with direct patch clamping of the yeast vacuolar membrane, we show that this yeast TRP-like gene is necessary for the YVC1 conductance. In physiological conditions, tens of micromolar cytoplasmic Ca2+ activates the YVC1 current carried by cations including Ca2+ across the vacuolar membrane. Immunodetection of a tagged YVC1 gene product indicates that YVC1 is primarily localized in the vacuole and not other intracellular membranes. Thus we have identified the YVC1 vacuolar/lysosomal cation-channel gene. This report has implications for the function of TRP channels in other organisms and the possible molecular identification of vacuolar/lysosomal ion channels in other eukaryotes.

Ligand-activated site-specific recombination in mice.

Current mouse gene targeting technology is unable to introduce somatic mutations at a chosen time and/or in a given tissue. We report here that conditional site-specific recombination can be achieved in mice using a new version of the Cre/lox system. The Cre recombinase has been fused to a mutated ligand-binding domain of the human estrogen receptor (ER) resulting in a tamoxifen-dependent Cre recombinase, Cre-ERT, which is activated by tamoxifen, but not by estradiol. Transgenic mice were generated expressing Cre-ERT under the control of a cytomegalovirus promoter. We show that excision of a chromosomally integrated gene flanked by loxP sites can be induced by administration of tamoxifen to these transgenic mice, whereas no excision could be detected in untreated animals. This conditional site-specific recombination system should allow the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting.

Elevation of intracellular calcium by muscarinic receptor activation induces a block of voltage-activated rat ether-à-go-go channels in a stably transfected cell line.

We have studied the properties of r-eag voltage-activated potassium channels in a stably transfected human embryonic kidney cell line. It was found that r-eag channels are rapidly and reversibly inhibited by a rise in intracellular calcium from 30 to 300 nM. The inhibition does not appear to depend on the activity of calcium-dependent kinases and phosphatases. The effect of calcium on r-eag channel activity was studied in inside-out membrane patches. Calcium inhibited r-eag channel activity with a mean IC50 of 67 nM. Activation of muscarinic receptors, generating calcium oscillations in the transfected cells, induced a synchronous inhibition of r-eag mediated outward currents. This shows that calcium can mediate r-eag current inhibition following muscarinic receptor activation. The data indicate that r-eag channels are calcium-inhibitable voltage-activated potassium channels.

Catalytic activity of the mouse guanine nucleotide exchanger mSOS is activated by Fyn tyrosine protein kinase and the T-cell antigen receptor in T cells.

mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutation and with a Src homology 2 deletion mutation resulted in a stimulation and suppression of the mSOS activity, respectively. The complex formations of Fyn-Shc, Shc-Grb2, and Grb2-mSOS were detected in the activated Fyn-transformed cells, whereas the SH2 deletion mutant of Fyn failed to form a complex with mSOS. Moreover, tyrosine phosphorylation of Shc was induced by the overexpression of the activated Fyn. These findings support the idea that Fyn activates the activity of mSOS bound to Grb2 through tyrosine phosphorylation of Shc. Unlike the current prevailing model, Fyn-induced activation of Ras might involve the stimulation of the catalytic guanine nucleotide exchange activity of mSOS.

Hypertonicity activates nonselective cation channels in mouse cortical collecting duct cells.

We investigated the effect of cell shrinkage on whole-cell currents of M-1 mouse cortical collecting duct cells. Addition of 100 mM sucrose to an isotonic NaCl bath solution induced cell shrinkage and increased whole-cell currents within 5-10 min by approximately 12-fold. The effect was reversible upon return to isotonic solution and could also be elicited by adding 100 mM urea or 50 mM NaCl. Replacement of bath Na+ by K+, Cs+, Li+, or Rb+ did not significantly affect the stimulated inward current, but replacement by N-methyl-D-glucamine reduced it by 88.1 +/- 1.3% (n = 34); this demonstrates that hypertonicity activates a nonselective alkali cation conductance. The activation was independent of extra- and intracellular Ca2+, but 1 or 10 mM ATP in the pipette suppressed it in a concentration-dependent manner, indicating that intracellular ATP levels may modulate the degree of channel activation. Flufenamic acid (0.1 mM) and gadolinium (0.1 mM) inhibited the stimulated current by 68.7 +/- 5.9% (n = 9) and 32.4 +/- 11.7% (n = 6), respectively, whereas 0.1 mM amiloride had no significant effect. During the early phase of hypertonic stimulation single-channel transitions could be detected in whole-cell current recordings, and a gradual activation of 30 and more individual channels with a single-channel conductance of 26.7 +/- 0.4 pS (n = 29) could be resolved. Thus, we identified the nonselective cation channel underlying the shrinkage-induced whole-cell conductance that may play a role in volume regulation.

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Activation of mitogen-activated protein kinases by vascular endothelial growth factor and basic fibroblast growth factor in capillary endothelial cells is inhibited by the antiangiogenic factor 16-kDa N-terminal fragment of prolactin.

A number of factors both stimulating and inhibiting angiogenesis have been described. In the current work, we demonstrate that the angiogenic factor vascular endothelial growth factor (VEGF) activates mitogen-activated protein kinase (MAPK) as has been previously shown for basic fibroblast growth factor. The antiagiogenic factor 16-kDa N-terminal fragment of human prolactin inhibits activation of MAPK distal to autophosphorylation of the putative VEGF receptor, Flk-1, and phospholipase C-gamma. These data show that activation and inhibition of MAPK may play a central role in the control of angiogenesis.

Electroadsorption of arsenic from natural water in granular activated carbon

The adsorption and electroadsorption of arsenic from a natural water has been studied in a filter-press electrochemical cell using a commercial granular activated carbon as adsorbent and Pt/Ti and graphite as electrodes. A significant reduction of the arsenic concentration is achieved when current is imposed between the electrodes, especially when the activated carbon was located in the vicinity of the anode. This enhancement can be explained in terms of the presence of electrostatic interactions between the polarized carbon surface and the arsenic ions, and changes in the distribution of most stable species of arsenic in solution due to As(III) to As(V) oxidation. In summary, electrochemical adsorption on a filter-press cell can be used for enhancement the arsenic remediation with activated carbon in the treatment of a real groundwater.

Tailoring the Surface Chemistry of Activated Carbon Cloth by Electrochemical Methods

This paper presents a systematic study of the effect of the electrochemical treatment (galvanostatic electrolysis in a filter-press electrochemical cell) on the surface chemistry and porous texture of commercial activated carbon cloth. The same treatments have been conducted over a granular activated carbon in order to clarify the effect of morphology. The influence of different electrochemical variables, such as the electrode polarity (anodic or cathodic), the applied current (between 0.2 and 1.0 A) and the type of electrolyte (HNO3 and NaCl) have also been analyzed. The anodic treatment of both activated carbons causes an increase in the amount of surface oxygen groups, whereas the cathodic treatment does not produce any relevant modification of the surface chemistry. The HNO3 electrolyte produced a lower generation of oxygen groups than the NaCl one, but differences in the achieved distribution of surface groups can be benefitial to selectively tune the surface chemistry. The porous texture seems to be unaltered after the electro-oxidation treatment. The validity of this method to introduce surface oxygen groups with a pseudocapacitive behavior has been corroborated by cyclic voltammetry. As a conclusion, the electrochemical treatment can be easily implemented to selectively and quantitatively modify the surface chemistry of activated carbons with different shapes and morphologies.

Electrochemical behaviour of activated carbons obtained via hydrothermal carbonization

Activated carbons were prepared by chemical activation of hydrochars, obtained by hydrothermal carbonisation (HTC) using low cost and abundant precursors such as rye straw and cellulose, with KOH. Hydrochars derived from rye straw were chemically activated using different KOH/precursor ratios, in order to assess the effect of this parameter on their electrochemical behaviour. In the case of cellulose, the influence of the hydrothermal carbonisation temperature was studied by fixing the activating agent/cellulose ratio. Furthermore, N-doped activated carbons were synthesised by KOH activation of hydrochars prepared by HTC from a mixture of glucose with melamine or glucosamine. In this way, N-doped activated carbons were prepared in order to evaluate the influence of nitrogen groups on their electrochemical behaviour in acidic medium. The results showed that parameters such as chemical activation or carbonisation temperature clearly affect the capacitance, since these parameters play a key role in the textural properties of activated carbons. Finally, symmetric capacitors based on activated carbon and N-doped activated carbon were tested at 1.3 V in a two-electrode cell configuration and the results revealed that N-groups improved the capacitance at high current density.

Aeration with a high-oxygen atmosphere in the activated sludge process /

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