Regulation of Orai1/STIM1 mediated ICRAC by intracellular pH.

Ca2+ release activated Ca2+ (CRAC) channels composed of two cellular proteins, Ca2+-sensing stromal interaction molecule 1 (STIM1) and pore-forming Orai1, are the main mediators of the Ca2+ entry pathway activated in response to depletion of intracellular Ca2+ stores. Previously it has been shown that the amplitude of CRAC current (ICRAC) strongly depends on extracellular and intracellular pH. Here we investigate the intracellular pH (pHi) dependence of ICRAC mediated by Orai1 and STIM1ectopically expressed in HEK293 cells. The results indicate that pHi affects not only the amplitude of the current, but also Ca2+ dependent gating of CRAC channels. Intracellular acidification changes the kinetics of ICRAC, introducing prominent re-activation component in the currents recorded in response to voltage steps to strongly negative potentials. ICRAC with similar kinetics can be observed at normal pHi if the expression levels of Orai1 are increased, relative to the expression levels of STIM1. Mutations in the STIM1 inactivation domain significantly diminish the dependence of ICRAC kinetics on pHi, but have no effect on pHi dependence of ICRAC amplitude, implying that more than one mechanism is involved in CRAC channel regulation by intracellular pH.

Curcumin inhibits activation of TRPM2 channels in rat hepatocytes.

Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca(2+) homeostasis, resulting in a sustained elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)]c) in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca(2+) entry through Transient Receptor Potential Melastatin 2 (TRPM2) channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca(2+)]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels.

The predominant role of IP3 type 1 receptors in activation of store-operated Ca²+ entry in liver cells.

Physiologically, hormone induced release of Ca²+ from intracellular stores occurs in response to inositol 1,4,5-trisphosphate (IP3) binding to its receptors expressed on the membranes of intracellular organelles, mainly endoplasmic reticulum. These IP3 receptors act as channels, releasing Ca²+ into the cytoplasmic space where it is responsible for regulating a host of distinct cellular processes. The depletion of intracellular Ca²+ stores leads to activation of store-operated Ca²+ channels on the plasma membrane which replenishes lost Ca²+ and sustain Ca²+ signalling. There are three isoforms of IP3 receptor, each exhibiting distinctive properties, however, little is known about the role of each isoform in the activation of store-operated Ca²+ entry. Recent evidence suggest that at least in some cell types the endoplasmic reticulum is not a homogeneous Ca²+ store, and there might be a sub-compartment specifically linked to the activation of store-operated Ca²+ channels, and Ca²+ release activated Ca²+ (CRAC) channel in particular. Furthermore, this sub-compartment might express only certain types of IP3 receptor but not the others. Here we show that H4IIE liver cells express all three types of IP3 receptor, but only type 1 and to a lesser extent type 3, but not type 2, participate in the activation of CRAC current (I(CRAC)), while type 1 and type 2, but not type 3, participate in observed Ca²+ release in response to receptor stimulation. Presented results suggest that in H4IIE rat liver cells the sub-compartment of intracellular Ca²+ store linked to the activation of I(CRAC) predominantly expresses type 1 IP3 receptors.

Properties of Orai1 mediated store-operated current depend on the expression levels of STIM1 and Orai1 proteins.

Two cellular proteins, stromal interaction molecule 1 (STIM1) and Orai1, are recently discovered essential components of the Ca2+ release activated Ca2+ (CRAC) channel. Orai1 polypeptides form the pore of the CRAC channel, while STIM1 plays the role of the endoplasmic reticulum Ca2+ sensor required for activation of CRAC current (I(CRAC)) by store depletion. It is not known, however, if the role of STIM1 is limited exclusively to Ca2+ sensing, or whether interaction between Orai1 and STIM1, either direct or indirect, also defines the properties of I(CRAC). In this study we investigated how the relative expression levels of ectopic Orai1 and STIM1 affect the properties of I(CRAC). The results show that cells expressing low Orai1 : STIM1 ratios produce I(CRAC) with strong fast Ca2+-dependent inactivation, while cells expressing high Orai1 : STIM1 ratios produce I(CRAC) with strong activation at negative potentials. Moreover, the expression ratio of Orai1 and STIM1 affects Ca2+, Ba2+ and Sr2+ conductance, but has no effect on the current in the absence of divalent cations. The results suggest that several key properties of Ca2+ channels formed by Orai1 depend on its interaction with STIM1, and that the stoichiometry of this interaction may vary depending on the relative expression levels of these proteins.

Oxygen sensitivity in the sheep adrenal medulla: role of SK channels.

The hypoxia-evoked secretion of catecholamines from the noninnervated fetal adrenal gland is essential for surviving intrauterine hypoxemia. The ion channels responsible for the initial depolarization that leads to catecholamine secretion have not been identified. Patch-clamp studies of adrenal chromaffin cells isolated from fetal and adult sheep revealed the presence of a Ca(2+)-dependent K(+) current that was reduced by hypoxia. Apamin, a blocker of small-conductance K(+) (SK) channels, reduced the Ca(2+)-dependent K(+) current, and the sensitivity of the channels to apamin indicated that the channels involved were of the SK2 subtype. In the presence of apamin, the hypoxia-evoked change in K(+) currents was largely eliminated. Both hypoxia and apamin blocked a K(+) current responsible for maintaining the resting potential of the cell, and the depolarization resulting from both led to an influx of Ca(2+). Simultaneous application of hypoxia and apamin did not potentiate the increase in cytosolic Ca(2+) concentration beyond that seen with either agent alone. Similar results were seen with curare, another blocker of SK channels. These results indicate that closure of SK2 channels would be the initiating event in the hypoxia-evoked catecholamine secretion in the adrenal medulla.

Temperature dependence of human muscle ClC-1 chloride channel.

1. In the present work we investigated the dependence on temperature of the ionic conductance and gating of human muscle ClC-1 chloride channels, transiently expressed in human embryonic kidney (HEK 293) cells. 2. At normal pH, ClC-1 currents deactivated at negative potentials with a double-exponential time course. The time constants of the exponential components, corresponding to the relaxations of the fast and slow gates, were temperature dependent with Q(10) values of approximately 3 and approximately 4, respectively. Current amplitude increased with increasing temperature with a Q(10) of approximately 1.6. 3. The voltage dependence of the two gating processes was shifted towards more positive potentials with increasing temperature. The half-saturation voltage (V(1/2)) of the steady-state open probability (P(o)) was shifted by approximately 23 and approximately 34 mV per 10 degrees C increase in temperature, for the fast and slow gate, respectively. 4. At low pH, the voltage dependence of ClC-1 was reversed and currents were activated by hyperpolarisation with a single-exponential time course. This type of gating in ClC-1 resembled the slow gating of the Torpedo ClC-0 homologue, but differed with respect to its kinetics and temperature dependence, with a Q(10) of gating relaxations at negative potentials of approximately 5. The Arrhenius plot of ClC-1 conductance at low pH had a clear break point at approximately 25 degrees C, with higher Q(10) values at lower temperatures. 5. The temperature sensitivity of relaxation and open probability of the slow gate, which in both ClC-0 and ClC-1 controls two pores simultaneously, implies that the slow gating of ClC-1 is mechanistically different from that of ClC-0.

Fast and slow gating of CLC-1: differential effects of 2-(4-chlorophenoxy) propionic acid and dominant negative mutations.

Our knowledge about ClC-1 muscle chloride channel gating, previously gained from single-channel recording and noise analysis, provides a theoretical basis for further analysis of macroscopic currents. In the present study, we propose a simple method of calculation of open probabilities (P(o)) of fast and slow gates from the relative amplitudes of ClC-1 inward current components. With this method, we investigated the effects of 2-(4-chlorophenoxy) propionic acid (CPP), a drug known to produce myotonia in animals, and dominant negative myotonic mutations, F307S and A313T, on fast and slow gating of ClC-1. We have shown that these mutations affected the P(o) of the slow gate, as expected from their mode of inheritance, and that CPP predominantly affected the fast gating process. CPP's action on the fast gating of mutant channels was similar to its effect in wild-type channels. Comparison of the effects of CPP and the mutations on fast and slow gating with the effects produced by reduction of external Cl(-) concentration suggested that CPP and mutations exert their action by affecting the transition of the channel from its closed to open state after Cl(-) binding to the gating site.

Interaction of hydrophobic anions with the rat skeletal muscle chloride channel ClC-1: effects on permeation and gating.

Permeation of a range of hydrophobic anions through the rat skeletal muscle chloride channel, rClC-1, expressed in Sf-9 (a Spodoptera frugiperda insect cell line) cells has been studied using the whole-cell patch-clamp technique. Bi-ionic reversal potentials measured with external application of foreign anions gave the following permeability sequence: Cl- (1) > benzoate (0.15) > hexanoate (0.12) > butyrate (0.09) > propionate (0.047) approximately formate (0.046). Anions with larger hydrophobic moieties were more permeant, which suggested that ClC-1 selectivity for hydrophobic anions is dominated by their interaction with a hydrophobic region in the external mouth of the pore. All anions studied when applied from outside show an apparently paradoxical voltage-dependent block of inward currents; this voltage-dependent block could be qualitatively described by a discrete-state permeation model with two binding sites and three barriers. Effects of the external anions with aliphatic side-chains on the apparent open probability (Po) suggested that they are unable to gate the channel, but can modulate ClC-1 gating, probably, by changing Cl- affinity to the gating site. Effects of internal application of benzoate, hexanoate or propionate mimicked those of increasing internal pH, and similarly depended on the channel protonation from the external side. Results for internal benzoate support the concept of a negatively charged cytoplasmic particle being involved in the ClC-1 gating mechanism sensitive to the internal pH.

Modulation of the gating of CIC-1 by S-(-) 2-(4-chlorophenoxy) propionic acid.

1. Using whole-cell patch-clamping and Sf-9 cells expressing the rat skeletal muscle chloride channel, rCIC-1, the cellular mechanism responsible for the myotonic side effects of clofibrate derivatives was examined. 2. RS-(+/-) 2-(4-chlorophenoxy)propionic acid (RS-(+/-) CPP) and its S-(-) enantiomer produced pronounced effects on CIC-1 gating. Both compounds caused the channels to deactivate more rapidly at hyperpolarizing potentials, which showed as a decrease in the time constants of both the fast and slow deactivating components of the whole cell currents. Both compounds also produced a concentration-dependent shift in the voltage dependence of channel apparent open probability to more depolarizing potentials, with an EC50 of 0.79 and 0.21 mM for the racemate and S-(-) enantiomer respectively. R-(+) CPP at similar concentrations had no effect on gating. RS-(+/-) CPP did not block the passage of Cl- through the pore of rCIC-1. 3. CIC-1 is gated by Cl- binding to a site within an access channel and S-(-) CPP alters gating of the channel by decreasing the affinity of this binding site for Cl-. Comparison of the EC50 for RS-(+/-) CPP and S-(-) CPP indicates that R-(+) CPP can compete with the S-(-) enantiomer for the site but that it is without biological activity. 4. RS-(+/-) CPP produced the same effect on rCIC-1 gating when added to the interior of the cell and in the extracellular solution. 5. S-(-) CPP modulates the gating of CIC-1 to decrease the membrane Cl- conductance (GCl), which would account for the myotonic side effects of clofibrate and its derivatives.

Permeation and block of the skeletal muscle chloride channel, ClC-1, by foreign anions.

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl- as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO3-, BrO3-); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl- at the regulatory binding site but impair Cl- passage through the channel pore (Br-, NO3-, ClO3-, I-, ClO4-, SCN-). The permeability sequence for rClC-1, SCN- approximately ClO4- > Cl- > Br- > NO3- approximately ClO3- > I- >> BrO3- > HCO3- >> methanesulfonate approximately cyclamate approximately glutamate, was different from the sequence determined for blocking potency and ability to shift the Popen curve, SCN- approximately ClO4- > I- > NO3- approximately ClO3- approximately methanesulfonate > Br- > cyclamate > BrO3- > HCO3- > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be approximately 4.5 A. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl- and SCN- or ClO4- suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.

Oxygen-sensing mechanisms are present in the chromaffin cells of the sheep adrenal medulla before birth.

1. The ability of the fetal adrenal medulla to respond directly to hypoxaemia and secrete catecholamines before the development of a functional innervation of the gland is essential for intrauterine survival. The cellular mechanisms involved in this response to low PO2 are not known, although the presence of oxygen-sensitive K+ channels in carotid body chemoreceptor cells and other sites suggests that these might underlie the chromaffin cell response. 2. Whole-cell patch-clamp techniques have been used to study K+ currents during normoxia and hypoxia in chromaffin cells isolated from the adrenal glands of fetal sheep. 3. Two types of chromaffin cells were observed, those with a fast inactivating K+ current and a larger capacitance and those with a delayed K+ current and smaller capacitance. No cell showed both types of current. The fast inactivating current showed voltage-dependent inactivation and was blocked by 1 mM 4-aminopyridine, characteristics of an IA-type current. The delayed current had two components, a TEA-sensitive, Ca2+-dependent current and a component with the kinetic behaviour of a delayed rectifier. 4. Both types of current were oxygen sensitive. The IA-type current was reduced by 27.4 +/- 3.2 % when the PO2 was reduced to about 15 mmHg. With the delayed current, hypoxia reduced the amplitude by 26.9 +/- 2.4 %, largely by reduction of the Ca2+-dependent component. 5. In the presence of hypoxia, reduction in the amplitude of these oxygen-sensitive K+ currents would increase the frequency and duration of action potentials, leading to increased activation of the L-type Ca2+ channels, influx of Ca2+ and the subsequent secretion of catecholamines.

Relevance of the D13 region to the function of the skeletal muscle chloride channel, ClC-1.

Although hydropathy analysis of the skeletal muscle chloride channel protein, ClC-1, initially predicted 13 potential membrane spanning domains (D1 to D13), later topological studies have suggested that domain D4 is extracellular and that D13, conserved in all eukaryotic ClC channels, is located within the extensive cytoplasmic tail that makes up the carboxyl terminus of the protein. We have examined the effect of deleting D13 (DeltaD13) and the function of the carboxyl tail by removing the final 72 (fs923X), 100 (fs895X), 125 (L869X), 398 (N596X), and 420 (Q574X) amino acids from rat ClC-1. Appropriate cDNA constructs were prepared and expressed using the baculovirus Sf9 insect cell system. Patch clamp analysis of chloride currents in Sf9 cells showed that only relatively insubstantial changes could be attributed to the expressed fs923X, fs895X, and DeltaD13 mutants compared with wild type rat ClC-1. For N596X and Q574X, however, adequate mRNA could be detected, but neither patch clamp nor polyacrylamide gel electrophoresis showed corresponding protein production. By contrast, expression of L869X was demonstrable by polyacrylamide gel electrophoresis, but no chloride conductance attributable to it could be detected. Overall, our results indicate that the domain D13 is dispensable, as are the final 100 amino acids, but not the final 125 amino acids or more, of the carboxyl tail. Some essential region of unknown significance, therefore, appears to reside in the 18 amino acids after D13, from Lys877 to Arg894.

pH-dependent interactions of Cd2+ and a carboxylate blocker with the rat C1C-1 chloride channel and its R304E mutant in the Sf-9 insect cell line.

1. Gating of the skeletal muscle chloride channel (ClC-1) is sensitive to extracellular pH. In this study, whole-cell recording of currents from wild-type (WT) ClC-1 and a mutant, R304E, expressed in the Sf-9 insect cell line was used to investigate further the nature of the pH-sensitive residues. 2. Extracellular Cd2+ produced a concentration-dependent block of WT ClC-1 with an IC50 of 1.0 +/- 0.1 mM and a Hill coefficient of 2.0 +/- 0.3. This block was sensitive to external pH, reducing at low pH, with an apparent pKa of 6.8 +/- 0.1 and a Hill coefficient for proton binding of 3.0 +/- 0.3. Anthracene-9-carboxylate (A-9-C) block of WT ClC-1 was also pH sensitive, increasing at low pH, with an apparent pKa of 6.4 +/- 0.1 and a Hill coefficient for proton binding of 1.0 +/- 0.2. 3. Compared with WT ClC-1, R304E had a lower affinity for Cd2+ (IC50, 3.0 +/- 0.3 mM) but it had a similar Hill coefficient for transition metal ion binding. The Hill coefficient for proton binding to the Cd2+ binding site was reduced to 1.4 +/- 0.3. In contrast, the A-9-C binding site in R304E showed the same pH sensitivity and affinity for the blocker as that seen in WT ClC-1. 4. ClC-1 has at least two binding sites for Cd2+, each of which has at least three residues which can be protonated. Binding of A-9-C is influenced by protonation of a single residue. Arg 304 is not sufficiently close to the A-9-C binding site to affect its characteristics, but it does. alter Cd2+ binding, indicating that transition metal ions and aromatic carboxylates interact with distinct sites. 5. The block of ClC-1 by transition metal ions and the apparent pKa of this block, together with the apparent pKa for A-9-C block and gating are all compatible with the involvement of His residues in the pore and gate of ClC-1.

Concentration and pH dependence of skeletal muscle chloride channel ClC-1.

1. The influence of Cl- concentration and pH on gating of the skeletal muscle Cl- channel, ClC-1, has been assessed using the voltage-clamp technique and the Sf-9 insect cell and Xenopus oocyte expression systems. 2. Hyperpolarization induces deactivating inward currents comprising a steady-state component and two exponentially decaying components, of which the faster is weakly voltage dependent and the slower strongly voltage dependent. 3. Open probability (Po) and kinetics depend on external but not internal Cl- concentration. 4. A point mutation, K585E, in human ClC-1, equivalent to a previously described mutation in the Torpedo electroplaque chloride channel, ClC-0, alters the I-V relationship and kinetics, but retains external Cl- dependence. 5. When external pH is reduced, the deactivating inward currents of ClC-1 are diminished without change in time constants while the steady-state component is enhanced. 6. In contrast, reduced internal pH slows deactivating current kinetics as its most immediately obvious action and the Po curve is shifted in the hyperpolarizing direction. Addition of internal benzoate at low internal pH counteracts both these effects. 7. A current activated by hyperpolarization can be revealed at an external pH of 5.5 in ClC-1, which in some ways resembles currents due to the slow gates of ClC-0. 8. Gating appears to be controlled by a Cl(-)-binding site accessible only from the exterior and, possibly, by modification of this site by external protonation. Intracellular hydroxyl ions strongly affect gating either allosterically or by direct binding and blocking of the pore, an action mimicked by intracellular benzoate.

Effects of water flow on transmembrane ionic currents in neurons of Helix pomatia and in squid giant axons.

1. The effects of water flow through the membrane produced by an osmotic gradient on the ionic currents in Helix neurons and in squid giant axons were studied. 2. Outward water flow had a marked effect on the ionic currents. 3. Cell volume diminution in hypertonic solution was accompanied by a decrease in the number of functioning ionic channels in the neurons. 4. Decrease of the tonicity of the external 10(-8) M TTX-containing solution leads to a transient recovery of the action potentials of the squid.