Adenosine stimulates Cl- channels of the nonpigmented (NPE) cells of the ciliary epithelium. We sought to identify the specific adenosine receptors mediating this action. Cl- channel activity in immortalized human (HCE) NPE cells was determined by monitoring cell volume in isotonic suspensions with the cationic ionophore gramicidin present. The A3-selective agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) triggered shrinkage (apparent Kd = 55 +/- 10 nM). A3-selective antagonists blocked IB-MECA-triggered shrinkage, and A3-antagonists (MRS-1097, MRS-1191, and MRS-1523) also abolished shrinkage produced by 10 microM adenosine when all four known receptor subtypes are occupied. The A1-selective agonist N6-cyclopentyladenosine exerted a small effect at 100 nM but not at higher or lower concentrations. The A2A agonist CGS-21680 triggered shrinkage only at high concentration (3 microM), an effect blocked by MRS-1191. IB-MECA increased intracellular Ca2+ in HCE cells and also stimulated short-circuit current across rabbit ciliary epithelium. A3 message was detected in both HCE cells and rabbit ciliary processes using RT-PCR. We conclude that human HCE cells and rabbit ciliary processes possess A3 receptors and that adenosine can activate Cl- channels in NPE cells by stimulating these A3 receptors. (+info)
A novel p64-related Cl- channel: subcellular distribution and nephron segment-specific expression.
(10/2847)
Several closely related proteins that have been implicated as chloride channels of intracellular membranes have recently been described. We report here the molecular cloning and characterization of a new member of this family from human cells. On the basis of sequence similarity, we conclude that this new protein represents the human version of a previously described protein from rat brain named p64H1. The human version of p64H1 (huH1) is a 28.7-kDa protein that shows an apparent molecular mass of 31 kDa by SDS-PAGE. A single 4.5-kb message is detected on Northern blots and is present in all tissues probed. The protein is expressed in an intracellular vesicular pattern in Panc-1 cells that is distinct from the endoplasmic reticulum, fluid-phase endocytic, and transferrin-recycling compartments, but which does colocalize with caveolin. In human kidney, huH1 is highly expressed in a diffuse pattern in the apical domain of proximal tubule cells. huH1 is expressed less abundantly in a vesicular pattern in glomeruli and distal nephron. (+info)
Effect of melittin on ion transport across cell membranes.
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Extensive work with melittin has shown that the venom has multiple effects, probably, as a result of its interaction with negatively changed phospholipids. It inhibits well known transport pumps such as the Na(+)-K(+)-ATPase and the H(+)-K(+)-ATPase. Melittin increases the permeability of cell membranes to ions, particularly Na+ and indirectly Ca2+, because of the Na(+)-Ca(2+)-exchange. This effect results in marked morphological and functional changes, particularly in excitable tissues such as cardiac myocytes. In some other tissues, e.g., cornea, not only Na+ but Cl- permeability is also increased by melittin. Similar effects to melittin on H(+)-K(+)-ATPase have been found with the synthetic amphipathic polypeptide Trp-3. (+info)
A single conductance pore for chloride ions formed by two cystic fibrosis transmembrane conductance regulator molecules.
(12/2847)
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)- and ATP-regulated chloride channel, whose gating process involves intra- or intermolecular interactions among the cytosolic domains of the CFTR protein. Tandem linkage of two CFTR molecules produces a functional chloride channel with properties that are similar to those of the native CFTR channel, including trafficking to the plasma membrane, ATP- and PKA-dependent gating, and a unitary conductance of 8 picosiemens (pS). A heterodimer, consisting of a wild type and a mutant CFTR, also forms an 8-pS chloride channel with mixed gating properties of the wild type and mutant CFTR channels. The data suggest that two CFTR molecules interact together to form a single conductance pore for chloride ions. (+info)
Block by fluoxetine of volume-regulated anion channels.
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1. We have used the whole-cell patch clamp technique to study the effect of fluoxetine, a commonly used antidepressant drug, on the volume-regulated anion channel (VRAC) in calf pulmonary artery endothelial (CPAE) cells. We also examined its effects on other Cl- channels, i.e. the Ca2(+)-activated Cl- current (I(Cl,Ca) and the cystic fibrosis transmembrane conductance regulator (CFTR) to assess the specificity of this compound for VRAC. 2. At pH 7.4 fluoxetine induced a fast and reversible block of the volume-sensitive chloride current (I(Cl,swell)), with a Ki value of 6.0+/-0.5 microM (n = 6-9). The blocking efficiency increased with increasing extracellular pH (Ki= 0.32+/-0.01 microM at pH 8.8, n = 3-9), indicating that the blockade is mediated by the uncharged form of fluoxetine. 3. Fluoxetine inhibited Ca2(+)-activated Cl(-) currents, I(Cl,Ca), activated by loading CPAE cells via the patch pipette with 1000 nM free Ca2+ (Ki= 10.7+/-1.6 microm at pH 7.4, n=3-5). The CFTR channel, transiently transfected in CPAE cells, was also inhibited with a Ki value of 26.9+/-9.4 microM at pH 7.4 (n = 3). 4. This study describes for the first time the effects of fluoxetine on anion channels. Our data reveal a potent block of VRAC at fluoxetine concentrations close to plasma concentrations. The results suggest a hydrophobic interaction with high affinity between uncharged fluoxetine and volume-activated chloride channels. Ca(2+)-activated Cl- currents and CFTR are also blocked by fluoxetine, revealing a novel characteristic of the drug as a chloride channel modulator. (+info)
Regulation of Cl- secretion in seawater fish (Dicentrarchus labrax) gill respiratory cells in primary culture.
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1. Primary cultures of sea bass (Dicentrarchus labrax) gill cells grown on permeable membranes form a highly differentiated tight epithelium composed of respiratory-like cells. This preparation was also found to provide a functional model for investigating the hormonal regulation of Cl- secretion. 2. In control conditions, i.e. in the absence of hormones or other stimuli, the cultured epithelium showed a short-circuit current (Isc) of 8.8 +/- 0.4 microA cm-2, a transepithelial potential (Vt) of 28.6 +/- 0.6 mV (serosal side positive), and a transepithelial resistance (Rt) of 5026 +/- 127 Omega cm2. Addition of 50 nM PGE2 caused a stimulation of Isc, Vt and transepithelial conductance, Gt. The increase in Isc was probably due to the elevation in Cl- secretion, since it could be correlated with the stimulation of serosal to mucosal 36Cl- flux. Application of the neurohypophyseal peptide arginine vasotocin (AVT; 50 nM) or the beta-adrenergic agonist isoproterenol (isoprenaline; 0. 5 microM) evoked a stimulation in Cl- secretion, as was shown by the increases in Isc and Gt. The excitatory effect of isoproterenol followed by the inhibitory action of propranolol, a beta-adrenergic antagonist, suggested the presence of beta-adrenergic receptors. Noradrenaline (0.1 microM) elicited a reduction in Isc, Vt and Gt, which was counterbalanced by the addition of phentolamine, an alpha-adrenergic antagonist. This suggested an activation of alpha-adrenergic receptors. 3. This study provides evidence for hormonal control of the Cl- secretion in sea bass gill respiratory cells in culture, involving AVT, prostaglandin (PGE2), and beta- and alpha-adrenergic receptors. (+info)
Modulation of the decay of Ca2+-activated Cl- currents in rabbit portal vein smooth muscle cells by external anions.
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1. The effects of external anions on the decay kinetics of Ca2+-activated Cl- currents (ICl(Ca)) were studied in smooth muscle cells isolated from rabbit portal vein using the perforated patch whole-cell voltage clamp technique. 2. In normal NaCl-containing external solution the decay of spontaneous Ca2+-activated Cl- currents (STICs) and Ca2+-activated Cl- 'tail' currents (Itail) was described by a single exponential with a time constant (tau) that was prolonged by external anions which are more permeable than Cl- (Br-, I- and SCN-) and accelerated by less permeant anions. However, intracellular I- did not affect the tau of STICs and Itail. 3. There was a positive correlation between the ability of an external anion to affect the decay tau of ICl(Ca) and its permeability relative to Cl-. 4. The voltage dependence of STIC and Itail decay was not affected by external or internal anions. 5. External permeating anions were not obligatory for activation of ICl(Ca) and STIC tau was not altered in Cl--free external solution. 6. Modulation of tau by mole fractions of SCN- and Cl- ions was fitted by a logistic curve, suggesting competition between SCN- and Cl- ions for a binding site. 7. In conclusion, external anions affect the decay of ICl(Ca) by a mechanism compatible with an interaction with a binding site which modulates Cl- channel kinetics. (+info)
Molecular cloning and biochemical characterization of a truncated, secreted member of the human family of Ca2+-activated Cl- channels.
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A novel family of chloride channel proteins has recently been discovered including two bovine (Lu-ECAM-1, bCLCA1), one murine (mCLCA1), and two human (hCLCA1 and hCLCA2) members. Here, we describe the cloning, expression, and molecular characterization of a truncated human homolog, tentatively named hCLCA3. It was cloned from a human spleen cDNA library and is expressed in numerous tissues including lung, trachea, spleen, thymus, and mammary gland as determined by reverse transcriptase-polymerase chain reaction. Unlike all previously known CLCA family members which consistently encode an approximately 125-kDa transmembrane protein that is cleaved to form a heterodimer of two proteins of approximately 90 and 35 kDa, the 3.6-kb hCLCA3 mRNA encodes a 37-kDa glycoprotein that corresponds to the N-terminal extracellular domain of its homologs. Moreover, when expressed in human embryonic kidney 293 or Chinese hamster ovary cells, this 37-kDa glycoprotein is secreted into the culture supernatant. These observations suggest that hCLCA3 is a structurally divergent member of the CLCA family of proteins and that it does not act as a channel protein but has distinct, yet unidentified functions. (+info)