SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Explore the source record for details and available documents.
The developmental changes of GABA(A) receptors were investigated in Meynert neurons freshly dissociated from day 0, 2 week-, and 6 month-old rats using both nystatin and gramicidin perforated patch recording modes under voltage-clamp conditions. The age-related changes in the current amplitude and threshold concentration in the concentration-response relationships for GABA indicated the developmental alteration of the GABA(A) receptor subunits and the channel density. The GABA-induced E(Cl-) measured by the gramicidin perforated patch mode shifted to more negative with development. The decay time constant of GABAergic inhibitory postsynaptic spontaneous currents (sIPSCs) in the synaptic active zone accelerated with aging. The GABA-induced currents were potentiated in a concentration dependent manner in the presence of benzodiazepine (BZP) agonists, diazepam (DZP) and zolpidem (ZPM). The potentiation rate of DZP on the GABA(A) response decreased with aging, but not in the case of ZPM, which demonstrated a stronger action in the aging rat neurons. These results suggested that the GABA(A) receptor x Cl- channel complexes may thus change both the assembly and interaction of subunits as well as their functional roles with aging.
Ca-activated Cl channels are an important component of olfactory transduction. Odor binding to olfactory receptors in the cilia of olfactory sensory neurons (OSNs) leads to an increase of intraciliary Ca concentration by Ca entry through cyclic nucleotide-gated (CNG) channels. Ca activates a Cl channel that leads to an efflux of Cl from the cilia, contributing to the amplification of the OSN depolarization. The molecular identity of this Cl channel remains elusive. Recent evidence has indicated that bestrophins are able to form Ca-activated Cl channels in heterologous systems. Here we have analyzed the expression of bestrophins in the mouse olfactory epithelium and demonstrated that only mouse bestrophin-2 (mBest2) was expressed. Single-cell RT-PCR showed that mBest2 was expressed in OSNs but not in supporting cells. Immunohistochemistry revealed that mBest2 was expressed on the cilia of OSNs, the site of olfactory transduction, and colocalized with the main CNGA2 channel subunit. Electrophysiological properties of Ca-activated Cl currents from native channels in dendritic knob/cilia of mouse OSNs were compared with those induced by the expression of mBest2 in HEK-293 cells. We found the same anion permeability sequence, small estimated single-channel conductances, a Ca sensitivity difference of one order of magnitude, and the same side-specific blockage of the two Cl channel blockers commonly used to inhibit the odorant-induced Ca-activated Cl current in OSNs, niflumic acid, and 4-acetamido-4'-isothiocyanato-stilben-2,2'-disulfonate (SITS). Therefore, our data suggest that mBest2 is a good candidate for being a molecular component of the olfactory Ca-activated Cl channel.
Chloride current was measured in cells isolated from the pulmonary epithelium of patients with cystic fibrosis. By applying the whole cell patch clamp method three types of chloride current were identified, i.e. a time-dependent, an activating with time, and an inactivating with time. The time-dependent component of ICl proved to be DIDS sensitive. The effect of the potassium current blocker sparteine and its analogue tedisamil on ICl was tested. both substances were found to be chloride current inhibitors, yet the effectiveness of sparteine was 10(4) times higher.
The cystic fibrosis transmembrane conductance regulator (CFTR) consists of two transmembrane domains (TMDs), TMD1 and TMD2, two cytoplasmic nucleotide binding domains (NBDs), NBD1 and NBD2, and a regulatory domain. To elucidate the complex function of the CFTR, deletion constructs encompassing the second half of the CFTR distal to the first transmembrane domain were expressed in Xenopus oocytes and IB3 cells (a cystic fibrosis cell line). Constructs containing the regulatory domain, the second transmembrane domain, and the second nucleotide binding domain formed constitutively active channels, which were further stimulated upon the addition of cAMP. On the other hand, a construct encompassing the second transmembrane domain and the second nucleotide binding domain was stimulated to a small but noticeable extent upon the addition of cAMP. The selectivity of the second-half construct was the same for iodide and chloride, in contrast to the selectivity of wild-type CFTR, which is Cl- > I-. However, both constructs displayed single-channel conductances that were significantly smaller than those displayed by the first half of the CFTR. We conclude that regions of the second transmembrane domain may contribute to the overall channel of the pore, although the first half of the CFTR may confer its selectivity.
Explore the source record for details and available documents.
The patch-clamp technique was used to study the effect of rapid pulses of gamma-aminobutyric acid (GABA) on excised membrane-patches in the outside-out configuration of the deep extensor abdominal muscle (DEAM) of crayfish. Channel currents reversed at the equilibrium potential of Cl- and were blocked by picrotoxin. Rare channel openings were elicited by 0.1 mM GABA, and a saturating open probability of 0.9 was reached with 10 nM GABA. The investigated channel was only sensitive to GABA and is different from a previously described GABAergic channel in crayfish that is in addition sensitive to acetylcholine and glutamate and shows three subconductance states.
Because persistent swelling causes cell damage and often results in cell death, volume regulation is an important physiological function in both neuronal and non-neuronal cells. Brain cell swelling has been observed not only in various pathological conditions but also during physiological synaptic transmissions. Volume-sensitive anion channels have been reported to play an important role in the regulatory volume decrease occurring after osmotic swelling in many cell types. In this study, using a two-photon laser scanning microscope and patch-clamp techniques, we found that mouse cortical neurons in primary culture exhibit regulatory volume decrease after transient swelling and activation of Cl- currents during exposure to a hypotonic solution. The regulatory volume decrease was inhibited by Cl- channel blockers or K+ channel blockers. Swelling-activated Cl- currents exhibited outward rectification, time-dependent inactivation at large positive potentials, a low-field anion permeability sequence, an intermediate unitary conductance and sensitivity to known blockers of volume-sensitive Cl- channels. Thus, it is concluded that the activity of the volume-sensitive outwardly rectifying Cl- channel plays a role in the control of cell volume in cortical neurons.
Explore the source record for details and available documents.
The Ca2+ and voltage dependence of Ca(2+)-activated Cl- currents in rat parotid acinar cells was examined with the whole-cell patch clamp technique. Acinar cells were dialyzed with buffered free Ca2+ concentrations ([Ca2+]i) from < 1 nM to 5 microM. Increasing [Ca2+]i induced an increase in Cl- current at all membrane potentials. In cells dialyzed with [Ca2+]i > 25 nM, depolarizing test pulses activated a Cl- current that was composed of an instantaneous and a slow monoexponential component. The steady-state current-voltage relationship showed outward rectification at low [Ca2+]i but became more linear as the [Ca2+]i increased because of a shift in Cl- channel activation toward more negative voltages. The Ca2+ dependence of steady-state channel activation at various membrane voltages was fit by the Hill equation. The apparent Kd and Hill coefficient obtained from this analysis were both functions of membrane potential. The Kd decreased from 417 to 63 nM between -106 and +94 mV, whereas the Hill coefficient was always > 1 and increased to values as large as 2.5 at large positive potentials. We found that a relatively simple mechanistic model can account for the channel steady-state and kinetic behavior. In this model, channel activation involves two identical, independent, sequential Ca2+ binding steps before a final Ca(2+)-independent transition to the conducting conformation. Channel activation proceeds sequentially through three closed states before reaching the open state. The Ca2+ binding steps of this model have a voltage dependence similar to that of the Kd from the Hill analysis. The simplest interpretation of our findings is that these channels are directly activated by Ca2+ ions that bind to sites approximately 13% into the membrane electric field from the cytoplasmic surface.
Rat and human pancreatic duct cells have small-conductance Cl- channels in their apical plasma membranes. These channels are regulated by secretin and adenosine 3',5'-cyclic monophosphate and may function in parallel with Cl(-)-HCO3- exchangers to allow HCO3- secretion from the duct cell. Using the patch-clamp technique, we have now determined the anion permeability sequence of the channel as NO3- greater than Br- approximately I- approximately Cl- much greater than HCO3- much greater than gluconate. From this we conclude 1) that anion permeation involves a weak interaction with charged sites inside the channel pore, 2) that because of the low HCO3-/Cl- permeability ratio it is unlikely that significant amounts of HCO3- could be secreted directly via the channel, and 3) that channel permeability may determine the anion selectivity of secretion. We also show that 5-nitro-2-(3-phenylpropylamino)benzoic acid blocks the small-conductance Cl- channel, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid has no effect.
The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by d-tubocurarine but not by alpha-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors.
We have described a preparation of Necturus maculosus gallbladder (NGB) epithelium yielding isolated cells that retain structural and functional polarity ("figure-eight" cells). These cells have a normal membrane voltage and remain polarized for several hours after isolation. Apical and basolateral membrane domains are differentially labeled with hydrophobic fluorescent dyes; freeze-fracture electron microscopy reveals two distinct membrane domains separated by tight junctions; ZO-1, Na+/H+ exchanger (NHE3), and Na(+)-K(+)-ATPase are present in the junctional, apical, and basolateral region, respectively; and cell-attached patch-clamp experiments reveal different K+ currents in the two membrane domains [R. J. Torres, G. A. Altenberg, J. A. Copello, G. Zampighi, and L. Reuss, Am. J. Physiol. 270 (Cell Physiol. 39): C1864-C1874, 1996]. Here, we show that NGB epithelial cells express a protein cross-reactive with an antibody against human cystic fibrosis transmembrane conductance regulator (CFTR). In figure-eight cells, immunoreactivity was restricted to the apical membrane domain. Using intracellular microelectrodes and a novel method of regional superfusion, we found that control cells have high K+ conductances in both membranes and a small basolateral Cl- conductance, similar to findings in the epithelium. Activation of adenylate cyclase with forskolin elicited a large apical membrane Cl- conductance and membrane depolarization. Whole cell patch-clamp studies yielded a forskolin-activated linear Cl- current, with high Cl-/aspartate selectivity. In conclusion, 1) figure-eight cells maintain the conductive membrane properties present in the epithelium, including polarized expression of adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- channels, and 2) the cAMP-activated Cl- conductance is underlied by a CFTR homologue.
OBJECTIVES: The cAMP-dependent Cl- conductance in heart is believed to be due to cardiac expression of the cystic fibrosis transmembrane conductance regulator (CFTR). While CFTR expressed in rabbit and guinea-pig heart (CFTRcardiac) is an alternatively spliced isoform of the epithelial gene product, little information is known regarding possible expression of CFTR in primate heart. In this study, we examined molecular expression of CFTR in human and simian atrium and ventricle and functional expression of cAMP-dependent Cl- currents in isolated human atrial and simian ventricular cells. METHODS: The reverse transcription polymerase chain reaction (RT-PCR) was performed on human and simian atrial and ventricular mRNA using primers designed to border regions of the CFTR gene product corresponding to transmembrane segments I-VI (TSI-VI), the first nucleotide binding domain (NBD1), transmembrane segments VII-XII (TSVII-XII), and the large cytoplasmic domain which includes the regulatory (R) domain and NBD1. Functional expression of CFTR Cl- channels in human atrial and simian ventricular myocytes was determined using whole-cell and giant inside-out patch-clamp techniques. RESULTS: Southern blot analysis of these RT-PCR products demonstrated expression of CFTR transcripts in human and simian atrial and ventricular tissue and revealed a novel pattern of expression compared to most animal species studies: both the exon 5 plus (unspliced) and exon 5 minus (spliced) CFTR transcripts are co-expressed in human and simian atrium and ventricle. Whole-cell experiments demonstrated a Cl- sensitive time-independent background conductance in both human atrial and simian ventricular myocytes that was activated by forskolin (FSK) and insensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). In inside-out patches utilizing the giant patch technique on human atrial myocytes, unitary Cl- sensitive channels resembling CFTR Cl- channels (approximately 14 pS conductance) were activated by the catalytic subunit of protein kinase A (PKA) in 3/12 patches examined. CONCLUSIONS: These results clearly demonstrate the molecular expression of CFTR Cl- channels and provide electrophysiological evidence consistent with functional expression of these channels in human atrial and simian ventricular myocardium.
Lanthanum was previously reported to potentiate the GABA-induced chloride currents reversibly by acting at a distinct site on the GABAA receptor-channel complex (Ma and Narahashi, 1993). We now report that all other lanthanides tested enhanced the GABA responses with greater efficiencies and produced inward currents by themselves. The rank order of efficacies of lanthanides to potentiate the GABA responses and to generate currents correlated inversely with the hydrated ionic radii of these ions. The GABA-induced currents were recorded from the rat dorsal root ganglion neurons in primary culture by the whole-cell patch-clamp technique. Lanthanides at 1 mM reversibly potentiated the GABA responses (times of control): lutetium (Lu3+) 12.4 +/- 0.1 > erbium (Er3+) 11.4 +/- 0.2 > terbium (Tb3+) 9.3 +/- 0.5 > europium (Eu3+) 5.4 +/- 0.4 > neodymium (Nd3+) 4.1 +/- 0.2 > cerium (Ce3+) 3.3 +/- 0.2 > lanthanum (La3+) 2.4 +/- 0.2 (n = 10). The enhancing effect of Tb3+ was dose dependent, with an EC50 356 +/- 12 microM (n = 10), and weakly voltage dependent, increasing with hyperpolarization. The amplitudes of currents induced by various lanthanides (1 mM) were (percentage of 10 microM GABA-induced current) Lu3+ 101 +/- 8 > Er3+ 57 +/- 8 > Tb3+ 33 +/- 3 > Eu3+ 20 +/- 2 > Nd3+ 14 +/- 2 approximately Ce3+ 13 +/- 0.6 > La3+ 10 +/- 0.2 (n = 4). Tb3+ reversibly induced currents in a dose-dependent manner with an EC50 of 863 +/- 0 microM (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)
Macroscopic and unitary currents through stretch-activated Cl- channels were examined in isolated human atrial myocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ and Ca2+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was reduced, application of positive pressure via the pipette activated membrane currents under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by 60 mV per 10-fold change in the external Cl- concentration, indicating that the current was Cl- selective. The current was inhibited by bath application of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 9-anthracenecarboxylic acid (9-AC). beta-Adrenergic stimulation failed to activate a Cl- current. In single channel recordings from outside-out patches, positive pressure in the pipette activated the unitary current with half-maximal activation of 14.7 mm Hg at +40 mV. The current-voltage relationship of single channel activity obtained in inside-out patches was linear in symmetrical Cl- solution with the averaged slope conductance of 8.6 +/- 0.7 pS (mean +/- SD, n = 10). The reversal potential shift of the channel by changing Cl- concentration was consistent with a Cl- selective channel. The open time distribution was best described by a single exponential function with mean open lifetime of 80.4 +/- 9.6 msec (n = 9), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 11.5 +/- 2.2 msec (n = 9) and that for the slow component of 170.2 +/- 21.8 msec (n = 9). Major changes in the single channel activity in response to pressure were caused by changes in the interburst interval. Single channel activity was inhibited by DIDS and 9-AC in a manner similar to whole-cell configuration. These results suggest that membrane stretch induced by applying pressure via the pipette activated a Cl- current in human atrial myocytes. The current was sensitive to Cl- channel blockers and exhibited membrane voltage-independent bursting opening without sensitive to beta-adrenergic stimulation.
Receptor binding studies have shown that the combination of some new quinolone antibacterial agents with 4-biphenylacetic acid (BPAA), a metabolite of fenbufen, inhibits GABAA receptors. In order to elucidate further the mechanism of these drug interactions, the effect of quinolone antibacterial agents on muscimol-stimulated 36Cl- uptake in rat cerebral cortical synaptoneurosomes was investigated in the absence or presence of BPAA. In the absence of BPAA, quinolones such as norfloxacin (NFLX) and enoxacin attenuated muscimol-stimulated 36Cl- uptake at 10 microM and above. In combination with 10 microM BPAA, the inhibitory effect of these drugs was potentiated and there was a parallel shift of the inhibition curves to the left for these drugs. BPAA alone (1 and 10 microM) did not affect basal or muscimol-stimulated 36Cl- uptake. Hybrid molecules of NFLX and BPAA were synthesized and their inhibitory potency was also investigated. Inhibition curves of muscimol-stimulated 36Cl- uptake revealed that a hybrid with a -CONH(CH2)3- chain between NFLX and BPAA (flexible structure) (1 nM-20 microM) inhibited muscimol-stimulated 36Cl- uptake more potently than did the combination of NFLX (10 nm-100 microM) and 10 microM BPAA. In contrast, another hybrid linked by -CONH-(stretched structure) exhibited a weak inhibitory effect at 10 microM. These results suggest that quinolones in combination with BPAA bind to GABAA receptors, thus inhibiting Cl- channel activity, and that the inhibitory potency of quinolones may be enhanced by an intermolecular interaction with BPAA.
The genetic disease cystic fibrosis (CF) causes decreased Cl- transport in several epithelia. cAMP-dependent regulation of apical membrane Cl- channels is defective in CF airway epithelia; as a result, CF epithelia fail to secrete Cl-. In contrast, Ca(2+)-stimulated Cl- secretion is intact in CF airway epithelia and thus has the potential to bypass the CF Cl- secretory defect. For a Cl- channel to govern Cl- secretion, it must be located in the apical membrane. To specifically investigate apical membrane Cl- channels, we studied cells grown on permeable filter supports and measured Cl- currents across the apical membrane. We found that Ca2+ and cAMP activate different Cl- channels in the apical membrane. (i) Ca(2+)-activated Cl- channels were present in the apical membrane of airway but not in intestinal epithelia. (ii) cAMP- but not Ca(2+)-activated Cl- channels were defective in CF airway epithelia. (iii) Ca(2+)- but not cAMP-activated Cl- channels were blocked by 4,4'-diisothiocyanato-2,2'-stilbenedisulfonate. (iv) Ca(2+)- and cAMP-activated apical channels had different anion permeabilities. (v) An increase in both second messengers produced an additive increase in Cl- current. These results also explain the puzzling observation that Ca(2+)-stimulated Cl- secretion is defective in CF intestine: the Ca(2+)-activated Cl- channels that could circumvent the Cl- secretory defect in CF airway are missing from the apical membrane of intestinal epithelia.