Activated Chloride Currents Research Articles

Identification of Chloride Channels CLCN3 and CLCN5 Mediating the Excitatory Cl- Currents Activated by Sphingosine-1-Phosphate in Sensory Neurons.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in numerous physiological and pathophysiological processes. We have previously reported a S1P-induced nocifensive response in mice by excitation of sensory neurons via activation of an excitatory chloride current. The underlying molecular mechanism for the S1P-induced chloride conductance remains elusive. In the present study, we identified two CLCN voltage-gated chloride channels, CLCN3 and CLCN5, which mediated a S1P-induced excitatory Cl− current in sensory neurons by combining RNA-seq, adenovirus-based gene silencing and whole-cell electrophysiological voltage-clamp recordings. Downregulation of CLCN3 and CLCN5 channels by adenovirus-mediated delivery of shRNA dramatically reduced S1P-induced Cl− current and membrane depolarization in sensory neurons. The mechanism of S1P-induced activation of the chloride current involved Rho GTPase but not Rho-associated protein kinase. Although S1P-induced potentiation of TRPV1-mediated ionic currents also involved Rho-dependent process, the lack of correlation of the S1P-activated Cl− current and the potentiation of TRPV1 by S1P suggests that CLCN3 and CLCN5 are necessary components for S1P-induced excitatory Cl− currents but not for the amplification of TRPV1-mediated currents in sensory neurons. This study provides a novel mechanistic insight into the importance of bioactive sphingolipids in nociception.

Ca2+/Calmodulin-Dependent Protein Kinase II γ-Dependent Serine727 Phosphorylation Is Required for TMEM16A Ca2+-Activated Cl- Channel Regulation in Cerebrovascular Cells.

TMEM16A is a critical component of Ca2+-activated chloride channels (CaCCs) and mediates basilar arterial smooth muscle cell (BASMC) proliferation in hypertensive cerebrovascular remodeling. CaMKII is a negative regulator of CaCC, and four CaMKII isoforms (α, β, γ and δ) are expressed in vasculature; however, it is unknown which and how CaMKII isoforms affect TMEM16A-associated CaCC and BASMC proliferation.Methods and Results:Patch clamp and small interfering RNA (siRNA) knockdown of different CaMKII isoforms revealed that only CaMKIIγ inhibited native Ca2+-activated chloride currents (ICl.Ca) in BASMCs. The TMEM16A overexpression evoked TMEM16A Cl-current and inhibited angiotensin II (Ang II)-induced proliferation in BASMCs. The co-immunoprecipitation and pull-down assay indicated an interaction between CaMKIIγ and TMEM16A protein. TMEM16A Cl-current was modulated by CaMKIIγ phosphorylation at serine residues in TMEM16A. Serine525 and Serine727 in TMEM16A were mutated to alanine, and only mutation at Ser727 (S727A) reversed the CaMKIIγ inhibition of the TMEM16A Cl-current. Phosphomimetic mutation S727D markedly decreased TMEM16A Cl-current and reversed TMEM16A-mediated suppression of BASMC proliferation, mimicking the inhibitory effects of CaMKIIγ on TMEM16A. A significant increase in CaMKIIγ isoform content was observed in parallel to the decrease of TMEM16A and ICl.Cain basilar artery proliferative remodeling in Ang II-infused mice. Serine 727 phosphorylation in TMEM16A by CaMKIIγ provides a new mechanism for regulating TMEM16A CaCC activity and Ang II-induced BASMC proliferation.

Emodin suppresses the nasopharyngeal carcinoma cells by targeting the chloride channels

Emodin is a natural anthraquinone derivative isolated from the Rheum palmatum. Recent studies demonstrated that emodin has anti-cancer activity in different kinds of human cancer cell lines. However, the underlying mechanism has not been very well studied. Our previous studies showed chloride channels is an important target of anti-cancer drugs. Therefore, the purpose of this research was aimed to explore the role of chloride channels involving in the anti-cancer activity of emodin. The proliferation, cell cycle arrest and apoptosis of poorly differentiated human nasopharyngeal carcinoma cells (CNE-2Z) and normal nasopharyngeal epithelial cells (NP69-SV40T) were detected by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide(MTT)and flow cytometry. The results indicated that emodin inhibited the CNE-2Z cell growth more significantly than NP69-SV40T cells and induced cell cycle arrest and apoptosis in CNE-2Z cells but not in NP69-SV40T cells. Chloride channel blocker 5-nitro-2-(3-phenylprop ylamino)-benzoate (NPPB) or tamoxifen both can prevent the apoptosis of CNE-2Z cells induced by emodin. Optical microscope and atomic force microscope (AFM) demonstrated that emodin can induce apoptotic volume decrease (AVD) and ultrastructure changes in CNE-2Z cell and inhibited by chloride channel blocker. These data could be a further evidence of chloride channel for preventing CNE-2Z cells from apoptosis induced by emodin. Whole cell patch clamp study also demonstrated that emodin can activate chloride channel in CNE-2Z cells but not in NP69-SV40T cells. Furthermore, the activated chloride currents can also be inhibited by chloride channel blockers indicating that chloride channel may be the potential target molecular of emodin exerting its anti-tumor efficiency in CNE-2Z cells.

TMEM16A overexpression contributes to tumor invasion and poor prognosis of human gastric cancer through TGF-β signaling.

TMEM16A is a newly identified calcium activated chloride channel, and has been reported to be overexpressed by various solid malignant cancers to promote proliferation and invasion, yet little is known about its role in gastric cancer(GC). Therefore, we investigated the role of TMEM16A in GC and its clinical significance by a retrospective analysis of 367 GC patients, and in vitro study was performed for validation and underlying molecular mechanism.TMEM16A was significantly upregulated and amplified in GC tissues, and its overexpression was positively correlated with disease stage, negatively with patient survival and identified as an independent prognostic factor for patient outcome. A negative correlation between TMEM16A and E-cadherin was found in 367 GC specimens. TMEM16A silencing significantly decreased calcium activated chloride currents, impaired TGF-β secretion, reduced E-cadherin expression, and inhibited the migration and invasion without affecting proliferation of GC cells (AGS and BGC-823). Supplement of TGF-β reverted the effects of TMEM16A silencing on E-cadherin expression, cell migration and invasion.In conclusion, TMEM16A promotes invasion and metastasis in GC, and might be a novel prognostic biomarker and potential therapeutic target in the treatment of GC.

TMEM16A is a Ca(2+) -activated Cl(-) channel expressed in the renal collecting duct.

In the renal collecting ducts, ATP stimulates a Ca(2+) -activated chloride current. The identity of the channel responsible for the current under physiological conditions is not known and it was hypothesized that TMEM16a is a relevant candidate in the renal collecting duct. The cortical collecting duct cell line M-1 was used as a model of the collecting duct. The ATP induced Ca(2+) signalling was imaged in cells loaded with Ca(2+) -sensitive fluorescent probes using confocal laser-scanning fluorescence microscopy. Chloride current was determined by mounting M-1 cell layers in Ussing chamber. The expression of TMEM16a in human kidney was tested by immunohistochemistry. M-1 cells displayed a transient increase in intracellular Ca(2+) concentration in response to 100 nm ATP. This response was completely blocked by addition of 100 μm suramin, indicating that ATP signals through purinergic P2 receptors. Apical addition of 100 nm ATP induced a Cl(-) current, which was blocked by suramin, DPC and the cysteine-modifying compound MTSET. M-1 cells were found to express TMEM16a at the mRNA and protein level. Functionally, it was found that knock-down of TMEM16a expression in M-1 cells inhibited the ATP induced Cl(-) -current. In human and mouse kidney sections, TMEM16a protein expression was localized to the collecting duct, and TMEM16a was found to be excreted in human urinary exosomes. TMEM16a is a Ca(2+) -activated Cl(-) channel expressed in the collecting ducts.

Disruption of vascular Ca2+-activated chloride currents lowers blood pressure

High blood pressure is the leading risk factor for death worldwide. One of the hallmarks is a rise of peripheral vascular resistance, which largely depends on arteriole tone. Ca2+-activated chloride currents (CaCCs) in vascular smooth muscle cells (VSMCs) are candidates for increasing vascular contractility. We analyzed the vascular tree and identified substantial CaCCs in VSMCs of the aorta and carotid arteries. CaCCs were small or absent in VSMCs of medium-sized vessels such as mesenteric arteries and larger retinal arterioles. In small vessels of the retina, brain, and skeletal muscle, where contractile intermediate cells or pericytes gradually replace VSMCs, CaCCs were particularly large. Targeted disruption of the calcium-activated chloride channel TMEM16A, also known as ANO1, in VSMCs, intermediate cells, and pericytes eliminated CaCCs in all vessels studied. Mice lacking vascular TMEM16A had lower systemic blood pressure and a decreased hypertensive response following vasoconstrictor treatment. There was no difference in contractility of medium-sized mesenteric arteries; however, responsiveness of the aorta and small retinal arterioles to the vasoconstriction-inducing drug U46619 was reduced. TMEM16A also was required for peripheral blood vessel contractility, as the response to U46619 was attenuated in isolated perfused hind limbs from mutant mice. Out data suggest that TMEM16A plays a general role in arteriolar and capillary blood flow and is a promising target for the treatment of hypertension.

Mechanism of CLCA1-Mediated CACC Activation in Chronic Lung Diseases

CLCA1 plays a central role in chronic inflammatory lung diseases (asthma and COPD). Overexpression of CLCA1 drives mucous overproduction and activates a Ca-activated chloride channel (CaCC), both through unknown mechanisms. Here we show that CLCA1 is a soluble secreted protein that gets cleaved into two fragments upon secretion. The cleavage is carried out by a novel internal zincin metalloprotease, located in the N-terminal domain. This self-cleavage reaction is required for the activation of chloride currents, as mutants with an impaired active site or cleavage site failed to increase currents above background levels. We observed that the N-terminal cleavage fragment is necessary and sufficient to activate the unknown CaCC, without directly proteolytically cleaving the channel. These results suggest a direct interaction between the channel and its modulator, CLCA1. Our current focus is on showing physical and functional association between these two molecules and identifying the CaCC by using pulldown experiments and functional patch clamp assays in the presence of siRNA and channel blockers. Latest results will be presented.

Divalent Cations Modulate TMEM16A Calcium-Activated Chloride Channels by a Common Mechanism

The gating of Ca²⁺-activated Cl⁻ channels is controlled by a complex interplay among [Ca²⁺](i), membrane potential and permeant anions. Besides Ca²⁺, Ba²⁺ also can activate both TMEM16A and TMEM16B. This study reports the effects of several divalent cations as regulators of TMEM16A channels stably expressed in HEK293T cells. Among the divalent cations that activate TMEM16A, Ca²⁺ is most effective, followed by Sr²⁺ and Ni²⁺, which have similar affinity, while Mg²⁺ is ineffective. Zn²⁺ does not activate TMEM16A but inhibits the Ca²⁺-activated chloride currents. Maximally effective concentrations of Sr²⁺ and Ni²⁺ occluded activation of the TMEM16A current by Ca²⁺, which suggests that Ca²⁺, Sr²⁺ and Ni²⁺ all regulate the channel by the same mechanism.

Differential effects of TGF-β1 on IL-5 and eotaxin-induced chloride currents in human blood eosinophils (P5130)

Abstract TGF-β1 is critical in the pathogenesis of chronic asthma and acts on eosinophils and other cells in the airways. Recently, we found TGF-β1 increases CLC3 expression in human blood eosinophils and induces migration. Here, we examined the interaction of TGF-β1 with IL-5 and eotaxin in the activation of chloride currents in human blood eosinophils. Eosinophils were purified (<99% pure, >98% viable) from the blood of healthy volunteers. Chloride current was measured at pH 7.4 by whole cell patch clamping in freshly isolated eosinophils using Axopatch 200B amplifier and analyzed by pClamp 10 software. Both eotaxin-1 and eotaxin-3 at low doses (0.075-10 ng/ml) increased outwardly rectified Cl- current in a time-dependent manner with maximum response at 500msec plus 100 mV. The eotaxin response at low doses (0.075-0.1 ng/ml) was slower in onset than at high dose. SB328437, a CCR3 antagonist (100 nM), inhibited eotaxin-induced Cl- current. Effect of eotaxin-3 was about 7-times larger than eotaxin-1. TGF-β1 (10ng/ml) decreased eotaxin-induced Cl- current. Incubation of eosinophils with IL-5 (10ng/ml) for 3hr decreased basal level of Cl- current. However, the addition of TGF-β1 with IL-5 significantly enhanced Cl- current. These data suggest differential effect of TGF-β1 in the activation and migration of human blood eosinophils in the presence of eotaxin or IL-5 in allergic airway inflammation and chronic asthma.

Mechanisms Involved in an Acidic pH‐conditioned NOXmediated Chloride Conductance in Nodose Sensory Neurons

We recently reported a novel prolonged low pH‐conditioned current in nodose sensory neurons following brief exposures to acidic extracellular pH. This current is similar to the Swell‐ Activated Chloride current, and mediated by NADPH oxidase (NOX) dependent hydrogen peroxide. Since NOX is known to be activated by intracellular (IC) alkaline pH, we hypothesized that low extracellular pH conditioning activates NOX by inducing IC alkalinity. We first buffered the IC pipette solution with 40mM HEPES, and found that the pH‐conditioned current was inhibited from 11.8±2.0 pA/pF to 3.4±0.7 pA/pF (n=6, p<0.01), indicating that an IC pH change is required for the current activation. The pH sensitive fluorescence dye 2′‐7′‐bis(carboxyethyl)‐5(6)‐ carboxyfluorescein showed significant but transient decreases in IC pH during the brief exposures to extracellular low pH (n=12). However, sustained reductions of IC pH to 7.0 and 6.0 did not elicit the current. Instead, increases in IC pH to 7.5, 7.6 and 8.0 dose‐dependently induced a similar current averaging 9.2±1.8, 16.3±4.1 and 30.6±9.7 pA/pF. Finally, inhibition of proton extrusion by blocking the Na+‐H+ exchanger with amiloride and proton channels with Zn2+ inhibited the low pH‐conditioned current (n=6). We therefore conclude that transient extracellular pH reductions can be transmitted intracellularly and activate Na+‐ H+ exchanger and proton channels. Our data suggest that the resulting activation of NOX and increase in chloride conductance involve IC alkalinization (NIH HL 14388).

Effects of oleic acid on ionic channels of plasma membranes of green alga Chara corallina

Extracellular application of 5–30 μM oleic acid (C18:1:9cis) produced a significant decrease in the amplitude of Ca2+ currents and Ca2+-activated chloride currents in plasma membrane of green alga Chara corallina. Introduction of 10 μM oleic acid intracellularly suppressed the development of the Ca2+ current. This inhibition of the Ca2+ current was reversible, and the Ca2+ conductivity of the cell membrane was restored after the removal of oleic acid. There were no changes in the non-specific leakage current, which indicates the intactness of the lipid component of the membrane. An increase of the oleic acid concentration up to 50–100 μM in the extracellular solution induced almost complete and irreversible suppression of inward currents and caused a substantial increase in the non-specific leakage current and conductivity of plasma membrane, suggesting its disruption. We suggest that the mechanism of cytotoxicity of oleic acid may be related to transport across plasma membrane of oleic acid and its complexes with α-lactalbumin (HAMLET and La-OA).

Regulation of renin secretion by renal juxtaglomerular cells

A major rate-limiting step in the renin-angiotensin-aldosterone system is the release of active renin from endocrine cells (juxtaglomerular (JG) cells) in the media layer of the afferent glomerular arterioles. The number and distribution of JG cells vary with age and the physiological level of stimulation; fetal life and chronic stimulation by extracellular volume contraction is associated with recruitment of renin-producing cells. Upon stimulation of renin release, labeled renin granules "disappear;" the number of granules decrease; cell membrane surface area increases in single cells, and release is quantal. Together, this indicates exocytosis as the predominant mode of release. JG cells release few percent of total renin content by physiological stimulation, and recruitment of renin cells is preferred to recruitment of granules during prolonged stimulation. Several endocrine and paracrine agonists, neurotransmitters, and cell swelling converge on the stimulatory cyclic AMP (cAMP) pathway. Renin secretion is attenuated in mice deficient in beta-adrenoceptors, prostaglandin E(2)-EP4 receptors, Gsα protein, and adenylyl cyclases 5 and 6. Phosphodiesterases (PDE) 3 and 4 degrade cAMP in JG cells, and PDE3 is inhibited by cyclic GMP (cGMP) and couples the cGMP pathway to the cAMP pathway. Cyclic AMP enhances K(+)-current in JG cells and is permissive for secretion by stabilizing membrane potential far from threshold that activates L-type voltage-gated calcium channels. Intracellular calcium paradoxically inhibits renin secretion likely through attenuated formation and enhanced degradation of cAMP; by activation of chloride currents and interaction with calcineurin. Connexin 40 is necessary for localization of JG cells in the vascular wall and for pressure- and macula densa-dependent suppression of renin release.

Airway smooth muscle electrophysiology in a state of flux?

Activation of chloride currents and release of internally sequestered Ca(2+) in airway smooth muscle have long been associated with excitation and contraction. Surprisingly, however, two recent publications (Deshpande DA, Wang WC, McIlmoyle EL, Robinett KS, Schillinger RM, An SS, Sham JS, Liggett SB. Nat Med 16: 1299-1304, 2010; Gallos G, Yim P, Chang S, Zhang Y, Xu D, Cook JM, Gerthoffer WT, Emala CW Sr. Am J Physiol Lung Cell Mol Physiol 302: L248-L256, 2012) have linked both events to relaxation. This begs a closer look at our understanding of airway smooth muscle electrophysiology and its contribution to excitation-contraction coupling. This Editorial Focus highlights those two aforementioned studies and several other equally paradoxical findings and proposes some possible reinterpretations of the data and/or new directions of research in which the answers might be found.

TMEM16A confers receptor-activated calcium-dependent chloride conductance

Calcium (Ca(2+))-activated chloride channels are fundamental mediators in numerous physiological processes including transepithelial secretion, cardiac and neuronal excitation, sensory transduction, smooth muscle contraction and fertilization. Despite their physiological importance, their molecular identity has remained largely unknown. Here we show that transmembrane protein 16A (TMEM16A, which we also call anoctamin 1 (ANO1)) is a bona fide Ca(2+)-activated chloride channel that is activated by intracellular Ca(2+) and Ca(2+)-mobilizing stimuli. With eight putative transmembrane domains and no apparent similarity to previously characterized channels, ANO1 defines a new family of ionic channels. The biophysical properties as well as the pharmacological profile of ANO1 are in full agreement with native Ca(2+)-activated chloride currents. ANO1 is expressed in various secretory epithelia, the retina and sensory neurons. Furthermore, knockdown of mouse Ano1 markedly reduced native Ca(2+)-activated chloride currents as well as saliva production in mice. We conclude that ANO1 is a candidate Ca(2+)-activated chloride channel that mediates receptor-activated chloride currents in diverse physiological processes.

Calcium-activated chloride current contributes to action potential alternations in left ventricular hypertrophy rabbit

T-wave alternans, characterized by a beat-to-beat change in T-wave morphology, amplitude, and/or polarity on the ECG, often heralds the development of lethal ventricular arrhythmias in patients with left ventricular hypertrophy (LVH). The aim of our study was to examine the ionic basis for a beat-to-beat change in ventricular repolarization in the setting of LVH. Transmembrane action potentials (APs) from epicardium and endocardium were recorded simultaneously, together with transmural ECG and contraction force, in arterially perfused rabbit left ventricular wedge preparation. APs and Ca(2+)-activated chloride current (I(Cl,Ca)) were recorded from left ventricular myocytes isolated from normal rabbits and those with renovascular LVH using the standard microelectrode and whole cell patch-clamping techniques, respectively. In the LVH rabbits, a significant beat-to-beat change in endocardial AP duration (APD) created beat-to-beat alteration in transmural voltage gradient that manifested as T-wave alternans on the ECG. Interestingly, contraction force alternated in an opposite phase ("out of phase") with APD. In the single myocytes of LVH rabbits, a significant beat-to-beat change in APD was also observed in both left ventricular endocardial and epicardial myocytes at various pacing rates. APD alternans was suppressed by adding 1 microM ryanodine, 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 100 microM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). The density of the Ca(2+)-activated chloride currents (I(Cl,Ca)) in left ventricular myocytes was significantly greater in the LVH rabbits than in the normal group. Our data indicate that abnormal intracellular Ca(2+) fluctuation may exert a strong feedback on the membrane I(Cl,Ca), leading to a beat-to-beat change in the net repolarizing current that manifests as T-wave alternans on the ECG.

Swelling activated chloride currents in the electrical activity of pulmonary vein cardiomyocytes

Pulmonary veins (PVs) contain cardiomyocytes with a high arrhythmogenicity for inducing atrial fibrillation. The swelling-activated outwardly rectifying Cl(-) currents (I(Cl,swell)) are important in the electrical activity of cardiomyocytes. This study was to investigate whether I(Cl,swell) play a role in the PV electrophysiological characteristics. A whole-cell patch clamp was used to investigate the action potentials and I(Cl,swell) in isolated rabbit single PV and atrial cardiomyocytes during immersion in isotonic (290-300 mosm L(-1)) and hypotonic (220-230 mosm L(-1)) solutions. The cell length and cell width were measured using confocal microscopy. Hypotonic solution induced larger I(Cl,swell) in the PV cardiomyocytes with pacemaker activity than those in the PV cardiomyocytes without pacemaker activity or atrial cardiomyocytes. Hypotonic solution shortened the action potential duration and increased the cell width to a greater extent in the PV cardiomyocytes than in the atrial cardiomyocytes. Moreover, hypotonic solution decreased the PV firing with a decrease in the transient inward currents and delayed after depolarizations. These findings suggest that the I(Cl,swell) plays an important role in the electrical activity of the PV cardiomyocytes.

Contribution of reverse Na+-Ca2+ exchange to spontaneous activity in interstitial cells of Cajal in the rabbit urethra.

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit regular Ca2+ oscillations that are associated with spontaneous transient inward currents (STICs) recorded under voltage clamp. Their frequency is known to be very sensitive to external Ca2+ concentration but the mechanism of this has yet to be elucidated. In the present study experiments were performed to assess the role of Na+-Ca2+ exchange (NCX) in this process. Membrane currents were recorded using the patch clamp technique and measurements of intracellular Ca2+ were made using fast confocal microscopy. When reverse mode NCX was enhanced by decreasing the external Na+ concentration [Na+]o from 130 to 13 mM, the frequency of global Ca2+ oscillations and STICs increased. Conversely, inhibition of reverse mode NCX by KB-R7943 and SEA0400 decreased the frequency of Ca2+ oscillations and STICs. Application of caffeine (10 mM) and noradrenaline (10 microM) induced transient Ca2+-activated chloride currents (I(ClCa)) at -60 mV due to release of Ca2+ from ryanodine- and inositol trisphosphate (IP3)-sensitive Ca2+ stores, respectively, but these responses were not blocked by KB-R7943 or SEA0400 suggesting that neither drug blocked Ca2+-activated chloride channels or Ca2+ release from stores. Intact strips of rabbit urethra smooth muscle develop spontaneous myogenic tone. This tone was relaxed by application of SEA0400 in a concentration-dependent fashion. Finally, single cell RT-PCR experiments revealed that isolated ICC from the rabbit urethra only express the type 3 isoform of the Na+-Ca2+ exchanger (NCX3). These results suggest that frequency of spontaneous activity in urethral ICC can be modulated by Ca2+ entry via reverse NCX.

The expression of wild-type pendrin (SLC26A4) in human embryonic kidney (HEK 293 Phoenix) cells leads to the activation of cationic currents

The SLC26A4 protein (pendrin) seems to be involved in the exchange of chloride with other anions, therefore being responsible for iodide organification in the thyroid gland and the conditioning of the endolymphatic fluid in the inner ear. Malfunction of SLC26A4 leads to Pendred syndrome, characterized by mild thyroid dysfunction often associated with goiter and/or prelingual deafness. The precise function of the SLC26A4 protein, however, is still elusive. An open question is still whether the SLC26A4-induced ion exchange mechanism is electrogenic or electroneutral. Recently, it has been shown that human pendrin expressed in monkey cells leads to chloride currents. We overexpressed the human SLC26A4 isoform in HEK293 Phoenix cells and measured cationic and anionic currents by the patch-clamp technique in whole cell configuration. Here we show that human pendrin expressed in human cells does not lead to the activation of chloride currents, but, in contrast, leads to an increase of cationic currents. Our experiments suggest that the SLC26A4-induced chloride transport is electroneutral when expressed in human cellular systems.

Structural features of phenol derivatives determining potency for activation of chloride currents via α1 homomeric and α1β heteromeric glycine receptors

Phenol derivatives constitute a family of neuroactive compounds. The aim of our study was to identify structural features that determine their modulatory effects at glycine receptors. We investigated the effects of four methylated phenol derivatives and two halogenated analogues on chloride inward currents via rat alpha(1) and alpha(1)beta glycine receptors, heterologously expressed in HEK 293. All compounds potentiated the effect of a submaximal glycine concentration in both alpha(1) homomeric and alpha(1)beta glycine receptors. While the degree of maximum potentiation of the glycine 10 microM effect in alpha(1)beta receptors was not different between the compounds, the halogenated compounds achieved half-maximum potentiating effects in the low microM range -- at more than 20-fold lower concentrations compared with their nonhalogenated analogues (P<0.0001). The coactivating effect was over-ridden by inhibitory effects at concentrations >300 microM in the halogenated compounds. Neither the number nor the position of the methyl groups significantly affected the EC(50) for coactivation. Only the bimethylated compounds 2,6 and 3,5 dimethylphenol (at concentrations >1000 microM) directly activated both alpha(1) and alpha(1)beta receptors up to 30% of the maximum response evoked by 1000 microM glycine. These results show that halogenation in the para position is a crucial structural feature for the potency of a phenolic compound to positively modulate glycine receptor function, while direct activation is only seen with high concentrations of compounds that carry at least two methyl groups. The presence of the beta subunit is not required for both effects.

Calcium accumulation in neurites and cell bodies of rat cerebellar granule cells in culture: effects on GABAA receptor function

Accumulation of calcium in rat cerebellar granule cells in culture was studied by two photon laser scanning microscopy. Depolarizations by high extracellular potassium induced short-lived increases in calcium in both cell bodies and neurites. However, although the increase in neurites subsided completely after the initial peak, in cell bodies there was a persistent plateau until the high potassium stimulus was removed. On the contrary, the calcium signal due to NMDA receptors activation was persistent in both cell bodies and neurites and remained until the agonist was present. The nature of these calcium signals provides an interpretation key for the effects of NMDA receptors activation on GABA(A) receptors. In particular, the persistent calcium increase in neurites may explain the decrease in GABA activated chloride currents which are related to activation of dendritic/synaptic GABA(A) receptors.