Search

A supramolecular nanoprodrug (DOX@GP5⊃Pro-NFA) was constructed based on the host–guest complexation of a chloride channel blocker prodrug (Pro-NFA) and glycosylated pillar[5]arenes (GP5), which could target chemoresistance cancer therapy.

Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (P(i)). P(i) diffuses into the sarcoplasmic reticulum (SR) where it is believed to forms insoluble Ca(2+) salts, thus contributing to the impairment of Ca(2+) release. Information on the P(i) entrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca(2+) spark frequency is correlated with Ca(2+) load of the SR and can be used to monitor this variable. We studied the effects of P(i) on Ca(2+) sparks in permeabilized fibres of the frog. Relative event frequency (f/f(ref)) rose with increasing [P(i)] reaching 2.54±1.6 at 5 mM and then decreased monotonically, reaching 0.09±0.03 at [P(i)] = 80 mM. Measurement of [Ca(2+)](SR) confirmed a decrease correlated with spark frequency at high [P(i)]. A large [Ca(2+)](SR) surge was observed upon P(i) removal. Anion channels are a putative path for P(i) into the SR. We tested the effect of chloride channel blocker 9-anthracenecarboxylic acid (9AC) on P(i) entrance. 400 μM 9AC applied to the cytoplasm produced a non-significant increase in spark frequency and reduced the P(i) effects on this parameter. Fibre treatment with 2 mM 9AC in the presence of high cytoplasmic Mg(2+) suppressed P(i) effects on [Ca(2+)](SR) and spark frequency up to 55 mM P(i). These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that it impairs Ca(2+) release by accumulating and precipitating with Ca(2+) inside the SR, thus contributing to myogenic fatigue.

Key points Accumulation of inorganic phosphate (Pi ) may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum (SR). Neither direct demonstration of this process nor definition of the entry pathway of Pi into SR are fully established. We showed that Pi promoted Ca2+ release at concentrations below 10 mm and decreased it at higher concentrations. This decrease correlated well with that of [Ca2+ ]SR . Pre-treatment of permeabilized myofibres with 2 mm Cl- channel blocker 9-anthracenecarboxylic acid (9AC) inhibited both effects of Pi . The biphasic dependence of Ca2+ release on [Pi ] is explained by a direct effect of Pi acting on the SR Ca2+ release channel, combined with the intra-SR precipitation of Ca2+ salts. The effects of 9AC demonstrate that Pi enters the SR via a Cl- pathway of an as-yet-undefined molecular nature. Abstract Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (Pi ). Pi diffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca2+ salts, thus contributing to the impairment of Ca2+ release. Information on the Pi entrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca2+ spark frequency is correlated with the Ca2+ load of the SR and can be used to monitor this variable. We studied the effects of Pi on Ca2+ sparks in permeabilized fibres of the frog. Relative event frequency (f/fref ) rose with increasing [Pi ], reaching 2.54 ± 1.6 at 5 mm, and then decreased monotonically, reaching 0.09 ± 0.03 at [Pi ] = 80 mm. Measurement of [Ca2+ ]SR confirmed a decrease correlated with spark frequency at high [Pi ]. A large [Ca2+ ]SR surge was observed upon Pi removal. Anion channels are a putative path for Pi into the SR. We tested the effect of the chloride channel blocker 9-anthracenecarboxylic acid (9AC) on Pi entrance. 9AC (400 µm) applied to the cytoplasm produced a non-significant increase in spark frequency and reduced the Pi effects on this parameter. Fibre treatment with 2 mm 9AC in the presence of high cytoplasmic Mg2+ suppressed the effects of Pi on [Ca2+ ]SR and spark frequency up to 55 mm [Pi ]. These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that Pi impairs Ca2+ release by accumulating and precipitating with Ca2+ inside the SR, thus contributing to myogenic fatigue.

The present study demonstrates the interaction of potential chloride channel blocker drug, namely, 9-methylanthroate (9MA) with dimyristoylphosphatidylcholine (DMPC) lipid bilayer membrane in the solid gel (SG, at 15 °C) and liquid crystalline (LC, at 37 °C) phases of the lipid. Our spectroscopic results reveal a relatively greater degree of partitioning of the drug molecules into the DMPC lipid membrane at 37 °C (LC phase) compared to that at 15 °C (SG phase). Furthermore, the study is extended to explore the interaction of a bile salt (sodium deoxycholate, NaDC) with the DMPC lipid bilayer membrane in the SG as well as LC phase utilizing the environment-sensitive photophysical characteristics of the drug. Our steady-state and time-resolved spectroscopic results are invoked to critically analyze all the possibilities of bile salt-induced modulation of the photophysical behavior of the lipid-bound drug e.g., (i) expulsion of the lipid-bound drug molecules to the bulk aqueous phase upon addition of the bile salt, (ii) redistribution of population of the drug molecules within the lipid membrane and (iii) penetration of water molecules into the lipid bilayer membrane and thereby altering the hydration structure of the membrane. Cumulatively, our results appear to converge on the possibility of bile salt-induced hydration of the interfacial region of the lipid membrane as the actuating mechanism of the lipid-bile salt interaction. It is imperative to state that the present study emphasizes on the interaction of DMPC lipid membrane with the bile salt (NaDC) only in the submicellar concentration regime of NaDC which involves no significant perturbation of the lipid vesicle structure.

The present study reveals the effect of various divalent ions (Ca2+, Mg2+and Zn2+) on the binding interaction of a prospective chloride channel blocker, 9-methylanthroate (9MA), with liposome membranes, namely, dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The liposome membranes DMPC and POPC differ in the unsaturation of the side-chain. The drug (9MA) is found to experience a greater degree of partitioning into the POPC lipid bilayer (containing unsaturated side-chain) in comparison to DMPC (containing saturated side-chain). The stronger 9MA-POPC binding interaction is found to be only nominally perturbed by the presence of metal salts. On the contrary, the 9MA-DMPC binding interaction is found to be significantly perturbed by the presence of metal salts and is manifested on the environment-responsive spectroscopic properties of the drug. The steady-state and picosecond-resolved fluorescence spectroscopic results reveal the effect of metal ions on DMPC bilayer to follow the trend Ca2+ < Mg2+ < Zn2+. This is also quantified by evaluating the partition coefficient of the drug into DMPC lipid in the presence of various divalent ions which is found to follow the same sequence. The degree of penetration of these cations has been rationalized on the basis of adsorption of cations on DMPC headgroup region resulting in dehydration of the headgroup along with shrinking of it.

The basis of the extraordinary sensitivity and frequency selectivity of the cochlea is a chloride‐sensitive protein called prestin which can produce an electromechanical response and which resides in the basolateral plasma membrane of outer hair cells (OHCs). The compound 9‐anthracenecarboxylic acid (9‐AC), an inhibitor of chloride channels, has been found to reduce the electromechanical response of the cochlea and the OHC mechanical impedance. To elucidate these 9‐AC effects, the functional electromechanical status of prestin was assayed by measuring the nonlinear capacitance of OHCs from the guinea‐pig cochlea and of prestin‐transfected human embryonic kidney 293 (HEK 293) cells. Extracellular application of 9‐AC caused reversible, dose‐dependent and chloride‐sensitive reduction in OHC nonlinear charge transfer, Q max. Prestin‐transfected cells also showed reversible reduction in Q max. For OHCs, intracellular 9‐AC application as well as reduced intracellular pH had no detectable effect on the reduction in Q max by extracellularly applied 9‐AC. In the prestin‐transfected cells, cytosolic application of 9‐AC approximately halved the blocking efficacy of extracellularly applied 9‐AC. OHC inside‐out patches presented the whole‐cell blocking characteristics. Disruption of the cytoskeleton by preventing actin polymerization with latrunculin A or by decoupling of spectrin from actin with diamide did not affect the 9‐AC‐evoked reduction in Q max. We conclude that 9‐AC acts on the electromechanical transducer principally by interaction with prestin rather than acting via the cytoskeleton, chloride channels or pH. The 9‐AC block presents characteristics in common with salicylate, but is almost an order of magnitude faster. 9‐AC provides a new tool for elucidating the molecular dynamics of prestin function.

The present study demonstrates a detailed characterization of the binding interaction of a potential chloride channel blocker 9-methyl anthroate (9-MA) with calf-thymus DNA. The modulated photophysical properties of the emissive molecule within the microheterogeneous bio-assembly have been spectroscopically exploited to monitor the drug-DNA binding interaction. Experimental results based on fluorescence and absorption spectroscopy aided with DNA-melting, viscometric and circular dichroism studies unambiguously establish the binding mode between the drug and DNA to be principally intercalative. Concomitantly, a discernible dependence of the mode of binding between the concerned moieties on the ionic strength of the medium is noteworthy. A dip-and-rise characteristic of the rotational relaxation profile of the drug within the DNA environment has been argued to be originating from a substantial difference in the lifetime as well as amplitude of the free and DNA bound drug molecule. In view of the prospective biological applications of the drug, the issue of facile dissociation of the intercalated drug from the DNA helix via a simple detergent-sequestration technique has also been unveiled. The utility of the present work resides in exploring the potential applicability of the fluorescence properties of 9-MA for studying its interactions with other relevant biological or biomimicking targets.

Interaction of a potential chloride channel blocker, 9-methyl anthroate (9-MA), has been studied with zwitterionic l-α-phosphatidylcholine (egg-PC) lipid vesicles, which ascertains the utility of the drug as an efficient molecular reporter for probing the microheterogeneous environment of lipid-bilayers. The effect of a non-ionic triblock co-polymer P123 on the stability of these drug-bound lipid-bilayers has also been investigated by means of steady state and time-resolved spectroscopic techniques exploiting the fluorescence properties of the drug. Experimental results reveal that the addition of P123 to the drug-bound lipid results in a preferential complexation of the drug with the Pluronic leaving the lipid vesicles aside, which has been attributed to a substantially stronger binding interaction of the drug with P123 than that with egg-PC. The result is of potential interest from a medical perspective owing to the context of excess drug desorption from bio-membranes.

The present work demonstrates a thorough description of the differential interactions of a potential chloride channel blocker, 9-methyl anthroate (9-MA), with a charged surfactant, sodium dodecyl sulfate (SDS), in the pre- and post-micellar regime along with the consequences of the addition of a hydrotropic salt in the post-micellar region. The modulated photophysical properties of the emissive probe within the biomimetic environment have been spectroscopically monitored to gain an insight into the binding interaction. The moderate strength of the binding phenomenon as revealed by Benesi–Hildebrand analysis, as well as the hindrance exerted by the binding process towards the aggregation of the surfactants, paves the way to rationalize the observation of the exclusion of the drug to the bulk after micelle formation to be an outcome of a free energy advantage in the thermodynamically favorable micellization process. A substantial increase in the bulk viscosity of the medium in the presence of the hydrotrope in the post-micellar region coupled with a non-covalent binding between the alkane chains of the salt and the hydrophobic drug has also been unveiled from steady state and time-resolved fluorimetric perspectives.

Indanyloxyacetic acid-94 (IAA-94), an intracellular chloride channel blocker, is shown to ablate cardioprotection rendered by ischemic preconditioning (IPC), N (6)-2-(4-aminophenyl) ethyladenosine or the PKC activator phorbol 12-myristate 13-acetate and cyclosporin A (CsA) in both ex-vivo and in-vivo ischemia-reperfusion (IR) injury. Thus signifying the role of the IAA-94 sensitive chloride channels in mediating cardio-protection upon IR injury. Although IAA-94 sensitive chloride currents are recorded in cardiac mitoplast, there is still a lack of understanding of the mechanism by which IAA-94 increases myocardial infarction (MI) by IR injury. Mitochondria are the key arbitrators of cell life and death pathways. Both oxidative stress and calcium overload in the mitochondria, elicit pathways resulting in the opening of mitochondrial permeability transition pore (mPTP) leading to cell death. Therefore, in this study we explored the role of IAA-94 in MI and in maintaining calcium retention capacity (CRC) of cardiac mitochondria after IR. IAA-94 inhibited the CRC of the isolated cardiac mitochondria in a concentration-dependent manner as measured spectrofluorimetrically using calcium green-5 N. Interestingly, IAA-94 did not change the mitochondrial membrane potential. Further, CsA a blocker of mPTP opening could not override the effect of IAA-94. We also showed for the first time that IAA-94 perfusion after ischemic event augments MI by reducing the CRC of mitochondria. To conclude, our results demonstrate that the mechanism of IAA-94 mediated cardio-deleterious effects is via modulating the mitochondria CRC, thereby playing a role in mPTP opening. These findings highlight new pharmacological targets, which can mediate cardioprotection from IR injury.

The purpose of this study was to investigate the effect of KCNQ (potassium channel, voltage-gated, KQT-like subfamily) openers in preventing myotonia caused by anthracene-9-carboxylic acid (9-AC, a chloride channel blocker). An animal model of myotonia can be elicited in murine skeletal muscle by 9-AC treatment. KCNQ openers, such as retigabine and flupirtine, can inhibit the increased twitch amplitude (0.1 Hz stimulation) and reduce the tetanic fade (20 Hz stimulations) observed in the presence of 9-AC. Furthermore, the prolonged twitch duration of skeletal muscle was also inhibited by retigabine or flupirtine. Lamotrigine (an anticonvulsant drug) has a lesser effect on the muscle twitch amplitude, tetanic fade, and prolonged twitch duration as compared with KCNQ openers. In experiments using intracellular recordings, retigabine and flupirtine clearly reduced the firing frequencies of repetitive action potentials induced by 9-AC. These data suggested that KCNQ openers prevent the myotonia induced by 9-AC, at least partly through enhancing potassium conductance in skeletal muscle. Taken together, these results indicate that KCNQ openers are potential alternative therapeutic agents for the treatment of myotonia.

Glioblastoma is the most common and aggressive primary malignant brain tumor. Temozolomide (TMZ), a chemotherapeutic agent combined with radiation therapy, is used as a standard treatment. The infiltrative nature of glioblastoma, however, interrupts effective treatment with TMZ and increases the tendency to relapse. Voltage-gated chloride channels have been identified as crucial regulators of glioma cell migration and invasion by mediating cell shape and volume change. Accordingly, chloride current inhibition by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), a chloride channel blocker, suppresses cell movement by diminishing the osmotic cell volume regulation. In this study, we developed a novel compound, TMZ conjugated with NPPB (TMZ-NPPB), as a potential anticancer drug. TMZ-NPPB blocked chloride currents in U373MG, a severely invasive human glioma cell line, and suppressed migration and invasion of U373MG cells. Moreover, TMZ-NPPB exhibited DNA modification activity similar to that of TMZ, and surprisingly showed remarkably enhanced cytotoxicity relative to TMZ by inducing apoptotic cell death via DNA damage. These findings indicate that TMZ-NPPB has a dual function in blocking both proliferation and migration of human glioma cells, thereby suggesting its potential to overcome challenges in current glioblastoma therapy.

Fipronil, a broad spectrum N-phenylpyrazole insecticide that inhibits GABAA-gated chloride channels, has been in use since the mid-1990s. A high affinity for insect compared to mammalian GABA receptors results in lower animal toxicity than other insecticides blocking this channel. To date, only two accidental cases of fipronil poisoning in humans have been published.We report seven patients with fipronil self-poisoning seen prospectively in Sri Lanka together with pharmacokinetics for four patients. Non-sustained generalized tonic-clonic seizures were seen in two patients (peak measured plasma fipronil concentrations 1600 and 3744 microg/L); both were managed with diazepam without complications. A patient with a peak measured plasma concentration of 1040 microg/L was asymptomatic throughout his stay. Plasma concentration was still high at discharge 3-4 days post-ingestion when the patients were well. Retrospective review of1000 pesticide poisoning deaths since 1995 found only one death from fipronil-based products. In contrast to the good outcome of the above cases, this patient required intubation and ventilation and had continuous fits despite therapy with barbiturates and benzodiazepines.Our experience with prospectively observed patients suggests that fipronil poisoning is characterized by vomiting, agitation, and seizures, and normally has a favorable outcome. Management should concentrate on supportive care and early treatment of seizures. However, further experience is needed to determine whether increased susceptibility to fipronil or larger doses can produce status epilepticus.

The Varroa mite is a primary driver behind periodical losses of honey bee colonies. These mites require honey bees for food and reproduction and, in turn, elicit physiological deficiencies and diseases that compromise colony health. Current acaricides for Varroa mite control, such as Apistan® (the pyrethroid tau-fluvalinate), CheckMite+® (the organophosphate coumaphos), and Apivar® (the formamidine amitraz) target the nervous system, can have adverse health effects on honey bees, and have limited effectiveness due to reported resistance issues. New target sites are needed to circumvent these obstacles in Varroa mite management, and voltage-gated chloride channels (VGCCs) are promising candidates due to their important role in the maintenance of nerve and muscle excitability in arthropod pests. Toxicological analysis of Varroa mites sensitive to tau-fluvalinate and coumaphos and Varroa mites with reduced sensitivity to these acaricides showed a significant increase in metabolic detoxification enzyme activities for the latter. Acetylcholinesterase activity in the Varroa mites exhibiting reduced mortality to coumaphos was significantly less sensitive to coumaphos-oxon compared to coumaphos-sensitive Varroa mites, which suggests target-site insensitivity to the acaricide. Voltage-gated chloride channel blocker DIDS had significantly greater field efficacy compared to Apistan® and CheckMite+® against Varroa mites from honey bee hives where tau-fluvalinate and coumaphos were observed to be ineffective, respectively. These data suggest that DIDS, and potentially other stilbene chemistries, might serve as candidates for continued field efficacy testing of alternative acaricides in apiaries where Apistan®- and CheckMite+® efficacy has been. reduced or lost for Varroa mites.

Chloride channels are known to be involved in the regulated volume decrease that occurs when the rat lens is exposed to hypotonic challenge. We now report that chloride channel blockage makes the rat lens gain water under isotonic conditions, suggesting that chloride and water fluxes may also play an important role under resting conditions. Histological comparison of hypotonically and isotonically swollen rat lenses revealed a significant difference: in the former, fibre cells were swollen from the periphery inwards, while in the latter, swollen fibre cells were confined to a discrete cortical zone which was located 150–200 µm from the lens surface with cells on either side of this zone appearing unaffected. This localised fibre cell swelling is remarkable because of its similarity to the situation in the diabetic rat lens.

The effects of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a potent anion transport blocker, on transmembrane action potentials (APs) and the sodium current (I[Na]) of guinea pig ventricular myocytes were examined by using conventional microelectrode and whole-cell patch-clamp recording techniques. In papillary muscle preparations, DIDS (> or =0.1 mM) suppressed the maximal upstroke velocity (.v[max]) of the AP without significant changes in other AP parameters. Extracellular application of DIDS on single cardiomyocytes isolated from the guinea pig ventricle markedly reduced the peak amplitude of the tetrodotoxin (TTX)-sensitive and voltage-activated sodium current. The concentration-dependent block of DIDS could be expressed by the Hill equation with a Hill coefficient of 0.97 and a dissociation constant of 0.15 mM at a holding potential of (VH) -120 mV. DIDS (0.1 mM) shifted the steady-state inactivation curve for I(Na) toward more negative potentials by 6.0 +/- 0.5 mV and the activation curve to more positive potentials by 5.0 +/- 1.0 mV, although the slope factors were unaffected. With repetitive depolarizing pulses from -120 mV, DIDS produced a use-dependent block on the I(Na). Recovery of I(Na) from inactivation was slowed (time constant = 245 ms, compared with 10 ms of control) in the presence of 0.1 mM DIDS. In the two-pulse experiments, DIDS produced two distinct phases of development of I(Na) block, the rapid phase (tau = 5 ms) caused by an open channel block, and the slower phase (tau = 382 ms) induced by an inactivated channel block. These results suggest that the Cl- transport blocker DIDS has a direct inhibitory effect on the cardiac sodium channel. DIDS-induced use dependence of I(Na) block may result from the interaction of the drug with sodium channels in both the open and inactivated channel states.

The effects of the anti-estrogen, anion channel blocker, tamoxifen on amino acid release from the ischemic rat cerebral cortex was investigated using a cortical cup technique. Tamoxifen (20 microM in artificial cerebrospinal fluid), applied topically, inhibited the ischemia-evoked efflux of aspartate, glutamate, taurine and phosphoethanolamine. Reductions in the ischemia-evoked levels of these amino acids suggest that tamoxifen may attenuate chloride-related osmotic cell swelling and the associated regulatory volume decrease (RVD) release of amino acids.

Background Tumor cells exfoliated during surgery for pancreatic cancer can cause peritoneal recurrence. Peritoneal lavage with distilled water has been performed during surgery, but there have been no systematic studies for its efficacy and no experimental data demonstrating the cytocidal effects of distilled water on pancreatic cancer cells. This study investigated the cytocidal effects of hypotonic shock and enhancement using chloride channel blocker in pancreatic cancer cells. Methods Three human pancreatic cancer cell lines, KP4-1, PK-1, and PK45-H, were exposed to distilled water, and the resultant morphological changes were observed under a differential interference contrast microscope connected to a high-speed video camera. Analysis of cell volume changes was performed using a high-resolution flow cytometer. To investigate the cytocidal effects of water, re-incubation of cells was performed after exposure to hypotonic solution. Additionally, the effects of 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a Cl − channel blocker, on cells during exposure to hypotonic solution were analyzed. Results Video recordings demonstrated that hypotonic shock induced cell swelling followed by cell rupture. Measurement of cell volume changes indicated that severe hypotonicity increased broken fragments of cancer cells within 5 min. Re-incubation experiments demonstrated the cytocidal effects of hypotonic shock. In all cell lines, treatment with NPPB increased cell volume by inhibiting regulatory volume decreases, which are observed during hypotonic shock, and enhanced the cytocidal effects of hypotonic solution. Conclusions These findings support the efficacy of peritoneal lavage with distilled water for pancreatic cancer and suggest that regulation of Cl − transport enhances the cytocidal effects of hypotonic shock.

Background Protection of pancreatic islets during isolation procedures is mandatory for successful islet transplantation. Chloride channel inhibition has been reported to prevent cell death induced by various stimuli. We examined the effects of the chloride channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt (DIDS) and extracellular Cl(-)-free conditions on islet isolation outcomes. Methods Experimental groups were created based on the collagenase solutions used for Wistar rat islet isolation: control group, Hanks' balanced salt solution; DIDS group, 200 μM DIDS; and Cl(-)-free group, sodium gluconate substituted for sodium chloride. We determined whether collagenase digestion induced the death of islet cells through Cl(-) influx into the cells. We then assessed islet yield and the viability of isolated islets. Results We observed an increase in intracellular Cl(-) concentration under collagenase digestion conditions using a Cl(-)-sensitive fluorescent dye and subsequent rupture of islet cells. Consequently, islet yields were significantly higher in the DIDS and Cl(-)-free groups than in the control group, and islet morphology of the former groups was preserved. Of streptozotocin-induced diabetic severe combined immunodeficiency mice transplanted with a marginal dose of islets, all seven mice in the DIDS group and six of the seven mice in the Cl(-)-free group became normoglycemic, compared with two of seven mice in the control group (control vs. DIDS, P=0.010; control vs. Cl(-) free, P=0.051). Conclusion Our results indicate that DIDS inhibition of Cl(-) influx into islets protects islets during digestion procedures, offering a new strategy for the improvement of islet isolation outcomes.

The voltage‐dependent chloride‐channel blocker anthracene‐9‐carboxylic acid (9AC) has been found to reduce the imaginary but not the real part of the mechanical impedance of the organ of Corti, suggesting that the effective stiffness of outer hair cells (OHCs) is reduced by 9AC. To examine whether 9AC interacts directly with the motor protein prestin to reduce the membrane component of the impedance, the patch‐clamp technique in whole‐cell configuration was used to measure the nonlinear capacitance (NLC) of isolated OHCs and, as control, prestin‐transfected human embryonic kidney 293 (HEK293) cells. Extracellular application of 9AC significantly reduced the NLC of both OHCs and HEK293 cells. Intracellular 9AC did not influence the blocking effect of the extracellular applied drug. These results suggest that 9AC interacts directly with prestin, reducing the effective stiffness of the motor, and that the interaction is extracellular.

Background and Purpose Calcium‐activated chloride channels (CaCCs) play varied physiological roles and constitute potential therapeutic targets for conditions such as asthma and hypertension. TMEM16A encodes a CaCC. CaCC pharmacology is restricted to compounds with relatively low potency and poorly defined selectivity. Anthracene‐9‐carboxylic acid (A9C), an inhibitor of various chloride channel types, exhibits complex effects on native CaCCs and cloned TMEM16A channels providing both activation and inhibition. The mechanisms underlying these effects are not fully defined. Experimental Approach Patch‐clamp electrophysiology in conjunction with concentration jump experiments was employed to define the mode of interaction of A9C with TMEM16A channels. Key Results In the presence of high intracellular Ca2+, A9C inhibited TMEM16A currents in a voltage‐dependent manner by entering the channel from the outside. A9C activation, revealed in the presence of submaximal intracellular Ca2+ concentrations, was also voltage‐dependent. The electric distance of A9C inhibiting and activating binding site was ~0.6 in each case. Inhibition occurred according to an open‐channel block mechanism. Activation was due to a dramatic leftward shift in the steady‐state activation curve and slowed deactivation kinetics. Extracellular A9C competed with extracellular Cl−, suggesting that A9C binds deep in the channel's pore to exert both inhibiting and activating effects. Conclusions and Implications A9C is an open TMEM16A channel blocker and gating modifier. These effects require A9C to bind to a region within the pore that is accessible from the extracellular side of the membrane. These data will aid the future drug design of compounds that selectively activate or inhibit TMEM16A channels.

4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) has been used as an inhibitor of anion transporters and channels since the early 1970s. A study in this issue shows that DIDS hydrolyzes in aqueous solution and then multimerizes to di-, tri-, tetra-, and pentameric polythioureas, which inhibit both the bacterial ClC-ec1 Cl(-)/H(+) exchanger and the mammalian ClC-Ka chloride channel 3-200 times more potently than DIDS itself. The DIDS tetra- and pentamer could potentially act as tethered blockers that simultaneously obstruct both chloride pathways in the dimeric CLC proteins.

We have previously shown that expression of a Ca2+-activated Cl- channel (mCLCA3 in mice and bCLCA1 in humans) is up-regulated along with goblet cell metaplasia and mucus overproduction in the lungs of interleukin 9 (IL9) transgenic mice, and in human primary lung cultures by IL4, IL13 and IL9. We show here that hCLCA1 expression in NCI-H292 cells specifically induces soluble gel-forming mucin production. Moreover, niflumic acid (NFA), a blocker of hCLCA1-dependent Cl- efflux, inhibits MUC5A/C production in these cells. NFA treatment during natural antigen-exposure, where mCLCA3 is greatly up-regulated in the lung, significantly reduces airway inflammation, goblet cell metaplasia and mucus overproduction in vivo. These data suggest that this Ca2+-activated Cl- channel plays an important role in epithelial-regulated inflammatory responses, including goblet cell metaplasia, and represents a potential novel therapeutic target for the control of mucus overproduction in chronic pulmonary disorders.

Chloride channels belong to diverse group of anion selective channels involved in different signaling processes. The present study was planned to investigate the involvement of chloride channels in crush injury-induced neuropathic pain in rats by using ivermectin, a ligand gated chloride channel opener and NPPB, a CaCC blocker. The effect of ivermectin (5, 10, 20 mg/kg i.p. or 50, 100 and 200 microg/rat by i.c.v. route) and NPPB (10, 20 and 40 mg/kg i.p.) was investigated on pain behavioural thresholds in crush injury-induced neuropathic pain rat model. Reduction in pain threshold by mechanical, thermal and cold stimuli confirmed the development of neuropathic pain in rats after crush injury. Ivermectin administered either by i.p. or i.c.v. route did not alter the pain threshold in mechanical, thermal and, cold allodynia tests in rats. NPPB (20 and 40 mg/kg i.p.) significantly reduced the pain threshold crush injury neuropathic pain model suggesting its hyperalgesic effect. The results showed that NPPB increased significantly the mechanical and thermal hyperalgesia in crush injury-induced neuropathic pain rat model, whereas ivermectin, either by i.p. or i.c.v. route of administration, has no effect on pain symptoms in this model. NPPB hyperalgesic effect is independent of CaCCs inhibition and may be due to blockade of Ca2+-activated K+ channel.

The present work demonstrates a detailed characterization of the interaction of a potential chloride channel blocker, 9-methyl anthroate (9-MA), with a model transport protein, Bovine Serum Albumin (BSA). The modulated photophysical properties of the emissive drug molecule within the microheterogeneous bio-environment of the protein have been exploited spectroscopically to monitor the probe-protein binding interaction. Apart from evaluating the binding constant, the probable location of the neutral molecule within the protein cavity (subdomain IB) is explored by an AutoDock-based blind docking simulation. The absence of the Red-Edge Effect has been corroborated by the enhanced lifetime of the probe, being substantially greater than the solvent reorientation time. A dip-and-rise characteristic of the rotational relaxation profile of the drug within the protein has been argued to originate from a significant difference in the lifetime as well as amplitude of the free and protein-bound drug molecule. Unfolding of the protein in the presence of the drug molecule has been probed by the decrease of the α-helical content, obtained via circular dichroism (CD) spectroscopy, which is also supported by the gradual loss of the esterase activity of the protein in the presence of the drug molecule.

A key mechanism for loading acetylcholine (ACh + ) into synaptic vesicles uses energy to transport H + into the vesicle interior and then exchanges H + for ACh + . This mechanism requires anions to follow the H + into the vesicles to prevent the building up of an overwhelming electrical gradient across the vesicle membrane. Frog nerve-muscle preparations were treated with hypertonic solution in which sodium gluconate was the major constituent, which substantially increases the sizes of the quanta by increasing their ACh + content. The Cl − channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) antagonized the increases in quantal size, so it seems likely that Cl − follows H + to prevent the buildup of a potential gradient across the vesicular membrane.