Oxidized glutathione (GSSG) inhibits epithelial sodium channel activity in primary alveolar epithelial cells.

Amiloride-sensitive epithelial Na(+) channels (ENaC) regulate fluid balance in the alveoli and are regulated by oxidative stress. Since glutathione (GSH) is the predominant antioxidant in the lungs, we proposed that changes in glutathione redox potential (Eh) would alter cell signaling and have an effect on ENaC open probability (Po). In the present study, we used single channel patch-clamp recordings to examine the effect of oxidative stress, via direct application of glutathione disulfide (GSSG), on ENaC activity. We found a linear decrease in ENaC activity as the GSH/GSSG Eh became less negative (n = 21; P < 0.05). Treatment of 400 µM GSSG to the cell bath significantly decreased ENaC Po from 0.39 ± 0.06 to 0.13 ± 0.05 (n = 8; P < 0.05). Likewise, back-filling recording electrodes with 400 µM GSSG reduced ENaC Po from 0.32 ± 0.08 to 0.17 ± 0.05 (n = 10; P < 0.05), thus implicating GSSG as an important regulatory factor. Biochemical assays indicated that oxidizing potentials promote S-glutathionylation of ENaC and irreversible oxidation of cysteine residues with N-ethylmaleimide blocked the effects of GSSG on ENaC Po. Additionally, real-time imaging studies showed that GSSG impairs alveolar fluid clearance in vivo as opposed to GSH, which did not impair clearance. Taken together, these data show that glutathione Eh is an important determinant of alveolar fluid clearance in vivo.

Receptor for advanced glycation end-products regulates lung fluid balance via protein kinase C-gp91(phox) signaling to epithelial sodium channels.

The receptor for advanced glycation end-products (RAGE), a multiligand member of the Ig family, may play a crucial role in the regulation of lung fluid balance. We quantified soluble RAGE (sRAGE), a decoy isoform, and advanced glycation end-products (AGEs) from the bronchoalveolar lavage fluid of smokers and nonsmokers, and tested the hypothesis that AGEs regulate lung fluid balance through protein kinase C (PKC)-gp91(phox) signaling to the epithelial sodium channel (ENaC). Human bronchoalveolar lavage samples from smokers showed increased AGEs (9.02 ± 3.03 µg versus 2.48 ± 0.53 µg), lower sRAGE (1,205 ± 292 pg/ml versus 1,910 ± 263 pg/ml), and lower volume(s) of epithelial lining fluid (97 ± 14 ml versus 133 ± 17 ml). sRAGE levels did not predict ELF volumes in nonsmokers; however, in smokers, higher volumes of ELF were predicted with higher levels of sRAGE. Single-channel patch clamp analysis of rat alveolar epithelial type 1 cells showed that AGEs increased ENaC activity measured as the product of the number of channels (N) and the open probability (Po) (NPo) from 0.19 ± 0.08 to 0.83 ± 0.22 (P = 0.017) and the subsequent addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.15 ± 0.07 (P = 0.01). In type 2 cells, human AGEs increased ENaC NPo from 0.12 ± 0.05 to 0.53 ± 0.16 (P = 0.025) and the addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.10 ± 0.03 (P = 0.013). Using molecular and biochemical techniques, we observed that inhibition of RAGE and PKC activity attenuated AGE-induced activation of ENaC. AGEs induced phosphorylation of p47(phox) and increased gp91(phox)-dependent reactive oxygen species production, a response that was abrogated with RAGE or PKC inhibition. Finally, tracheal instillation of AGEs promoted clearance of lung fluid, whereas concomitant inhibition of RAGE, PKC, and gp91(phox) abrogated the response.

Acute effects of cigarette smoke extract on alveolar epithelial sodium channel activity and lung fluid clearance.

Cigarette smoke contains high levels of reactive species. Moreover, cigarette smoke can induce cellular production of oxidants. The purpose of this study was to determine the effect of cigarette smoke extract (CSE)-derived oxidants on epithelial sodium channel (ENaC) activity in alveolar type 1 (T1) and type 2 (T2) cells and to measure corresponding rates of fluid clearance in mice receiving a tracheal instillation of CSE. Single-channel patch clamp analysis of T1 and T2 cells demonstrate that CSE exposure increases ENaC activity (NPo), measured as the product of the number of channels (N) and a channels open probability (Po), from 0.17 ± 0.07 to 0.34 ± 0.10 (n = 9; P = 0.04) in T1 cells. In T2 cells, CSE increased NPo from 0.08 ± 0.03 to 0.35 ± 0.10 (n = 9; P = 0.02). In both cell types, addition of tetramethylpiperidine and glutathione attenuated CSE-induced increases in ENaC NPo. Biotinylation and cycloheximide chase assays indicate that CSE-derived ROS increases channel activity, in part, by maintaining cell surface expression of the α-ENaC subunit. In vivo studies show that tracheal instillation of CSE promoted alveolar fluid clearance after 105 minutes compared with vehicle control (n = 10/group; P < 0.05).

Ethanol alters alveolar fluid balance via Nadph oxidase (NOX) signaling to epithelial sodium channels (ENaC) in the lung.

Chronic alcohol consumption is associated with increased incidence of ICU-related morbidity and mortality, primarily from acute respiratory distress syndrome (ARDS). However, the mechanisms involved are unknown. One explanation is that alcohol regulates epithelial sodium channels (ENaC) via oxidant signaling to promote a pro- injury environment. We used small rodent models to mimic acute and chronic alcohol consumption and tested the hypothesis that ethanol (EtOH) would affect lung fluid clearance by up-regulating ENaC activity in the lung. Fluorescence labeling of rat lung slices and in vivo mouse lung revealed an increase in ROS production in response to acute EtOH exposure. Using western blots and fluorescein-5-maleimide labeling, we conclude that EtOH exposure modifies cysteines of α-ENaC while data from single channel patch clamp analysis confirm that 0.16% EtOH increased ENaC activity in rat alveolar cells. In vivo lung fluid clearance demonstrated a latent increase in fluid clearance in mice receiving EtOH diet. Ethanol mice given a tracheal instillation of LPS demonstrated early lung fluid clearance compared to caloric control mice and C57Bl/6 mice. Standard biochemical techniques reveal that chronic EtOH consumption resulted in greater protein expression of the catalytic gp91(phox) subunit and the obligate Rac1 protein. Collectively these data suggest that chronic EtOH consumption may lead to altered regulation of ENaC, contributing to a 'pro-injury' environment in the alcohol lung.

H2O2 regulates lung epithelial sodium channel (ENaC) via ubiquitin-like protein Nedd8.

Redundancies in both the ubiquitin and epithelial sodium transport pathways allude to their importance of proteolytic degradation and ion transport in maintaining normal cell function. The classical pathway implicated in ubiquitination of the epithelial sodium channel (ENaC) involves Nedd4-2 regulation of sodium channel subunit expression and has been studied extensively studied. However, less attention has been given to the role of the ubiquitin-like protein Nedd8. Here we show that Nedd8 plays an important role in the ubiquitination of ENaC in alveolar epithelial cells. We report that the Nedd8 pathway is redox-sensitive and that under oxidizing conditions Nedd8 conjugation to Cullin-1 is attenuated, resulting in greater surface expression of α-ENaC. This observation was confirmed in our electrophysiology studies in which we inhibited Nedd8-activating enzyme using MLN4924 (a specific Nedd8-activating enzyme inhibitor) and observed a marked increase in ENaC activity (measured as the product of the number of channels (N) and the open probability (Po) of a channel). These results suggest that ubiquitination of lung ENaC is redox-sensitive and may have significant implications for our understanding of the role of ENaC in pulmonary conditions where oxidative stress occurs, such as pulmonary edema and acute lung injury.

Calretinin regulates Ca2+-dependent inactivation and facilitation of Ca(v)2.1 Ca2+ channels through a direct interaction with the α12.1 subunit.

Voltage-gated Ca(v)2.1 Ca(2+) channels undergo dual modulation by Ca(2+), Ca(2+)-dependent inactivation (CDI), and Ca(2+)-dependent facilitation (CDF), which can influence synaptic plasticity in the nervous system. Although the molecular determinants controlling CDI and CDF have been the focus of intense research, little is known about the factors regulating these processes in neurons. Here, we show that calretinin (CR), a Ca(2+)-binding protein highly expressed in subpopulations of neurons in the brain, inhibits CDI and enhances CDF by binding directly to α(1)2.1. Screening of a phage display library with CR as bait revealed a highly basic CR-binding domain (CRB) present in multiple copies in the cytoplasmic linker between domains II and III of α(1)2.1. In pulldown assays, CR binding to fusion proteins containing these CRBs was largely Ca(2+)-dependent. α(1)2.1 coimmunoprecipitated with CR antibodies from transfected cells and mouse cerebellum, which confirmed the existence of CR-Ca(v)2.1 complexes in vitro and in vivo. In HEK293T cells, CR significantly decreased Ca(v)2.1 CDI and increased CDF. CR binding to α(1)2.1 was required for these effects, because they were not observed upon substitution of the II-III linker of α(1)2.1 with that from the Ca(v)1.2 α(1) subunit (α(1)1.2), which lacks the CRBs. In addition, coexpression of a protein containing the CRBs blocked the modulatory action of CR, most likely by competing with CR for interactions with α(1)2.1. Our findings highlight an unexpected role for CR in directly modulating effectors such as Ca(v)2.1, which may have major consequences for Ca(2+) signaling and neuronal excitability.

Compensatory regulation of Cav2.1 Ca2+ channels in cerebellar Purkinje neurons lacking parvalbumin and calbindin D-28k.

Ca(v)2.1 channels regulate Ca(2+) signaling and excitability of cerebellar Purkinje neurons. These channels undergo a dual feedback regulation by incoming Ca(2+) ions, Ca(2+)-dependent facilitation and inactivation. Endogenous Ca(2+)-buffering proteins, such as parvalbumin (PV) and calbindin D-28k (CB), are highly expressed in Purkinje neurons and therefore may influence Ca(v)2.1 regulation by Ca(2+). To test this, we compared Ca(v)2.1 properties in dissociated Purkinje neurons from wild-type (WT) mice and those lacking both PV and CB (PV/CB(-/-)). Unexpectedly, P-type currents in WT and PV/CB(-/-) neurons differed in a way that was inconsistent with a role of PV and CB in acute modulation of Ca(2+) feedback to Ca(v)2.1. Ca(v)2.1 currents in PV/CB(-/-) neurons exhibited increased voltage-dependent inactivation, which could be traced to decreased expression of the auxiliary Ca(v)beta(2a) subunit compared with WT neurons. Although Ca(v)2.1 channels are required for normal pacemaking of Purkinje neurons, spontaneous action potentials were not different in WT and PV/CB(-/-) neurons. Increased inactivation due to molecular switching of Ca(v)2.1 beta-subunits may preserve normal activity-dependent Ca(2+) signals in the absence of Ca(2+)-buffering proteins in PV/CB(-/-) Purkinje neurons.

Endogenous and exogenous Ca2+ buffers differentially modulate Ca2+-dependent inactivation of Ca(v)2.1 Ca2+ channels.

Voltage-gated Ca2+ channels undergo a negative feedback regulation by Ca2+ ions, Ca2+-dependent inactivation, which is important for restricting Ca2+ signals in nerve and muscle. Although the molecular details underlying Ca2+-dependent inactivation have been characterized, little is known about how this process might be modulated in excitable cells. Based on previous findings that Ca2+-dependent inactivation of Ca(v)2.1 (P/Q-type) Ca2+ channels is suppressed by strong cytoplasmic Ca2+ buffering, we investigated how factors that regulate cellular Ca2+ levels affect inactivation of Ca(v)2.1 Ca2+ currents in transfected 293T cells. We found that inactivation of Ca(v)2.1 Ca2+ currents increased exponentially with current amplitude with low intracellular concentrations of the slow buffer EGTA (0.5 mm), but not with high concentrations of the fast Ca2+ buffer BAPTA (10 mm). However, when the concentration of BAPTA was reduced to 0.5 mm, inactivation of Ca2+ currents was significantly greater than with an equivalent concentration of EGTA, indicating the importance of buffer kinetics in modulating Ca2+-dependent inactivation of Ca(v)2.1. Cotransfection of Ca(v)2.1 with the EF-hand Ca2+-binding proteins, parvalbumin and calbindin, significantly altered the relationship between Ca2+ current amplitude and inactivation in ways that were unexpected from behavior as passive Ca2+ buffers. We conclude that Ca2+-dependent inactivation of Ca(v)2.1 depends on a subplasmalemmal Ca2+ microdomain that is affected by the amplitude of the Ca2+ current and differentially modulated by distinct Ca2+ buffers.

Synaptic shunting by a baseline of synaptic conductances modulates responses to inhibitory input volleys in cerebellar Purkinje cells.

When processing synaptic input in vivo, large neurons in the brain must cope with thousands of events each second. Much work has focused on the specific processing of synchronous excitatory input volleys, both in cerebellar and cerebral cortical research. Here we pursue the question of how a continuous background of ongoing 'noise' inputs interacts with the processing of synchronous inhibitory input volleys. Specifically we examine the processing of inhibitory input transients in cerebellar Purkinje cells, which by inducing pauses in Purkinje cell spike activity may lead to a disinhibition of the deep cerebellar nuclei and thus to cerebellar motor command signals. We use the technique of dynamic clamping in vitro to simulate controlled patterns of in vivo like background inputs. We use electrical stimulation of inhibitory interneurons in the deep or upper molecular layer to create inhibitory input transients that lead to spike pauses in Purkinje cell activity. These pauses were much longer in the absence than in the presence of background inputs applied with dynamic clamping. We found that a significant amount of the synaptic current elicited by electrical stimulation was shunted by the background inputs. The overall amount of background conductance as well as the pattern of background inputs modulated spike pause duration in a specific manner. This modulation by shunting may be employed in vivo to evaluate the salience of specific sensory input received by cerebellar cortex.