NMDA receptor-BK channel coupling regulates synaptic plasticity in the barrel cortex.

Postsynaptic N-methyl-D-aspartate receptors (NMDARs) are crucial mediators of synaptic plasticity due to their ability to act as coincidence detectors of presynaptic and postsynaptic neuronal activity. However, NMDARs exist within the molecular context of a variety of postsynaptic signaling proteins, which can fine-tune their function. Here, we describe a form of NMDAR suppression by large-conductance Ca2+- and voltage-gated K+ (BK) channels in the basal dendrites of a subset of barrel cortex layer 5 pyramidal neurons. We show that NMDAR activation increases intracellular Ca2+ in the vicinity of BK channels, thus activating K+ efflux and strong negative feedback inhibition. We further show that neurons exhibiting such NMDAR-BK coupling serve as high-pass filters for incoming synaptic inputs, precluding the induction of spike timing-dependent plasticity. Together, these data suggest that NMDAR-localized BK channels regulate synaptic integration and provide input-specific synaptic diversity to a thalamocortical circuit.

Subunit-specific inhibition of BK channels by piperine.

Piperine is the principal alkaloid present in black pepper and is well-known for its diverse pharmacological effects, including inhibition of different ion channels. Large conductance Ca2+-activated K+ channels (BK) are widely expressed across several tissues and play a vital role in many physiological functions. In this study, we investigated the pharmacological effects of piperine on various BK channel subunit compositions (BKα, BKαß1,4, BKαγ1,3) expressed in HEK293T cells. Piperine in zero Ca2+ reversibly inhibited currents from the pore-forming BKα channels in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) of 4.8 µM. Elevating the internal Ca2+ concentration from 0 to 100 µM significantly attenuated the inhibitory effects of piperine on BKα channels. The mutation G311S in the pore domain failed to alter the modulatory effects of piperine, whereas deletion of the entire cytoplasmic domain from BKα channels ablated its inhibitory effects. Addition of either BKß1 or ß4 regulatory subunits did not alter the efficacy of piperine on BKα channels. Interestingly, co-expression of either BKγ1 or BKγ3 subunits greatly diminished the ability of piperine to inhibit BKα channels. Our findings demonstrate that piperine is a potent natural modulator of BKα/BKαß1,4 subunits but not BKαγ1,3 subunits. The mechanism of piperine modulation appeared to be allosteric and differs from that of other BK pore blockers (paxilline, peptide toxins, and quaternary ammonium compounds). Together, our results unravel the potential of piperine to inhibit BK channels, providing a new tool to explore mechanisms underlying the effects of regulatory subunits.

Effects of Chronic Kidney Disease on Nanomechanics of the Endothelial Glycocalyx Are Mediated by the Mineralocorticoid Receptor.

Endothelial mechanics control vascular reactivity and are regulated by the mineralocorticoid receptor (MR) and its downstream target, the epithelial Na+ channel (ENaC). Endothelial dysfunction is a hallmark of chronic kidney disease (CKD), but its mechanisms are poorly understood. We hypothesized that CKD disrupts endothelial mechanics in an MR/ENaC-dependent process. METHODS: Primary human endothelial cells were cultured with uremic serum derived from children with stage 3-5 (predialysis) CKD or adult hemodialysis (HD) patients or healthy controls. The height and stiffness of the endothelial glycocalyx (eGC) and cortex were monitored by atomic force microscopy (AFM) using an ultrasensitive mechanical nanosensor. RESULTS: In a stage-dependent manner, sera from children with CKD induced a significant increase in eGC and cortex stiffness and an incremental reduction of the eGC height. AFM measurements were significantly associated with individual pulse wave velocity and serum concentrations of gut-derived uremic toxins. Serum from HD patients increased MR expression and mechanical stiffness of the endothelial cortex, an effect reversed by MR and ENaC antagonists, decreased eNOS expression and NO bioavailability, and augmented monocyte adhesion. CONCLUSION: These data indicate progressive structural damage of the endothelial surface with diminishing kidney function and identify the MR as a mediator of CKD-induced endothelial dysfunction.

SGK1.1 limits brain damage after status epilepticus through M current-dependent and independent mechanisms.

Epilepsy is a neurological condition associated to significant brain damage produced by status epilepticus (SE) including neurodegeneration, gliosis and ectopic neurogenesis. Reduction of these processes constitutes a useful strategy to improve recovery and ameliorate negative outcomes after an initial insult. SGK1.1, the neuronal isoform of the serum and glucocorticoids-regulated kinase 1 (SGK1), has been shown to increase M-current density in neurons, leading to reduced excitability and protection against seizures. For this study, we used 4-5 months old male transgenic C57BL/6 J and FVB/NJ mice expressing near physiological levels of a constitutively active form of the kinase controlled by its endogenous promoter. Here we show that SGK1.1 activation potently reduces levels of neuronal death (assessed using Fluoro-Jade C staining) and reactive glial activation (reported by GFAP and Iba-1 markers) in limbic regions and cortex, 72 h after SE induced by kainate, even in the context of high seizure activity. This neuroprotective effect is not exclusively through M-current activation but is also directly linked to decreased apoptosis levels assessed by TUNEL assays and quantification of Bim and Bcl-xL by western blot of hippocampal protein extracts. Our results demonstrate that this newly described antiapoptotic role of SGK1.1 activation acts synergistically with the regulation of cellular excitability, resulting in a significant reduction of SE-induced brain damage in areas relevant to epileptogenesis.

SGK1.1 Reduces Kainic Acid-Induced Seizure Severity and Leads to Rapid Termination of Seizures.

Approaches to control epilepsy, one of the most important idiopathic brain disorders, are of great importance for public health. We have previously shown that in sympathetic neurons the neuronal isoform of the serum and glucocorticoid-regulated kinase (SGK1.1) increases the M-current, a well-known target for seizure control. The effect of SGK1.1 activation on kainate-induced seizures and neuronal excitability was studied in transgenic mice that express a permanently active form of the kinase, using electroencephalogram recordings and electrophysiological measurements in hippocampal brain slices. Our results demonstrate that SGK1.1 activation leads to reduced seizure severity and lower mortality rates following status epilepticus, in an M-current-dependent manner. EEG is characterized by reduced number, shorter duration, and early termination of kainate-induced seizures in the hippocampus and cortex. Hippocampal neurons show decreased excitability associated to increased M-current, without altering basal synaptic transmission or other neuronal properties. Altogether, our results reveal a novel and selective anticonvulsant pathway that promptly terminates seizures, suggesting that SGK1.1 activation can be a potent factor to secure the brain against permanent neuronal damage associated to epilepsy.

Interactions of divalent cations with calcium binding sites of BK channels reveal independent motions within the gating ring.

Large-conductance voltage- and calcium-activated K+ (BK) channels are key physiological players in muscle, nerve, and endocrine function by integrating intracellular Ca2+ and membrane voltage signals. The open probability of BK channels is regulated by the intracellular concentration of divalent cations sensed by a large structure in the BK channel called the "gating ring," which is formed by four tandems of regulator of conductance for K+ (RCK1 and RCK2) domains. In contrast to Ca2+ that binds to both RCK domains, Mg2+, Cd2+, or Ba2+ interact preferentially with either one or the other. Interaction of cations with their binding sites causes molecular rearrangements of the gating ring, but how these motions occur remains elusive. We have assessed the separate contributions of each RCK domain to the cation-induced gating-ring structural rearrangements, using patch-clamp fluorometry. Here we show that Mg2+ and Ba2+ selectively induce structural movement of the RCK2 domain, whereas Cd2+ causes motions of RCK1, in all cases substantially smaller than those elicited by Ca2+ By combining divalent species interacting with unique sites, we demonstrate that RCK1 and RCK2 domains move independently when their specific binding sites are occupied. Moreover, binding of chemically distinct cations to both RCK domains is additive, emulating the effect of fully occupied Ca2+ binding sites.

Functional validation of Ca2+-binding residues from the crystal structure of the BK ion channel.

BK channels are dually regulated by voltage and Ca2+, providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca2+ concentration is sensed by a large cytoplasmic region in the channel known as "gating ring", which is formed by four tandems of regulator of conductance for K+ (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca2+ coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca2+. Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca2+ in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca2+ sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca2+ coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca2+ binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca2+ binding sites regulating the Ca2+ dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Plasma membrane insertion of epithelial sodium channels occurs with dual kinetics.

The epithelial sodium channel (ENaC) constitutes the rate-limiting step for Na(+) transport across electrically tight epithelia. Regulation of ENaC activity is critical for electrolyte and extracellular volume homeostasis, as well as for lung liquid clearance and colon Na(+) handling. ENaC activity is tightly controlled by a combination of mechanisms involving changes in open probability and plasma membrane abundance. The latter reflects a combination in channel biosynthesis and trafficking to and from the membrane. Studying ENaC trafficking with different techniques in a variety of expression systems has yielded inconsistent results, indicating either fast or slow rates of insertion and retrieval, which range from the order of minutes to several hours. Here, we use Xenopus oocytes as ENaC expression system to study channel insertion rate in the membrane using two different techniques under comparable conditions: (1) confocal microscopy coupled to fluorescence recovery after photobleaching (FRAP) measurements; and (2) fluorescent bungarotoxin (BTX) binding to ENaC subunits modified to include BTX binding sites (BBSs) in their extracellular domain, a technique that has not been previously used to study ENaC trafficking. Our confocal-FRAP data indicate a fast rate of ENaC incorporation to the membrane in a process conditioned by channel subunit composition. On the other hand, BTX binding experiments indicate much slower channel insertion rates, with matching slow ENaC retrieval rates. The data support a model that includes fast recycling of endocytosed ENaC with parallel incorporation of newly synthesized channels at a slower rate.

State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels.

Large-conductance voltage- and calcium-dependent potassium channels (BK, "Big K+") are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a "gating-ring" structure has been proposed to transduce Ca(2+) binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductance. Fluorescence resonance energy transfer (FRET) between fluorescent protein inserts indicates that Ca(2+) binding produces structural changes of the gating ring that are much larger than those predicted by current X-ray crystal structures of isolated gating rings.

Molecular components of nitrate and nitrite efflux in yeast.

Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast Hansenula polymorpha was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either SSU2 or NAR1 along with the nitrate reductase gene YNR1 showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known Saccharomyces cerevisiae sulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation.

The neuronal serum- and glucocorticoid-regulated kinase 1.1 reduces neuronal excitability and protects against seizures through upregulation of the M-current.

The M-current formed by tetramerization of Kv7.2 and Kv7.3 subunits is a neuronal voltage-gated K(+) conductance that controls resting membrane potential and cell excitability. In Xenopus laevis oocytes, an increase in Kv7.2/3 function by the serum- and glucocorticoid-regulated kinase 1 (SGK1) has been reported previously (Schuetz et al., 2008). We now show that the neuronal isoform of this kinase (SGK1.1), with distinct subcellular localization and modulation, upregulates the Kv7.2/3 current in Xenopus oocytes and mammalian human embryonic kidney HEK293 cells. In contrast to the ubiquitously expressed SGK1, the neuronal isoform SGK1.1 interacts with phosphoinositide-phosphatidylinositol 4,5-bisphosphate (PIP(2)) and is distinctly localized to the plasma membrane (Arteaga et al., 2008). An SGK1.1 mutant with disrupted PIP(2) binding sites produced no effect on Kv7.2/3 current amplitude. SGK1.1 failed to modify the voltage dependence of activation and did not change activation or deactivation kinetics of Kv7.2/3 channels. These results suggest that the kinase increases channel membrane abundance, which was confirmed with flow cytometry assays. To evaluate the effect of the kinase in neuronal excitability, we generated a transgenic mouse (Tg.sgk) expressing a constitutively active form of SGK1.1 (S515D). Superior cervical ganglion (SCG) neurons isolated from Tg.sgk mice showed a significant increase in M-current levels, paralleled by reduced excitability and more negative resting potentials. SGK1.1 effect on M-current in Tg.sgk-SCG neurons was counteracted by muscarinic receptor activation. Transgenic mice with increased SGK1.1 activity also showed diminished sensitivity to kainic acid-induced seizures. Altogether, our results unveil a novel role of SGK1.1 as a physiological regulator of the M-current and neuronal excitability.

Heterogeneous nuclear ribonucleoprotein A2/B1 is a tissue-specific aldosterone target gene with prominent induction in the rat distal colon.

The steroid hormone aldosterone enhances transepithelial Na(+) reabsorption in tight epithelia and is crucial to achieve extracellular volume homeostasis and control of blood pressure. One of the main transport pathways regulated by aldosterone involves the epithelial Na(+) channel (ENaC), which constitutes the rate-limiting step of Na(+) reabsorption in parts of the distal nephron and the collecting duct, the distal colon, and sweat and salivary glands. Although these epithelial tissues share the same receptor for aldosterone (mineralocorticoid receptor, MR), and the same transport system (ENaC), it has become clear that the molecular mechanisms involved in the modulation of channel activity are tissue-specific. Recent evidence suggests that aldosterone controls transcription and also translation of ENaC subunits in some cell types. A possible pathway for translational regulation is binding of regulatory proteins to ENaC subunit mRNAs, such as the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). In this study, we examined whether hnRNP A2/B1 is an aldosterone-target gene in vivo. Our data show that physiological levels of aldosterone markedly induce hnRNP A2/B1 expression in an early and sustained manner in the late distal colon epithelium but not in other aldosterone-target tissues. The effect depends on MR but not on glucocorticoid receptor activity. We also demonstrate that the genomic region upstream of hnRNP A2/B1 contains aldosterone-responsive elements involved in the control of gene expression. We hypothesize that hnRNP A2/B1 is involved in the tissue-specific regulation of ENaC biosynthesis and may coordinate the response of other genes relevant for transepithelial Na(+) reabsorption by aldosterone.

Differential N termini in epithelial Na+ channel δ-subunit isoforms modulate channel trafficking to the membrane.

The epithelial Na(+) channel (ENaC) is a heteromultimeric ion channel that plays a key role in Na(+) reabsorption across tight epithelia. The canonical ENaC is formed by three analogous subunits, α, ß, and γ. A fourth ENaC subunit, named δ, is expressed in the nervous system of primates, where its role is unknown. The human δ-ENaC gene generates at least two splice isoforms, δ(1) and δ(2) , differing in the N-terminal sequence. Neurons in diverse areas of the human and monkey brain differentially express either δ(1) or δ(2) , with few cells coexpressing both isoforms, which suggests that they may play specific physiological roles. Here we show that heterologous expression of δ(1) in Xenopus oocytes and HEK293 cells produces higher current levels than δ(2) . Patch-clamp experiments showed no differences in single channel current magnitude and open probability between isoforms. Steady-state plasma membrane abundance accounts for the dissimilarity in macroscopic current levels. Differential trafficking between isoforms is independent of ß- and γ-subunits, PY-motif-mediated endocytosis, or the presence of additional lysine residues in δ(2)-N terminus. Analysis of δ(2)-N terminus identified two sequences that independently reduce channel abundance in the plasma membrane. The δ(1) higher abundance is consistent with an increased insertion rate into the membrane, since endocytosis rates of both isoforms are indistinguishable. Finally, we conclude that δ-ENaC undergoes dynamin-independent endocytosis as opposed to αßγ-channels.

The epithelial sodium channel δ-subunit: new notes for an old song.

Amiloride-sensitive epithelial Na(+) channels (ENaCs) can be formed by different combinations of four homologous subunits, named α, ß, γ, and δ. In addition to providing an apical entry pathway for transepithelial Na(+) reabsorption in tight epithelia such as the kidney distal tubule and collecting duct, ENaCs are also expressed in nonepithelial cells, where they may play different functional roles. The δ-subunit of ENaC was originally identified in humans and is able to form amiloride-sensitive Na(+) channels alone or in combination with ß and γ, generally resembling the canonical kidney ENaC formed by α, ß, and γ. However, δ differs from α in its tissue distribution and channel properties. Despite the low sequence conservation between α and δ (37% identity), their similar functional characteristics provide an excellent model for exploring structural correlates of specific ENaC biophysical and pharmacological properties. Moreover, the study of cellular mechanisms modulating the activity of different ENaC subunit combinations provides an opportunity to gain insight into the regulation of the channel. In this review, we examine the evolution of ENaC genes, channel subunit composition, the distinct functional and pharmacological features that δ confers to ENaC, and how this can be exploited to better understand this ion channel. Finally, we briefly consider possible functional roles of the ENaC δ-subunit.

Thiol-reactive compounds from garlic inhibit the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key factor in the transepithelial movement of sodium, and consequently salt and water homeostasis in various organs. Dysregulated activity of ENaC is associated with human diseases such as hypertension, the salt-wasting syndrome pseudohypoaldosteronism type 1, cystic fibrosis, pulmonary oedema or intestinal disorders. Therefore it is important to identify novel compounds that affect ENaC activity. This study investigated if garlic (Allium sativum) and its characteristic organosulfur compounds have impact on ENaCs. Human ENaCs were heterologously expressed in Xenopus oocytes and their activity was measured as transmembrane currents by the two-electrode voltage-clamp technique. The application of freshly prepared extract from 5g of fresh garlic (1% final concentration) decreased transmembrane currents of ENaC-expressing oocytes within 10 min. This effect was dose-dependent and irreversible. It was fully sensitive to the ENaC-inhibitor amiloride and was not apparent on native control oocytes. The effect of garlic was blocked by dithiothreitol and l-cysteine indicating involvement of thiol-reactive compounds. The garlic organosulsur compounds S-allylcysteine, alliin and diallyl sulfides had no effect on ENaC. By contrast, the thiol-reactive garlic compound allicin significantly inhibited ENaC to a similar extent as garlic extract. These data indicate that thiol-reactive compounds which are present in garlic inhibit ENaC.

SGK1.1 isoform is involved in nociceptive modulation, offering a protective effect against noxious cold stimulus in a sexually dimorphic manner.

The serum and glucocorticoid-regulated kinase 1 (SGK1) is a widely expressed protein in the Central Nervous System (CNS), involved in regulating the activity of a wide variety of ion channels and transporters and physiological functions, such as neuronal excitability. SGK1.1 is a neuronal splice isoform of SGK1, expressed exclusively in the CNS, distributed in brain and cerebellum, that decreases neuronal excitability via up-regulation of M-current, linked to Kv7.2/3 potassium channels. Strategies to maintain increased SGK1.1 activity could be helpful in decreasing neuronal hyperexcitability, as occurs in neuropathic pain. Transgenic mice overexpressing SGK1.1 (B6.Tg.sgk1) offer a particularly relevant opportunity to assess the physiological involvement of this protein in nociception. Behavior and physiological nociception were evaluated in male and female B6.Tg.sgk1 and wild-type mice (B6.WT), characterizing nociceptive thresholds to different nociceptive stimuli (thermal, chemical and mechanical), as well as the electrophysiological properties of cutaneous sensory Aδ-fibres isolated from the saphenous nerve. The acute antinociceptive effect of morphine was also evaluated. Compared with B6.WT animals, male and female B6.Tg.sgk1 mice showed increased spontaneous locomotor activity. Regarding nociception, there were no differences between transgenic and wild-type mice in heat, chemical and mechanical thresholds, but interestingly, male B6.Tg.sgk1 mice were less sensitive to cold stimulus; B6.Tg.sgk1 animals showed lower sensitivity to morphine. Electrophysiological properties of cutaneous primary afferent fibres were maintained. This is the first demonstration that the SGK1.1 isoform is involved in nociceptive modulation, offering a protective effect against noxious cold stimulus in a sexually dimorphic manner. B6.Tg.sgk1 mice offer a particularly relevant opportunity to further analyze the involvement of this protein in nociception, and studies in models of chronic, neuropathic pain are warranted.

Activation of SGK1.1 Upregulates the M-current in the Presence of Epilepsy Mutations.

In the central nervous system, the M-current plays a critical role in regulating subthreshold electrical excitability of neurons, determining their firing properties and responsiveness to synaptic input. The M-channel is mainly formed by subunits Kv7.2 and Kv7.3 that co-assemble to form a heterotetrametric channel. Mutations in Kv7.2 and Kv7.3 are associated with hyperexcitability phenotypes including benign familial neonatal epilepsy (BFNE) and neonatal epileptic encephalopathy (NEE). SGK1.1, the neuronal isoform of the serum and glucocorticoids-regulated kinase 1 (SGK1), increases M-current density in neurons, leading to reduced excitability and protection against seizures. Herein, using two-electrode voltage clamp on Xenopus laevis oocytes, we demonstrate that SGK1.1 selectively activates heteromeric Kv7 subunit combinations underlying the M-current. Importantly, activated SGK1.1 increases M-channel activity in the presence of two different epilepsy mutations found in Kv7.2, R207W and A306T. In addition, proximity ligation assays in the N2a cell line allowed us to address the effect of these mutations on Kv7-SGK1.1-Nedd4 molecular associations, a proposed pathway underlying augmentation of M-channel activity by SGK1.1.

Kv1.3 Channel Inhibition Limits Uremia-Induced Calcification in Mouse and Human Vascular Smooth Muscle.

Chronic kidney disease (CKD) significantly increases cardiovascular risk. In advanced CKD stages, accumulation of toxic circulating metabolites and mineral metabolism alterations triggers vascular calcification, characterized by vascular smooth muscle cell (VSMC) transdifferentiation and loss of the contractile phenotype. Phenotypic modulation of VSMC occurs with significant changes in gene expression. Even though ion channels are an integral component of VSMC function, the effects of uremia on ion channel remodeling has not been explored. We used an in vitro model of uremia-induced calcification of human aorta smooth muscle cells (HASMCs) to study the expression of 92 ion channel subunit genes. Uremic serum-induced extensive remodeling of ion channel expression consistent with loss of excitability but different from the one previously associated with transition from contractile to proliferative phenotypes. Among the ion channels tested, we found increased abundance and activity of voltage-dependent K+ channel Kv1.3. Enhanced Kv1.3 expression was also detected in aorta from a mouse model of CKD. Pharmacological inhibition or genetic ablation of Kv1.3 decreased the amount of calcium phosphate deposition induced by uremia, supporting an important role for this channel on uremia-induced VSMC calcification.

The neuronal-specific SGK1.1 kinase regulates {delta}-epithelial Na+ channel independently of PY motifs and couples it to phospholipase C signaling.

The δ-subunit of the epithelial Na(+) channel (ENaC) is expressed in neurons of the human and monkey central nervous system and forms voltage-independent, amiloride-sensitive Na(+) channels when expressed in heterologous systems. It has been proposed that δ-ENaC could affect neuronal excitability and participate in the transduction of ischemic signals during hypoxia or inflammation. The regulation of δ-ENaC activity is poorly understood. ENaC channels in kidney epithelial cells are regulated by the serum- and glucocorticoid-induced kinase 1 (SGK1). Recently, a new isoform of this kinase (SGK1.1) has been described in the central nervous system. Here we show that δ-ENaC isoforms and SGK1.1 are coexpressed in pyramidal neurons of the human and monkey (Macaca fascicularis) cerebral cortex. Coexpression of δßγ-ENaC and SGK1.1 in Xenopus oocytes increases amiloride-sensitive current and channel plasma membrane abundance. The kinase also exerts its effect when δ-subunits are expressed alone, indicating that the process is not dependent on accessory subunits or the presence of PY motifs in the channel. Furthermore, SGK1.1 action depends on its enzymatic activity and binding to phosphatidylinositol(4,5)-bisphosphate. Physiological or pharmacological activation of phospholipase C abrogates SGK1.1 interaction with the plasma membrane and modulation of δ-ENaC. Our data support a physiological role for SGK1.1 in the regulation of δ-ENaC through a pathway that differs from the classical one and suggest that the kinase could serve as an integrator of different signaling pathways converging on the channel.

The dopamine transporter is differentially regulated after dopaminergic lesion.

The dopamine transporter (DAT) is a transmembrane glycoprotein responsible for dopamine (DA) uptake, which has been shown to be involved in DA-cell degeneration in Parkinson's disease (PD). At the same time, some studies suggest that DAT may be regulated in response to dopaminergic injury. We have investigated the mechanisms underlying DAT regulation after different degrees of dopaminergic lesion. DAT is persistently down-regulated in surviving midbrain DA-neurons after substantial (62%) loss of striatal DA-terminals, and transiently after slight (11%) loss of DA-terminals in rats. Transient DAT down-regulation consisted of a decrease of glycosylated (mature) DAT in the plasma membrane with accumulation of non-glycosylated (immature) DAT in the endoplasmic reticulum-Golgi (ERG) compartment, and recovery of the normal expression pattern 5 days after lesion. DAT redistribution to the ERG was also observed in HEK cells expressing rat DAT exposed to MPP(+), but not after exposure to DAT-unrelated neurotoxins. In contrast to other midbrain DA-cells, those in the ventrolateral region of the substantia nigra do not regulate DAT and degenerate shortly after slight DA-lesion. These data suggest that DAT down-regulation is a post-translational event induced by DA-analogue toxins, consisting of a stop in its glycosylation and trafficking to the plasma membrane. Its persistence after substantial DA-lesion may act as a compensatory mechanism helping maintain striatal DA levels. The fact that neurons which do not regulate DAT die shortly after lesion suggests a relationship between DAT down-regulation and neuroprotection.