Phosphatases maintain low catalytic activity of SGK1: DNA damage resets the balance in favor of phosphorylation.

The serum- and glucocorticoid-induced kinase 1 (SGK1) promotes cell survival under stress conditions and facilitates the emergence of drug resistance in cancer. The underlying mechanisms of these observations are not fully understood. In this study, we found that SGK1 activity is suppressed by the action of the S/T phosphatases PP5 and PP2A, which constantly dephosphorylate SGK1. Using newly developed anti-phospho SGK1 antibodies and inhibitors of phosphatases, we determined that the high degree of dephosphorylation is caused by two factors: the tendency of SGK1 to unfold, which makes it dependent on Hsp90 chaperone complexes composed of four proteins, Hsp90/CDC37/PP5/SGK1, and where the phosphatase PP5 persistently dephosphorylates SGK1 within the complex. SGK1 binding to PP2A regulatory subunits B55γ and B55δ brings PP2A catalytic subunit close to exposed SGK1 phosphoresidues. A further association of phosphorylated pS37-FAM122A-an endogenous inhibitor of PP2A-to the holoenzyme diminishes dephosphorylation of SGK1 mediated by PP2A. Our study also reveals that genotoxic stress can reverse the dominant impact of phosphatases over kinases by activating the DNA-dependent protein kinase, which enhances mTORC2 activity directed to SGK1. Thus, our results provide insight into a molecular pathway that enables SGK1 to gain phosphorylation and catalytic activity and promote cell survival, potentially diminishing the efficacy of cancer treatments. As the DNA damage response operates in many cancer cells and is further induced by chemotherapies, the findings of this study could have significant implications for the development of novel cancer therapies targeting SGK1.

Lipid droplets and autophagosomes together with chaperones fine-tune expression of SGK1.

Serum-glucocorticoid-induced kinase-1 (SGK1) regulates ion homeostasis and promotes survival under stress conditions. The expression of SGK1 is under transcriptional and post-translational regulations that are frequently altered in cancer and immune disorders. We report that an N-terminal amphipathic alpha-helix determines SGK1 expression levels through two distinct mechanisms. It tethers SGK1 to intracellular organelles generating a large pool of membrane-bound SGK1, which is differentially stabilized in lipid droplets (LD) in fed conditions or degraded in the endoplasmic reticulum by ER-phagy in starvation. Association of the α-helix to organelles does not depend on dedicated receptors or special phospholipids rather, it is intrinsic to its physicochemical properties and depends on the presence of bulky hydrophobic residues for attachment to LDs. The second mechanism is recruitment of protein-chaperones that recognize the α-helix as an unfolded protein promoting survival of the cytosolic SGK1 fraction. Together, the findings unveil an unexpected link between levels of energy storage and abundance of SGK1 and how changes in calorie intake could be used to modulate SGK1 expression, whereas the inhibition of molecular chaperones could serve as an additional enhancer in the treatment of malignancies and autoimmune disorders with high levels of SGK1 expression.

A method for activation of endogenous acid-sensing ion channel 1a (ASIC1a) in the nervous system with high spatial and temporal precision.

Protons activate acid-sensing ion channel 1a (ASIC1a) in the central nervous system (CNS) although the impact of such activation on brain outputs remains elusive. Progress elucidating the functional roles of ASIC1a in the CNS has been hindered by technical difficulties of achieving acidification with spatial and temporal precision. We have implemented a method to control optically the opening of ASIC1a in brain slices and also in awake animals. The light-driven H(+) pump ArchT was expressed in astrocytes of mouse cortex by injection of adenoviral vectors containing a strong and astrocyte-specific promoter. Illumination with amber light acidified the surrounding interstitium and led to activation of endogenous ASIC1a channels and firing of action potentials in neurons localized in close proximity to ArchT-expressing astrocytes. We conclude that this optogenetic method offers a minimally invasive approach that enables examining the biological consequences of ASIC1a currents in any structure of the CNS and in the modulation of animal behaviors.

The neuronal-specific SGK1.1 (SGK1_v2) kinase as a transcriptional modulator of BAG4, Brox, and PPP1CB genes expression.

The Serum- and Glucocorticoid-induced Kinase 1, SGK1, exhibits a broad range of cellular functions that include regulation of the number of ion channels in plasma membrane and modulation of signaling pathways of cell survival. This diversity of functions is made possible by various regulatory processes acting upon the SGK1 gene, giving rise to various isoforms: SGK1_v1-5, each with distinct properties and distinct aminotermini that serve to target proteins to different subcellular compartments. Among cellular effects of SGK1 expression is to indirectly modulate gene transcription by phosphorylating transcriptional factors of the FOXO family. Here we examined if SGK1.1 (SGK1_v2; NM_001143676), which associates primarily to the plasma membrane, is also able to regulate gene expression. Using a differential gene expression approach we identified six genes upregulated by SGK1.1 in HeLa cells. Further analysis of transcript and protein levels validated two genes: BCL2-associated athanogene 4 (BAG-4) and Brox. The results indicate that SGK1.1 regulates gene transcription upon a different set of genes some of which participate in cell survival pathways (BAG-4) and others in intracellular vesicular traffic (Brox).

Impact of recovery from desensitization on acid-sensing ion channel-1a (ASIC1a) current and response to high frequency stimulation.

BACKGROUND: Consecutive proton stimulation reduces ASIC1a peak currents leading to silencing of channels. RESULTS: Kinetic analysis using a fast perfusion system shows that human ASIC1a has two desensitized states with markedly different stabilities. CONCLUSION: High frequency trains of short stimuli prevent desensitization. SIGNIFICANCE: The results predict steady ASIC1a responses to high frequency release of protons as in synaptic transmission. ASIC1a is a neuronal sodium channel activated by external H(+) ions. To date, all the characterization of ASIC1a has been conducted applying long H(+) stimuli lasting several seconds. Such experimental protocols weaken and even silence ASIC1a currents to repetitive stimulation. In this work, we examined ASIC1a currents by methods that use rapid application and removal of H(+). We found that brief H(+) stimuli, <100 ms, even if applied at high frequency, prevent desensitization thereby generate full and steady peak currents of human ASIC1a. Kinetic analysis of recovery from desensitization of hASIC1a revealed two desensitized states: short- and long-lasting with time constants of τ(Ds) ≤0.5 and τ(Dl) = 229 s, while in chicken ASIC1a the two desensitized states have similar values τ(D) 4.5 s. It is the large difference in stability of the two desensitized states that makes hASIC1a desensitization more pronounced and complex than in cASIC1a. Furthermore, recovery from desensitization was unrelated to cytosolic variations in pH, ATP, PIP(2), or redox state but was dependent on the hydrophobicity of key residues in the first transmembrane segment (TM1). In conclusion, brief H(+)-stimuli maintain steady the magnitude of peak currents thereby the ASIC1a channel is well poised to partake in high frequency signals in the brain.

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.

A flexible GAS belt responds to pore mutations changing the ion selectivity of proton-gated channels.

Proton-gated ion channels conduct mainly Na+ to induce postsynaptic membrane depolarization. Finding the determinants of ion selectivity requires knowledge of the pore structure in the open conformation, but such information is not yet available. Here, the open conformation of the hASIC1a channel was computationally modeled, and functional effects of pore mutations were analyzed in light of the predicted structures. The open pore structure shows two constrictions of similar diameter formed by the backbone of the GAS belt and, right beneath it, by the side chains of H28 from the reentrant loop. Models of nonselective mutant channels, but not those that maintain ion selectivity, predict enlargement of the GAS belt, suggesting that this motif is quite flexible and that the loss of stabilizing interactions in the central pore leads to changes in size/shape of the belt. Our results are consistent with the "close-fit" mechanism governing selectivity of hASIC1a, wherein the backbone of the GAS substitutes at least part of the hydration shell of a permeant ion to enable crossing the pore constriction.

Asn415 in the beta11-beta12 linker decreases proton-dependent desensitization of ASIC1.

Neurons of the mammalian nervous system express the proton-sensing ion channel ASIC1. Low concentrations of protons in the normal range of extracellular pH, pH 7.4-7.3, shut the pore by a conformational transition referred as steady-state desensitization. Therefore, the potential of local acidification to open ASIC1 relies on proton affinity for desensitization. This property is important physiologically and also can be exploited to develop strategies to increase or decrease the channel response to protons. In a previous study (Li, T., Yang, Y., and Canessa, C. M. (2010) J. Biol. Chem. 285, 22706-22712), we found that Leu-85 in the ß1-ß2 linker of the extracellular domain decreases the apparent proton affinity for steady-state desensitization and retards openings, slowing down the time course of the macroscopic currents. Here, we show that Asn-415 in the ß11-ß12 linker works together with the ß1-ß2 linker to stabilize a closed conformation that delays transition from the closed to the desensitized state. Substitutions of Asn-415 for Cys, Ser, or Gly render ASIC1 responsive to small increases in proton concentrations near the baseline physiological pH.

Leu85 in the beta1-beta2 linker of ASIC1 slows activation and decreases the apparent proton affinity by stabilizing a closed conformation.

Acid-sensing ion channels (ASICs) are proton-activated channels expressed in neurons of the central and peripheral nervous systems where they modulate neuronal activity in response to external increases in proton concentration. The size of ASIC1 currents evoked by a given local acidification is determined by the number of channels in the plasma membrane and by the apparent proton affinities for activation and steady-state desensitization of the channel. Thus, the magnitude of the pH drop and the value of the baseline pH both are functionally important. Recent characterization of ASIC1s from an increasing number of species has made evident that proton affinities of these channels vary across vertebrates. We found that in species with high baseline plasma pH, e.g. frog, shark, and fish, ASIC1 has high proton affinity compared with the mammalian channel. The beta1-beta2 linker in the extracellular domain, specifically by the substitution M85L, determines the interspecies differences in proton affinities and also the time course of ASIC1 macroscopic currents. The mechanism underlying these observations is a delay in channel opening after application of protons, most likely by stabilizing a closed conformation that decreases the apparent affinity to protons and also slows the rise and decay phases of the current. Together, the results suggest evolutionary adaptation of ASIC1 to match the value of the species-specific plasma pH. At the molecular level, adaptation is achieved by substitutions of nonionizable residues rather than by modification of the channel proton sensor.

A brain-specific SGK1 splice isoform regulates expression of ASIC1 in neurons.

Neurodegenerative diseases and noxious stimuli to the brain enhance transcription of serum- and glucocorticoid-induced kinase-1 (SGK1). Here, we report that the SGK1 gene encodes a brain-specific additional isoform, SGK1.1, which exhibits distinct regulation, properties, and functional effects. SGK1.1 decreases expression of the acid-sensing ion channel-1 (ASIC1); thereby, SGK1.1 may limit neuronal injury associated to activation of ASIC1 in ischemia. Given that neurons express at least two splice isoforms, SGK1 and SGK1.1, driven by distinct promoters, any changes in SGK1 transcript level must be examined to define the isoform induced by each stimulus or neurological disorder.

An arginine residue in the outer segment of hASIC1a TM1 affects both proton affinity and channel desensitization.

Acid-sensing ion channels (ASICs) respond to changes in pH in the central and peripheral nervous systems and participate in synaptic plasticity and pain perception. Understanding the proton-mediated gating mechanism remains elusive despite the of their structures in various conformational states. We report here that R64, an arginine located in the outer segment of the first transmembrane domain of all three isoforms of mammalian ASICs, markedly impacts the apparent proton affinity of activation and the degree of desensitization from the open and preopen states. Rosetta calculations of free energy changes predict that substitutions of R64 in hASIC1a by aromatic residues destabilize the closed conformation while stabilizing the open conformation. Accordingly, F64 enhances the efficacy of proton-mediated gating of hASIC1a, which increases the apparent pH50 and facilitates channel opening when only one or two subunits are activated. F64 also lengthens the duration of opening events, thus keeping channels open for extended periods of time and diminishing low pH-induced desensitization. Our results indicate that activation of a proton sensor(s) with pH50 equal to or greater than pH 7.2-7.1 opens F64hASIC1a, whereas it induces steady-state desensitization in wildtype channels due to the high energy of activation imposed by R64, which prevents opening of the pore. Together, these findings suggest that activation of a high-affinity proton-sensor(s) and a common gating mechanism may mediate the processes of activation and steady-state desensitization of hASIC1a.

Structure and analysis of nanobody binding to the human ASIC1a ion channel.

ASIC1a is a proton-gated sodium channel involved in modulation of pain, fear, addiction, and ischemia-induced neuronal injury. We report isolation and characterization of alpaca-derived nanobodies (Nbs) that specifically target human ASIC1a. Cryo-electron microscopy of the human ASIC1a channel at pH 7.4 in complex with one of these, Nb.C1, yielded a structure at 2.9 Å resolution. It is revealed that Nb.C1 binds to a site overlapping with that of the Texas coral snake toxin (MitTx1) and the black mamba venom Mambalgin-1; however, the Nb.C1-binding site does not overlap with that of the inhibitory tarantula toxin psalmotoxin-1 (PcTx1). Fusion of Nb.C1 with PcTx1 in a single polypeptide markedly enhances the potency of PcTx1, whereas competition of Nb.C1 and MitTx1 for binding reduces channel activation by the toxin. Thus, Nb.C1 is a molecular tool for biochemical and structural studies of hASIC1a; a potential antidote to the pain-inducing component of coral snake bite; and a candidate to potentiate PcTx1-mediated inhibition of hASIC1a in vivo for therapeutic applications.

Two residues in the extracellular domain convert a nonfunctional ASIC1 into a proton-activated channel.

Acid-sensing ion channels (ASICs) are proton-activated sodium channels of the nervous system. Mammals express four ASICs, and orthologs of these genes have been found in all chordates examined to date. Despite a high degree of sequence conservation of all ASICs across species, the response to a given increase in external proton concentration varies markedly: from large and slowly inactivating inward currents to no detectable currents. The underlying bases of this functional variability and whether it stems from differences in proton-binding sites or in structures that translate conformational changes have not been determined yet. We show here that the ASIC1 ortholog of an early vertebrate, lamprey ASIC1, does not respond to protons; however, only two amino acid substitutions for the corresponding ones in rat ASIC1, Q77L and T85L, convert lamprey ASIC1 into a highly sensitive proton-activated channel with apparent H(+) affinity of pH(50) 7.2. Addition of C73H increases the magnitude of the currents by fivefold, and W64R confers desensitization similar to that of the mammalian counterpart. Most amino acid substitutions in these four positions increase the rates of opening and closing the pore, whereas only few, namely, the ones in rat ASIC1, slow the rates. The four residues are located in a contiguous segment made by the beta1-beta2-linker, beta1-strand, and the external segment of the first transmembrane helix. We conclude that the segment thus defined modulates the kinetics of opening and closing the pore and that fast kinetics of desensitization rather than lack of acid sensor accounts for the absence of proton-induced currents in the parent lamprey ASIC1.

Multiple translational isoforms give functional specificity to serum- and glucocorticoid-induced kinase 1.

Serum- and glucocorticoid-induced kinase 1 is a ubiquitous kinase that regulates diverse processes such as ion transport and cell survival. We report that a single SGK1 mRNA produces isoforms with different N-termini owing to alternative translation initiation. The long isoforms, 49 and 47 kDa, are the most abundant, localize to the ER membrane, exhibit rapid turnover, their expression is decreased by ER stress, activate the epithelial sodium channel (ENaC) and translocate FoxO3a transcriptional factors from the nucleus to the cytoplasm. The short isoforms, 45 and 42 kDa, localize to the cytoplasm and nucleus, exhibit long half-life and phosphorylate glycogen synthase kinase-3beta. The data indicate that activation of Sgk1 in different cellular compartments is key to providing functional specificity to Sgk1 signaling pathways. We conclude that the distinct properties and functional specialization of Sgk1 given by the N-terminus confer versatility of function while maintaining the same core kinase domain.

Simple chordates exhibit a proton-independent function of acid-sensing ion channels.

Acid-sensing ion channels (ASICs) constitute a family of neuron-specific voltage-insensitive sodium channels gated by extracellular protons. Functions of ASICs in mammals include nociception, mechanosensation, and modulation of synaptic transmission. However, the role protons play in mediating the effects of ASICs remains elusive. We have examined ASICs from the ascidian Ciona intestinalis, a simple chordate organism whose nervous system in the larval stage exhibits high similarity to that of higher vertebrates. We found that the ascidian genome contains a single ASIC gene that gives rise to two splice forms analogous to the mammalian ASIC1 and ASIC2. CiASIC is expressed in most neurons of the larva but is absent in the adult. Despite high sequence similarity with mammalian counterparts, CiASIC is proton-insensitive when examined in heterologous systems or in larval neurons; the latter rules out the possibility that proton sensitivity is conferred by accessory proteins or particular factors present only in Ciona neurons. Down-regulation of the CiASIC transcript by double-stranded RNA disrupted the regular pattern of larval swimming, implying that proton-independent mechanisms mediate the effects of ASIC in vivo. Together the data identify ASIC as a highly conserved channel distinctive of chordate nervous systems and show that protons are not essential for ASIC function.

A valve-like mechanism controls desensitization of functional mammalian isoforms of acid-sensing ion channels.

ASICs are proton-gated sodium channels expressed in neurons. Structures of chicken ASIC1 in three conformations have advanced understanding of proton-mediated gating; however, a molecular mechanism describing desensitization from open and pre-open states (steady-state desensitization or SSD) remains elusive. A distinct feature of the desensitized state is an 180o rotation of residues L415 and N416 in the ß11- ß12 linker that was proposed to mediate desensitization; whether and how it translates into desensitization has not been explored yet. Using electrophysiological measurements of injected Xenopus oocytes, we show that Q276 in ß9 strand works with L415 and N416 to mediate both types of desensitization in ASIC1a, ASIC2a and ASIC3. Q276 functions as a valve that enables or restricts rotation of L415 and N416 to keep the linker compressed, its relaxation lengthens openings and leads to sustained currents. At low proton concentrations, the proposed mechanism working in only one of three subunits of the channel is sufficient to induce SSD.

Gating of acid-sensitive ion channel-1: release of Ca2+ block vs. allosteric mechanism.

The acid-sensitive ion channels (ASICs) are a family of voltage-insensitive sodium channels activated by external protons. A previous study proposed that the mechanism underlying activation of ASIC consists of the removal of a Ca2+ ion from the channel pore (Immke and McCleskey, 2003). In this work we have revisited this issue by examining single channel recordings of ASIC1 from toadfish (fASIC1). We demonstrate that increases in the concentration of external protons or decreases in the concentration of external Ca2+ activate fASIC1 by progressively opening more channels and by increasing the rate of channel opening. Both maneuvers produced similar effects in channel kinetics, consistent with the former notion that protons displace a Ca2+ ion from a high-affinity binding site. However, we did not observe any of the predictions expected from the release of an open-channel blocker: decrease in the amplitude of the unitary currents, shortening of the mean open time, or a constant delay for the first opening when the concentration of external Ca2+ was decreased. Together, the results favor changes in allosteric conformations rather than unblocking of the pore as the mechanism gating fASIC1. At high concentrations, Ca2+ has an additional effect that consists of voltage-dependent decrease in the amplitude of unitary currents (EC50 of 10 mM at -60 mV and pH 6.0). This phenomenon is consistent with voltage-dependent block of the pore but it occurs at concentrations much higher than those required for gating.

Single channel properties of rat acid-sensitive ion channel-1alpha, -2a, and -3 expressed in Xenopus oocytes.

The mammalian nervous system expresses proton-gated ion channels known as acid-sensing ion channels (ASICs). Depending on their location and specialization some neurons express more than one type of ASIC where they may form homo- or heteromeric channels. Macroscopic characteristics of the ASIC currents have been described, but little is known at the single channel level. Here, we have examined the properties of unitary currents of homomeric rat ASIC1alpha, ASIC2a, and ASIC3 expressed in Xenopus oocytes with the patch clamp technique. We describe and characterize properties unique to each of these channels that can be used to distinguish the various types of ASIC channels expressed in mammalian neurons. The amplitudes of the unitary currents in symmetrical Na(+) are similar for the three types of channels (23-18 pS) and are not voltage dependent. However, ASIC1alpha exhibits three subconductance states, ASIC2a exhibits only one, and ASIC3 none. The kinetics of the three types of channels are different: ASIC1alpha and ASIC2a shift between modes of activity, each mode has different open probability and kinetics. In contrast, the kinetics of ASIC3 are uniform throughout the burst of activity. ASIC1alpha, ASIC2a, and ASIC3 are activated by external protons with apparent pH(50) of 5.9, 5.0, and 5.4, respectively. Desensitization in the continual presence of protons is fast and complete in ASIC1alpha and ASIC3 (2.0 and 4.5 s(-1), respectively) but slow and only partial in ASIC2a (0.045 s(-1)). The response to external Ca(2+) also differs: micro M concentrations of extracellular Ca(2+) are necessary for proton gating of ASIC3 (EC(50) = 0.28 micro M), whereas ASIC1alpha and ASIC2a do not require Ca(2+). In addition, Ca(2+) inhibits ASIC1alpha (K(D) = 9.2 +/- 2 mM) by several mechanisms: decrease in the amplitude of unitary currents, shortening of the burst of activity, and decrease in the number of activated channels. Contrary to previous reports, our results indicate that the Ca(2+) permeability of ASIC1alpha is very small.

Effects of aldosterone on biosynthesis, traffic, and functional expression of epithelial sodium channels in A6 cells.

The collecting duct regulates Na(+) transport by adjusting the abundance/activity of epithelial Na(+) channels (ENaC). In this study we have investigated the synthesis, degradation, endocytosis, and activity of ENaC and the effects of aldosterone on these processes using endogenous channels expressed in the A6 cell line. Biochemical studies were performed with a newly raised set of specific antibodies against each of the three subunits of the amphibian ENaC. Our results indicate simultaneous transcription and translation of alpha, beta, and gamma subunits and enhancement of both processes by aldosterone: two- and fourfold increase, respectively. The biosynthesis of new channels can be followed by acquisition of endoglycosidase H-resistant oligosacharides in alpha and beta subunits and, in the case of alpha, by the appearance of a form resistant to reducing agents. The half-life of the total pool of subunits (t(1/2) 40-70 min) is longer than the fraction of channels in the apical membrane (t(1/2) 12-17 min). Aldosterone induces a fourfold increase in the abundance of the three subunits in the apical membrane without significant changes in the open probability, kinetics of single channels, or in the rate of degradation of ENaC subunits. Accordingly, the aldosterone response could be accounted by an increase in the abundance of apical channels due, at least in part, to de novo synthesis of subunits.