The acid-sensitive ion channel 1 (ASIC1␣ or BNaC2a) is the most abundant of all mammalian proton-gated ion channels and the one that has the broadest distribution in the nervous system. Hallmarks of ASIC1␣ are gating by external protons and rapid desensitization. In sensory neurons ASIC1 may constitute a nociceptor for pain induced by local acidification, whereas in central neurons it may modulate synaptic activity. To gain insight into the functional roles of ASIC1, we cloned and examined the properties of the evolutionarily distant species toadfish (Opsanus tau), ϳ420-million year divergent from mammals. Analysis of the protein sequence from fish ASIC1 revealed 76% amino acid identity with the rat orthologue. The regions of highest conservation are the second transmembrane domain and the ectodomain, whereas the amino and carboxyl termini and first transmembrane domain are poorly conserved. At the functional level, fish ASIC1 is gated by external protons with a half-maximal activation at pH o 5.6 and a halfmaximal inactivation at pH o 7.30. The fish differs from the rat channel on having a 25-fold faster rate of desensitization. Functional studies of chimeras made from rat and fish ASIC1 indicate that the extracellular domain specifically, a cluster of three residues, confers the faster desensitization rate to the fish ASIC1.The acid-sensitive ion channels (ASICs) 1 constitute a subfamily of the large epithelial sodium channel (ENaC)/DEG family of ion channels (17, 27). The ASIC1␣ protein (or BNaC2a) is the most abundant of all mammalian proton-gated ion channels and the one that is expressed in most neurons of the central and peripheral nervous systems (1, 2). The mammalian ASIC1, ASIC2, and ASIC3 are all activated by protons but the degree and rate of desensitization markedly differ in each type of channel. For instance, rat ASIC1 and ASIC3 exhibit rapid and complete desensitization at pH o 5.0, whereas ASIC2 has a slow and incomplete desensitization, leaving a substantial component of persistent current in the continual presence of protons (29). Indeed, most of the functional differences in the currents generated by the ASICs can be attributed to differences in desensitization rate, suggesting that this property may be important in conferring specificity to the response of the various ASICs in different regions of the nervous system. Numerous functions have been proposed for ASIC1 including a role in sensory transduction, specifically in nociception (pain induced by ischemia and inflammation) (7,9,15,20,21,24), and as modulators of synaptic transmission and long term plasticity (2, 28). For many of these functions, in particular nociception, proton sensitivity plays a fundamental role in the physiology of these channels.In order to gain more insight in the structure-function of these channels, we cloned and examined the functional properties of ASIC1 from an evolutionarily distant species, the fish Opsanus tau. Comparison of the properties of the fish and mammalian ASIC1 revealed significant differences, pri...

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.alternative promoter ͉ Proton-activated channel ͉ serum-and glucocorticoid-induced kinase

The acid-sensitive ion channel 1 (ASIC1) is a neuronal Na + channel insensitive to changes in membrane potential but is gated by external protons. Proton sensitivity is believed to be essential for the role of ASIC1 in modulating synaptic transmission and nociception in the mammalian nervous system. To examine the structural determinants that confer proton sensitivity, we cloned and functionally characterized ASIC1 from different species of the chordate lineage: lamprey, shark, toadfish and chicken. We observed that ASIC1s from early vertebrates (lamprey and shark) were proton insensitive in spite of a high degree of amino acid conservation (66-67%) with their mammalian counterparts. Sequence analysis showed that proton-sensitive ASIC1s could not be distinguished from proton-insensitive channels by any signature in the protein sequence. Chimeras made with rat ASIC1 (rASIC1) and lamprey or shark indicated that most of the ectodomain of rASIC1 was required to confer proton sensitivity and the distinct kinetics of activation and desensitization of the rat channel. Proton-sensitive chimeras contained the segment D 78 -E 136 , together with residues D 351 , Q 358 and E 359 of the rat sequence. However, none of the functional chimeras containing only part of the rat extracellular domain retained the kinetics of activation and desensitization of rASIC1, suggesting that residues distributed in several regions of the ectodomain contribute to allosteric changes underlying activation and desensitization. The results also demonstrate that gating by protons is not a feature common to all ASIC1 channels. Proton sensitivity arose recently in evolution, implying that agonists different from protons activate ASIC1 in lower vertebrates.

The serum‐ and glucocorticoid‐induced kinase‐1 (sgk1) increases the activity of a number of epithelial ion channels and transporters. The present study examines the distribution and subcellular localization of sgk1 protein in the rat kidney and the regulation of levels of expression induced by steroids. The results indicate that the kidney expresses predominantly the sgk1 isoform with a distribution restricted to the thick ascending limb of Henle, distal convoluted, connecting and cortical collecting tubules. Within cells, sgk1 strongly associates with the microsomal fraction of homogenates and it colocalizes with the Na+,K+‐ATPase to the basolateral membrane. Analysis of the levels of expression of sgk1 by Western blotting and immunohistochemistry indicates constitutive high expression under basal conditions. Approximately half of the basal level is maintained by glucocorticoids whereas physiological fluctuations of aldosterone produce minor changes in sgk1 abundance in adrenal‐intact animals. These results do not support the notion that physiological changes of aldosterone concentration turn the expression of sgk1 ‘on and off’ in the mammalian kidney. Additionally, localization of sgk1 to the basolateral membrane indicates that the effects mediated by sgk1 do not require a direct interaction with the ion channels and transporters whose activity is modulated, since most of these proteins are located in the apical membrane of renal epithelial cells.

Aberrations in the mTOR (mechanistic target of rapamycin) axis are frequently reported in cancer. Using publicly available tumor genome sequencing data, we identified several point mutations in MTOR and its upstream regulator RHEB (Ras homolog enriched in brain) in patients with clear cell renal cell carcinoma (ccRCC), the most common histology of kidney cancer. Interestingly, we found a prominent cluster of hyperactivating mutations in the FAT (FRAP-ATM-TTRAP) domain of mTOR in renal cell carcinoma that led to an increase in both mTORC1 and mTORC2 activities and led to an increased proliferation of cells. Several of the FAT domain mutants demonstrated a decreased binding of DEPTOR (DEP domain containing mTOR-interacting protein), while a subset of these mutations showed altered binding of the negative regulator PRAS40 (proline rich AKT substrate 40). We also identified a recurrent mutation in RHEB in ccRCC patients that leads to an increase in mTORC1 activity. In vitro characterization of this RHEB mutation revealed that this mutant showed considerable resistance to TSC2 (Tuberous Sclerosis 2) GAP (GTPase activating protein) activity, though its interaction with TSC2 remained unaltered. Mutations in the FAT domain of MTOR and in RHEB remained sensitive to rapamycin, though several of these mutations demonstrated residual mTOR kinase activity after treatment with rapamycin at clinically relevant doses. Overall, our data suggests that point mutations in the mTOR pathway may lead to downstream mTOR hyperactivation through multiple different mechanisms to confer a proliferative advantage to a tumor cell.

Tamoxifen is widely used to treat estrogen receptor (ER)-positive breast cancer. Recent findings that tamoxifen and its derivative 4-dehydroxy-tamoxifen (OHT) can exert ER-independent cytotoxic effects have prompted the initiation of clinical trials to evaluate its use in ER-negative malignancies. For example, tamoxifen and OHT exert cytotoxic effects in malignant peripheral nerve sheath tumors (MPNSTs) where estrogen is not involved. In this study, we gained insights into the ER-independent cytotoxic effects of OHT by studying how it kills MPNST cells. Although caspases were activated following OHT treatment, caspase inhibition provided no protection from OHT-induced death. Rather, OHT-induced death in MPNST cells was associated with autophagic induction and attenuated by genetic inhibition of autophagic vacuole formation. Mechanistic investigations revealed that OHT stimulated K-Ras degradation through autophagy induction, which is critical for survival of MPNST cells. Similarly, we found that OHT induced K-Ras degradation in breast, colon, glioma and pancreatic cancer cells. Our findings describe a novel mechanism of autophagic death triggered by tamoxifen and OHT in tumor cells that may be more broadly useful clinically in cancer treatment.

. Expression of ENaC and serum-and glucocorticoid-induced kinase 1 in the rat intestinal epithelium. Am J Physiol Gastrointest Liver Physiol 286: G663-G670, 2004. First published November 20, 2003 10.1152/ ajpgi.00364.2003.-Increase in epithelium sodium channel (ENaC) activity induced by aldosterone in the distal tubule of the kidney has been attributed to serum-and glucocorticoid-induced kinase 1 (sgk1). The distal colon constitutes another classical aldosterone-responsive epithelium that expresses both ENaC and sgk1 in an aldosteronedependent manner. However, the site of expression and the temporal relationship of the aldosterone induction of these two proteins have not been investigated. Here, we examined the distribution and abundance of sgk1 in the rat intestine under basal conditions and after changes in the concentration of aldosterone and glucocorticoids. Results indicate that sgk1 is expressed in the distal colon and also in the ileum and jejunum. Abundance of sgk1 was high in control animals, and it did not change significantly after sodium depletion or after a single dose of aldosterone; however, it decreased after adrenalectomy. In contrast, the three subunits of ENaC were markedly induced in the distal colon by acute and chronic increases in aldosterone levels. Results indicate differential regulation of sgk and ENaC subunits by aldosterone in the distal colon. Distribution of sgk1 in the intestine beyond the aldosterone-responsive segments suggests that sgk1 may additionally regulate other sodium transporters in the intestinal epithelium. aldosterone; sodium depletion; distal colon; amiloride; serum-and glucocorticoid-induced kinase 1 DISTAL SEGMENTS OF THE renal tubule (21,24,28) and the epithelium of the distal colon (5, 32) respond to aldosterone by increasing amiloride-sensitive transepithelial sodium reabsorption. Epithelium sodium channel (ENaC), comprised of ␣-, ␤-and ␥-subunits, mediates the effects of aldosterone in both tissues, although the mechanisms underlying the response differ in the kidney and colon. Under basal conditions, neither the distal renal tubule nor the distal colon of the rat exhibits significant ENaC activity. However, the kidney expresses mRNA and protein of the three subunits; aldosterone increases transcription and translation of only the ␣-subunit by approximately twofold (4,20). The distal colon expresses ␣-mRNA and very low levels of ␤-and ␥-subunits; aldosterone increases the expression of mRNA of the three subunits, but the effect is 10-fold larger for ␤-and ␥-subunits than for ␣-subunits (4, 12, 18, 22).These observations imply that the early aldosterone response (29), which takes place within the first 2 h of administration of the hormone, differs in these tissues. In the cortical collecting tubule of the kidney, aldosterone may increase ENaC activity by translocation of channels to the apical membrane (19), by reducing the rate of endocytosis (10), or by activating previously silent channels (14). In the colon, however, aldosterone must first induce synthesi...

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.