Tatjana Coric

Distribution and regulation of expression of serum‐ and glucocorticoid‐induced kinase‐1 in the rat kidney

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.

Proton sensitivity of ASIC1 appeared with the rise of fishes by changes of residues in the region that follows TM1 in the ectodomain of the channel

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 D78–E136, together with residues D351, Q358 and E359 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.

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.

4-Hydroxytamoxifen Induces Autophagic Death through K-Ras Degradation

Tamoxifen is widely used to treat estrogen receptor-positive breast cancer. Recent findings that tamoxifen and its derivative 4-hydroxytamoxifen (OHT) can exert estrogen receptor-independent cytotoxic effects have prompted the initiation of clinical trials to evaluate its use in estrogen receptor-negative malignancies. For example, tamoxifen and OHT exert cytotoxic effects in malignant peripheral nerve sheath tumors (MPNST) where estrogen is not involved. In this study, we gained insights into the estrogen receptor-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 autophagic degradation of K-Ras, 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 OHT in tumor cells that may be more broadly useful clinically in cancer treatment.

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

Acid‐sensing ion channels (ASICs) con stitute a family of neuron‐specific voltage‐insensitive sodium channels gated by extracellular protons. Functions of ASICs in mammals include nociception, mech‐ anosensation, 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 coun terparts, CiASIC is proton‐insensitive when examined in heterologous systems or in larval neurons;the latter rules out the possibility that proton sensitivity is con ferred 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 ner vous systems and show that protons are not essential for ASIC function.— Coric, T., Passamaneck, Y. J., Zhang, P., Di Gregorio, A., and Canessa, C. M. Simple chordates exhibit a proton‐independent function of acid sensing ion channels. FASEB J. 22, 1914–1923 (2008)

High-throughput RNA interference screening: tricks of the trade.

The process of validating an assay for high-throughput screening (HTS) involves identifying sources of variability and developing procedures that minimize the variability at each step in the protocol. The goal is to produce a robust and reproducible assay with good metrics. In all good cell-based assays, this means coefficient of variation (CV) values of less than 10% and a signal window of fivefold or greater. HTS assays are usually evaluated using Z′ factor, which incorporates both standard deviation and signal window. A Z′ factor value of 0.5 or higher is acceptable for HTS. We used a standard HTS validation procedure in developing small interfering RNA (siRNA) screening technology at the HTS center at Southern Research. Initially, our assay performance was similar to published screens, with CV values greater than 10% and Z′ factor values of 0.51 ± 0.16 (average ± standard deviation). After optimizing the siRNA assay, we got CV values averaging 7.2% and a robust Z′ factor value of 0.78 ± 0.06 (average ± standard deviation). We present an overview of the problems encountered in developing this whole-genome siRNA screening program at Southern Research and how equipment optimization led to improved data quality.

Effects of extracellular calcium on fASIC1 currents.

Abstract: Fish ASIC1 (fASIC1) cloned from Opsanus tau, unlike the rat ASICs, requires Ca2+ in the extracellular preconditioning solution (pH 7.4) to be activated. Here we show that fASIC1 is interacting with Ca2+ in the same way as mammalian ASICs: extracellular Ca2+ is increasing the proportion of channels available for activation by stabilizing the closed state of the channel; in the activation process Ca2+ is released; H+ compete for the binding site of Ca2+ making the gating mechanism both Ca2+ and H+ dependent; H+ stabilizes the desensitized state; Ca2+ blocks the fASIC1 channel; and the affinity of the block is also modulated by H+. The “Ca2+ activation requirement” of fASIC1 reflects its greater affinity for steady‐state desensitization by H+ compared to mammalian ASIC1.

Acoustic Droplet Ejection Technology and Its Application in High-Throughput RNA Interference Screening

The development of acoustic droplet ejection (ADE) technology has resulted in many positive changes associated with the operations in a high-throughput screening (HTS) laboratory. Originally, this liquid transfer technology was used to simply transfer DMSO solutions of primarily compounds. With the introduction of Labcyte’s Echo 555, which has aqueous dispense capability, the application of this technology has been expanded beyond its original use. This includes the transfer of many biological reagents solubilized in aqueous buffers, including siRNAs. The Echo 555 is ideal for siRNA dispensing because it is accurate at low volumes and a step-down dilution is not necessary. The potential for liquid carryover and cross-contamination is eliminated, as no tips are needed. Herein, we describe the siRNA screening platform at Southern Research’s HTS Center using the ADE technology. With this technology, an siRNA library can be dispensed weeks or even months in advance of the assay itself. The protocol has been optimized to achieve assay parameters comparable to small-molecule screening parameters, and exceeding the norm reported for genomewide siRNA screens.