Protein In Xenopus Oocytes Research Articles

FRI289 It Takes A Village: Musashi And Its Associated Proteins In The Adenohypophysis

Abstract Disclosure: K.L. Bronson: None. L. Hardy: None. A. Lagasse: None. A.M. MacNicol: None. Musashi is an RNA-binding protein that regulates stem and progenitor cell fates. Interestingly, we have reported expression of Musashi pituitary stem cells as expected but also in the differentiated anterior pituitary hormone-producing cell lineages as well as. The adult anterior pituitary relies on the functional cellular plasticity of its hormone-producing cell populations to maintain endocrine homeostasis when physiological demands change. Both Musashi isoforms (Musashi1 and Musashi2) are highly conserved across species and have two N-terminal RNA-recognition motifs (RRMs) and a disordered C-terminus containing two regulatory phosphorylation sites. Musashi can selectively activate or repress the translation of mRNAs depending on the transcript and cellular context. While Musashi phosphorylation is required for translational activation, Musashi does not possess inherent enzymatic activity; therefore, co-associated proteins must mediate Musashi-directed translational control. Musashi immunoprecipitation studies initially identified 50 co-associated proteins in Xenopus oocytes, but only a subset of these were directly implicated in Musashi-dependent translational activation (e.g. Elavl1 and Pabp4). The identity of Musashi-associated proteins in the anterior pituitary and their requirement for Musashi function are not known. To this end, we have immunoprecipitated Musashi from whole male mouse pituitaries for mass spectrometry analysis. 206 proteins significantly (adj p-value < 0.05) associated with Musashi1 and Musashi2, and seven of these overlapped with the Xenopus dataset: Elavl1, Pabp4, Hnrnpc, Mov10, Pura, Rps27, and Upf1. Three proteins from the mouse mass spectrometry dataset were selected for further analyses. Sam68 is a multi-functional protein that sensitizes ovaries to FSH and LH, as well as mediates the activation of leptin and insulin signaling in a variety of tissues. Ago2 is a component of the RNA-induced silencing complex that is necessary for miRNA-directed translational control and is known to associate with Musashi1. The AU-rich element binding protein Elavl1 (HuR) contributes to Musashi-dependent activation of target mRNAs in Xenopus and also stabilizes Gnrhr mRNA in LβT2 cells and sensitizes them to GnRH signaling. We will present our ongoing studies on the requirement for these protein interactions for Musashi-directed mRNA translational regulation. Presentation: Friday, June 16, 2023

MtNPF6.5 mediates chloride uptake and nitrate preference in Medicago roots.

The preference for nitrate over chloride through regulation of transporters is a fundamental feature of plant ion homeostasis. We show that Medicago truncatula MtNPF6.5, an ortholog of Arabidopsis thaliana AtNPF6.3/NRT1.1, can mediate nitrate and chloride uptake in Xenopus oocytes but is chloride selective and that its close homologue, MtNPF6.7, can transport nitrate and chloride but is nitrate selective. The MtNPF6.5 mutant showed greatly reduced chloride content relative to wild type, and MtNPF6.5 expression was repressed by high chloride, indicating a primary role for MtNPF6.5 in root chloride uptake. MtNPF6.5 and MtNPF6.7 were repressed and induced by nitrate, respectively, and these responses required the transcription factor MtNLP1. Moreover, loss of MtNLP1 prevented the rapid switch from chloride to nitrate as the main anion in nitrate-starved plants after nitrate provision, providing insight into the underlying mechanism for nitrate preference. Sequence analysis revealed three sub-types of AtNPF6.3 orthologs based on their predicted substrate-binding residues: A (chloride selective), B (nitrate selective), and C (legume specific). The absence of B-type AtNPF6.3 homologues in early diverged plant lineages suggests that they evolved from a chloride-selective MtNPF6.5-like protein.

In Vitro Reconstitutions and Functional Characterizations of Fibrillar Centers of Nucleoli

The nucleolus is the largest membraneless biomolecular condensate in cells. It plays a central role in the synthesis of ribosomal subunits. Nucleoli form via spontaneous phase transitions that are driven by key nucleolar protein and RNA molecules. Each nucleolus has a core-shell architecture that is organized around an inner fibrillar center (FC) and an outer granular component (GC). The most abundant nucleolar proteins are nucleophosmin 1 (NPM1) in the GC and nucleolin (NCL) in the FC. We quantified the spatial distribution of NCL and NPM1 by co-expressing these proteins in Xenopus oocytes. As expected, we find that NCL is concentrated in the FC of the nucleolus and depleted in the GC, with the converse being true of NPM1. We used in vitro reconstitutions to show that GFP-tagged NCL forms condensates in the presence of a key binding partner protein Ly-1 Antibody Reactive (LYAR) and RNA. NCL has the curious feature of undergoing auto-proteolysis and LYAR inhibits this auto-proteolysis of NCL. The condensates formed using the minimal ternary system show an intriguing layered structure with NCL at the core of the condensate. Therefore, the minimal system recapitulates the structural organization of bona fide FCs. We report results from a detailed analysis of the driving forces that control the assembly of minimal FCs. Further, we query the impact of designed changes to intrinsically disordered RG-rich domains in NCL on the phase behavior, material properties, and functions of reconstituted FCs. Our results pave the way for understanding how condensates form, how they are regulated, and how material properties impact functions controlled by condensates such as nucleoli.

Specialized stellate cells offer a privileged route for rapid water flux in Drosophila renal tubule

Insects are highly successful, in part through an excellent ability to osmoregulate. The renal (Malpighian) tubules can secrete fluid faster on a per-cell basis than any other epithelium, but the route for these remarkable water fluxes has not been established. In Drosophila melanogaster, we show that 4 genes of the major intrinsic protein family are expressed at a very high level in the fly renal tissue: the aquaporins (AQPs) Drip and Prip and the aquaglyceroporins Eglp2 and Eglp4 As predicted from their structure, and by their transport function by expressing these proteins in Xenopus oocytes, Drip, Prip, and Eglp2 show significant and specific water permeability, whereas Eglp2 and Eglp4 show very high permeability to glycerol and urea. Knockdowns of any of these genes result in impaired hormone-induced fluid secretion. The Drosophila tubule has 2 main secretory cell types: active cation-transporting principal cells, wherein the aquaglyceroporins localize to opposite plasma membranes, and small stellate cells, the site of the chloride shunt conductance, with these AQPs localizing to opposite plasma membranes. This suggests a model in which osmotically obliged water flows through the stellate cells. Consistent with this model, fluorescently labeled dextran, an in vivo marker of membrane water permeability, is trapped in the basal infoldings of the stellate cells after kinin diuretic peptide stimulation, confirming that these cells provide the major route for transepithelial water flux. The spatial segregation of these components of epithelial water transport may help to explain the unique success of the higher insects in regulating their internal environments.

Expression of LRRC8/VRAC Currents in Xenopus Oocytes: Advantages and Caveats

Volume-regulated anion channels (VRACs) play a role in controlling cell volume by opening upon cell swelling. Apart from controlling cell volume, their function is important in many other physiological processes, such as transport of metabolites or drugs, and extracellular signal transduction. VRACs are formed by heteromers of the pannexin homologous protein LRRC8A (also named Swell1) with other LRRC8 members (B, C, D, and E). LRRC8 proteins are difficult to study, since they are expressed in all cells of our body, and the channel stoichiometry can be changed by overexpression, resulting in non-functional heteromers. Two different strategies have been developed to overcome this issue: complementation by transient transfection of LRRC8 genome-edited cell lines, and reconstitution in lipid bilayers. Alternatively, we have used Xenopus oocytes as a simple system to study LRRC8 proteins. Here, we have reviewed all previous experiments that have been performed with VRAC and LRRC8 proteins in Xenopus oocytes. We also discuss future strategies that may be used to perform structure-function analysis of the VRAC in oocytes and other systems, in order to understand its role in controlling multiple physiological functions.

Ammonium Conductance in a Mouse Thick Ascending Limb Cell Line

NH4+ is a key buffer component that regulates blood pH. The kidneys excrete NH4+ to urine as they produce HCO3−, and the mechanism by which NH4+ excretion results in net acid excretion involves a series of sophisticated NH4+ transport processes in different parts of the nephron. One of the nephron segments that play key roles in NH4+ excretion is the thick ascending limb (TAL). In the luminal membrane of the TAL tubules, the Na/K/2Cl cotransporter is the major fraction of the active NH4+ flux. Nonetheless, in vitro studies reveal that K/NH4 exchange and NH4+ conductance, whose molecular identities are presently unknown, can contribute to the TAL NH4+ transport by 35–50%. The two pathways exhibit biophysical and pharmacological characteristics that distinguish them from other NH4+‐transporting proteins. Despite such physiological and functional significance, our understanding of these pathways is limited. Especially, the electrophysiological properties of the NH4+ conductance have been unknown.In this study, we examined the NH4+conductance in the TAL cell line ST‐1 to determine its basic electrophysiological properties such as the amounts of NH4+current produced in cells, current‐voltage (I–V) relationship, and reversal potential of the current. Whole cell patch clamp was performed to measure the current evoked by NH4Cl in the presence of BaCl2, tetraethylammonium and BAPTA. Application of 20 mM NH4Cl induced an inward current with mean amplitude of −272 ± 79 pA (n = 9). In I–V relationships, NH4Cl application caused the I–V curve to shift down in an inward direction. The difference in curves before and after NH4Cl application, which corresponds to the NH4Cl‐mediated currents at different voltages, was progressively larger at more negative potentials. The reversal potential was +15 mV, more positive than a calculated equilibrium potential for Cl−, thus indicating that the current is not due to Cl−but to NH +. We then expressed ST‐1 total proteins in Xenopus oocytes and performed two‐electrode voltage clamp. Application of NH4Cl in the presence of BaCl2 caused the I–V curve to be steeper in an inward direction. Interestingly, removing Na+ from the bath solution caused the NH4+ conductance to disappear. Na+removal additionally led to a partial reduction of endogenous oocyte conductance, which was abolished by bumetanide. At pH 6.4, the NH4+conductance was still produced while endogenous oocyte conductance was not detected. In conclusion, we, for the first time, report the electrophysiological properties of the NH4+conductance in the ST‐1 cells. This conductance is Na+‐dependent, different from the previously reported Cl−‐dependent NH4+conductance in isolated TAL tubules.Support or Funding InformationThis work was supported by the Emory physiology department bridge fund to I.C.

SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways.

Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis.

Cellular Uptake of Imatinib into Leukemic Cells Is Independent of Human Organic Cation Transporter 1 (OCT1)

In addition to mutated BCR-ABL1 kinase, the organic cation transporter 1 (OCT1, encoded by SLC22A1) has been considered to contribute to imatinib resistance in patients with chronic myeloid leukemia (CML). As data are conflicting as to whether OCT1 transports imatinib and may serve as a clinical biomarker, we used a combination of different approaches including animal experiments to elucidate comprehensively the impact of OCT1 on cellular imatinib uptake. Transport of imatinib was studied using OCT1-expressing Xenopus oocytes, mammalian cell lines (HEK293, MDCK, V79) stably expressing OCT1, human leukemic cells, and Oct1-knockout mice. OCT1 mRNA and protein expression were analyzed in leukemic cells from patients with imatinib-naïve CML as well as in cell lines. Transport and inhibition studies showed that overexpression of functional OCT1 protein in Xenopus oocytes or mammalian cell lines did not lead to an increased cellular accumulation of imatinib. The CML cell lines (K562, Meg-01, LAMA84) and leukemic cells from patients expressed neither OCT1 mRNA nor protein as demonstrated by immunoblotting and immunofluorescence microscopy, yet they showed a considerable imatinib uptake. Oct1 deficiency in mice had no influence on plasma and hepatic imatinib concentrations. These data clearly demonstrate that cellular uptake of imatinib is independent of OCT1, and therefore OCT1 is apparently not a valid biomarker for imatinib resistance.

Intracellular Trafficking of FXYD1 (Phospholemman) and FXYD7 Proteins in Xenopus Oocytes and Mammalian Cells

FXYD proteins are a group of short single-span transmembrane proteins that interact with the Na(+)/K(+) ATPase and modulate its kinetic properties. This study characterizes intracellular trafficking of two FXYD family members, FXYD1 (phospholemman (PLM)) and FXYD7. Surface expression of PLM in Xenopus oocytes requires coexpression with the Na(+)/K(+) ATPase. On the other hand, the Na(+)/Ca(2+) exchanger, another PLM-interacting protein could not drive it to the cell surface. The Na(+)/K(+) ATPase-dependent surface expression of PLM could be facilitated by either a phosphorylation-mimicking mutation at Thr-69 or a truncation of three terminal arginine residues. Unlike PLM, FXYD7 could translocate to the cell surface of Xenopus oocytes independently of the coexpression of α1β1 Na(+)/K(+) ATPase. The Na(+)/K(+) ATPase-independent membrane translocation of FXYD7 requires O-glycosylation of at least two of three conserved threonines in its ectodomain. Subsequent experiments in mammalian cells confirmed the role of conserved extracellular threonine residues and demonstrated that FXYD7 protein, in which these have been mutated to alanine, is trapped in the endoplasmic reticulum and Golgi apparatus.

A New Human NHERF1 Mutation Decreases Renal Phosphate Transporter NPT2a Expression by a PTH-Independent Mechanism

BackgroundThe sodium-hydrogen exchanger regulatory factor 1 (NHERF1) binds to the main renal phosphate transporter NPT2a and to the parathyroid hormone (PTH) receptor. We have recently identified mutations in NHERF1 that decrease renal phosphate reabsorption by increasing PTH-induced cAMP production in the renal proximal tubule.MethodsWe compared relevant parameters of phosphate homeostasis in a patient with a previously undescribed mutation in NHERF1 and in control subjects. We expressed the mutant NHERF1 protein in Xenopus Oocytes and in cultured cells to study its effects on phosphate transport and PTH-induced cAMP production.ResultsWe identified in a patient with inappropriate renal phosphate reabsorption a previously unidentified mutation (E68A) located in the PDZ1 domain of NHERF1.We report the consequences of this mutation on NHERF1 function. E68A mutation did not modify cAMP production in the patient. PTH-induced cAMP synthesis and PKC activity were not altered by E68A mutation in renal cells in culture. In contrast to wild-type NHERF1, expression of the E68A mutant in Xenopus oocytes and in human cells failed to increase phosphate transport. Pull down experiments showed that E68A mutant did not interact with NPT2a, which robustly interacted with wild type NHERF1 and previously identified mutants. Biotinylation studies revealed that E68A mutant was unable to increase cell surface expression of NPT2a.ConclusionsOur results indicate that the PDZ1 domain is critical for NHERF1- NPT2a interaction in humans and for the control of NPT2a expression at the plasma membrane. Thus we have identified a new mechanism of renal phosphate loss and shown that different mutations in NHERF1 can alter renal phosphate reabsorption via distinct mechanisms.

Probing S4 Length Changes during Gating with LRET

Voltage-activated proteins containing a Voltage Sensor Domain (VSD) respond to changes in the membrane potential by transferring across the electric field several positively charged residues (gating charges) located in the fourth transmembrane segment (S4). Even though the mechanistic details of gating charge translocation and S4 re-arrangement are presently unclear, a prevailing hypothesis suggests that the S4 segment adopts a 310 helix conformation during gating, thus aligning the gating charges and facilitating their transport across the membrane electric field. If a whole typical S4 segment were to change its conformation from an α-helix to a 310 helix, its length would stretch by about 8 Å. Here we tested the existence of such transition by measuring the length of the S4 segment during gating using the LRET technique. We used the VSD domain of the Ciona intestinalis Voltage-Sensitive Phosphatase (CiVSP), truncated from its phosphatase and phospholipid-binding domains, to genetically encode a lanthanide (Tb3+)-binding-tag at the extracellular end of S4 and the red fluorescent protein mCherry at its intracellular end. We expressed the protein in Xenopus oocytes from which we recorded gating currents using the cut-open and two-electrode voltage-clamp techniques. The distance-dependent efficiency of energy transfer between the two probes was measured at steady-state voltages and also during voltage pulse protocols of varying amplitudes and durations. Our technique could only detect distance changes larger than 2.5 Å. We found that in our CiVSP construct, the results are not consistent with the expected length change if the whole S4 were converted (and remained converted) from a 310 helix to an α-helix (or vice-versa) when the membrane potential is changed from −100 to +80 mV. Supported by NIH GM68044-07 and GM30376-30S1.

Role of an apical K,Cl cotransporter in urine formation by renal tubules of the yellow fever mosquito (Aedes aegypti)

The K,Cl cotransporters (KCCs) of the SLC12 superfamily play critical roles in the regulation of cell volume, concentrations of intracellular Cl(-), and epithelial transport in vertebrate tissues. To date, the role(s) of KCCs in the renal functions of mosquitoes and other insects is less clear. In the present study, we sought molecular and functional evidence for the presence of a KCC in renal (Malpighian) tubules of the mosquito Aedes aegypti. Using RT-PCR on Aedes Malpighian tubules, we identified five alternatively spliced partial cDNAs that encode putative SLC12-like KCCs. The majority transcript is AeKCC1-A(1); its full-length cDNA was cloned. After expression of the AeKCC1-A protein in Xenopus oocytes, the Cl(-)-dependent uptake of (86)Rb(+) is 1) activated by 1 mM N-ethylmaleimide and cell swelling, 2) blocked by 100 μM dihydroindenyloxyalkanoic acid (DIOA), and 3) dependent upon N-glycosylation of AeKCC1-A. In Aedes Malpighian tubules, AeKCC1 immunoreactivity localizes to the apical brush border of principal cells, which are the predominant cell type in the epithelium. In vitro physiological assays of Malpighian tubules show that peritubular DIOA (10 μM): 1) significantly reduces both the control and diuretic rates of transepithelial fluid secretion and 2) has negligible effects on the membrane voltage and input resistance of principal cells. Taken together, the above observations indicate the presence of a KCC in the apical membrane of principal cells where it participates in a major electroneutral transport pathway for the transepithelial secretion of fluid in this highly electrogenic epithelium.

Biochemical Characterization of Pumilio1 and Pumilio2 in Xenopus Oocytes

Precise control of the timing of translational activation of dormant mRNAs stored in oocytes is required for normal progression of oocyte maturation. We previously showed that Pumilio1 (Pum1) is specifically involved in the translational control of cyclin B1 mRNA during Xenopus oocyte maturation, in cooperation with cytoplasmic polyadenylation element-binding protein (CPEB). It was reported that another Pumilio, Pumilio2 (Pum2), exists in Xenopus oocytes and that this protein regulates the translation of RINGO mRNA, together with Deleted in Azoospermia-like protein (DAZL). In this study, we characterized Pum1 and Pum2 biochemically by using newly produced antibodies that discriminate between them. Pum1 and Pum2 are bound to several key proteins involved in translational control of dormant mRNAs, including CPEB and DAZL, in immature oocytes. However, Pum1 and Pum2 themselves have no physical interaction. Injection of anti-Pum1 or anti-Pum2 antibody accelerated CPEB phosphorylation, cyclin B1 translation, and oocyte maturation. Pum1 phosphorylation coincides with the dissociation of CPEB from Pum1 and the translational activation of cyclin B1 mRNA, a target of Pum1, whereas Pum2 phosphorylation occurred at timing earlier than that for Pum1. Some, but not all, of cyclin B1 mRNAs release the deadenylase PARN during oocyte maturation, whereas Pum1 remains associated with the mRNA. On the basis of these findings, we discuss the functions of Pum1 and Pum2 in translational control of mRNAs during oocyte maturation.

Can one ‘Bad Egg’ really spoil the batch?

The oocyte from the South African clawed frog Xenopus laevis is a versatile expression system particularly suitable for the study of membrane transporters and channels. Due to minimal background activity and very high signal-to-noise ratios, oocytes are often used to evaluate the function, structure and signal transduction pathways of a variety of different proteins. One such group of proteins are the water channels, or aquaporins. Of particular note, studies in Xenopus oocytes have contributed to our understanding of an inherited form of nephrogenic diabetes insipidus. However, as highlighted by Leduc-Nadeau et al. (2010) in a recent issue of The Journal of Physiology, caution should be taken in interpreting the results of studies on membrane proteins in Xenopus oocytes.

The 2009 Lindau Nobel Laureate Meeting: Peter Agre, Chemistry 2003

Peter Agre, born in 1949 in Northfield Minnesota, shared the 2003 Nobel Prize in Chemistry with Roderick MacKinnon for his discovery of aquaporins, the channel proteins that allow water to cross the cell membrane.Agre's interest medicine was inspired by the humanitarian efforts of the Medical Missionary program run by the Norwegians of his home community in Minnesota. Hoping to provide new treatments for diseases affecting the poor, he joined a cholera laboratory during medical school at Johns Hopkins. He found that he enjoyed biomedical research, and continued his laboratory studies for an additional year after medical school.Agre completed his clinical training at Case Western Hospitals of Cleveland and the University of North Carolina, and returned to Johns Hopkins in 1981. There, his serendipitous discovery of aquaporins was made while pursuing the identity of the Rhesus (Rh) antigen.For a century, physiologists and biophysicists had been trying to understand the mechanism by which fluid passed across the cell's plasma membrane. Biophysical evidence indicated a limit to passive diffusion of water, suggesting the existence of another mechanism for water transport across the membrane. The putative "water channel," however, could not be identified.In 1988, while attempting to purify the 30kDa Rh protein, Agre and colleagues began investigating a 28 kDa contaminant that they believed to be a proteolytic fragment of the Rh protein. Subsequent studies over the next 3-4 years revealed that the contaminant was a membrane-spanning oligomeric protein, unrelated to the Rh antigen, and that it was highly abundant in renal tubules and red blood cells. Still, they could not assign a function to it.The breakthrough came following a visit with his friend and former mentor John Parker. After Agre described the properties of the mysterious 28 kDa protein, Parker suggested that it might be the long-sought-after water channel. Agre and colleagues tested this idea by expressing the protein in Xenopus oocytes, which typically have low water permeability. When the test oocytes were placed in a hypotonic solution, they swelled and exploded, thus revealing the function of the unknown protein as a water channel, which they named aquaporin.The Nobel Prize enabled Agre to take his research and scientific interests in new directions. He felt that over the years his work had continually taken him further from his original interests in third-world diseases, so he shifted his focus back in that direction. He now serves as the director of the Malaria institute at Johns Hopkins where he has applied his knowledge to the study of the malarial parasite and the Anopheles mosquito, which both express aquaporins. In addition, since winning the Nobel Prize, he has enjoyed increased opportunities for bringing science to the public and for "encouraging young people to go into science."

Renal and Brain Isoforms of WNK3 Have Opposite Effects on NCCT Expression

Mutations in the WNK kinases WNK1 and WNK4 cause a rare familial form of hypertension (Gordon syndrome) by increasing expression of the thiazide-sensitive co-transporter NCCT in the kidney. Regulation of NCCT expression involves a scaffold of proteins composed of several kinases, including the third member of the WNK kinase family, WNK3. This protein, expressed in several tissues including kidney and brain, displays splice variation around exons 18 and 22. We expressed these proteins in Xenopus oocytes and found that the renal isoform of WNK3 increased but the brain isoform decreased NCCT expression and activity. Introduction of a kinase-inactivating mutation into renal WNK3 reversed its action on NCCT, and the same mutation in the brain isoforms led to loss of function. We also studied the effect of phosphorylation of a key NCCT threonine (T58) on the effects of WNK3/4 coexpression; NCCT mutants with a T58A or T58D substitution had the same surface expression as T58 but had significantly altered transporter activity; however, both isoforms of WNK3 as well as WNK4 still modulated expression of these NCCT mutants. Finally, experiments using kinase-dead STE20/SPS1-related proline/alanine-rich kinase (SPAK), a putative downstream target for WNKs, revealed that brain WNK3 acts in tandem with SPAK, whereas renal WNK3 seems to upregulate NCCT through a SPAK-independent pathway. Taken together, these results suggest that the C-terminal motifs contributed by exons 18 and 22 play an important role in the actions of WNK3 isoforms on NCCT.

Differential modulation of cardiac potassium channels by Grb adaptor proteins

Scaffolding growth factor receptor-bound (Grb) adaptor proteins are components of macromolecular signaling complexes at the plasma membrane and thus are putative regulators of ion channel activity. The present study aimed to define the impact of Grb adaptor proteins on the function of cardiac K + channels. To this end channel proteins were coinjected with the adaptor proteins in Xenopus oocytes and channel activity analyzed with two-electrode voltage-clamp. It is shown that coexpression of Grb adaptor proteins can reduce current amplitudes of coexpressed channels. Grb7 and 10 significantly inhibited functional currents generated by hERG, Kv1.5 and Kv4.3 channels. Only Grb10 significantly inhibited KCNQ1/KCNE1 K + channels, and only Grb7 reduced Kir2.3 activity, whereas neither Grb protein significantly affected the closely related Kir2.1 and Kir2.2 channels. The present observations for the first time provide evidence for a selective and modulatory role of Grb adaptor proteins in the functional expression of cardiac K + channels.

Expression of ENaC and other Transport Proteins in Xenopus Oocytes is Modulated by Intracellular Na+

The expression of the epithelial Na⁺ channel (ENaC) is tissue-specific and dependent on a variety of mediators and interacting proteins. Here we examined the role of intracellular Na⁺ ([Na⁺]_i) as a modulator of the expression of rat ENaC in Xenopus laevis oocytes. We manipulated [Na⁺]_i of ENaC-expressing oocytes in the range of 0-20 mM by incubating in extracellular solutions of different [Na⁺]_o. Electrophysiological, protein biochemical and fluorescence optical methods were used to determine the effects of different [Na⁺]_i on ENaC expression and membrane abundance. In voltage-clamp experiments we found that amiloride-sensitive ENaC current (I_ami) and conductance (G_ami) peak at a [Na⁺]_i of ∼10 mM Na⁺, but were significantly reduced in 5 mM and 20 mM [Na⁺]_i. Fluorescence intensity of EGFP-ENaC-expressing oocytes also followed a bell-shaped curve with a maximum at ∼ 10 mM [Na⁺]_i. In Western blot experiments with specific anti-ENaC antibodies the highest protein expression was found in ENaC-expressing oocytes with [Na⁺]_i of 10-15 mM. Since ENaC is also highly permeable for Li⁺, we incubated ENaC-expressing oocytes in different Li⁺ concentrations and found a peak of I_ami and G_ami with 5 mM Li⁺. The influence of [Na⁺]_i on the expression is not ENaC-specific, since expression of a Cl^- channel (CFTR) and a Na⁺/glucose cotransporter (SGLT1) showed the same dependence on [Na⁺]_i. We conclude that specific concentrations of Na⁺ and Li⁺ influence the expression and abundance of ENaC and other transport proteins in the plasma membrane in Xenopus laevis oocytes. Furthermore, we suggest the existence of a general mechanism dependent on monovalent cations that optimizes the expression of membrane proteins.

Expression cloning and radiotracer uptakes in Xenopus laevis oocytes

This protocol describes the method of expression cloning of heterologous proteins using Xenopus laevis oocytes and the functional characterization of membrane proteins using radiotracer assays. It can be used to isolate proteins for which sequence data is unavailable and to characterize the functions of proteins. A cDNA library is generated, that is, translated into proteins in Xenopus oocytes, and the function of these proteins is assessed by a radiotracer assay. Their large size, high degree of expression and ease of handling makes Xenopus oocytes an optimal tool for the expression and characterization of protein function when compared with traditional expression systems, such as Escherichia coli, yeast or eukaryotic cell lines. The expected results of this technique include the following: functional identification of novel proteins; molecular (kinetic) characterization of protein function; and determination of functionally relevant residues and domains of membrane proteins, including transporters, ion channels and receptors. The identification of novel proteins can take several months, whereas functional characterization in Xenopus oocytes can take 1 week.

Functional and structural characterization of the zebrafish Na+-sulfate cotransporter 1 (NaS1) cDNA and gene (slc13a1)

Sulfate plays an essential role during growth, development and cellular metabolism. In this study, we characterized the function and structure of the zebrafish (Danio rerio) Na+-sulfate cotransporter 1 (NaS1) cDNA and gene (slc13a1). Zebrafish NaS1 encodes a protein of 583 amino acids with 13 putative transmembrane domains. Expression of zebrafish NaS1 protein in Xenopus oocytes led to Na+-sulfate cotransport, which was significantly inhibited by thiosulfate, selenate, molybdate, and tungstate. Zebrafish NaS1 transport kinetics were: V(max) = 1,731.670 +/- 92.853 pmol sulfate/oocyte.hour and K(m) = 1.414 +/- 0.275 mM for sulfate and V(max) = 307.016 +/- 32.992 pmol sulfate/oocyte x hour, K(m) = 24.582 +/- 4.547 mM and n (Hill coefficient) = 1.624 +/- 0.354 for sodium. Zebrafish NaS1 mRNA is developmentally expressed in embryos from day 1 postfertilization and in the intestine, kidney, brain, and eye of adult zebrafish. The zebrafish NaS1 gene slc13a1 contains 15 exons spanning 8,716 bp. Characterization of the zebrafish NaS1 contributes to a greater understanding of sulfate transporters in a well-defined genetic model and will allow the elucidation of evolutionary and functional relationships among vertebrate sulfate transporters.