Injection Of cRNA Research Articles

A heterozygous SPRY4 variant identified in female infertility characterized by reduced oocyte potential and early embryonic arrest.

Can novel genetic factors contributing to early embryonic arrest in infertile patients be identified, along with the underlying mechanisms of the pathogenic variant? We identified a heterozygous variant in the SPRY4 (sprouty RTK signaling antagonist 4) in infertile patients and conducted in vitro and in vivo studies to investigate the effects of the variant/deletion, highlighting its critical role in female reproductive health. SPRY4 acts as a negative regulator of receptor tyrosine kinases (RTKs) and functions as a tumor suppressor. Its abnormal expression can lead to recurrent miscarriage by affecting trophoblast function. In mice, Spry4 knockout (KO) leads to craniofacial anomalies and growth defects. A human study links the SPRY4 variant to a male patient with isolated hypogonadotropic hypogonadism (IHH), hypothetically impacting gonadotropin-releasing hormone (GnRH) neurons, and causing reproductive dysfunctions. SPRY4 is thus potentially integral in regulating endocrine homeostasis and reproductive function. To date, no study has reported SPRY4 variants associated with female fertility, and a causal relationship has not been established with functional evidence. Whole-exome sequencing (WES) was performed in 392 infertile women who suffered from primary infertility of unknown reason, and the heterozygous SPRY4 variant were identified in one independent family. The infertile patients presenting were recruited from July 2017 to November 2023. Women diagnosed with primary infertility were recruited from the Reproduction Center of Zhongshan Hospital, Fudan University. Genomic DNA was extracted from peripheral blood for WES analysis. The SPRY4 variant were identified through WES, in silico analysis, and variant screening. All variants were confirmed by Sanger sequencing. The effects of the variants were investigated in human embryonic kidney (HEK) 293T (HEK293T) cells via western blotting, and in mouse oocytes and embryos through complementary RNA (cRNA) injection, RNA sequencing, fluorescence, absorbance, and RT-qPCR assays. Gene function was further examined in Spry4 KO mice via histology, western blotting, ELISA, and RT-qPCR assays. We identified a missense heterozygous pathogenic variant in SPRY4 (GRCh38, GenBank: NM_030964.5, c.157C>T p.(Arg53Trp), rs200531302) that reduces SPRY4 protein levels in HEK293T cells and disrupts the redox system and mitochondrial function in mouse oocyte, and perturbs developmental potential in mouse embryos. These phenotypes could be partially reversed by the exogenous addition of Nrf1 cRNA. Additionally, Spry4-/- mice exhibit ovarian oxidative stress and decreased ovarian function. Due to the limited WES data and population, we identified only one family with a SPRY4 mutation. The deeper mechanism and therapeutic strategy should be further investigated through mutant mice and recovery experiment. Our study has identified a pathogenic variant in SPRY4 associated with early embryonic arrest in humans. These findings enhance our understanding of the role of SPRY4 in early embryonic development and present a new genetic marker for female infertility. This work was supported by the National Natural Science Foundation of China (82071643 and 82171655) and Natural Science Foundation of Shanghai (22ZR1456200). None of the authors have any competing interests. N/A.

O-057 Newly identified female mutations causing failed fertilization

Abstract Over the past decade, advancements in next-generation sequencing technology, alongside reduced costs, and the rising demand for infertility medical care, have resulted in the discovery of new genes and genetic variants linked to infertility conditions. Phenotypes of ICSI failure, such as oocyte maturation arrest (OMA), fertilization failure (FF) and embryo developmental arrest (EDA), can now be attributed to male or female genetic causes. This represents a transformative shift in patient treatment, allowing the identification of some cases previously classified as unexplained infertility, thereby reducing patient stress. Moreover, it facilitates personalized treatment recommendations, as well as paves the way for the exploration of novel treatment options, thus expanding research opportunities. This talk will review the genetic causes of FF following ICSI, with the main focus on the newly identified female causes, as well as give an update on current diagnostic tests and treatment options for this infertility condition. FF after ICSI still occurs in 1-3% of ICSI cycles, primarily due to oocyte activation deficiency (OAD). Defects in the male PLCζ protein, responsible of inducing calcium oscillations during oocyte activation, have long been known as the leading cause of OAD. Recent discoveries have identified genetic variants in the male genes ACTL7A, ACTL9 and IQCN, which also reduce PLCζ protein content in sperm cells and thus, contribute to FF after ICSI. Additionally, for the first time in 2018, mutations in a female gene (WEE2) crucial for oocyte activation were identified. WEE2 is an oocyte kinase that acts downstream the calcium release during fertilization. Interestingly, genetic variants in other female genes, such as PATL2, TUBB8 and CDC20 associated mainly to OMA and TLE6 and NLRP5 associated mainly to EDA, have also been described to cause failed fertilization in some patients. Determining whether the deficiency lies in sperm- or oocyte-related genes is crucial for tailoring the most effective treatment for FF after ICSI. For sperm-related factors, assisted oocyte activation (AOA), which consists in the artificial induction of calcium oscillations using calcium ionophores, has proven highly beneficial. However, most patients with oocyte-factors do not benefit from AOA (e.g. patients with WEE2 variants). In this regard, wild-type cRNA injection of the defected protein has been shown to rescue fertilization. A more generalist approach, such as maternal spindle transfer, which consists in replacing poor-quality oocyte cytoplasm with healthy cytoplasm from a donor oocyte, represents a potential treatment for these patients, as recently demonstrated in infertile females with repeated IVF failures. Nevertheless, further research into the safety of these procedures is needed before clinical application, and for now, gamete donation should be considered when facing female-related FF.

KAT8 functions in redox homeostasis and mitochondrial dynamics during mouse oocyte meiosis progression.

As a histone acetyltransferase, lysine acetyltransferase 8 (KAT8) participates in diverse biological processes. However, the effect of KAT8 on oocyte maturation in mice remains unclear. In this study, we found that mouse oocytes overexpressing Kat8-OE induced maturation failure manifested reduced rates of GVBD and first polar body emission. In addition, immunostaining results revealed that Kat8 overexpressing oocytes showed inappropriate mitochondrial distribution patterns, overproduction of reactive oxygen species (ROS), accumulation of phosphorylated γH2AX, hyperacetylation of α-tubulin, and severely disrupted spindle/chromosome organization. Moreover, we revealed that Kat8 overexpression induced a decline in SOD1 proteins and KAT8's interaction with SOD1 in mouse ovaries via immunoprecipitation. Western blotting data confirmed that Kat8-OE induced downregulation of SOD1 expression, which is a key factor for the decline of oocyte quality in advanced maternal age. Also, the injection of Myc-Sod1 cRNA could partially rescue maternal age-induced meiotic defects in oocytes. In conclusion, our data demonstrated that high level of KAT8 inhibited SOD1 activity, which in turn induced defects of mitochondrial dynamics, imbalance of redox homeostasis, and spindle/chromosome disorganization during mouse oocyte maturation.

Exonic genetic variants associated with unexpected fertilization failure and zygotic arrest after ICSI: a systematic review.

Fertilization failure (FF) and zygotic arrest after ICSI have a huge effect on both patients and clinicians, but both problems are usually unexpected and cannot be properly diagnosed. Fortunately, in recent years, gene sequencing has allowed the identification of multiple genetic variants underlying failed ICSI outcomes, but the use of this approach is still far from routine in the fertility clinic. In this systematic review, the genetic variants associated with FF, abnormal fertilization and/or zygotic arrest after ICSI are compiled and analyzed. Forty-seven studies were included. Data from 141 patients carrying 121 genetic variants affecting 16 genes were recorded and analyzed. In total, 27 variants in PLCZ1 (in 50 men) and 26 variants in WEE2 (in 24 women) are two of the factors related to oocyte activation failure that could explain a high percentage of male-related and female-related FF. Additional variants identified were reported in WBP2NL, ACTL9, ACTLA7, and DNAH17 (in men), and TUBB8, PATL2, TLE6, PADI6, TRIP13, BGT4, NLRP5, NLRP7, CDC20 and ZAR1 (in women). Most of these variants are pathogenic or potentially pathogenic (89/121, 72.9%), as demonstrated by experimental and/or in silico approaches. Most individuals carried bi-allelic variants (89/141, 63.1%), but pathogenic variants in heterozygosity have been identified for PLCZ1 and TUBB8. Clinical treatment options for affected individuals, such as chemical-assisted oocyte activation (AOA) or PLCZ1 cRNA injection in the oocyte, are still experimental. In conclusion, a genetic study of known pathogenic variants may help in diagnosing recurrent FF and zygotic arrest and guide patient counselling and future research perspectives.

The COVID-19 viral 3a protein forms a potassium channel that can be inhibited by antiarrhythmic drugs

Funding AcknowledgementsType of funding sources: Public Institution(s). Main funding source(s): Deutsche Gesellschaft für Kardiologie /German Cardiac Society Joachim Herz Stiftung /Joachim Herz FoundationBackground/IntroductionSupraventricular and ventricular arrhythmias can often be observed in patients with COVID-19 infection. Both, the clinically observed increase in cardiac biomarkers as well as histological studies indicate virus replication within cardiomyocytes. The 3a open reading frame of the viral genome encodes for a transmembrane protein that is transported to the cell membrane where it can serve as a potassium channel.PurposeThe aim of this study was to investigate whether COVID-19 infected induced pluripotent stem cell (iPSC)-derived cardiomyocytes also express the 3a protein and whether the potassium currents that are conducted through the 3a protein can be inhibited by clinically used antiarrhythmic drugs.Methods and ResultsiPSC-derived cardiomyocytes were infected with COVID-19 and subsequently subjected to immunoblotting, where expression of the 3a protein could be observed. Plasmid DNA, encoding the COVID-19 3a protein, was generated by gene synthesis and used for in vitro transcription of cRNA. 3–5 days after intracytoplasmic injection of the 3a protein cRNA into Xenopus laevis oocytes, potassium currents could be measured by two-electrode voltage clamp recordings. While class I and class IV antiarrhythmic drugs showed only minor effects on the potassium currents of the 3a protein, a robust inhibition by several beta-blockers and by class III antiarrhythmic drugs could be observed. The strongest effects were found with dofetilide (58.1 % inhibition at 100 µM) and amiodarone (50.1 % inhibition at 100 µM, IC50 level 4.7 µM). An in silico docking analysis, based on the recently revealed crystal structure of the 3a protein, identified the amino acid residues K61 and D142 as part of the binding site of amiodarone. After deactivation of these amino acid residues by site-directed mutagenesis, the inhibition by amiodarone was significantly attenuated.ConclusionThe COVID-19 viral 3a protein is expressed in COVID-19-infected iPS-derived cardiomyocytes and forms a potassium channel that can be inhibited by antiarrhythmic drugs.

Maternal Prkce expression in mature oocytes is critical for the first cleavage facilitating maternal‐to‐zygotic transition in mouse early embryos

ObjectivesEarly embryo development is dependent on the regulation of maternal messages stored in the oocytes during the maternal‐to‐zygote transition. Previous studies reported variability of oocyte competence among different inbred mouse strains. The present study aimed to identify the maternal transcripts responsible for early embryonic development by comparing transcriptomes from oocytes of high‐ or low‐ competence mouse strains.Materials and Methods In vitro fertilization embryos from oocytes of different mouse strains were subject to analysis using microarrays, RNA sequencing, real‐time quantitative PCR (RT‐qPCR) analysis, Western blotting, and immunofluorescence. One candidate gene, Prkce, was analysed using Prkce knockout mice, followed by a cRNA rescue experiment.ResultsThe fertilization and 2‐cell rate were significantly higher for FVB/NJ (85.1% and 82.0%) and DBA/2J (79.6% and 76.7%) inbred mouse strains than those for the MRL/lpr (39.9% and 35.8%) and 129S3 (35.9% and 36.6%) strains. Thirty‐nine differentially expressed genes (DEGs) were noted, of which nine were further verified by RT‐qPCR. Prkce knockout mice showed a reduced 2‐cell rate (Prkce +/+ 80.1% vs. Prkce −/− 32.4%) that could be rescued by Prkce cRNA injection (2‐cell rate reached 76.7%). Global transcriptional analysis revealed 143 DEGs in the knockout mice, which were largely composed of genes functioning in cell cycle regulation.ConclusionsThe transcription level of maternal messages such as Prkce in mature oocytes is associated with different 2‐cell rates in select inbred mouse strains. Prkce transcript levels could serve as a potential biomarker to characterize high‐quality mature oocytes.

Inositol-1,4,5-Trisphosphate Receptor-1 and -3 and Ryanodine Receptor-3 May Increase Ooplasmic Ca2+ During Quail Egg Activation.

We previously reported that egg activation in Japanese quail is driven by two distinct types of intracellular Ca2+ ([Ca2+]i): transient elevations in [Ca2+]i induced by phospholipase Czeta 1 (PLCZ1) and long-lasting spiral-like Ca2+ oscillations by citrate synthase (CS) and aconitate hydratase 2 (ACO2). Although the blockade of inositol 1,4,5-trisphosphate receptors (ITPRs) before microinjections of PLCZ1, CS, and ACO2 cRNAs only prevented transient increases in [Ca2+]i, a microinjection of an agonist of ryanodine receptors (RYRs) induced spiral-like Ca2+ oscillations, indicating the involvement of both ITPRs and RYRs in these events. In this study, we investigated the isoforms of ITPRs and RYRs responsible for the expression of the two types of [Ca2+]i increases. RT-PCR and western blot analyses revealed that ITPR1, ITPR3, and RYR3 were expressed in ovulated eggs. These proteins were degraded 3 h after the microinjection of PLCZ1, CS, and ACO2 cRNAs, which is the time at which egg activation was complete. However, degradation of ITPR1 and ITPR3, but not RYR3, was initiated 30 min after a single injection of PLCZ1 cRNA, corresponding to the time of the initial Ca2+ wave termination. In contrast, RYR3 degradation was observed 3 h after the microinjection of CS and ACO2 cRNAs. These results indicate that ITPRs and RYR3 differentially mediate in creases in [Ca2+]i during egg activation in Japanese quail, and that downregulation of ITPRs and RYR3-mediated events terminate the initial Ca2+ wave and spiral-like Ca2+ oscillations, respectively.

Deletion of TRPV3 and CaV3.2 T-type channels in mice undermines fertility and Ca2+ homeostasis in oocytes and eggs.

Ca2+ influx during oocyte maturation and after sperm entry is necessary to fill the internal Ca2+ stores and for complete egg activation. We knocked out the transient receptor potential vanilloid member 3 (TRPV3) and the T-type channel, CaV3.2, to determine their necessity for maintaining these functions in mammalian oocytes/eggs. Double-knockout (dKO) females were subfertile, their oocytes and eggs showed reduced internal Ca2+ stores, and, following sperm entry or Plcz (also known as Plcz1) cRNA injection, fewer dKO eggs displayed Ca2+ responses compared to wild-type eggs, which were also of lower frequency. These parameters were rescued and/or enhanced by removing extracellular Mg2+, suggesting that the residual Ca2+ influx could be mediated by the TRPM7 channel, consistent with the termination of divalent-cation oscillations in dKO eggs by a TRPM7 inhibitor. In total, we demonstrated that TRPV3 and CaV3.2 mediate the complete filling of the Ca2+ stores in mouse oocytes and eggs. We also showed that they are required for initiating and maintaining regularly spaced-out oscillations, suggesting that Ca2+ influx through PM ion channels dictates the periodicity and persistence of Ca2+ oscillations during mammalian fertilization.

Functional Characterization of the Arabidopsis Abscisic Acid Transporters NPF4.5 and NPF4.6 in Xenopus Oocytes.

Few proteins have been characterized as abscisic acid transporters. Several of them are NRT1/PRT Family (NPF) transporters which have been characterized in yeast using reporter systems. Because several members of the NPF4 subfamily members were identified in yeast as ABA transporters, here, we screened for ABA transport activity the seven members of the NPF4 subfamily in Xenopus oocytes using cRNA injection and 3H-ABA accumulation. The ABA transport capacities of NPF4.2, NPF4.5, NPF4.6, and NPF4.7 were confirmed. The transport properties of NPF4.5 and NPF4.6 were studied in more detail. Both ABA transporter activities are pH-dependent and slightly pH-dependent apparent Km around 500 μM. There is no competitive inhibition of the ABA-analogs pyrabactin and quinabactin on ABA accumulation demonstrating a different selectivity compared to the ABA receptors. Functional expression of these ABA transporters in Xenopus oocyte is an opportunity to start structure–function studies and also to identify partner proteins of these hormone transporters.

Leishmania heme uptake involves LmFLVCRb, a novel porphyrin transporter essential for the parasite.

Leishmaniasis comprises a group of neglected diseases caused by the protozoan parasite Leishmania spp. As is the case for other trypanosomatid parasites, Leishmania is auxotrophic for heme and must scavenge this essential compound from its human host. In mammals, the SLC transporter FLVCR2 mediates heme import across the plasma membrane. Herein we identify and characterize Leishmania major FLVCRb (LmFLVCRb), the first member of the FLVCR family studied in a non-metazoan organism. This protein localizes to the plasma membrane of the parasite and is able to bind heme. LmFLVCRb levels in Leishmania, which are modulated by overexpression thereof or the abrogation of an LmFLVCRb allele, correlate with the ability of the parasite to take up porphyrins. Moreover, injection of LmFLVCRb cRNA to Xenopus laevis oocytes provides these cells with the ability to take up heme. This process is temperature dependent, requires monovalent ions and is inhibited at basic pH, characteristics shared by the uptake of heme by Leishmania parasites. Interestingly, LmFLVCRb is essential as CRISPR/Cas9-mediated knockout parasites were only obtained in the presence of an episomal copy of the gene. In addition, deletion of just one of the alleles of the LmFLVCRb gene markedly impairs parasite replication as intracellular amastigotes as well as its virulence in an in vivo model of cutaneous leishmaniasis. Collectively, these results show that Leishmania parasites can rescue heme through plasma membrane transporter LFLVCRb, which could constitute a novel target for therapeutic intervention against Leishmania and probably other trypanosomatid parasites in which FLVCR genes are also present.

Novel phospholipase C zeta 1 mutations associated with fertilization failures after ICSI.

Are phospholipase C zeta 1 (PLCZ1) mutations associated with fertilization failure (FF) after ICSI? New mutations in the PLCZ1 sequence are associated with FFs after ICSI. FF occurs in 1-3% of ICSI cycles, mainly due to oocyte activation failure (OAF). The sperm PLCζ/PLCZ1 protein hydrolyzes phosphatidylinositol (4, 5)-bisphosphate in the oocyte, leading to intracellular calcium release and oocyte activation. To date, few PLCZ1 point mutations causing decreased protein levels or activity have been linked to FF. However, functional alterations of PLCζ/PLCZ1 in response to both described and novel mutations have not been investigated. We performed a study including 37 patients presenting total or partial FF (fertilization rate (FR), ≤25%) after ICSI occurring between 2014 and 2018. Patients were divided into two groups based on oocyte evaluation 19h post ICSI: FF due to a defect in oocyte activation (OAF, n = 22) and FF due to other causes ('no-OAF', n = 15). Samples from 13 men with good fertilization (FR, >50%) were used as controls. PLCζ/PLCZ1 protein localization and levels in sperm were evaluated by immunofluorescence and western blot, respectively. Sanger sequencing on genomic DNA was used to identify PLCZ1 mutations in exonic regions. The effect of the mutations on protein functionality was predicted in silico using the MODICT algorithm. Functional assays were performed by cRNA injection of wild-type and mutated forms of PLCZ1 into human in vitro matured metaphase II oocytes, and fertilization outcomes (second polar body extrusion, pronucleus appearance) scored 19h after injection. In the OAF group, 12 (54.6%) patients carried at least one mutation in the PLCZ1 coding sequence, one patient out of 15 (6.7%) in the no-OAF group (P < 0.05) and none of the 13 controls (P < 0.05). A total of six different mutations were identified. Five of them were single-nucleotide missense mutations: p.I120M, located at the end of the EF-hand domain; p.R197H, p.L224P and p.H233L, located at the X catalytic domain; and p.S500L, located at the C2 domain. The sixth mutation, a frameshift variant (p.V326K fs*25), generates a truncated protein at the X-Y linker region. In silico analysis with MODICT predicted all the mutations except p.I120M to be potentially deleterious for PLCζ/PLCZ1 activity. After PLCZ1 cRNA injection, a significant decrease in the percentage of activated oocytes was observed for three mutations (p.R197H, p.H233L and p.V326K fs*25), indicating a deleterious effect on enzymatic activity. PLCZ1 protein localization and expression levels in sperm were similar across groups. FRs were restored (to >60%) in patients carrying PLCZ1 mutations (n = 10) after assisted oocyte activation (AOA), with seven patients achieving pregnancy and live birth. Caution should be exerted when comparing the cRNA injection results with fertilization outcomes after ICSI, especially in patients presenting mutations in heterozygosis. PLCZ1 mutations were found in high frequency in patients presenting OAF. Functional analysis of three mutations in human oocytes confirms alteration of PLCζ/PLCZ1 activity and their likely involvement in impaired oocyte activation. Our results suggest that PLCZ1 gene sequencing could be useful as a tool for the diagnosis and counseling of couples presenting FF after ICSI due to OAF. This work was supported by intramural funding of Clínica EUGIN, by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 049 to M. T.-M. and GENCAT 2015 DI 048 to D. C.-B.) and by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness to A. F.-V. No competing interest declared.

In vivo masculinizing function of the Ostrinia furnacalis Masculinizer gene

The Masculinizer gene (Masc) encodes a CCCH tandem zinc finger protein essential for masculinization and dosage compensation in the silkworm Bombyx mori. Previously we identified a Masc orthologue from the crambid Ostrinia furnacalis (OfMasc) and observed its masculinizing activity in the B. mori cultured cell line BmN-4. However, the role of OfMasc in masculinization of O. furnacalis has not been assessed. In this study, we unexpectedly discovered that all of the male larvae that escaped from Wolbachia-induced embryonic male-killing by OfMasc cRNA injection expressed the female-type splicing variants of O. furnacalis doublesex (Ofdsx). To clarify the role of OfMasc in the masculinization process in vivo, we established a system to monitor both sex chromosome- and dsx splicing-based sexes from a single O. furnacalis embryo. Using this system, we investigated the effects of OfMasc knockdown in early embryos on Ofdsx splicing and found that depletion of OfMasc mRNA in male embryos induced the production of the female-type splicing variants of Ofdsx. This result indicates that OfMasc is required for masculinization in O. furnacalis, and that the Masc protein possesses masculinizing activity in an insect species that is phylogenetically distant from Bombycidae.

Homozygous Mutations in WEE2 Cause Fertilization Failure and Female Infertility

Fertilization is a fundamental process of development and is a prerequisite for successful human reproduction. In mice, although several receptor proteins have been shown to play important roles in the process of fertilization, only three genes have been shown to cause fertilization failure and infertility when deleted invivo. In clinical practice, some infertility case subjects suffer from recurrent failure of invitro fertilization and intracytoplasmic sperm injection attempts due to fertilization failure, but the genetic basis of fertilization failure in humans remains largely unknown. Wee2 is a key oocyte-specific kinase involved in the control of meiotic arrest in mice, but WEE2 has not been associated with any diseases in humans. In this study, we identified homozygous mutations in WEE2 that are responsible for fertilization failure in humans. All four independent affected individuals had homozygous loss-of-function missense mutations or homozygous frameshift protein-truncating mutations, and the phenotype of fertilization failure was shown to follow a Mendelian recessive inheritance pattern. All four mutations significantly decreased the amount of WEE2 protein invitro and in affected individuals' oocytes invivo, and they all led to abnormal serine phosphorylation of WEE2 and reduced tyrosine 15 phosphorylation of Cdc2 invitro. In addition, injection of WEE2 cRNA into affected individuals' oocytes rescued the fertilization failure phenotype and led to the formation of blastocysts invitro. This work presents a novel gene responsible for human fertilization failure and has implications for future therapeutic treatments for infertility cases.

SGK3 Sensitivity of Large-Conductance Ca2+-Activated K+ Channel

Background/Aims: Large conductance Ca²⁺-activated K⁺ channels (maxi K⁺ channels or BK channels) are rapidly activated by increase of cytosolic Ca²⁺ activity. The channels participate in the regulation of diverse functions including neuronal excitation and cell volume. The BK channels may be modified by kinases. Channel regulating kinases include the serum & glucocorticoid inducible kinase 3 (SGK3). The present study explored whether SGK3 modifies the activity of BK channels. Methods: cRNA encoding the Ca²⁺ insensitive BK channel mutant BK^{M513I+Δ899-903} was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type SGK3, constitutively active ^S419DSGK3, or catalytically inactive ^K191NSGK3. K⁺ channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel activity in BK^{M513I+Δ899-903} expressing oocytes was significantly increased by co-expression of SGK3 or active ^S419DSGK3, but not by coexpression of inactive ^K191NSGK3. Conclusion: SGK3 is a novel positive regulator of BK channels, and thus participates in the regulation of cell volume and excitability.

Up-Regulation of the Excitatory Amino Acid Transporters EAAT1 and EAAT2 by Mammalian Target of Rapamycin

Background: The excitatory amino-acid transporters EAAT1 and EAAT2 clear glutamate from the synaptic cleft and thus terminate neuronal excitation. The carriers are subject to regulation by various kinases. The EAAT3 isoform is regulated by mammalian target of rapamycin (mTOR). The present study thus explored whether mTOR influences transport by EAAT1 and/or EAAT2. Methods: cRNA encoding wild type EAAT1 (SLC1A3) or EAAT2 (SLC1A2) was injected into Xenopus oocytes without or with additional injection of cRNA encoding mTOR. Dual electrode voltage clamp was performed in order to determine electrogenic glutamate transport (I_EAAT). EAAT2 protein abundance was determined utilizing chemiluminescence. Results: Appreciable I_EAAT was observed in EAAT1 or EAAT2 expressing but not in water injected oocytes. I_EAAT was significantly increased by coexpression of mTOR. Coexpression of mTOR increased significantly the maximal I_EAAT in EAAT1 or EAAT2 expressing oocytes, without significantly modifying affinity of the carriers. Moreover, coexpression of mTOR increased significantly EAAT2 protein abundance in the cell membrane. Conclusions: The kinase mTOR up-regulates the excitatory amino acid transporters EAAT1 and EAAT2.

Up-Regulation of Excitatory Amino Acid Transporters EAAT3 and EAAT4 by Lithium Sensitive Glycogen Synthase Kinase GSK3ß

Background: Cellular uptake of glutamate by the excitatory amino-acid transporters (EAATs) decreases excitation and thus participates in the regulation of neuroexcitability. Kinases impacting on neuronal function include Lithium-sensitive glycogen synthase kinase GSK3ß. The present study thus explored whether the activities of EAAT3 and/or EAAT4 isoforms are sensitive to GSK3ß. Methods: cRNA encoding wild type EAAT3 (SLC1A1) or EAAT4 (SLC1A6) was injected into Xenopus oocytes without or with additional injection of cRNA encoding wild type GSK3ß or the inactive mutant ^K85AGSK3ß. Dual electrode voltage clamp was performed in order to determine glutamate-induced current (I_EAAT). Results: Appreciable I_EAAT was observed in EAAT3 or EAAT4 expressing but not in water injected oocytes. I_EAAT was significantly increased by coexpression of GSK3ß but not by coexpression of ^K85AGSK3ß. Coexpression of GSK3ß increased significantly the maximal I_EAAT in EAAT3 or EAAT4 expressing oocytes, without significantly modifying apparent affinity of the carriers. Lithium (1 mM) exposure for 24 hours decreased I_EAAT in EAAT3 and GSK3ß expressing oocytes to values similar to I_EAAT in oocytes expressing EAAT3 alone. Lithium did not significantly modify I_EAAT in oocytes expressing EAAT3 without GSK3ß. Conclusions: Lithium-sensitive GSK3ß is a powerful regulator of excitatory amino acid transporters EAAT3 and EAAT4.

Up-Regulation of the Large-Conductance Ca2+-Activated K+ Channel by Glycogen Synthase Kinase GSK3β

Background/Aims: The pleotropic functions of the large conductance Ca²⁺-activated K⁺ channels (maxi K⁺ channel or BK channels) include regulation of neuronal excitation and cell volume. Kinases participating in those functions include the glycogen synthase kinase GSK3 ß which is under negative control of protein kinase B (PKB/Akt). GSK3ß is inhibited by the antidepressant Lithium. The present study thus explored whether GSK3ß modifies the activity of BK channels. Methods: cRNA encoding the Ca²⁺ insensitive BK channel mutant BK^{M513I+Δ899-903} was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type GSK3ß, inactive ^K85RGSK3ß, or wild-type GSK3ß with wild-type PKB. K⁺ channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel activity in BK^{M513I+Δ899-903} expressing oocytes was significantly increased by co-expression of GSK3ß, but not by co-expression of ^K85RGSK3ß. The effect of wild type GSK3ß was abrogated by additional co-expression of wild-type PKB and by 24 hours incubation with Lithium (1 mM). Disruption of channel insertion into the cell membrane by brefeldin A (5 µM) was followed by a decline of the current to a similar extent in oocytes expressing BK and GSK3ß and in oocytes expressing BK alone. Conclusion: GSK3ß may up-regulate BK channels, an effect disrupted by Lithium or additional expression of PKB and possibly participating in the regulation of cell volume and excitability.

OSR1 and SPAK Sensitivity of Large-Conductance Ca2+ Activated K+ Channel

Background/Aims: The oxidative stress-responsive kinase 1 (OSR1) and the serine/threonine kinases SPAK (SPS1-related proline/alanine-rich kinase) are under the control of WNK (with-no-K [Lys]) kinases. OSR1 and SPAK participate in diverse functions including cell volume regulation and neuronal excitability. Cell volume and neuronal excitation are further modified by the large conductance Ca²⁺-activated K⁺ channels (maxi K⁺ channel or BK channels). An influence of OSR1 and/or SPAK on BK channel activity has, however, never been shown. The present study thus explored whether OSR1 and/or SPAK modify the activity of BK channels. Methods: cRNA encoding the Ca²⁺ insensitive BK channel mutant BK^{M513I+Δ899-903} was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type OSR1 or wild-type SPAK, constitutively active ^T185EOSR1, catalytically inactive ^D164AOSR1, constitutively active ^T233ESPAK or catalytically inactive ^D212ASPAK. K⁺ channel activity was measured utilizing dual electrode voltage clamp. Results: BK channel activity in BK^{M513I+Δ899-903} expressing oocytes was significantly decreased by co-expression of OSR1 or SPAK. The effect of wild-type OSR1/SPAK was mimicked by ^T185EOSR1 and ^T233ESPAK, but not by ^D164AOSR1 or ^D212ASPAK. Conclusions: OSR1 and SPAK suppress BK channels, an effect possibly contributing to cell volume regulation and neuroexcitability.

SGK3 Sensitivity of Voltage Gated K+ Channel Kv1.5 (KCNA5)

Background: The serum & glucocorticoid inducible kinase isoform SGK3 is a powerful regulator of several transporters, ion channels and the Na⁺/K⁺ ATPase. Targets of SGK3 include the ubiquitin ligase Nedd4-2, which is in turn a known regulator of the voltage gated K⁺ channel K_v1.5 (KCNA5). The present study thus explored whether SGK3 modifies the activity of the voltage gated K⁺ channel KCNA5, which participates in the regulation of diverse functions including atrial cardiac action potential, activity of vascular smooth muscle cells, insulin release and tumour cell proliferation. Methods: cRNA encoding KCNA5 was injected into Xenopus oocytes with and without additional injection of cRNA encoding wild-type SGK3, constitutively active ^S419DSGK3, inactive ^K191NSGK3 and/or wild type Nedd4-2. Voltage gated K⁺ channel activity was quantified utilizing dual electrode voltage clamp. Results: Voltage gated current in KCNA5 expressing Xenopus oocytes was significantly enhanced by wild-type SGK3 and ^S419DSGK3, but not by ^K191NSGK3. SGK3 was effective in the presence of ouabain (1 mM) and thus did not require Na⁺/K⁺ ATPase activity. Coexpression of Nedd4-2 decreased the voltage gated current in KCNA5 expressing Xenopus oocytes, an effect largely reversed by additional coexpression of SGK3. Conclusion: SGK3 is a positive regulator of KCNA5, which is at least partially effective by abrogating the effect of Nedd4-2.

Caveolin-1 Sensitivity of Excitatory Amino Acid Transporters EAAT1, EAAT2, EAAT3, and EAAT4.

Excitatory amino acid transporters EAAT1 (SLC1A3), EAAT2 (SLC1A2), EAAT3 (SLC1A1), and EAAT4 (SLC1A6) serve to clear L-glutamate from the synaptic cleft and are thus important for the limitation of neuronal excitation. EAAT3 has previously been shown to form complexes with caveolin-1, a major component of caveolae, which participate in the regulation of transport proteins. The present study explored the impact of caveolin-1 on electrogenic transport by excitatory amino acid transporter isoforms EAAT1-4. To this end cRNA encoding EAAT1, EAAT2, EAAT3, or EAAT4 was injected into Xenopus oocytes without or with additional injection of cRNA encoding caveolin-1. The L-glutamate (2mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1-, EAAT2-, EAAT3-, or EAAT4-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of caveolin-1. Caveolin-1 decreased significantly the maximal transport rate. Treatment of EAATs-expressing oocytes with brefeldin A (5µM) was followed by a decrease in conductance, which was similar in oocytes expressing EAAT together with caveolin-1 as in oocytes expressing EAAT1-4 alone. Thus, caveolin-1 apparently does not accelerate transporter protein retrieval from the cell membrane. In conclusion, caveolin-1 is a powerful negative regulator of the excitatory glutamate transporters EAAT1, EAAT2, EAAT3, and EAAT4.