Glucocorticoid-Induced Leucine Zipper (GILZ) in Cardiovascular Health and Disease.

Glucocorticoids (GCs) are essential in regulating functions and homeostasis in many biological systems and are extensively used to treat a variety of conditions associated with immune/inflammatory processes. GCs are among the most powerful drugs for the treatment of autoimmune and inflammatory diseases, but their long-term usage is limited by severe adverse effects. For this reason, to envision new therapies devoid of typical GC side effects, research has focused on expanding the knowledge of cellular and molecular effects of GCs. GC-induced leucine zipper (GILZ) is a GC-target protein shown to mediate several actions of GCs, including inhibition of the NF-κB and MAPK pathways. GILZ expression is not restricted to immune cells, and it has been shown to play a regulatory role in many organs and tissues, including the cardiovascular system. Research on the role of GILZ on endothelial cells has demonstrated its ability to modulate the inflammatory cascade, resulting in a downregulation of cytokines, chemokines, and cellular adhesion molecules. GILZ also has the capacity to protect myocardial cells, as its deletion makes the heart, after a deleterious stimulus, more susceptible to apoptosis, immune cell infiltration, hypertrophy, and impaired function. Despite these advances, we have only just begun to appreciate the relevance of GILZ in cardiovascular homeostasis and dysfunction. This review summarizes the current understanding of the role of GILZ in modulating biological processes relevant to cardiovascular biology.

Biophysical and Structural Methods to Study the bHLHZip Region of Human c-MYC.

The C-terminal region of the c-MYC transcription factor consists of approximately 100 amino acids that in its native state does not adopt a stable structure. When this region binds to the obligatory partner MAX via a coupled folding-and-binding mechanism, it forms a basic-helix-loop-helix-leucine zipper (bHLHZip) heterodimeric complex. The C-terminal region of MYC is the target for numerous drug discovery programs for direct MYC inhibition via blocking the dimerization event and/or binding to DNA, and a proper understanding of the partially folded, dynamic nature of the heterodimeric complex is essential to these efforts. The bHLHZip motif also drives protein-protein interactions with cofactors that are crucial for both transcriptional repression and activation of MYC target genes. Targeting these interactions could potentially provide a means of developing alternative approaches to halt MYC functions; however, the molecular mechanism of these regulatory interactions is poorly understood. Herein we provide methods to produce high-quality human c-MYC C-terminal by itself and in complex MAX, and how to study them using Nuclear Magnetic Resonance spectroscopy and X-ray crystallography. Our protein expression and purification protocols have already been used to study interactions with cofactors.

Increased Glucocorticoid Receptor Alpha Expression and Signaling in Critically Ill Coronavirus Disease 2019 Patients.

OBJECTIVES: Critical illness is characterized by increased serum cortisol concentrations and bioavailability resulting from the activation of the hypothalamic-pituitary-adrenal axis, which constitutes an essential part of the stress response. The actions of glucocorticoids are mediated by a ubiquitous intracellular receptor protein, the glucocorticoid receptor. So far, data on coronavirus disease 2019 and glucocorticoid receptor alpha expression are lacking. DESIGN: Prospective observational study. SETTING: One academic multidisciplinary ICU. SUBJECTS: Twenty-six adult coronavirus disease 2019 patients; 33 adult noncoronavirus disease 2019 patients, matched for age, sex, and disease severity, constituted the control group. All patients were steroid-free. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Glucocorticoid receptor alpha, glucocorticoid-inducible leucine zipper expression, and serum cortisol were measured on ICU admission. In coronavirus disease 2019 patients, glucocorticoid receptor alpha and glucocorticoid-inducible leucine zipper messenger RNA expression were upregulated (4.7-fold, p < 0.01 and 14-fold, p < 0.0001, respectively), and cortisol was higher (20.3 vs 14.3 µg/dL, p < 0.01) compared with the control group. CONCLUSIONS: ICU coronavirus disease 2019 patients showed upregulated glucocorticoid receptor alpha and glucocorticoid-inducible leucine zipper expression, along with cortisol levels, compared with ICU noncoronavirus disease 2019 patients. Thus, on ICU admission, critical coronavirus disease 2019 appears to be associated with hypercortisolemia, and increased synthesis of glucocorticoid receptor alpha and induced proteins.

Methods of Expression, Purification, and Preparation of the c-Myc b-HLH-LZ for Its Biophysical Characterization.

The b-HLH-LZ domain of c-Myc is a key target for the development of cancer therapies by blunting its binding to DNA with cell penetrant b-HLH-LZs and/or by stabilizing it into a state that cannot recognize Max to activate and amplify transcription of oncogenic genes. Although recent milestones have been reached with DNA binding blunting of c-Myc with the cell penetrant b-HLH-LZ Omomyc, the targeting of its b-HLH-LZ with small molecules, peptides, or proteins is lagging. As reviewed recently, the main problem relies in the intrinsically disordered nature of the b-HLH-LZ of c-Myc. This greatly complicates the classical approach of targeting a docking site with inhibitors. The solution state methods such as NMR are progressing towards the characterization of the ensembles of structures or states the b-HLH-LZ can adopt. However, the delicate balance that dictates the population of these dynamically interchanging states relies on its primary structure and the weak polar, electrostatic and hydrophobic interactions allowed. In this context, it is of the utmost importance to study the b-HLH-LZ of c-Myc in its WT background and avoid the use of tags such as His-tags. These tags could disrupt the balance of forces which could alter the conformational and physical transitions and states it can undergo and adopt. Here, we describe a robust protocol to express the WT b-HLH-LZ in E. coli and purify it, without the need of tags, to obtain the required quantities for solution state biophysical characterization such as NMR.

Oil Body Formation in Marchantia polymorpha Is Controlled by MpC1HDZ and Serves as a Defense against Arthropod Herbivores.

The origin of a terrestrial flora in the Ordovician required adaptation to novel biotic and abiotic stressors. Oil bodies, a synapomorphy of liverworts, accumulate secondary metabolites, but their function and development are poorly understood. Oil bodies of Marchantia polymorpha develop within specialized cells as one single large organelle. Here, we show that a class I homeodomain leucine-zipper (C1HDZ) transcription factor controls the differentiation of oil body cells in two different ecotypes of the liverwort M. polymorpha, a model genetic system for early divergent land plants. In flowering plants, these transcription factors primarily modulate responses to abiotic stress, including drought. However, loss-of-function alleles of the single ortholog gene, MpC1HDZ, in M. polymorpha did not exhibit phenotypes associated with abiotic stress. Rather, Mpc1hdz mutant plants were more susceptible to herbivory, and total plant extracts of the mutant exhibited reduced antibacterial activity. Transcriptomic analysis of the mutant revealed a reduction in expression of genes related to secondary metabolism that was accompanied by a specific depletion of oil body terpenoid compounds. Through time-lapse imaging, we observed that MpC1HDZ expression maxima precede oil body formation, indicating that MpC1HDZ mediates differentiation of oil body cells. Our results indicate that M. polymorpha oil bodies, and MpC1HDZ, are critical for defense against herbivory, but not for abiotic stress tolerance. Thus, C1HDZ genes were co-opted to regulate separate responses to biotic and abiotic stressors in two distinct land plant lineages.

Characterization of gonadal glucocorticoid-induced leucine zipper (GILZ) protein expression during sex change in the protogynous orange-spotted grouper, Epinephelus coioides.

Steroid hormones have been proven as a key drive of sex change in sequentially hermaphroditic organisms. However, the upstream mechanism of sex steroid hormones regulation that affect sex change remain unknown. The main glucocorticoid in teleost fish is cortisol, which both regulates steroidogenesis and has antistress action. Thus, cortisol might be one of the prime factors in sex change. In this study, the glucocorticoid-induced leucine zipper (GILZ) gene, was proven to have a dramatic effect in orange-spotted groupers (Epinephelus coioides) during sex change at the early stage of gonadal transition. The specific action of the GILZ protein is at the pouch-shaped proliferative spermatogonia instead of the degenerative oocyte at the onset of sex change. Immunohistochemical (IHC) evidence revealed that GILZ performs intensively at undifferentiated spermatogonia in the early testis stage. These results imply that cortisol provokes a rise of GILZ through regulation caused by steroid hormones leading to sex change.

The glucocorticoid-induced leucine zipper mediates statin-induced muscle damage.

Statins, the most prescribed class of drugs for the treatment of hypercholesterolemia, can cause muscle-related adverse effects. It has been shown that the glucocorticoid-induced leucine zipper (GILZ) plays a key role in the anti-myogenic action of dexamethasone. In the present study, we aimed to evaluate the role of GILZ in statin-induced myopathy. Statins induced GILZ expression in C2C12 cells, primary murine myoblasts/myotubes, primary human myoblasts, and in vivo in zebrafish embryos and human quadriceps femoris muscle. Gilz induction was mediated by FOXO3 activation and binding to the Gilz promoter, and could be reversed by the addition of geranylgeranyl, but not farnesyl, pyrophosphate. Atorvastatin decreased Akt phosphorylation and increased cleaved caspase-3 levels in myoblasts. This effect was reversed in myoblasts from GILZ knockout mice. Similarly, myofibers isolated from knockout animals were more resistant toward statin-induced cell death than their wild-type counterparts. Statins also impaired myoblast differentiation, and this effect was accompanied by GILZ induction. The in vivo relevance of our findings was supported by the observation that gilz overexpression in zebrafish embryos led to impaired embryonic muscle development. Taken together, our data point toward GILZ as an essential mediator of the molecular mechanisms leading to statin-induced muscle damage.

Could GILZ Be the Answer to Glucocorticoid Toxicity in Lupus?

Glucocorticoids (GC) are used globally to treat autoimmune and inflammatory disorders. Their anti-inflammatory actions are mainly mediated via binding to the glucocorticoid receptor (GR), creating a GC/GR complex, which acts in both the cytoplasm and nucleus to regulate the transcription of a host of target genes. As a result, signaling pathways such as NF-κB and AP-1 are inhibited, and cell activation, differentiation and survival and cytokine and chemokine production are suppressed. However, the gene regulation by GC can also cause severe side effects in patients. Systemic lupus erythematosus (SLE or lupus) is a multisystem autoimmune disease, characterized by a poorly regulated immune response leading to chronic inflammation and dysfunction of multiple organs, for which GC is the major current therapy. Long-term GC use, however, can cause debilitating adverse consequences for patients including diabetes, cardiovascular disease and osteoporosis and contributes to irreversible organ damage. To date, there is no alternative treatment which can replicate the rapid effects of GC across multiple immune cell functions, effecting disease control during disease flares. Research efforts have focused on finding alternatives to GC, which display similar immunoregulatory actions, without the devastating adverse metabolic effects. One potential candidate is the glucocorticoid-induced leucine zipper (GILZ). GILZ is induced by low concentrations of GC and is shown to mimic the action of GC in several inflammatory processes, reducing immunity and inflammation in in vitro and in vivo studies. Additionally, GILZ has, similar to the GC-GR complex, the ability to bind to both NF-κB and AP-1 as well as DNA directly, to regulate immune cell function, while potentially lacking the GC-related side effects. Importantly, in SLE patients GILZ is under-expressed and correlates negatively with disease activity, suggesting an important regulatory role of GILZ in SLE. Here we provide an overview of the actions and use of GC in lupus, and discuss whether the regulatory mechanisms of GILZ could lead to the development of a novel therapeutic for lupus. Increased understanding of the mechanisms of action of GILZ, and its ability to regulate immune events leading to lupus disease activity has important clinical implications for the development of safer anti-inflammatory therapies.

Basic-leucine zipper 17 and Hmg-CoA reductase degradation 3A are involved in salt acclimation memory in Arabidopsis.

Salt acclimation, which is induced by previous salt exposure, increases the resistance of plants to future exposure to salt stress. However, little is known about the underlying mechanism, particularly how plants store the "memory" of salt exposure. In this study, we established a system to study salt acclimation in Arabidopsis thaliana. Following treatment with a low concentration of salt, seedlings were allowed to recover to allow transitory salt responses to subside while maintaining the sustainable effects of salt acclimation. We performed transcriptome profiling analysis of these seedlings to identify genes related to salt acclimation memory. Notably, the expression of Basic-leucine zipper 17 (bZIP17) and Hmg-CoA reductase degradation 3A (HRD3A), which are important in the unfolded protein response (UPR) and endoplasmic reticulum-associated degradation (ERAD), respectively, increased following treatment with a low concentration of salt and remained at stably high levels after the stimulus was removed, a treatment which improved plant tolerance to future high-salinity challenge. Our findings suggest that the upregulated expression of important genes involved in the UPR and ERAD represents a "memory" of the history of salt exposure and enables more potent responses to future exposure to salt stress, providing new insights into the mechanisms underlying salt acclimation in plants.

Molecular interactions of the γ-clade homeodomain-leucine zipper class I transcription factors during the wheat response to water deficit.

The γ-clade of class I homeodomain-leucine zipper (HD-Zip I) transcription factors (TFs) constitute members which play a role in adapting plant growth to conditions of water deficit. Given the importance of wheat (Triticum aestivum L.) as a global food crop and the impact of water deficit upon grain yield, we focused on functional aspects of wheat drought responsive HD-Zip I TFs. While the wheat γ-clade HD-Zip I TFs share significant sequence similarities with homologous genes from other plants, the clade-specific features in transcriptional response to abiotic stress were detected. We demonstrate that wheat TaHDZipI-3, TaHDZipI-4, and TaHDZipI-5 genes respond differentially to a variety of abiotic stresses, and that proteins encoded by these genes exhibit pronounced differences in oligomerisation, strength of DNA binding, and trans-activation of an artificial promoter. Three-dimensional molecular modelling of the protein-DNA interface was conducted to address the ambiguity at the central nucleotide in the pseudo-palindromic cis-element CAATNATTG that is recognised by all three HD-Zip I proteins. The co-expression of these genes in the same plant tissues together with the ability of HD-Zip I TFs of the γ-clade to hetero-dimerise suggests a role in the regulatory mechanisms of HD-Zip I dependent transcription. Our findings highlight the complexity of TF networks involved in plant responses to water deficit. A better understanding of the molecular complexity at the protein level during crop responses to drought will enable adoption of efficient strategies for production of cereal plants with enhanced drought tolerance.

The identification of Cucumis sativus Glabrous 1 (CsGL1) required for the formation of trichomes uncovers a novel function for the homeodomain-leucine zipper I gene.

The spines and bloom of cucumber (Cucumis sativus L.) fruit are two important quality traits related to fruit market value. However, until now, none of the genes involved in the formation of cucumber fruit spines and bloom trichomes has been identified. Here, the characterization of trichome development in wild-type (WT) cucumber and a spontaneous mutant, glabrous 1 (csgl1) controlled by a single recessive nuclear gene, with glabrous aerial organs, is reported. Via map-based cloning, CsGL1 was isolated and it was found that it encoded a member of the homeodomain-leucine zipper I (HD-Zip I) proteins previously identified to function mainly in the abiotic stress responses of plants. Tissue-specific expression analysis indicated that CsGL1 was strongly expressed in trichomes and fruit spines. In addition, CsGL1 was a nuclear protein with weak transcriptional activation activity in yeast. A comparative analysis of the digital gene expression (DGE) profile between csgl1 and WT leaves revealed that CsGL1 had a significant influence on the gene expression profile in cucumber, especially on genes related to cellular process, which is consistent with the phenotypic difference between csgl1 and the WT. Moreover, two genes, CsMYB6 and CsGA20ox1, possibly involved in the formation of cucumber trichomes and fruit spines, were characterized. Overall, the findings reveal a new function for the HD-Zip I gene subfamily, and provide some candidate genes for genetic engineering approaches to improve cucumber fruit external quality.

Emerging functions of mammalian and plant mTERFs.

Organellar gene expression (OGE) is crucial for plant development, respiration and photosynthesis, but the mechanisms that control it are still largely unclear. Thus, OGE requires various nucleus-encoded proteins that promote transcription, splicing, trimming and editing of organellar RNAs, and regulate their translation. In mammals, members of the mitochondrial transcription termination factor (mTERF) family play important roles in OGE. Intriguingly, three of the four mammalian mTERFs do not actually terminate transcription, as their designation suggests, but appear to function in antisense transcription termination and ribosome biogenesis. During the evolution of land plants, the mTERF family has expanded to approximately 30 members, but knowledge of their function in photosynthetic organisms remains sparse. Here, we review recent advances in the characterization of mterf mutants in mammals and photosynthetic organisms, focusing particularly on the progress made in elucidating their molecular functions in the last two years. This article is part of a Special Issue entitled: Chloroplast biogenesis.

The N-terminal leucine-zipper motif in PTRF/cavin-1 is essential and sufficient for its caveolae-association.

PTRF/cavin-1 is a protein of two lives. Its reported functions in ribosomal RNA synthesis and in caveolae formation happen in two different cellular locations: nucleus vs. plasma membrane. Here, we identified that the N-terminal leucine-zipper motif in PTRF/cavin-1 was essential for the protein to be associated with caveolae in plasma membrane. It could counteract the effect of nuclear localization sequence in the molecule (AA 235-251). Deletion of this leucine-zipper motif from PTRF/cavin-1 caused the mutant to be exclusively localized in nuclei. The fusion of this leucine-zipper motif with histone 2A, which is a nuclear protein, could induce the fusion protein to be exported from nucleus. Cell migration was greatly inhibited in PTRF/cavin-1(-/-) mouse embryonic fibroblasts (MEFs). The inhibited cell motility could only be rescued by exogenous cavin-1 but not the leucine-zipper motif deleted cavin-1 mutant. Plasma membrane dynamics is an important factor in cell motility control. Our results suggested that the membrane dynamics in cell migration is affected by caveolae associated PTRF/cavin-1.

Control of cotton fibre elongation by a homeodomain transcription factor GhHOX3.

Cotton fibres are unusually long, single-celled epidermal seed trichomes and a model for plant cell growth, but little is known about the regulation of fibre cell elongation. Here we report that a homeodomain-leucine zipper (HD-ZIP) transcription factor, GhHOX3, controls cotton fibre elongation. GhHOX3 genes are localized to the 12th homoeologous chromosome set of allotetraploid cotton cultivars, associated with quantitative trait loci (QTLs) for fibre length. Silencing of GhHOX3 greatly reduces (>80%) fibre length, whereas its overexpression leads to longer fibre. Combined transcriptomic and biochemical analyses identify target genes of GhHOX3 that also contain the L1-box cis-element, including two cell wall loosening protein genes GhRDL1 and GhEXPA1. GhHOX3 interacts with GhHD1, another homeodomain protein, resulting in enhanced transcriptional activity, and with cotton DELLA, GhSLR1, repressor of the growth hormone gibberellin (GA). GhSLR1 interferes with the GhHOX3-GhHD1 interaction and represses target gene transcription. Our results uncover a novel mechanism whereby a homeodomain protein transduces GA signal to promote fibre cell elongation.

Simulations of potentials of mean force for separating a leucine zipper dimer and the basic region of a basic region leucine zipper dimer.

Basic region leucine zipper (bZIP) transcription factors involved in DNA recognition are dimeric proteins. The monomers consist of two subdomains, a leucine zipper sequence responsible for dimerization and a highly basic DNA recognition sequence. Leucine zippers are strongly dimerized, and in a bZIP, the basic region can, in the absence of DNA, undergo extensive relative monomer-to-monomer fluctuations. In this work, LZ and bZIP potentials of mean force (PMFs), which provide free energies along reaction coordinates, are simulated with a distance replica exchange method. The method uses restraint potentials to provide sampling along a reaction coordinate and enhances configuration space exploration by exchanging information between neighboring restraint potential configurations. Restraint potentials that are constructed from sums over a number of atom distances are employed. Their use requires a modification of the Weighted Histogram Analysis Method (WHAM) procedure to combine and unbias the data from the different restraint-potential-biased window densities to provide a PMF. These methods are first used to obtain a PMF for separating a leucine zipper (GCN4-p1) of the yeast transcriptional activator GCN4. The PMF indicates a very strong binding free energy that only weakens when the monomers are separated by about 12 Å, which is about 6 Å beyond their bound, dimer equilibrium distance. PMFs are also obtained for separating the basic subdomain monomer parts of the GCN4 bZIP transcriptional factor, in the absence of DNA. In a monomer separation range spanning the open, crystal-based structure to closer configurations, the basic subdomain PMF is quite flat, implying essentially thermal sampling in this distance range. A PMF generated starting from a "collapsed" state, taken from a previous simulation ( J. Phys. Chem. B 2012 , 116 , 6071 ), where collapsed refers to the feature that the basic subdomain monomers are also effectively dimerized, shows that this state is bound in free energy, though much less so than the leucine zipper dimer.

Controlled localization of functionally active proteins to inclusion bodies using leucine zippers.

Inclusion bodies (IBs) are typically non-functional particles of aggregated proteins. However, some proteins in fusion with amyloid-like peptides, viral coat proteins, and cellulose binding domains (CBDs) generate IB particles retaining the original functions in cells. Here, we attempted to generate CBD IBs displaying functional leucine zipper proteins (LZs) as bait for localizing cytosolic proteins in E. coli. When a red fluorescent protein was tested as a target protein, microscopic observations showed that the IBs red-fluoresced strongly. When different LZ pairs with KDs of 8-1,000 µM were tested as the bait and prey, the localization of the red fluorescence appeared to change following the affinities between the LZs, as observed by fluorescence imaging and flow cytometry. This result proposed that LZ-tagged CBD IBs can be applied as an in vivo matrix to entrap cytosolic proteins in E. coli while maintaining their original activities. In addition, easy detection of localization to IBs provides a unique platform for the engineering and analyses of protein-protein interactions in E. coli.

Macrophage migration inhibitory factor inhibits the antiinflammatory effects of glucocorticoids via glucocorticoid-induced leucine zipper.

OBJECTIVE: Glucocorticoids remain a mainstay in the treatment of rheumatoid arthritis (RA). Dose-dependent adverse effects highlight the need for therapies that regulate glucocorticoid sensitivity to enable dosage reduction. Macrophage migration inhibitory factor (MIF) is a proinflammatory protein that has been implicated in the pathogenesis of RA; it impairs glucocorticoid sensitivity via MAPK phosphatase 1 (MKP-1) inhibition. The intracellular protein glucocorticoid-induced leucine zipper (GILZ) mimics the effects of glucocorticoids in models of RA, but whether it represents a target for the modulation of glucocorticoid sensitivity remains unknown. We undertook this study to investigate whether GILZ is involved in the regulation of glucocorticoid sensitivity by MIF. METHODS: GILZ expression was studied in the presence and absence of MIF, and the role of GILZ in the MIF-dependent regulation of the glucocorticoid sensitivity mediator MKP-1 was studied at the level of expression and function. RESULTS: GILZ expression was significantly inhibited by endogenous MIF, both basally and during responses to glucocorticoid treatment. The effects of MIF on GILZ were dependent on the expression and Akt-induced nuclear translocation of the transcription factor FoxO3A. GILZ was shown to regulate the expression of MKP-1 and consequent MAPK phosphorylation and cytokine release. CONCLUSION: MIF exerts its effects on MKP-1 expression and MAPK activity through inhibitory effects on GILZ. These findings suggest a previously unsuspected interaction between MIF and GILZ and identify GILZ as a potential target for the therapeutic regulation of glucocorticoid sensitivity.

RETINA-specific expression of Kcnv2 is controlled by cone-rod homeobox (Crx) and neural retina leucine zipper (Nrl).

Cone dystrophy with supernormal rod response (CDSRR) is an autosomal recessive disorder that leads to progressive retinal degeneration with a distinct electroretinogram (ERG) phenotype. CDSRR patients show reduced sensitivity to dim light, augmented response to suprathreshold light and reduced response to flicker. The disorder is caused by mutations in the KCNV2 gene, which encodes the Kv11.1 subunit of a voltage-gated potassium channel. Here, we studied the retina-specific expression and cis-regulatory activity of the murine Kcnv2 gene using electroporation of explanted retinas. Using qRT-PCR profiling of early postnatal retinas, we showed that Kcnv2 expression increased towards P14, which marks the beginning of visual activity in mice. In vivo electroporation of GFP-Kcnv2 expressing plasmids revealed that Kv11.1 localizes to the inner segment membranes of adult P21 photoreceptors. Using bioinformatic prediction and chromatin immunoprecipitation (ChIP), we identified two Crx binding sites (CBS) and one Nrl binding site (NBS) in the Kcnv2 promoter. Reporter electroporation of the wild type promoter region induced strong DsRed expression, indicating high regulatory activity, whereas shRNA-mediated knockdown of Crx and Nrl resulted in reduced Kcnv2 promoter activity and low endogenous Kcnv2 mRNA expression in the retina. Site-directed mutagenesis of the CBS and NBS demonstrated that CBS2 is crucial for Kcnv2 promoter activity. We conclude that nucleotide changes in evolutionary conserved CBS could impact retina-specific expression levels of Kcnv2.