Viswanathan Raghuram

Slc26a9—Anion Exchanger, Channel and Na+ Transporter

The SLC26 gene family encodes anion transporters with diverse functional attributes: (a) anion exchanger, (b) anion sensor, and (c) anion conductance (likely channel). We have cloned and studied Slc26a9, a paralogue expressed mostly in lung and stomach. Immunohistochemistry shows that Slc26a9 is present at apical and intracellular membranes of lung and stomach epithelia. Using expression in Xenopus laevis oocytes and ion-sensitive microelectrodes, we discovered that Slc26a9 has a novel function not found in any other Slc26 proteins: cation coupling. Intracellular pH and voltage measurements show that Slc26a9 is a nCl−-HCO3− exchanger, suggesting roles in gastric HCl secretion or pulmonary HCO3− secretion; Na+ electrodes and uptakes reveal that Slc26a9 has a cation dependence. Single-channel measurements indicate that Slc26a9 displays discrete open and closed states. These experiments show that Slc26a9 has three discrete physiological modes: nCl−-HCO3− exchanger, Cl− channel, and Na+-anion cotransporter. Thus, the Slc26a9 transporter channel is uniquely suited for dynamic and tissue-specific physiology or regulation in epithelial tissues.

Systems-level identification of PKA-dependent signaling in epithelial cells

Significance Maintenance of homeostasis is dependent on intercellular communication via secreted hormones that bind G protein-coupled receptors. Many of these receptors activate an enzyme called protein kinase A (PKA) that modifies cell function by covalently attaching phosphate groups to proteins. To comprehensively identify PKA substrates, we used genome editing (CRISPR-Cas9) to delete PKA from kidney epithelial cells followed by large-scale mass spectrometry to measure phosphorylation changes throughout the proteome; 229 PKA target sites were identified, many previously unrecognized. Surprisingly, PKA deletion caused seemingly paradoxical phosphorylation increases at many sites, indicating secondary activation of one or more mitogen-activated kinases. The data, coupled with transcriptomics and standard proteomics, identified a signaling network that explains the effects of PKA that regulate cellular functions. G protein stimulatory α-subunit (Gαs)-coupled heptahelical receptors regulate cell processes largely through activation of protein kinase A (PKA). To identify signaling processes downstream of PKA, we deleted both PKA catalytic subunits using CRISPR-Cas9, followed by a “multiomic” analysis in mouse kidney epithelial cells expressing the Gαs-coupled V2 vasopressin receptor. RNA-seq (sequencing)–based transcriptomics and SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics revealed a complete loss of expression of the water-channel gene Aqp2 in PKA knockout cells. SILAC-based quantitative phosphoproteomics identified 229 PKA phosphorylation sites. Most of these PKA targets are thus far unannotated in public databases. Surprisingly, 1,915 phosphorylation sites with the motif x-(S/T)-P showed increased phosphooccupancy, pointing to increased activity of one or more MAP kinases in PKA knockout cells. Indeed, phosphorylation changes associated with activation of ERK2 were seen in PKA knockout cells. The ERK2 site is downstream of a direct PKA site in the Rap1GAP, Sipa1l1, that indirectly inhibits Raf1. In addition, a direct PKA site that inhibits the MAP kinase kinase kinase Map3k5 (ASK1) is upstream of JNK1 activation. The datasets were integrated to identify a causal network describing PKA signaling that explains vasopressin-mediated regulation of membrane trafficking and gene transcription. The model predicts that, through PKA activation, vasopressin stimulates AQP2 exocytosis by inhibiting MAP kinase signaling. The model also predicts that, through PKA activation, vasopressin stimulates Aqp2 transcription through induction of nuclear translocation of the acetyltransferase EP300, which increases histone H3K27 acetylation of vasopressin-responsive genes (confirmed by ChIP-seq).

Letter to the editor: “Systems biology versus reductionism in cell physiology”

to the editor: The following is a response to the editorial comment of Prihandoko and Tobin (15) about our recent paper in American Journal of Physiology-Cell Physiology (2), which addresses a key question in modeling of signaling networks: How to assign the protein kinases (from the entire 521-member kinome list) that are responsible for each measurable phosphorylation event in a given cell type. In our study, we used vasopressin-stimulated phosphorylation of the water channel protein, aquaporin-2, at serine-256 as an example because of its importance to the physiology of collecting duct principal cells. We thank Prihandoko and Tobin for their thorough and well thought out summary of our paper. We write now to provide additional clarification regarding the epistemological approach, which was based on a systems biological framework rather than on reductionist principles. Understanding the two ways of doing experiments is aided by a bit of history.

Genome-Wide Mapping of DNA Accessibility and Binding Sites for CREB and C/EBPβ in Vasopressin-Sensitive Collecting Duct Cells

Background Renal water excretion is controlled by vasopressin, in part through regulation of the transcription of the aquaporin-2 gene (Aqp2).Methods To identify enhancer regions likely to be involved in the regulation of Aqp2 and other principal cell-specific genes, we used several next generation DNA-sequencing techniques in a well characterized cultured cell model of collecting duct principal cells (mpkCCD). To locate enhancers, we performed the assay for transposase-accessible chromatin using sequencing (ATAC-Seq) to identify accessible regions of DNA and integrated the data with data generated by chromatin immunoprecipitation followed by next generation DNA-sequencing (ChIP-Seq) for CCCTC binding factor (CTCF) binding, histone H3 lysine-27 acetylation, and RNA polymerase II.Results We identified two high-probability enhancers centered 81 kb upstream and 5.8 kb downstream from the Aqp2 transcriptional start site. Motif analysis of these regions and the Aqp2 promoter identified several potential transcription factor binding sites, including sites for two b-ZIP transcription factors: CCAAT/enhancer binding protein-β (C/EBPβ) and cAMP-responsive element binding protein (CREB). To identify genomic binding sites for both, we conducted ChIP-Seq using well characterized antibodies. In the presence of vasopressin, C/EBPβ, a pioneer transcription factor critical to cell-specific gene expression, bound strongly at the identified enhancer downstream from Aqp2 However, over multiple replicates, we found no detectable CREB binding sites within 390 kb of Aqp2 Thus, any role for CREB in the regulation of Aqp2 gene transcription is likely to be indirect.Conclusions The analysis identified two enhancer regions pertinent to transcriptional regulation of the Aqp2 gene and showed C/EBPβ (but not CREB) binding.