Susan J. Anderson

Heteromeric assembly of acid-sensitive ion channel and epithelial sodium channel subunits.

Amiloride-sensitive ion channels are formed from homo- or heteromeric combinations of subunits from the epithelial Na+ channel (ENaC)/degenerin superfamily, which also includes the acid-sensitive ion channel (ASIC) family. These channel subunits share sequence homology and topology. In this study, we have demonstrated, using confocal fluorescence resonance energy transfer microscopy and co-immunoprecipitation, that ASIC and ENaC subunits are capable of forming cross-clade intermolecular interactions. We have also shown that combinations of ASIC1 with ENaC subunits exhibit novel electrophysiological characteristics compared with ASIC1 alone. The results of this study suggest that heteromeric complexes of ASIC and ENaC subunits may underlie the diversity of amiloride-sensitive cation conductances observed in a wide variety of tissues and cell types where co-expression of ASIC and ENaC subunits has been observed.

The Role of Pre-H2 Domains of α- and δ-Epithelial Na + Channels in Ion Permeation, Conductance, and Amiloride Sensitivity

Epithelial Na+ channels (ENaC) regulate salt and water re-absorption across the apical membrane of absorptive epithelia such as the kidney, colon, and lung. Structure-function studies have suggested that the second transmembrane domain (M2) and the adjacent pre- and post-M2 regions are involved in channel pore formation, cation selectivity, and amiloride sensitivity. Because Na+ selectivity, unitary Na+ conductance (γNa), and amiloride sensitivity of δ-ENaC are strikingly different from those of α-ENaC, the hypothesis that the pre-H2 domain may contribute to these characterizations has been examined by swapping the pre-H2, H2, and both (pre-H2+H2) domains of δ- and α-ENaCs. Whole-cell and single channel results showed that the permeation ratio of Li+ and Na+ (PLi/PNa) for the swap α chimeras co-expressed with βγ-ENaC in Xenopus oocytes decreased significantly. In contrast, the ratio of PLi/PNa for the swap δ constructs was not significantly altered. Single channel studies confirmed that swapping of the H2 and the pre-H2+H2 domains increased the γNa of α-ENaC but decreased the γNa of δ-ENaC. A significant increment in the apparent inhibitory dissociation constant for amiloride (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{i}^{\mathrm{amil}}\) \end{document}) was observed in the α chimeras by swapping the pre-H2, H2, and pre-H2+H2 domains. In contrast, a striking decline of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{i}^{\mathrm{amil}}\) \end{document} was obtained in the chimeric δ constructs with substitution of the H2 and pre-H2+H2 domains. Our results demonstrate that the pre-H2 domain, combined with the H2 domain, contributes to the PLi/PNa ratio, single channel Na+ conductance, and amiloride sensitivity of α- and δ-ENaCs.

The role of pre-H2 domains of alpha-and delta-ENaC in ion permeation, conductance, and amiloride sensitivity

Epithelial Na+ channels (ENaC) regulate salt and water re-absorption across the apical membrane of absorptive epithelia such as the kidney, colon, and lung. Structure-function studies have suggested that the second transmembrane domain (M2) and the adjacent pre- and post-M2 regions are involved in channel pore formation, cation selectivity, and amiloride sensitivity. Because Na+ selectivity, unitary Na+ conductance (γNa), and amiloride sensitivity of δ-ENaC are strikingly different from those of α-ENaC, the hypothesis that the pre-H2 domain may contribute to these characterizations has been examined by swapping the pre-H2, H2, and both (pre-H2+H2) domains of δ- and α-ENaCs. Whole-cell and single channel results showed that the permeation ratio of Li+ and Na+ (PLi/PNa) for the swap α chimeras co-expressed with βγ-ENaC in Xenopus oocytes decreased significantly. In contrast, the ratio of PLi/PNa for the swap δ constructs was not significantly altered. Single channel studies confirmed that swapping of the H2 and the pre-H2+H2 domains increased the γNa of α-ENaC but decreased the γNa of δ-ENaC. A significant increment in the apparent inhibitory dissociation constant for amiloride (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{i}^{\mathrm{amil}}\) \end{document}) was observed in the α chimeras by swapping the pre-H2, H2, and pre-H2+H2 domains. In contrast, a striking decline of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{i}^{\mathrm{amil}}\) \end{document} was obtained in the chimeric δ constructs with substitution of the H2 and pre-H2+H2 domains. Our results demonstrate that the pre-H2 domain, combined with the H2 domain, contributes to the PLi/PNa ratio, single channel Na+ conductance, and amiloride sensitivity of α- and δ-ENaCs.

The Transcription Elongation Factor Spt5 Influences Transcription by RNA Polymerase I Positively and Negatively

Spt5p is a universally conserved transcription factor that plays multiple roles in eukaryotic transcription elongation. Spt5p forms a heterodimer with Spt4p and collaborates with other transcription factors to pause or promote RNA polymerase II transcription elongation. We have shown previously that Spt4p and Spt5p also influence synthesis of ribosomal RNA by RNA polymerase (Pol) I; however, previous studies only characterized defects in Pol I transcription induced by deletion of SPT4. Here we describe two new, partially active mutations in SPT5 and use these mutant strains to characterize the effect of Spt5p on Pol I transcription. Genetic interactions between spt5 and rpa49Δ mutations together with measurements of ribosomal RNA synthesis rates, rDNA copy number, and Pol I occupancy of the rDNA demonstrate that Spt5p plays both positive and negative roles in transcription by Pol I. Electron microscopic analysis of mutant and WT strains confirms these observations and supports the model that Spt4/5 may contribute to pausing of RNA polymerase I early during transcription elongation but promotes transcription elongation downstream of the pause(s). These findings bolster the model that Spt5p and related homologues serve diverse critical roles in the control of transcription.

Efficient transcription by RNA polymerase I using recombinant core factor.

Transcription of ribosomal DNA by RNA polymerase I is a central feature of eukaryotic ribosome biogenesis. Since ribosome synthesis is closely linked to cell proliferation, there is a need to define the molecular mechanisms that control transcription by RNA polymerase I. To fully define the factors that control RNA polymerase I activity, biochemical analyses using purified transcription factors are essential. Although such assays exist, one limitation is the low abundance and difficult purification strategies required for some of the essential transcription factors for RNA polymerase I. Here, we describe a new method for expression and purification of the three subunit core factor complex from Escherichia coli. We demonstrate that the recombinant material is more active than yeast-derived core factor in assays for RNA polymerase I transcription in vitro. Finally, we use recombinant core factor to differentiate between two opposing models for the role of the TATA-binding protein in transcription by RNA polymerase I.

Two PKC consensus sites on human acid-sensing ion channel 1b differentially regulate its function

Human acid-sensing ion channel 1b (hASIC1b) is a H(+)-gated amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker psalmotoxin-1 decrease the migration and proliferation of glioma cells. PKC also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1bs COOH terminus has been shown to interact with protein interacting with C kinase (PICK)1, which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine, to prevent phosphorylation, and to glutamic acid or aspartic acid, to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating the modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild type (WT), whereas S40E, S499A, and S499D currents were decreased. The PKC activators PMA and phorbol 12,13-dibutyrate inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A but had no effect on S499A or S40A/S499A. PKC activators or the inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information that may help in designing potentially beneficial therapies in their treatment.

The SWI/SNF Chromatin Remodeling Complex Influences Transcription by RNA Polymerase I in Saccharomyces cerevisiae

SWI/SNF is a chromatin remodeling complex that affects transcription initiation and elongation by RNA polymerase II. Here we report that SWI/SNF also plays a role in transcription by RNA polymerase I (Pol I) in Saccharomyces cerevisiae. Deletion of the genes encoding the Snf6p or Snf5p subunits of SWI/SNF was lethal in combination with mutations that impair Pol I transcription initiation and elongation. SWI/SNF physically associated with ribosomal DNA (rDNA) within the coding region, with an apparent peak near the 5′ end of the gene. In snf6Δ cells there was a ∼2.5-fold reduction in rRNA synthesis rate compared to WT, but there was no change in average polymerase occupancy per gene, the number of rDNA gene repeats, or the percentage of transcriptionally active rDNA genes. However, both ChIP and EM analyses showed a small but reproducible increase in Pol I density in a region near the 5′ end of the gene. Based on these data, we conclude that SWI/SNF plays a positive role in Pol I transcription, potentially by modifying chromatin structure in the rDNA repeats. Our findings demonstrate that SWI/SNF influences the most robust transcription machinery in proliferating cells.