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Human acid-sensing ion channel 1b (hASIC1b) is a [H.sup.+]-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, hASIC1b's 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. two-electrode voltage clamp; Xenopus laevis oocytes; phorbol 12-myristate 13-acetate; chelerythrine; mutagenesis; phorbol 12,13-dibutyrate; protein kinase C

Despite extensive work in the field of glioblastoma research no significant increase in survival rates for this devastating disease has been achieved. It is known that disturbance of intracellular [Ca.sup.2+] ([[Ca.sup.2+]].sub.i]) and intracellular pH ([pH.sub.i]) regulation could be involved in tumor formation. The sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA) is a major regulator of [[Ca.sup.2+]].sub.i]. We have investigated the effect of inhibition of SERCA by thapsigargin (TG) on [[Ca.sup.2+]].sub.i] and pHi in human primary glioblastoma multiforme (GBM) cells and GBM cell lines, compared with normal human astrocytes, using the fluorescent indicators fura-2 and BCECF, respectively. Basal [[Ca.sup.2+]].sub.i] was higher in SK-MG-1 and U87 MG but not in human primary GBM cells compared with normal astrocytes. However, in tumor cells, TG evoked a much larger and faster [[Ca.sup.2+]].sub.i] increase than in normal astrocytes. This increase was prevented in nominally [Ca.sup.2+]-free buffer and by 2-APB, an inhibitor of storeoperated [Ca.sup.2+] channels. In addition, TG-activated [Ca.sup.2+] influx, which was sensitive to 2-APB, was higher in all tumor cell lines and primary GBM cells compared with normal astrocytes. The p[H.sub.i] was also elevated in tumor cells compared with normal astrocytes. TG caused acidification of both normal and all GBM cells, but in the tumor cells, this acidification was followed by an amiloride- and 5-(N,N-hexamethylene)-amiloride-sensitive recovery, indicating involvement of a [Na.sup.+]/[H.sup.+] exchanger. In summary, inhibition of SERCA function revealed a significant divergence in intracellular [Ca.sup.2+] homeostasis and pH regulation in tumor cells compared with normal human astrocytes. fura-2; BCECF; store-operated calcium channels

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Despite extensive work in the field of glioblastoma research no significant increase in survival rates for this devastating disease has been achieved. It is known that disturbance of intracellular Ca2+ ([Ca2+]i) and intracellular pH (pHi) regulation could be involved in tumor formation. The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a major regulator of [Ca2+]i. We have investigated the effect of inhibition of SERCA by thapsigargin (TG) on [Ca2+]i and pHi in human primary glioblastoma multiforme (GBM) cells and (IBM cell lines, compared with normal human astrocytes, using the fluorescent indicators fura-2 and BCECF, respectively. Basal [Ca2+]i was higher in SK-MG-1 and U87 MG but not in human primary GBM cells compared with normal astrocytes. However, in tumor cells, TO evoked a much larger and faster [Ca2+]i increase than in normal astrocytes. This increase was prevented in nominally Ca2+-free buffer and by 2-APB, an inhibitor of store-operated Ca2+ channels. In addition, TG-activated Ca2+ influx, which was sensitive to 2-APB, was higher in all tumor cell lines and primary GBM cells compared with normal astrocytes. The pHi was also elevated in tumor cells compared with normal astrocytes. TG caused acidification of both normal and all GBM cells, but in the tumor cells, this acidification was followed by an amiloride- and 5-(N,N-hexamethylene)-amiloride-sensitive recovery, indicating involvement of a Na+/H+ exchanger. In summary, inhibition of SERCA function revealed a significant divergence in intracellular Ca2+ homeostasis and pH regulation in tumor cells compared with normal human astrocytes. [ABSTRACT FROM AUTHOR]

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 coexpressed 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 (Kiamil) was observed in the α chimeras by swapping the pre-H2, H2, and pre-H2+H2 domains. In contrast, a striking decline of Kimil was obtained in the chimeric 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. [ABSTRACT FROM AUTHOR]

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 (gamma(Na)), and amiloride sensitivity of delta-ENaC are strikingly different from those of alpha-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 delta- and alpha-ENaCs. Whole-cell and single channel results showed that the permeation ratio of Li(+) and Na(+) (P(Li)/P(Na)) for the swap alpha chimeras co-expressed with betagamma-ENaC in Xenopus oocytes decreased significantly. In contrast, the ratio of P(Li)/P(Na) for the swap delta constructs was not significantly altered. Single channel studies confirmed that swapping of the H2 and the pre-H2+H2 domains increased the gamma(Na) of alpha-ENaC but decreased the gamma(Na) of delta-ENaC. A significant increment in the apparent inhibitory dissociation constant for amiloride (K(i)(amil)) was observed in the alpha chimeras by swapping the pre-H2, H2, and pre-H2+H2 domains. In contrast, a striking decline of K(i)(amil) was obtained in the chimeric delta 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 P(Li)/P(Na) ratio, single channel Na(+) conductance, and amiloride sensitivity of alpha- and delta-ENaCs.