SUR1-TRPM4 and AQP4 form a heteromultimeric complex that amplifies ion/water osmotic coupling and drives astrocyte swelling.

Astrocyte swelling occurs after central nervous system injury and contributes to brain swelling, which can increase mortality. Mechanisms proffered to explain astrocyte swelling emphasize the importance of either aquaporin-4 (AQP4), an astrocyte water channel, or of Na+ -permeable channels, which mediate cellular osmolyte influx. However, the spatio-temporal functional interactions between AQP4 and Na+ -permeable channels that drive swelling are poorly understood. We hypothesized that astrocyte swelling after injury is linked to an interaction between AQP4 and Na+ -permeable channels that are newly upregulated. Here, using co-immunoprecipitation and Förster resonance energy transfer, we report that AQP4 physically co-assembles with the sulfonylurea receptor 1-transient receptor potential melastatin 4 (SUR1-TRPM4) monovalent cation channel to form a novel heteromultimeric water/ion channel complex. In vitro cell-swelling studies using calcein fluorescence imaging of COS-7 cells expressing various combinations of AQP4, SUR1, and TRPM4 showed that the full tripartite complex, comprised of SUR1-TRPM4-AQP4, was required for fast, high-capacity transmembrane water transport that drives cell swelling, with these findings corroborated in cultured primary astrocytes. In a murine model of brain edema involving cold-injury to the cerebellum, we found that astrocytes newly upregulate SUR1-TRPM4, that AQP4 co-associates with SUR1-TRPM4, and that genetic inactivation of the solute pore of the SUR1-TRPM4-AQP4 complex blocked in vivo astrocyte swelling measured by diolistic labeling, thereby corroborating our in vitro functional studies. Together, these findings demonstrate a novel molecular mechanism involving the SUR1-TRPM4-AQP4 complex to account for bulk water influx during astrocyte swelling. These findings have broad implications for the understanding and treatment of AQP4-mediated pathological conditions.

TonEBP is inhibited by RNA helicase A via interaction involving the E'F loop.

TonEBP [TonE (tonicity-responsive enhancer)-binding protein] is a transcriptional activator of the Rel family like NF-kappaB (nuclear factor kappaB) and NFAT (nuclear factor of activated T-cells). TonEBP plays a key role in the protection of cells in the kidney medulla from the deleterious effects of hyperosmolality. This is achieved by enhancing expression of HSP70 (heat-shock protein 70) and other genes whose products drive cellular accumulation of organic osmolytes. TonEBP is stimulated by ambient hypertonicity via multiple pathways that regulate nuclear translocation and transactivation. In the present paper, we report that TonEBP is associated in vivo with RHA (RNA helicase A). The N- and C-termini of RHA bound the E'F loop of the DNA-binding domain of TonEBP. The interaction was not affected by DNA binding or dimerization of TonEBP. Overexpression of RHA inhibited the activity of TonEBP; however, catalytic activity of RHA was dispensable for the inhibition. When the ambient tonicity was raised, the TonEBP-RHA interaction decreased, suggesting that dissociation of RHA is a pathway to stimulate TonEBP. We conclude that the E'F loop of TonEBP interacts with RHA like NFAT and NF-kappaB interact with AP1 (activator protein 1) and the high-mobility group protein HMG-I(Y) respectively. While RHA interacts with and stimulates other transcription factors such as CREB (cAMP-response-element-binding protein), NF-kappaB and mineralocorticoid receptor, it inhibits TonEBP.

Factors affecting the catalytic epoxidation of olefins by iron porphyrin complexes and H2O2 in protic solvents.

The catalytic epoxidation of cyclohexene by iron(III) porphyrin complexes and H2O2 has been investigated in alcohol solvents to understand factors affecting the catalyst activity in protic solvents. The yields of cyclohexene oxide and the Fe(III/II) reduction potentials of iron porphyrin complexes were significantly affected by the protic solvents, and there was a close correlation between the product yields and the reduction potentials of the iron porphyrin catalysts. The role of alcohol solvents was proposed to control the electronic nature of iron porphyrin complexes that determines the catalyst activity in the epoxidation of olefins by H2O2. We have also demonstrated that an electron-deficient iron porphyrin complex can catalyze the epoxidation of olefins by H2O2 under conditions of limiting substrate with high conversion efficiency in a solvent mixture of CH3OH and CH2Cl2.

Mouse TonEBP-NFAT5: expression in early development and alternative splicing.

Tonicity-responsive enhancer binding protein (TonEBP)- nuclear factor of activated T cell family 5 is a DNA binding protein that plays a key role in the response of cells to hypertonicity. However, TonEBP is expressed and active in tissues that are in an isotonic milieu. To explore the biological role of TonEBP, we cloned mouse TonEBP that shares 92% of amino acids with the human counterpart. TonEBP is expressed in embryonic stem cells and throughout the stages of fetal development. Immunohistochemical analysis shows expression of TonEBP in most, if not all, developing tissues, including the brain, colon, heart, muscle, and eyes. Widespread alternative splicing in exons 2-4 was detected throughout development and in different adult tissues. As a result, four different polypeptides are produced with different lengths at the NH(2) terminus. Two of the isoforms differ in their ability to stimulate transcription. In conclusion, the presence of TonEBP mRNA during mouse embryogenesis suggests that TonEBP functions at all stages of mouse development, as well as in isotonic adult tissues.