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Author(s): Michael Seiler [1]; Akihide Yoshimi [2]; Rachel Darman [1]; Betty Chan [1]; Gregg Keaney [1]; Michael Thomas [1]; Anant A Agrawal [1]; Benjamin Caleb [1]; Alfredo Csibi [1]; Eckley [...]

In nerve and muscle cells, the voltage-gated opening and closing of cation-selective ion channels is accompanied by the translocation of l2-14 elementary charges across the membrane's electric field. Although most of these charges are carried by residues in the S4 helix of the gating module of these channels, the precise nature of their physical movement is currently the topic of spirited debate. Broadly speaking, two classes of models have emerged: those that suggest that small-scale motions can account for the extensive charge displacement, and those that invoke a much larger physical movement. In the most recent incarnation of the latter type of model, which is based on structural and functional data from the archaebacterial [K.sup.+] channel KvAP, a 'voltage-sensor paddle' comprising a helix-turn-helix of S3-S4 translocates ~20 [Angstrom] through the bilayer during the gating cycle (Jiang, Y., A. Lee, J. Chen, V. Ruta, M. Cadene, B.T. Chair, and R. MacKinnon. 2003. Nature. 423:33-41; Jiang, Y., V. Ruta, J. Chen, A. Lee, and R. MacKinnon. 2003. Nature. 423:42-48.; Ruta, V.,J. Chen, and R. MacKinnon. 2005. Cell. 123:463-475). We used two methods to test for analogous motions in the Shaker [K.sup.+] channel, each examining the aqueous exposure of residues near S3. In the first, we employed a pore-blocking maleimide reagent (Blaustein, R.O., P.A. Cole, C. Williams, and C. Miller. 2000. Nat. Struct. Biol. 7:309-311) to probe for state-dependent changes in the chemical reactivity of substituted cysteines; in the second, we tested the state-dependent accessibility of a tethered biotin to external streptavidin (Qiu, X.Q,., K.S. Jakes, A. Finkelstein, and S.L. Slatin. 1994. J. Biol. Chem. 269:7483-7488; Slatin, S.L., X.Q. Qiu, K.S. Jakes, and A. Finkelstein. 1994. Nature. 371:158-161). In both types of experiments, residues predicted to lie near the top of $3 did not exhibit any change in aqueous exposure during the gating cycle. This lack of state dependence argues against large-scale movements, either axially or radially, of Shaker's S3-S4 voltage-sensor paddle.

Euryhaline teleosts such as Atlantic killifish (Fundulus heteroclitus) are able to acclimate to changing environmental salinity by tightly regulating NaCI absorption and secretion across their gills. Many studies have examined the mechanisms responsible for long-term (days) salinity acclimation; however, much remains unknown about the mechanisms of acute (hours) salinity acclimation. In this study, we tested the hypotheses that phosphorylation of the Na+-K+-Cl- cotransporter (NKCC1) located in the basolateral membrane of the gill plays a role in acute salinity acclimation and that changes in NKCC1 phosphorylation are mediated by a cAMP-protein kinase A (cAMP-PKA) pathway. Using a phospho-specific antibody, we determined the time course of changes in total and phosphorylated NKCC1 protein during acclimation to water of various salinities. Long-term (⩾14 days) acclimation of killifish to seawater (SW) and 2× SW resulted in 4- to 6-fold and 5- to 8-fold increases, respectively, in total gill NKCC1 protein relative to fish maintained in freshwater (FW). NKCC1 was found to be between 20% and 70% activated in fish, with lower average activation in fish acclimated to SW and 2× SW compared with FW fish. Increases and decreases in the fractional level of NKCC1 phosphorylation were seen within 1 h of transfer of fish to water of higher and lower salinity, respectively, consistent with a regulatory role of phosphorylation prior to an increase in the biosynthesis of NKCC1; large changes in protein expression of NKCC1 were observed over periods of hours to days. We found that NKCC1 phosphorylation is acutely regulated in the killifish gill in response to changing environmental salinity and that phosphorylation in excised gills increases in response to forskolin stimulation of the cAMP-PKA pathway. The role of phosphorylation is further underscored by the observation that mRNA expression of sterile 20 (Ste20)-related proline-alanine-rich kinase (SPAK) changes with salinity acclimation, being 2.7-fold greater in SW-acclimated killifish relative to FW fish. Overall, these results demonstrate an important role of NKCC1 phosphorylation in the gill of Atlantic killifish during acute salinity acclimation. [ABSTRACT FROM AUTHOR]

In nerve and muscle cells, the voltage-gated opening and closing of cation-selective ion channels is accompanied by the translocation of 12–14 elementary charges across the membrane's electric field. Although most of these charges are carried by residues in the S4 helix of the gating module of these channels, the precise nature of their physical movement is currently the topic of spirited debate. Broadly speaking, two classes of models have emerged: those that suggest that small-scale motions can account for the extensive charge displacement, and those that invoke a much larger physical movement. In the most recent incarnation of the latter type of model, which is based on structural and functional data from the archaebacterial K+, channel KvAP, a ‘voltage-sensor paddle’ comprising a helix-turn-helix of S3–S4 translocates ~20 Å through the bilayer during the gating cycle (Jiang, Y., A. Lee, J. Chen, V. Ruta, M. Cadene, B.T. Chait, and R. MacKinnon. 2003. Nature. 423:33–41; Jiang, Y., V. Ruta, J. Chen, A. Lee, and R. MacKinnon. 2003. Nature. 423:42–48.; Ruta, V., J. Chen, and R. MacKinnon. 2005. Cell. 123:463–475). We used two methods to test for analogous motions in the Shaker K+ channel, each examining the aqueous exposure of residues near S3. In the first, we employed a pore-blocking maleimide reagent (Blaustein, R.O., P.A. Cole, C. Williams, and C. Miller. 2000. Nat. Struct. Biol. 7:309–311) to probe for state-dependent changes in the chemical reactivity of substituted cysteines; in the second, we tested the state-dependent accessibility of a tethered biotin to external streptavidin (Qiu, X.Q., K.S. Jakes, A. Finkelstein, and S.L. Slatin. 1994. J. Biol. Chem. 269:7483–7488; Slatin, S.L., X.Q. Qiu, K.S. Jakes, and A. Finkelstein. 1994. Nature. 371:158–161). In both types of experiments, residues predicted to lie near the top of S3 did not exhibit any change in aqueous exposure during the gating cycle. This lack of state dependence argues against large-scale movements, either axially or radially, of Shaker's S3-S4 voltage-sensor paddle. [ABSTRACT FROM AUTHOR]

DNMT3A (DNA methyltransferase 3A) is a de novo DNA methyltransferase responsible for establishing CpG methylation patterns within the genome. DNMT3A activity is essential for normal development, and its dysfunction has been linked to developmental disorders and cancer. DNMT3A is frequently mutated in myeloid malignancies with the majority of mutations occurring at Arg-882, where R882H mutations are most frequent. The R882H mutation causes a reduction in DNA methyltransferase activity and hypomethylation at differentially-methylated regions within the genome, ultimately preventing hematopoietic stem cell differentiation and leading to leukemo-genesis. Although the means by which the R882H DNMT3A mutation reduces enzymatic activity has been the subject of several studies, the precise mechanism by which this occurs has been elusive. Herein, we demonstrate that in the context of the full-length DNMT3A protein, the R882H mutation stabilizes the formation of large oligomeric DNMT3A species to reduce the overall DNA methyltransferase activity of the mutant protein as well as the WT-R882H complex in a dominant-negative manner. This shift in the DNMT3A oligomeric equilibrium and the resulting reduced enzymatic activity can be partially rescued in the presence of oligomer-disrupting DNMT3L, as well as DNMT3A point mutations along the oligomer-forming interface of the catalytic domain. In addition to modulating the oligomeric state of DNMT3A, the R882H mutation also leads to a DNA-binding defect, which may further reduce enzymatic activity. These findings provide a mechanistic explanation for the observed loss of DNMT3A activity associated with the R882H hot spot mutation in cancer. [ABSTRACT FROM AUTHOR]

Tubular acid-base transport regulates systemic acid-base balance. Transepithelial acid-base transport across nephron segments requires the coordinated control of intracellular pH and cellular volume by transporters of protons and bicarbonate. Bicarbonate transporter polypeptides are encoded by at least two gene families, SLC4 and SLC26. The SLC4 gene family includes at least three Na()+)-independent chloride-bicarbonate exchanger genes and multiple Na(+)-bicarbonate cotransporter and Na(+)-dependent anion exchanger genes. The most extensively studied among them are the Na(+)-independent anion exchangers, AE1, AE2, and AE3, all of which are expressed in kidney. The AE1 gene encodes eAE1 (band 3), the major intrinsic protein of the erythrocyte, as well as kAE1, the basolateral Cl/HCO3 exchanger of the acid-secreting Type A intercalated cell. Mutations in AE1 are responsible for some forms of heritable distal renal tubular acidosis. The widely expressed AE2 anion exchanger participates in recovery from alkaline load and in regulatory cell volume increase following shrinkage. AE2 can also be regulated by ammonium ion. These properties are not shared by the closely related AE1 anion exchanger. Less is known about AE3 in kidney. Structure-function studies of recombinant proteins involving chimeras, deletions, and point mutations have delineated regions of AE2 which are important in exhibition of the regulatory properties absent from AE1. These include regions of the transmembrane domain and the N-terminal cytoplasmic domain. Noncontiguous regions in the middle of the N-terminal cytoplasmic domain are of particular importance for acute regulation by several types of stimulus.