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The voltage-gated proton channel 1 ( HV1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. HV1 has been identified in a wide range of eukaryotes throughout the animal kingdom, with the exception of insects. Therefore, it has been proposed that insects do not possess an HV1 channel. In the present study, we report the existence of an HV1-type proton channel in insects. We searched insect transcriptome shotgun assembly ( TSA) sequence databases and found putative HV1 orthologues in various polyneopteran insects. To confirm that these putative HV1 orthologues were functional channels, we studied the HV1 channel of Nicoletia phytophila (Np HV1), an insect of the Zygentoma order, in more detail. Np HV1 comprises 239 amino acids and is 33% identical to the human voltage-gated proton channel 1. Patch clamp measurements in a heterologous expression system showed proton selectivity, as well as pH- and voltage-dependent gating. Interestingly, Np HV1 shows slightly enhanced pH-dependent gating compared to the human channel. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, lead to a loss of proton-selective conduction, confirming the importance of this aspartate residue in voltage-gated proton channels. Database Nucleotide sequence data have been deposited in the GenBank database under accession number . [ABSTRACT FROM AUTHOR]

Abstract: The promising use of mesenchymal stromal cells (MSC) in regenerative technologies accounts for necessity of detailed study of their physiology. Proliferation and differentiation of multipotent cells often involve changes in their metabolic state. In the present study, we analyzed the expression of ATP-sensitive potassium (KATP) channels in MSC and upon in vitro differentiation. KATP channels are present in many cells and regulate a variety of cellular functions by coupling cell metabolism with membrane potential. Kir6.1, Kir6.2 and SUR2A were expressed in undifferentiated MSC, whereas SUR2B and SUR1 were not detected on cDNA and protein level. Upon adipogenic differentiation Kir6.1 and SUR2A showed a significant reduction of the amount of mRNA by 84% and 95%, respectively, whereas Kir6.2 expression was unchanged. Osteogenic differentiation strongly up-regulated Kir6.2 mRNA (28-fold) whereas Kir6.1 and SUR2A showed no significant change in expression. Quantitative Western blot analysis and immunofluorescence staining confirmed the elevated expression of Kir6.2 upon osteogenic differentiation. Taken together, expression changes of KATP channels may contribute to in vitro differentiation of MSC and represent changes in the metabolic state of the developing tissue. [Copyright &y& Elsevier]

K+-channels fulfill several important functions in the mammalian kidney such as volume regulation, recirculation and secretion of K+ ions, and maintaining the resting potential. In this study we used immunocytochemical methods, in situ hybridization, and nephron segment-specific RT-PCR to obtain a detailed picture of the cellular localization of two tandem pore domain potassium (K2P) channels, THIK-1 (K2P13.1, KCNK13) and THIK-2 (K2P12.1, KCNK12). Monospecific antibodies against C-terminal domains of rat THIK-1 and THIK-2 proteins (GST-fusion proteins) were raised in rabbits, freed from cross-reactivity, and affinity purified. All antibodies were validated by Western blot analysis, competitive ELISA, and preabsorption experiments. The expression of THIK channels in specific nephron segments was confirmed by double staining with marker proteins. Results indicate that in rat and mouse THIK-1 and THIK-2 were expressed in the proximal tubule (PT), thick ascending limb (TAL), connecting tubule (CNT), and cortical collecting duct (CCD). In human kidney THIK-1 and THIK-2 were localized in PT, TAL and CCD. Immunostaining of rat tissue revealed an intracellular expression of THIK-1 and THIK-2 throughout the identified nephron segments. However in mouse kidney THIK-2 was identified in basolateral membranes. Overall, the glomerulus, thin limbs and medullary collecting ducts were devoid of THIK-1 and THIK-2 signal. In summary, THIK-1 and THIK-2 are abundantly expressed in the proximal and distal nephron of the mammalian kidney. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The potassium channels TASK-1 and TASK-3 show high sequence homology but differ in their sensitivity to extracellular divalent cations. Heterologous expression in HEK293 cells showed that the single-channel conductance of TASK-3 increased approximately four-fold after removal of external divalent cations, whereas the conductance of TASK-1 was unaffected. Replacing the glutamate at position 70 of TASK-3 by a lysine or arginine residue abolished the sensitivity to divalent cations. The reverse mutation in TASK-1 (K70E) induced sensitivity to divalent cations. The organic polycations spermine and ruthenium red modulated the conductance of TASK-3 in a similar way as Ca2+ or Mg2+. Our data suggest that these effects were mediated by shielding of the negative charges in the extracellular loops of TASK-3. Whole-cell currents carried by TASK-3 channels were inhibited by spermine and ruthenium red even in the presence of external divalent cations. These data suggest that, in addition to their effect on single-channel conductance, spermine and ruthenium red decreased the open probability of TASK-3 channels, probably by binding to residue E70. The standing outward current in thalamocortical relay neurons, which is largely carried by TASK channels, was also inhibited by divalent cations and spermine. Using the differential sensitivity of TASK-1 and TASK-3 to divalent cations and spermine we found that about 20% of the standing outward current in thalamocortical relay neurons flows through TASK-3 channels. We conclude from our results that inhibition of TASK-3 channels may contribute to the neuromodulatory effect of spermine released from neurons during repetitive activity or during hypoxia. [ABSTRACT FROM AUTHOR]

Studies the evolutionary link between prokaryotic and eukaryotic potassium channels. Discussion on prokaryotic potassium channel structures lacking a putative NAD...-binding domain; Summary of prokaryotic potassium channels; Evolutionary scheme.

The importance of five tyrosine residues of Escherichia coli asparaginase H (EcA2) for catalysis and protein stability was examined by site-directed mutagenesis, chemical modification of wildtype and variant enzymes, and by thermodynamic studies of protein denaturation. While the tyrosine residue Y25 is directly involved in catalysis, the hydroxyl groups of residues Y181, Y250, Y289 and Y326 are not necessary for EcA2 activity. However. residues Y181 and Y326 are crucial for stabilization of the native EcA2 tetramer, pH titration curves showed that the active-site residue Y25 has a normal pKa, while the C-terminal Y326 is unusually acidic. ¹H-NMR signals of a peculiar ligand-sensitive tyrosine residue were assigned to Y25. These and other data suggest that a peptide loop (residues 14-27) which shields the active site during catalysis is highly flexible in the free enzyme. [ABSTRACT FROM AUTHOR]

The amino acid sequence and tertiary structure of Wolinella succinogenes L-asparaginase were determined, and were compared with the structures of other type-II bacterial L-asparaginases. Each chain of this homotetrameric enzyme consists of 330 residues. The amino acid sequence is 40–50% identical to the sequences of related proteins from other bacterial sources, and all residues previously shown to be crucial for the catalytic action of these enzymes are identical. Differences between the amino acid sequence of W. succinogenes L-asparaginase and that of related enzymes are discussed in terms of the possible influence on the substrate specificity. The overall fold of the protein subunit is almost identical to that observed for other L-asparaginases. Two fragments in each subunit, a very highly flexible loop (≈20 amino acids) that forms part of the active site, and the N-terminus (two amino acids), are not defined in the structure. The orientation of Thr14, a residue probably involved in the catalytic activity, indicates the absence of ligand in the active-site pocket. The rigid part of the active site, which includes the asparaginase triad Thr93-Lys166-Asp94, is structurally very highly conserved with equivalent regions found in other type-II bacterial L-asparaginases. [ABSTRACT FROM AUTHOR]

Site-specific mutagenesis was used to replace the three histidine residues of Escherichia coli asparaginase II (EcA2) with other amino acids. The following enzyme variants were studied: [H87A]EcA2, [H87L]EcA2, [H87K]EcA2, [H183L]EcA2 and [H197L]EcA2. None of the mutations substantially affected the Km for L-aspartic acid β-hydroxamate or impaired aspartate binding. The relative activities towards L-Ash, L-Gln, and l-aspartic acid β-hydroxamate were reduced to the same extent, with residual activities exceeding 10% of the wild-type values. These data do not support a number of previous reports suggesting that histidine residues are essential for catalysis. Spectroscopic characterization of the modified enzymes allowed the unequivocal assignment of the histidine resonances in ¹H-NMR spectra of asparaginase II. A histidine signal previously shown to disappear upon aspartate binding is due to His183, not to the highly conserved His87. The fact that [H183L]EcA2 has normal activity but greatly reduced stability in the presence of urea suggests that His183 is important for the stabilization of the native asparaginase tetramer. ¹H-NMR and fluorescence spectroscopy indicate that His87 is located in the interior of the protein, possibly adjacent to the active site. [ABSTRACT FROM AUTHOR]