Identification of specific pore residues mediating KCNQ1 inactivation. A novel mechanism for long QT syndrome

KCNQ1 inactivation bears electrophysiological characteristics different from classical N- and C-type inactivation in Shaker-like potassium channels. However, the molecular site of KCNQ1 inactivation has not yet been determined. KCNQ2 channels do not exert a fast inactivation in contrast to KCNQ1 channels. By expressing functional chimeras between KCNQ1 and KCNQ2 in Xenopus oocytes, we mapped the region of this inactivation to transmembrane domain S5 and the pore loop H5 and finally narrowed down the site to positions Gly(272) and Val(307) in KCNQ1. Exchanging these two amino acids individually with the analogous KCNQ2 residue abolished inactivation. Furthermore, a KCNQ1-like inactivation was introduced into KCNQ2 by mutagenesis in the corresponding region, confirming its relevance for the inactivation process. As KCNQ1 inactivation involves the regions S5 and H5, it exhibits a geography distinct from N- or C-type inactivation. Native cardiac I(Ks) channels comprising KCNQ1 and accessory MinK subunits do not inactivate because of the functional interaction of KCNQ1 with MinK. Mutations in KCNQ1 can lead to long QT1 syndrome, an inherited form of arrhythmia. The long QT1 mutant KCNQ1(L273F) displays a pronounced KCNQ1 inactivation. Here we show that when expressing mutant I(Ks) channels formed from KCNQ1(L273F) and MinK, MinK association no longer eliminates KCNQ1 inactivation. This results in smaller repolarizing currents in the heart and therefore represents a novel mechanism leading to long QT syndrome.     

Constitutively active mitogen-activated protein kinase kinase 6 (MKK6) or salicylate induces spontaneous 3T3-L1 adipogenesis

Although much has been learned regarding the importance of p38 mitogen-activated protein kinase in inflammatory and stress responses, relatively little is known concerning its role in differentiation processes. Recently, we demonstrated that p38 mitogen-activated protein kinase activity is necessary for the differentiation of 3T3-L1 fibroblasts into adipocytes (Engelman, J. A., Lisanti, M. P., and Scherer, P. E. (1998) J. Biol. Chem. 273, 32111-32120). p38 activity is high during the initial stages of differentiation but decreases drastically as the fibroblasts undergo terminal differentiation into adipocytes. However, it remains unknown whether activation of p38 is sufficient to stimulate adipogenesis and whether the down-regulation of p38 activity in mature adipocytes is critical for maintaining adipocyte homeostasis. In this report, we have directly addressed these questions by analyzing 3T3-L1 cell lines harboring a specific upstream activator of p38 (a constitutively active mitogen-activated protein kinase kinase 6 (MKK6) mutant, MKK6(Glu)) under the control of an inducible promoter. Induction of MKK6(Glu) in 3T3-L1 fibroblasts spurs adipocyte conversion in the absence of the hormonal mixture normally required for efficient differentiation of wild-type cells. However, activation of p38 in adipocytes leads to cell death. Furthermore, treatment of 3T3-L1 fibroblasts with salicylate, a potent stimulator of p38, produces adipocyte-specific changes consistent with those observed with induction of MKK6(Glu). Expression of MKK6(Glu) in NIH-3T3 fibroblasts (cells that do not differentiate into adipocytes under normal conditions) is capable of converting these fibroblasts into lipid-laden fat cells following hormonal stimulation. Thus, p38 activation has pro-adipogenic effects in multiple fibroblast cell lines.     

DNA sequence similarity requirements for interspecific recombination in Bacillus

Gene transfer in bacteria is notoriously promiscuous. Genetic material is known to be transferred between groups as distantly related as the Gram positives and Gram negatives. However, the frequency of homologous recombination decreases sharply with the level of relatedness between the donor and recipient. Several studies show that this sexual isolation is an exponential function of DNA sequence divergence between recombining substrates. The two major factors implicated in producing the recombinational barrier are the mismatch repair system and the requirement for a short region of sequence identity to initiate strand exchange. Here we demonstrate that sexual isolation in Bacillus transformation results almost exclusively from the need for regions of identity at both the 5' and 3' ends of the donor DNA strand. We show that, by providing the essential identity, we can effectively eliminate sexual isolation between highly divergent sequences. We also present evidence that the potential of a donor sequence to act as a recombinogenic, invasive end is determined by the stability (melting point) of the donor-recipient complex. These results explain the exponential relationship between sexual isolation and sequence divergence observed in bacteria. They also suggest a model for rapid spread of novel adaptations, such as antibiotic resistance genes, among related species.     

The Occurrence of 4-Amino-4-Deoxy-Arabinose in LPS of Supersusceptible Strain Pseudomonas aeruginosa Z61: Noncorrelation with Polymyxin Resistance

Lipopolysaccharides (LPS) extracted from the supersusceptible strain Pseudomonas aeruginosa Z61 were compared with LPS from other strains with varying antimicrobial susceptibilities. The presence of 4-amino-4-deoxy-arabinose (4-AraN) in P. aeruginosa Z61 LPS was confirmed by gas-liquid chromatography/mass spectrometry (GLC-MS) and quantitated by high-performance liquid chromatography (HPLC). Z61 LPS (compared with wild-type strain PAO1) has reduced amounts of rhamnose and higher concentrations of hydroxy fatty acids, 4-AraN, and phosphates. ³¹P Nuclear magnetic resonance revealed that Z61 LPS phosphates are configured in monophosphates, phosphodiesters, pyrophosphomonoesters, and glycosidic pyrophosphodiester groups. The presence of 4-AraN in P. aeruginosa LPS did not correlate with antimicrobial resistance. Received: 31 August 1998 / Accepted: 5 November 1998     

AVP V1 receptor-mediated decrease in Cl- efflux and increase in dark cell number in choroid plexus epithelium

The cerebrospinalfluid (CSF)-generating choroid plexus (CP) has manyV₁ binding sites for argininevasopressin (AVP). AVP decreases CSF formation rate and choroidal bloodflow, but little is known about how AVP alters ion transport across theblood-CSF barrier. Adult rat lateral ventricle CP was loaded with³⁶Cl,exposed to AVP for 20 min, and then placed in isotope-free artificial CSF to measure release of³⁶Cl.Effect of AVP at 10¹² to10⁷ M on theCl efflux rate coefficient(in s¹) was quantified.Maximal inhibition (by 20%) ofCl extrusion at10⁹ M AVP was prevented bythe V₁ receptor antagonist[-mercapto-,-cyclopentamethyleneproprionyl¹,O-Me-Tyr²,Arg⁸]vasopressin.AVP also increased by more than twofold the number of dark and possiblydehydrated but otherwise morphologically normal choroid epithelialcells in adult CP. The V₁ receptorantagonist prevented this AVP-induced increment in dark cell frequency.In infant rats (1 wk) with incomplete CSF secretory ability,10⁹ M AVP altered neitherCl efflux nor dark cellfrequency. The ability of AVP to elicit functional and structuralchanges in adult, but not infant, CP epithelium is discussed in regardto ion transport, CSF secretion, intracranial pressure, and hydrocephalus.

     

Alteration of Phosphoinositide Degradation by Cytosolic and Membrane-Bound Phospholipases after Forebrain Ischemia - Reperfussion in Gerbil: Effects of Amyloid Beta Peptide

The reperfusion of previously ischemic brain is associated with exacerbation of cellular injury. Reperfusion occasionally potentates release of intracellular enzymes, influx of Ca²⁺, breakdown of membrane phospholipids, accumulation of amyloid precursor protein or amyloid -(like) proteins, and apolipoprotein E. In this study, the effect of reperfusion injury on the activity of cerebral cortex enzymes acting on phosphatidyl [³H] inositol (PI) and [^l4C-arachidonoyl] PI was investigated. Moreover the effect of amyloid 25–35 on PI degradation by phospholipase(s) of normoxic brain and subjected to ichemia-reperfussion injury was determined. Brain ischemia in gerbils (Meriones unguiculatus) was induced by ligation of both common carotid arteries for 5 min and then brains were perfused for 15 min, 2 h and 7 days. Statistically significant activation of enzyme(s) involved in phosphatidylinositol degradation in gerbils subjected to ischemia-reperfusion injury was observed. Nearly all gerbils showed a higher activity of cytosolic PI phos-pholipase C (PLC) at 15 min after ischemia. Concomitantly, the significant enhancement of the level of DAG and AA radioactivity at this short reperfusion time confirmed the active PI degradation by phospholipase(s) in cerebral cortex and hippocampus. After a prolonged reperfusion time of 7 days after ischemia, both cytosolic and membrane-bound forms of PI-PLC were activated. The question arises if alteration of membranes by the degradation of phospholipids occurring after an ischemic episode potentates the effect of A on membrane-bound enzymes. A neuro-toxic fragment of amyloid, A 25–35, incubated in the presence of endogenous Ca²⁺, increased significantly the PI-PLC activity of normoxic brain. In its non-aggregated form, A 25–35 activates PI-PLC but in the aggregated form the enzymatic activity decreased. Thus, A 25–35 exerts a similar effect on the membrane-bound PI-PLC from normoxic brain or subjected to ischemia reperfussion injury. We conclude that the degradation of phosphatidylinositol by cytosolic phosphoinositide-phospholipase C may contribute to the pathophysiology of delayed neuronal death following cerebral ischemia. Thus, a specific inhibitor of this enzyme(s) may offer therapeutic strategies to protect the brain from damage triggered by ischemia. Ischemia-reperfusion injury had no effect on A-evoked alterations of synaptic plasma membrane-bound PI-PLC.     

Domain mapping of the Rad51 paralog protein complexes

The five human Rad51 paralogs are suggested to play an important role in the maintenance of genome stability through their function in DNA double-strand break repair. These proteins have been found to form two distinct complexes in vivo, Rad51B–Rad51C–Rad51D–Xrcc2 (BCDX2) and Rad51C–Xrcc3 (CX3). Based on the recent Pyrococcus furiosus Rad51 structure, we have used homology modeling to design deletion mutants of the Rad51 paralogs. The models of the human Rad51B, Rad51C, Xrcc3 and murine Rad51D (mRad51D) proteins reveal distinct N-terminal and C-terminal domains connected by a linker region. Using yeast two-hybrid and co-immunoprecipitation techniques, we have demonstrated that a fragment of Rad51B containing amino acid residues 1–75 interacts with the C-terminus and linker of Rad51C, residues 79–376, and this region of Rad51C also interacts with mRad51D and Xrcc3. We have also determined that the N-terminal domain of mRad51D, residues 4–77, binds to Xrcc2 while the C-terminal domain of mRad51D, residues 77–328, binds Rad51C. By this, we have identified the binding domains of the BCDX2 and CX3 complexes to further characterize the interaction of these proteins and propose a scheme for the three-dimensional architecture of the BCDX2 and CX3 paralog complexes.