Blockade of hERG K؉ channels in the heart is an unintentional side effect of many drugs and can induce cardiac arrhythmia and sudden death. It has become common practice in the past few years to screen compounds for hERG channel activity early during the drug discovery process. Understanding the molecular basis of drug binding to hERG is crucial for the rational design of medications devoid of this activity. We previously identified 2 aromatic residues, Tyr-652 and Phe-656, located in the S6 domain of hERG, as critical sites of interaction with structurally diverse drugs. Here, Tyr-652 and Phe-656 were systematically mutated to different residues to determine how the physicochemical properties of the amino acid side group affected channel block by cisapride, terfenadine, and MK-499. The potency for block by all three drugs was well correlated with measures of hydrophobicity, especially the twodimensional approximation of the van der Waals hydrophobic surface area of the side chain of residue 656. For residue 652, an aromatic side group was essential for high affinity block, suggesting the importance of a cation-interaction between Tyr-652 and the basic tertiary nitrogen of these drugs. hERG also lacks a Pro-Val-Pro motif common to the S6 domain of most other voltagegated K ؉ channels. Introduction of Pro-Val-Pro into hERG reduced sensitivity to drugs but also altered channel gating. Together, these findings assign specific residues to receptor fields predicted by pharmacophore models of hERG channel blockers and provide a refined molecular understanding of the drug binding site. Long QT syndrome (LQTS)1 is a disorder of ventricular repolarization that predisposes affected individuals to cardiac arrhythmia and sudden death. Inherited LQTS is caused by mutations in K ϩ or Na ϩ ion channel genes or ankyrin-B (1, 2). Acquired LQTS is more common and can be induced as an unintended and rare side effect of treatment with many structurally diverse medications. In the past few years, several commonly used drugs (e.g. terfenadine, cisapride, sertindole, thioridazine, grepafloxacin) were withdrawn from the market, or their approved use was severely restricted, when it was discovered that they caused arrhythmia or were associated with unexplained sudden death, albeit very infrequently (3). The molecular basis of drug-induced LQTS is block of human ether-a-go-go related gene (hERG) channels that conduct I Kr , the rapid delayed rectifier K ϩ current important for repolarization of cardiac action potentials (4, 5). A reduction in I Kr prolongs the action potential duration of ventricular myocytes, lengthens the QT interval and increases dispersion as measured by ECG recordings, and increases the risk of torsades de pointes, a ventricular tachyarrhythmia that can degenerate into fibrillation and cause sudden death. In a laboratory setting, it is possible to induce arrhythmia in animals with drugs that block voltage-gated K ϩ (Kv) channels other than hERG. However, in clinical practice, drug-induced LQTS is always attributable...

The Agrobacterium tumefaciens virB7 gene product is a lipoprotein whose function is required for the transmission of oncogenic T-DNA to susceptible plant cells. Three lines of study provided evidence that VirB7 interacts with and stabilizes other VirB proteins during the assembly of the putative T-complex transport apparatus. First, a precise deletion of virB7 from the pTiA6NC plasmid of wild-type strain A348 was correlated with significant reductions in the steady-state levels of several VirB proteins, including VirB4, VirB9, VirB10, and VirB11; trans expression of virB7 in the ⌬virB7 mutant partially restored the levels of these proteins, and trans coexpression of virB7 and virB8 fully restored the levels of these proteins to wild-type levels. Second, modulation of VirB7 levels resulted in corresponding changes in the levels of other VirB proteins in the following cell types: (i) a ⌬virB7 mutant expressing virB7 and virB8 from isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible P lac and other virB genes from acetosyringone (AS)-inducible P virB ; (ii) a ⌬virB operon mutant expressing virB7 and virB8 from P lac and virB9, virB10, and virB11 from P virB ; and (iii) a ⌬virB operon mutant expressing virB7 from IPTG-inducible P lac and virB9 from an AS-inducible P virB . Third, the synthesis of a VirB7::PhoA fusion protein in strain A348 was correlated with a significant reduction in the steady-state levels of VirB4, VirB5, and VirB7 through VirB11; these cells also exhibited a severely attenuated virulence phenotype, indicating that synthesis of the fusion protein perturbs the assembly of VirB proteins into a stabilized protein complex required for T-complex transport. Extracts of AS-induced cells electrophoresed under nonreducing conditions possessed undetectable levels of the 32-kDa VirB9 and 4.5-kDa VirB7 monomers and instead possessed a 36-kDa complex that cross-reacted with both VirB7 and VirB9 antisera and accumulated as a function of virB7 expression. Our results are consistent with a model in which VirB7 stabilizes VirB9 by formation of a covalent intermolecular cross-link; in turn, the VirB7-VirB9 heterodimer promotes the assembly of a functional T-complex transport machinery.The Agrobacterium tumefaciens ϳ9.5-kb virB operon encodes 11 proteins which are proposed to assemble into a transport apparatus required for the transmission of a complex of DNA and protein (T-complexes) to susceptible plant cells (for recent reviews, see references 11, 23, 26, 50, and 51). Recent genetic analyses have demonstrated that the products of virB2 through virB11 are essential for this transport process whereas the product of virB1 serves to enhance the efficiency of nucleoprotein transfer (6).Considerable efforts in several laboratories are being directed toward a structural definition of the putative virB-encoded T-complex transport apparatus. Initial studies have focused on defining the subcellular localizations and topologies of the VirB proteins. DNA sequence analyses of the virB operon showed that most of the VirB p...

The Agrobacterium tumefaciens virB7 gene product contains a typical signal sequence ending with a consensus signal peptidase II cleavage site characteristic of bacterial lipoproteins. VirB7 was shown to be processed as a lipoprotein by (i) in vivo labeling of native VirB7 and a VirB7::PhoA fusion with [ 3 H]palmitic acid and (ii) inhibition of VirB7 processing by globomycin, a known inhibitor of signal peptidase II. A VirB7 derivative sustaining a Ser substitution for the invariant Cys-15 residue within the signal peptidase II cleavage site could not be visualized immunologically and failed to complement a ⌬virB7 mutation, establishing the importance of this putative lipid attachment site for VirB7 maturation and function. VirB7 partitioned predominantly with outer membrane fractions from wild-type A348 cells as well as a ⌬virB operon derivative transformed with a virB7 expression plasmid. Expression of virB7 fused to phoA, the alkaline phosphatase gene of Escherichia coli, gave rise to high alkaline phosphatase activities in E. coli and A. tumefaciens cells, providing genetic evidence for the export of VirB7 in these hosts. VirB7 was shown to be intrinsically resistant to proteinase K; by contrast, a VirB7::PhoA derivative was degraded by proteinase K treatment of A. tumefaciens spheroplasts and remained intact upon treatment of whole cells. Together, the results of these studies favor a model in which VirB7 is topologically configured as a monotopic protein with its amino terminus anchored predominantly to the outer membrane and with its hydrophilic carboxyl domain located in the periplasmic space. Parallel studies of VirB5, VirB8, VirB9, and VirB10 established that each of these membrane-associated proteins also contains a large periplasmic domain whereas VirB11 resides predominantly or exclusively within the interior of the cell.Agrobacterium tumefaciens incites disease by processing a segment of its genome (T-DNA) into a translocation-competent nucleoprotein particle (T-complex) which is then delivered across the bacterial envelope to a susceptible plant cell (for recent reviews, see references 28 and 81). In recent years, studies of two early events in the infection process, T-DNA processing and T-complex transport across the A. tumefaciens envelope, and parallel studies of analogous events associated with the conjugative transfer of the IncP, IncN, and IncW broad-host-range plasmids have provided strong evidence that these interkingdom and interbacterial DNA transfer systems are evolutionarily related (see reference 40). Both the T-DNA and RP4 (an IncP␣ plasmid) processing reactions have been examined in detail. These studies have shown that transfer is initiated by a strand-and site-specific cleavage at nick sites found within T-DNA border sequences and transfer origins (oriT), respectively. The nick sites of these elements are highly conserved, as are the active-site motifs of the relaxases, VirD2 and TraI, that catalyze the nicking events at T-DNA borders and oriT of RP4, respectively (48, 49). Purifie...

Polynomial deformations of the Heisenberg algebra are studied in detail. Natural realizations of them are given by the higher order susy partners of the harmonic oscillator for even order polynomials. Here, it is shown that the susy partners of the radial oscillator play a similar role when the order of the polynomial is odd. Indeed, it will be proved that the general systems ruled by such a kind of algebras, in the quadratic and cubic cases, involve Painlevé transcendents of type IV and V, respectively.

The Agrobacterium tumefaciens VirB7 lipoprotein contributes to the stabilization of VirB proteins during biogenesis of the putative T-complex transport apparatus. Here, we report that stabilization of VirB7 itself is correlated with its ability to form disulfide cross-linked homodimers via a reactive Cys-24 residue. Three types of f8-mercaptoethanol-dissociable complexes were visualized