Modulation of cardiac sodium channel variants by Fyn tyrosine kinase

Spannungabhängige Natriumkanäle sind sehr große Membranproteine. In Säugetieren werden sie von zehn Genen kodiert. Natriumkanäle spielen eine wichtige Rolle in erregbaren Zellen und sie sind deshalb ein wichtiger Angriffspunkt für Arzneistoffe und Toxine. NaV1.5 ist der spannungsabhängige Natriumkanal im Herzen, wo er für die rasche Depolarisationsphase im Aktionspotential verantwortlich ist und auch an der Reizweiterleitung beteiligt ist. Von den neun Spleißvarianten von NaV1.5 sind vier funktionell. Die Spleißvariante ΔQ1077 und die Q1077Present Variante des kardialen Natriumkanals sind in 45% bzw. 25% der Bevölkerung anzutreffen. Kardiale Natriumkanäle werden durch unterschiedliche intrazelluläre Proteine moduliert. Tyrosinkinasen sind eine der potenten Modulatoren verschiedener Ionenkanäle. Fyn, das zur Src Tyrosinkinase-Familie gehört, moduliert verschiedene Natriumkanal-Isoformen auf unterschiedliche Weise. Die mit den Q1077Present und ΔQ1077 Varianten erhaltenen Ergebnisse zeigten unterschiedliche Inaktivierung in Gegenwart katalytisch aktiver FynCA. Während die Q1077Present Variante die Inaktivierungskurve in die depolarisierende Richtung verschob, trat die halbmaximale Inaktivierung bei ΔQ1077 bei negativeren Potentialen auf, ähnlich wie beim neuronalen Natriumkanal NaV1.2. Die durch FynCA bedingte Verschiebung der Inaktivierungskurve in der Q1077Present Variante war vergleichbar mit der durch FynCA hervorgerufenen Modulation in der hH1 polymorphen kardialen Natriumkanal-Variante. Mit der Kinase-toten Mutante FynKD kam es weder bei ΔQ1077 noch bei Q1077Present zu einer Verschiebung der Inaktivierungskurven. Weiters konnte in beiden Varianten die durch FynCA bedingte Verschiebung der Inaktivierungskurven durch den Src Tyrosinkinase-Hemmer PP2 aufgehoben werden. Die Aktivierungkurven beider Varianten, Q1077Present und ΔQ1077, wurden durch FynCA nicht beeinflusst. Eine Sequenzanalyse aller Natriumkanal-Isoformen zeigte, dass Glutamin in Position 1077 in einer der drei für NaV1.5 einzigartigen Sequenzen in der intrazellulären Schleife liegt, die die Domänen DII und DIII verbindet und die "Natriumionentransport-assoziierte" Region bildet. Eine Punktmutation in Position 1077 von Glutamin zu Alanin, Lysin, Tyrosin oder Prolin verschob die Inaktivierungskurven in Gegenwart von FynCA zu negativeren Potentialen. Es konnte eine lineare Korrelation zwischen dem Ausmaß der Verschiebung der Inaktivierungskurven durch FynCA und der hydrophoben Eigenschaft der Aminosäuren-Seitenketten beobachtet werden. Mögliche SH2 und SH3 Bindungsstellen für Fyn im kardialen Natriumkanal wurden ebenfalls identifiziert. Fünf spezifische, prolinreiche Motive, die als SH3 Bindungsstelle für Fyn im kardialen Natriumkanal fungieren könnten, wurden aufgefunden. Mögliche SH2 Bindungsmotive für Fyn wurden im C-Terminus von NaV1.5 identifiziert. Diese Daten tragen zum Verständnis der Rolle des Q1077 und den angrenzenden Aminosäuren bei. Die intrazelluläre Schleife LDII-DIII des kardialen Natriumkanals, die sich wesentlich von der anderer Natriumkanal-Isoformen unterscheidet, bildet eine der "Natriumionentransport-assoziierten" Regionen. Deshalb spielt LDII-DIII eine wichtige Rolle bei der Bindung intrazellulärer Signalmoleküle an den Natriumkanal.Voltage-gated sodium channels are very large membrane proteins. They are encoded by ten genes in mammals. Sodium channels are a crucial component of excitable tissues; hence, they are a target for various drugs and toxins. NaV1.5 is the cardiac voltage-gated sodium channel involved in the rapid depolarizing phase of the cardiac action potential. This isoform is also involved in the propagation of electrical impulses in the heart. Out of nine Nav1.5 splice variants, four of them were functional. The splice variant ∆Q1077 and Q1077Present variant of NaV1.5 are present in 45% and 25 % of human population, respectively. Cardiac sodium channels are modulated by different intracellular proteins. Tyrosine kinases are one of the potent intracellular modulators of ion channels. Fyn, a member of the Src tyrosine kinase family, modulates sodium channel isoforms distinctly. Results on the Q1077Present and the ΔQ1077 variants show different steady-state inactivation in presence of catalytic active FynCA. While the Q1077Present variant shifted the inactivation curve to more depolarizing potentials, the ΔQ1077 variant shifted the inactivation curve to more hyperpolarizing potentials, similar to neuronal sodium channel NaV1.2. The Fyn-induced shift in inactivation of Q1077Present was similar to the modulation observed in the hH1 polymorphic cardiac sodium channel variant with Fyn. The Src tyrosine kinase inhibitor PP2 reversed back the shifts caused by FynCA in both Q1077Present and ΔQ1077 cardiac sodium channel variants. Moreover, the kinase dead mutant FynKD did not shift both the ΔQ1077 and Q1077Present inactivation curves. The activation curves of both Q1077Present and ΔQ1077 were not affected by active Fyn. Sequence analysis of all the sodium channel isoforms reveal that the Q1077glutamine is present in one the three unique sequences in the intracellular loop connecting the domain DII and DIII, which forms the ‘sodium ion transport associated’ region. Point mutation at position 1077 from glutamine to alanine, lysine, tyrosine and proline shifted the inactivation curves to more hyperpolarizing potentials with FynCA. Linear correlation was observed between the hyperpolarizing shift caused by FynCA and the hydrophobicity of the amino acids’ side chains. Putative SH2 and SH3 binding sites for Fyn in the cardiac sodium channel were also determined. Five unique proline-rich motifs were identified, which are potential SH3 binding sites for Fyn in the cardiac sodium channel. Putative SH2 binding motifs for Fyn were found in the C-terminal of Nav1.5. These data will be pertinent in understanding the role of Q1077 and its surrounding amino acids. The intracellular loop LDII-DIII, which is distinct in cardiac sodium channels from other sodium channel isoforms, forms one of the ‘sodium ion transport associated’ regions. Thus, LDII-DIII plays a pivotal role in regulating cardiac sodium channel binding to various intracellular signaling molecules

Stereoselective synthesis of protected peptides containing an anti β‐Hydroxy tyrosine

Protected peptides containing an anti b-hydroxy tyrosine are synthesized in a straightforward and highly efficient manner through the direct and stereoselective addition of N-azidoacetyl-4-isopropyl-1,3-thiazolidine-2-thione to dialkyl acetals catalyzed by a nickel(II) complex, the forging of an amide bond by removal of the chiral auxiliary with an amino ester, and final coupling with a third amino acid

Synthesis of Chondramide A Analogues with Modified β‐Tyrosine and Their Biological Evaluation

Starting from cinnamates 9 , obtained by Wittig reaction or Heck coupling, the diols 17 were prepared by asymmetric dihydroxylation. This was followed by a regioselective substitution of the 3‐OH group with hydrazoic acid under Mitsunobu conditions. Methylation of the 2‐OH group and reduction of the azide group led to the β‐tyrosine derivatives 8 . Condensation with the dipeptide acid 6 furnished the tripeptide part of the chondramides. The derived acids 21 were combined with the hydroxy ester 7 to the esters 22 . Cleavage of the tert ‐butyl groups and intramolecular lactam formation gave rise to the chondramide A analogues 2 b – k . Growth inhibition assays showed most of the analogues to be biologically active. Some of them even reach the activity of jasplakinolide. It can be concluded that the 4‐position of the aryl ring in the β‐tyrosine of chondramide A tolerates structural modifications quite well. SUB ‐ units : Ten different chondramide A analogues were prepared with different substituents at the 3‐amino‐2‐methoxy‐propanoate subunit. The modified β‐tyrosines were obtained from the corresponding cinnamates. From Mitsunobu esterification, acyclic depsipeptides were obtained that were cyclized by macrolactam formation. Almost all analogues were at least as active as chondramide A itself. However, an amide group reduced the cytotoxicity significantly.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/88032/1/13349_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/88032/2/chem_201101978_sm_miscellaneous_information.pd

Immobilization of pyrene - adorned N - heterocyclic carbene complexes of rhodium (I) on reduced graphene oxide and study of catalytic activity

Twopyrene-tagged N-heterocyclic carbene (NHC) complexes of rhodium(I) wereobtained and characterized. Thetwo complexes were supported onto reduced graphene oxide (rGO), generating two new materials in which the molecular complexes are immob ilized by p–p stacking interactions onto the surfaceofthe solid. The catalytic activity of both complexes and solid hybrid materials were studied in the 1,4-addition of phenylboronic acid to cyclohex-2-one, and in the hydrosilylation of terminal alkynes. The studies showed that for both reactions,the dimetallic complex displayed better catalytic performances than the monometallic one. This accounted for both the reactions performed in homogeneous conditions and for the reactions performed with the solid. In the case of the addition of phenylboronic acid to cyclohexanone,the solid containing the dimetallic catalystcould be effectively recycled up to five times, with negligible loss of activity,whereas the monometallic catalyst rapidlybecame inactive. In the hydrosilylation of terminal alkynes, the selectivity towards the b-(Z)-vinylsilane was improved if the immobilized dimetallic catalyst was used, although the catalyst startedtolose activity after the second run

Cyclodepsipeptides from Marine Sponges: Natural Agents for Drug Research

A number of natural products from marine sponges, such as cyclodepsipeptides, have been identified. The structural characteristics of this family of cyclic peptides include various unusual amino acid residues and unique N-terminal polyketide-derived moieties. Papuamides are representatives of a class of marine sponge derived cyclic depsipeptides, including callipeltin A, celebesides A and B, homophymine A, mirabamides, microspinosamide, neamphamide A and theopapuamides. They are thought to have cytoprotective activity against HIV-1 in vitro by inhibiting viral entry. Jasplakinolide, a representative member of marine sponge-derived cyclodepsipeptides that include arenastatin A, geodiamolides, homophymines, spongidepsin and theopapuamides, is a potent inducer of actin polymerization in vitro. Although actin dynamics is essential for tumor metasasis, no actin targeting drugs have been used in clinical trials due to their severe cytotoxicity. Nonetheless, the actin cytoskeleton remains a potential target for anti-cancer drug development. These features imply the use of cyclodepsipeptides as molecular models in drug research

Role of the Interaction Motif in Maintaining the Open Gate of an Open Sodium Channel

Voltage-gated sodium channels undergo transitions between open, closed, and inactivated states, enabling regulation of the translocation of sodium ions across membranes. A recently published crystal structure of the full-length prokaryotic NavMs crystal structure in the activated open conformation has revealed the presence of a novel motif consisting of an extensive network of salt bridges involving residues in the voltage sensor, S4-S5 linker, pore, and C-terminal domains. This motif has been proposed to be responsible for maintaining an open conformation that enables ion translocation through the channel. In this study, we have used long-time molecular dynamics calculations without artificial restraints to demonstrate that the interaction network of full-length NavMs indeed prevents a rapid collapse and closure of the gate, in marked difference to earlier studies of the pore-only construct in which the gate had to be restrained to remain open. Interestingly, a frequently discussed “hydrophobic gating” mechanism at nanoscopic level is also observed in our simulations, in which the discontinuous water wire close to the gate region leads to an energetic barrier for ion conduction. In addition, we demonstrate the effects of in silico mutations of several of the key residues in the motif on the open channel’s stability and functioning, correlating them with existing functional studies on this channel and homologous disease-associated mutations in human sodium channels; we also examine the effects of truncating/removing the voltage sensor and C-terminal domains in maintaining an open gate

Cyclodepsipeptides from Marine Sponges: Natural Agents for Drug Research

A number of natural products from marine sponges, such as cyclodepsipeptides, have been identified. The structural characteristics of this family of cyclic peptides include various unusual amino acid residues and unique N-terminal polyketide-derived moieties. Papuamides are representatives of a class of marine sponge derived cyclic depsipeptides, including callipeltin A, celebesides A and B, homophymine A, mirabamides, microspinosamide, neamphamide A and theopapuamides. They are thought to have cytoprotective activity against HIV-1 in vitro by inhibiting viral entry. Jasplakinolide, a representative member of marine sponge-derived cyclodepsipeptides that include arenastatin A, geodiamolides, homophymines, spongidepsin and theopapuamides, is a potent inducer of actin polymerization in vitro. Although actin dynamics is essential for tumor metasasis, no actin targeting drugs have been used in clinical trials due to their severe cytotoxicity. Nonetheless, the actin cytoskeleton remains a potential target for anti-cancer drug development. These features imply the use of cyclodepsipeptides as molecular models in drug research