Structural changes during HCN channel gating defined by high affinity metal bridges

Hyperpolarization-activated cyclic nucleotide–sensitive nonselective cation (HCN) channels are activated by membrane hyperpolarization, in contrast to the vast majority of other voltage-gated channels that are activated by depolarization. The structural basis for this unique characteristic of HCN channels is unknown. Interactions between the S4–S5 linker and post-S6/C-linker region have been implicated previously in the gating mechanism of HCN channels. We therefore introduced pairs of cysteines into these regions within the sea urchin HCN channel and performed a Cd2+-bridging scan to resolve their spatial relationship. We show that high affinity metal bridges between the S4–S5 linker and post-S6/C-linker region can induce either a lock-open or lock-closed phenotype, depending on the position of the bridged cysteine pair. This suggests that interactions between these regions can occur in both the open and closed states, and that these regions move relative to each other during gating. Concatenated constructs reveal that interactions of the S4–S5 linker and post-S6/C-linker can occur between neighboring subunits. A structural model based on these interactions suggests a mechanism for HCN channel gating. We propose that during voltage-dependent activation the voltage sensors, together with the S4–S5 linkers, drive movement of the lower ends of the S5 helices around the central axis of the channel. This facilitates a movement of the pore-lining S6 helices, which results in opening of the channel. This mechanism may underlie the unique voltage dependence of HCN channel gating

Inositol 1,4,5-trisphosphate receptors and their protein partners as signalling hubs.

Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are expressed in nearly all animal cells, where they mediate the release of Ca(2+) from intracellular stores. The complex spatial and temporal organization of the ensuing intracellular Ca(2+) signals allows selective regulation of diverse physiological responses. Interactions of IP3 Rs with other proteins contribute to the specificity and speed of Ca(2+) signalling pathways, and to their capacity to integrate information from other signalling pathways. In this review, we provide a comprehensive survey of the proteins proposed to interact with IP3 Rs and the functional effects that these interactions produce. Interacting proteins can determine the activity of IP3 Rs, facilitate their regulation by multiple signalling pathways and direct the Ca(2+) that they release to specific targets. We suggest that IP3 Rs function as signalling hubs through which diverse inputs are processed and then emerge as cytosolic Ca(2+) signals.This work was supported by the Biotechnology and Biological Sciences Research Council (L0000075) and the Wellcome Trust (101844).This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1113/JP27113

Polemologija i ksenologija: Valdenfels i žaoka stranog

After explaining why phenomenology of the alien cannot be counted among traditional philosophical disciplines, the author explores why all of European history can be read as the “shading of the alien” (Verblendung des Fremden), although not in the sense of mere disregarding, neglecting or denying of the alien, but disciplining it, manipulating and exploiting it. The alien has not been forgotten for centuries, it was always in the European focus, but only as an instance through which the sense of power was traditionally constructed. Following the basic presumptions of Bernhard Waldenfels’ phenomenology of the alien the article presents the shading of the alien as analogous to the process of its naturalization. As if the tradition of European colonialism can be best understood in the key of maître et possesseur de’l étranger. That is to say, the European legacy shows, in an extraordinary manner, that the alien can be transformed into a resource, from which we can appropriate and assimilate everything. A crucial insight for Waldenfels is also that strangeness is not reducible to a narrow segment of reality, whether it is culture, religion or art-based, because strangeness is a radical dimension that transcends all regions.Nakon razjašnjavanja zbog čega fenomenologija stranog ne može biti ubrojana među tradicionalne filozofske discipline, autor ispituje zašto bi celokupna evropska istorija mogla biti čitana kao „zasenčenje stranog“, ali ne u smislu pukog odbacivanja, zanemarivanja ili poricanja stranog, nego njegovog disciplinovanja, manipulisanja, eksploatisanja. Strano nije bilo vekovima zaboravljeno, ono je uvek bilo u evropskom fokusu, ali samo kao instanca posredstvom koje je tradicionalno konstruisan osećaj moći. Prateći osnovne pretpostavke fenomenologije stranog Bernharda Valdenfelsa, članak uočava zasenčenje stranog kao analogno procesu njegove naturalizacije. Kao da tradicija evropskog kolonijalizma može biti najbolje shvaćena zahvaljujući ključu gospodar i vlasnik stranog. Rečju, evropsko nasleđe na izuzetan način pokazuje da strano može biti transformisano u resurs od kojeg možemo da prisvojimo i asimilujemo sve što nam može biti od koristi. Valdenfelsov presudan uvid glasi da stranost ne može biti svedena na uzak segment realnosti, bila ona vezana za kulturu, umetnost ili religiju, jer je ona radikalna dimenzija koja transcendira sve regije

Reversal of HCN Channel Voltage Dependence via Bridging of the S4–S5 Linker and Post-S6

Voltage-gated ion channels possess charged domains that move in response to changes in transmembrane voltage. How this movement is transduced into gating of the channel pore is largely unknown. Here we show directly that two functionally important regions of the spHCN1 pacemaker channel, the S4–S5 linker and the C-linker, come into close proximity during gating. Cross-linking these regions with high-affinity metal bridges or disulfide bridges dramatically alters channel gating in the absence of cAMP; after modification the polarity of voltage dependence is reversed. Instead of being closed at positive voltage and activating with hyperpolarization, modified channels are closed at negative voltage and activate with depolarization. Mechanistically, this reversal of voltage dependence occurs as a result of selectively eliminating channel deactivation, while retaining an existing inactivation process. Bridging also alters channel activation by cAMP, showing that interaction of these two regions can also affect the efficacy of physiological ligands

Über das Problem der phänomenologischen Geschichte der Philosophie

»Phänomenologische Geschichte der Philosophie?« bearbeitet manche Grundprobleme Husserls Phänomenologie, dargestellt auf Grundlage seiner Kritik an der Geschichte der Philosophie. Nach einleitender Untersuchung der Möglichkeiten und des Rahmens solcher Auslegung Husserls Phänomenologie, wurde die Grundcharakteristik Husserls Interpretation der Philosophiegeschichte betrachtet. Trotz Husserls anfänglichen Gesichtspunktes, daß der Philosoph ein absoluter Anfänger ist, hat sich herausgestellt, daß die Phänomenologie doch keine urgeschichtliche Bildung ist, und daß ihre Selbstbesinnung sich in der strukturellen Verbindung mit ihrer Besinnung der Philosophiegeschichte bewegt. Der Kulminationspunkt der philosophischgeschichtlichen Untersuchungen der Phänomenologie stellt die Einsicht in der Notwendigkeit der Befragung über das Sinn dar, das in der philosophischen Begrifflichkeit sedimentiert wurde

Der geschichtliche Status der Philosophie Hegels innerhalb des Heideggerschen Entwurfs der Destruktion

U članku se najprije tematizira diskrepancija između oštre kritike fenomenologije Edmunda Husserla u Heideggerovim ranim freiburškim i marburškim godinama, s jedne strane, i afirmativnog spominjanja Husserlova imena u "Bitku i vremenu", s druge strane. Ovaj "okret" može se interpretirati na temelju tumačenja prema kojem Heidegger u svojem glavnom djelu nije ostao pri stavu o nedostatnosti fenomenologije, zato što mu je ona služila kao polazište za izlaganje vlastite filozofske pozicije. Nakon kritičkog razmatranja onih pozicija prema kojima je Heidegger kroz kritiku Husserla ciljao na ontološku kritiku spekulativnoga idealizma, autor propituje razloge odgađanja razračunavanja s Hegelom, koje je Heidegger iznio u svojem habilitacijskom spisu. Provedba ovog razračunavanja na temelju nacrta destrukcije povijesti ontologije od početka se nije trebala razvijati sistematski, budući da je prevladavanje sistematskog izlaganja povijesti filozofije i povijesti uopće bilo postavljeno u središte poželjnih učinaka destrukcije

A genetically encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling.

BACKGROUND: Intrabodies enable targeting of proteins in live cells, but generating specific intrabodies against the thousands of proteins in a proteome poses a challenge. We leverage the widespread availability of fluorescently labelled proteins to visualize and manipulate intracellular signalling pathways in live cells by using nanobodies targeting fluorescent protein tags. RESULTS: We generated a toolkit of plasmids encoding nanobodies against red and green fluorescent proteins (RFP and GFP variants), fused to functional modules. These include fluorescent sensors for visualization of Ca2+, H+ and ATP/ADP dynamics; oligomerising or heterodimerising modules that allow recruitment or sequestration of proteins and identification of membrane contact sites between organelles; SNAP tags that allow labelling with fluorescent dyes and targeted chromophore-assisted light inactivation; and nanobodies targeted to lumenal sub-compartments of the secretory pathway. We also developed two methods for crosslinking tagged proteins: a dimeric nanobody, and RFP-targeting and GFP-targeting nanobodies fused to complementary hetero-dimerizing domains. We show various applications of the toolkit and demonstrate, for example, that IP3 receptors deliver Ca2+ to the outer membrane of only a subset of mitochondria and that only one or two sites on a mitochondrion form membrane contacts with the plasma membrane. CONCLUSIONS: This toolkit greatly expands the utility of intrabodies and will enable a range of approaches for studying and manipulating cell signalling in live cells