Investigations on structure and functional properties of CNG channels

Ion channels are allosteric proteins that play a fundamental role for cells; they regulate ion fluxes across biological membranes by residing in any of three functionally distinct states: deactivated (closed), activated (open) or inactivated (closed). In addition to selecting permeant ions, channels undergo conformational changes that open and close their ion-permeable pores, a process referred to as gating. On the basis of their primary structure the ion channels are classified into a relatively small number of discrete gene families. Cyclic nucleotide gated (CNG) and hyperpolarization-activated, cyclic nucleotide-gated (HCN), together with K+, Na+, Ca2+ and transient receptor potential (TRP) channels belong to the superfamily of voltage gated channels. All members of this family are tetrameric or pseudotetrameric and share a common pore domain that contains two transmembrane (TM) segments - referred to as M1 and M2, or S5 and S6. As in all the voltage-gated channels, CNG channels have four additional TM segments per subunit: S1\u2013S4 that, correspond to the sensor for the allosteric modulation of gating by membrane voltage. CNG channels are mainly activated by binding of cyclic nucleotides, but, in the presence of symmetrical ionic conditions, current-voltage (I-V) relationship depends, in a complex way, on the radius of permeating ion and has been suggested that both the pore and S4 helix contribute to the observed rectification. CNG channels are essential for visual and olfactory transduction: in rod photoreceptors CNG channels open in dark conditions and are responsible for a steady inward current that flows through them keeping the membrane depolarized. In vertebrates, seven members of the CNG channel gene family have been identified that, according to sequence similarity, are grouped into two subtypes, CNGA (CNGA1-CNGA5) and CNGB (CNGB1 and CNGB3). Only CNGA subunits - except the A4 subunit - can form cyclic nucleotide-activated homomeric channels, while CNGB1 and CNGB3 are modulatory and cannot form functional homomeric channels. CNG channels are only slightly voltage-dependent and, for opening, require the binding of cGMP or cAMP to a receptor site located in their C-terminal. In the presence of a saturating agonist, CNG channel gating is voltage independent and it is not known why CNG channels are voltage-insensitive despite harbouring the S4-type voltage sensor. Moreover, unlike many K+ channels, cyclic nucleotide-gated (CNG) channels are known not to inactivate. While electrophysiology combined with mutagenesis have identified the channel pore and the binding domain for cyclic nucleotides (CNs), conformational changes associated with gating have remained elusive. During my PhD course I focused my energies on investigating the structural and functional properties of different domains of the CNGA1 channels, combining results from different techniques like homology modelling, single molecule force spectroscopy (SMFS), molecular dynamics (MD) simulations, and X-ray crystallography with my own data from electrophysiology and site-directed mutagenesis in order to get better insights of fundamental properties of the channels, like permeation and channel gating. The main findings of my PhD thesis are the following: I. I showed that current rectification, in presence of large organic cations, like ethylammonium and dimethylammonium, strongly depends on two voltage-dependent transitions, of which, only the first, is sensible to mutation of charge residues in the S4 helix. These results indicate the existence of at least two distinct mechanisms underlying rectification in CNG channels: a restricted motion of the S4 helix, together with an inefficient coupling to the channel gate that render CNGA1 channels poorly sensitive to voltage in the presence of physiological Na+ and K+. II. has been demonstrated that, contrary to previous belief, when extracellular pH is decreased from 7.4 to 6 or lower, wild-type CNGA1 channels inactivate in a voltage-dependent manner. This mechanism is reminiscent of the C-type inactivation observed in K+ channels. Low pH inactivation may represent an unrecognized endogenous mechanism that regulate CNG physiological functions in diverse tissues. III. combining electrophysiology with molecular dynamics (MD) simulations, and X-ray crystallography has been demonstrated that the pore of CNG channels is highly flexible and, mainly due to the side chain of Glu66 in the selectivity filter and to the prolines in the outer vestibule, capable to coordinate different ionic species. This flexibility underlies the coupling between gating and permeation and the poor ionic selectivity of CNG channels. IV. by mean of electrophysiology combined with single-molecule force spectroscopy (SMFS), mutagenesis and bioinformatics have been obtained better insights about conformational changes associated with gating and, in particular, about the mechanical coupling between the voltage sensor and the binding domain. We revealed putative electrostatic interactions between an aspartate in the C-linker and an arginine in the S4-S5 linker. We hypothesized a possible mechanism, possibly shared with HCN channels, by which these electrostatic interactions determine the motion of S5, leading to conformational rearrangements of residues flanking the pore so that the lumen of the pore widens and the channel opens

Pressure induced magnetic phase separation in La0.75_{0.75}Ca0.25_{0.25}MnO3_{3} manganite

The pressure dependence of the Curie temperature T$_{C}(P)$ in La$_{0.75}$Ca$_{0.25}$MnO$_{3}$ was determined by neutron diffraction up to 8 GPa, and compared with the metallization temperature T$_{IM}(P)$ \cite{irprl}. The behavior of the two temperatures appears similar over the whole pressure range suggesting a key role of magnetic double exchange also in the pressure regime where the superexchange interaction is dominant. Coexistence of antiferromagnetic and ferromagnetic peaks at high pressure and low temperature indicates a phase separated regime which is well reproduced with a dynamical mean-field calculation for a simplified model. A new P-T phase diagram has been proposed on the basis of the whole set of experimental data.Comment: 5 pages, 4 figure

High pressure Raman study of La1-xCaxMnO3-δ manganites

We report a high-pressure Raman study on two members of the La1-xCaxMnO3-δ manganite family (x = 0.20, δ = 0 and δ = 0.08). The results obtained for the δ = 0 sample show a different behavior in the low and high pressure regime which is ascribed to the onset of a new pressure-activated interaction previously invoked in other manganite compounds. The comparison of our results with literature data gives further support to the identification of the Jahn-Teller sensitive stretching mode and shows that pressure-induced octahedral symmetrization is more effective in systems exhibiting a lower metallic character. On the contrary the new interaction sets in at a pressure which decreases on increasing the metallic character of the system indicating an important role of the Mn-Mn hopping integral in its activatio

High pressure Raman study of La1−x_{1-x}Cax_xMnO3−δ_{3-\delta} manganites

We report on a high-pressure Raman study on two members of the La$_{1-x}$Ca$_x$MnO$_{3-\delta}$ manganite family ($x=0.20$, $\delta=0$ and $\delta=0.08$). The results obtained for the $\delta=0$ sample show a different behavior in the low and high pressure regimes ascribed to the onset of a new pressure-activated interaction previously invoked in other manganite compounds. The comparison of our results with literature data gives further support to the identification of the Jahn-Teller active stretching mode and shows that pressure-induced octahedral symmetrization is more effective in systems exhibiting a lower metallic character. On the contrary the new interaction sets in at pressure which decreases on increasing the metallic character of the system indicating a relevant role of the Mn-Mn hopping integral in its activation.Comment: 4 pages, 3 figure, submitted to Phys. Rev.

Evidence of a pressure-induced metallization process in monoclinic VO2_2

Raman and combined trasmission and reflectivity mid infrared measurements have been carried out on monoclinic VO$_2$ at room temperature over the 0-19 GPa and 0-14 GPa pressure ranges, respectively. The pressure dependence obtained for both lattice dynamics and optical gap shows a remarkable stability of the system up to P*$\sim$10 GPa. Evidence of subtle modifications of V ion arrangements within the monoclinic lattice together with the onset of a metallization process via band gap filling are observed for P$>$P*. Differently from ambient pressure, where the VO$_2$ metal phase is found only in conjunction with the rutile structure above 340 K, a new room temperature metallic phase coupled to a monoclinic structure appears accessible in the high pressure regime, thus opening to new important queries on the physics of VO$_2$.Comment: 5 pages, 3 figure

Electrodynamics near the Metal-to-Insulator Transition in V3O5

The electrodynamics near the metal-to-insulator transitions (MIT) induced, in V3O5 single crystals, by both temperature (T) and pressure (P) has been studied by infrared spectroscopy. The T- and P-dependence of the optical conductivity may be explained within a polaronic scenario. The insulating phase at ambient T and P corresponds to strongly localized small polarons. Meanwhile the T-induced metallic phase at ambient pressure is related to a liquid of polarons showing incoherent dc transport, in the P-induced metallic phase at room T strongly localized polarons coexist with partially delocalized ones. The electronic spectral weight is almost recovered, in both the T and P induced metallization processes, on an energy scale of 1 eV, thus supporting the key-role of electron-lattice interaction in the V3O5 metal-to-insulator transition.Comment: 7 pages, 5 figure

The influence of the net rainfall mixed Curve Number – Green Ampt procedure in flood hazard mapping: a case study in Central Italy

A net rainfall estimation procedure, referred to as Curve-Number For Green-Ampt (CN4GA), combining the Soil Conservation Service - Curve Number (SCS-CN) method and the Green and Ampt (GA) infiltration equation was recently developed, aiming to distribute at subdaily time resolution the information provided by the SCS-CN method. The initial abstraction and the total volume of rainfall provided by the SCS-CN method are used to identify the ponding time and to quantify the hydraulic conductivity parameter of the GA equation, whereas the GA infiltration model distributes the total volume of the rainfall excess provided by the SCS-CN method. In this study we evaluate the proposed procedure with reference to a real case comparing the flood mapping obtained applying the event-based approach for two different net rainfall scenarios: the proposed CN4GA and the common SCS-CN. Results underline that the net rainfall estimation step can affect the final flood mapping result

Pressure dependence of the charge-density-wave gap in rare-earth tri-tellurides

We investigate the pressure dependence of the optical properties of CeTe$_3$, which exhibits an incommensurate charge-density-wave (CDW) state already at 300 K. Our data are collected in the mid-infrared spectral range at room temperature and at pressures between 0 and 9 GPa. The energy for the single particle excitation across the CDW gap decreases upon increasing the applied pressure, similarly to the chemical pressure by rare-earth substitution. The broadening of the bands upon lattice compression removes the perfect nesting condition of the Fermi surface and therefore diminishes the impact of the CDW transition on the electronic properties of $R$Te$_3$.Comment: 5 pages, 4 figure

Relationship between TLR4 signalling alterations and effective human cytomegalovirus infection

Toll-like receptors (TLR), the main class of immune-sensor molecules triggering the innate immunity pathways, are known to be involved in the infection of different RNA and DNA viruses, including herpesviruses. Human cytomegalovirus (HCMV) is a widespread human beta-herpesvirus that infects 80–90 % of the world’s population and it can cause severe and even fatal diseases in immunocompromised patients and it is also responsible for birth defects as a consequence of congenital infection. Aim of this review is to discuss the existing data regarding the role of TLRs in HCMV concentrating mainly on TLR4. A better understanding in this relationship could be exploited for the development of efficient early diagnosis methodologies and anti viral therapies.Toll-подібні рецептори (TLR) як основний клас молекул імунних датчиків, що регулюють сигнальні шляхи вродженого імунітету, також беруть участь у зараженні різними РНК- і ДНК-вірусами, з-поміж яких віруси герпесу. Цитомегаловірус людини – широко розповсюджений бета-герпесвірус людини, їм заражаено приблизно 80–90 % населення земної кулі, що призводить до серйозних і навіть смертельних захворювань у пацієнтів з імунодефіцитом та є причиною спадкових патологій через вроджені інфекції. Мета цього огляду полягає в обговоренні існуючих даних щодо ролі TLR, і особливо TLR4, в цитомегаловірусній інфекції. Краще розуміння цього може бути використано для розробки ефективних методик ранньої діагностики і противірусних препаратів.Toll-подобные рецепторы (TLR) как основной класс молекул иммунных датчиков, регулирующих сигнальные пути врожденного иммунитета, участвуют также в заражении различными РНК- и ДНК-вирусами, включая вирусы герпеса. Цитомегаловирус человека – широко распространенный бета-герпесвирус человека, им заражаено примерно 80–90 % населения земного шара, что приводит к серьезным и даже смертельным заболеваниям у пациентов с иммунодефицитом и является причиной наследственных патологий из-за врожденной инфекции. Цель представленного обзора состоит в обсуждении существующих данных о роли TLR, и особенно TLR4, в цитомегаловирусной инфекции. Лучшее понимание этого может быть использовано для разработки эффективных методик ранней диагностики и противовирусных препаратов

Argentina, 1985, further proof of the transformations of the cultural industries

Argentina, 1985 resulta un producto cultural de gran relevancia para explorar las transformaciones de la industria del cine en particular y de las industrias culturales en general. En ese sentido, este trabajo pretende analizar la película en relación con las características generales de las industrias culturales desde la perspectiva teórica de la Economía Política de la Comunicación y, específicamente, identificar cómo el desarrollo tecnológico y los procesos de globalización del sector afectaron sus formas de producción y distribución. A partir de una estrategia metodológica cualitativa de revisión bibliográfica, este artículo muestra cómo Argentina 1985 adopta ciertas características clásicas de las industrias culturales como el reciclaje y la aplicación del star system, entre otros, al mismo tiempo que evidencia cómo los avances tecnológicos determinaron su producción y distribución a través del conglomerado digital Amazon.Argentina, 1985 is a cultural product of great relevance to explore the transformations of the film industry in particular and the cultural industries in general. In this sense, this paper aims to analyze the film in relation to the general characteristics of the cultural industries from the theoretical perspective of the Political Economy of Communication and, specifically, to identify how technological development and the processes of globalization of the sector affected its forms of production and distribution. Based on a qualitative methodological strategy of bibliographic review, this article shows how Argentina 1985 adopts certain classic characteristics of the cultural industries such as recycling and the application of the star system, among others, while at the same time demonstrating how technological advances determined its production and distribution through the digital conglomerate Amazon.Facultad de Periodismo y Comunicación Socia