Time-resolved evolution of coherent structures in turbulent channels

El objetivo de esta tesis es estudiar la dinámica de la capa logarítmica de flujos turbulentos de pared. En concreto, proponemos un nuevo modelo estructural utilizando diferentes tipos de estructuras coherentes: sweeps, eyecciones, grupos de vorticidad y streaks. La herramienta utilizada es la simulación numérica directa de canales turbulentos. Desde los primeros trabajos de Theodorsen (1952), las estructuras coherentes han jugado un papel fundamental para entender la organización y dinámica de los flujos turbulentos. A día de hoy, datos procedentes de simulaciones numéricas directas obtenidas en instantes no contiguos permiten estudiar las propiedades fundamentales de las estructuras coherentes tridimensionales desde un punto de vista estadístico. Sin embargo, la dinámica no puede ser entendida en detalle utilizando sólo instantes aislados en el tiempo, sino que es necesario seguir de forma continua las estructuras. Aunque existen algunos estudios sobre la evolución temporal de las estructuras más pequeñas a números de Reynolds moderados, por ejemplo Robinson (1991), todavía no se ha realizado un estudio completo a altos números de Reynolds y para todas las escalas presentes de la capa logarítmica. El objetivo de esta tesis es llevar a cabo dicho análisis. Los problemas más interesantes los encontramos en la región logarítmica, donde residen las cascadas de vorticidad, energía y momento. Existen varios modelos que intentan explicar la organización de los flujos turbulentos en dicha región. Uno de los más extendidos fue propuesto por Adrian et al. (2000) a través de observaciones experimentales y considerando como elemento fundamental paquetes de vórtices con forma de horquilla que actúan de forma cooperativa para generar rampas de bajo momento. Un modelo alternativo fué ideado por del Álamo & Jiménez (2006) utilizando datos numéricos. Basado también en grupos de vorticidad, planteaba un escenario mucho más desorganizado y con estructuras sin forma de horquilla. Aunque los dos modelos son cinemáticamente similares, no lo son desde el punto de vista dinámico, en concreto en lo que se refiere a la importancia que juega la pared en la creación y vida de las estructuras. Otro punto importante aún sin resolver se refiere al modelo de cascada turbulenta propuesto por Kolmogorov (1941b), y su relación con estructuras coherentes medibles en el flujo. Para dar respuesta a las preguntas anteriores, hemos desarrollado un nuevo método que permite seguir estructuras coherentes en el tiempo y lo hemos aplicado a simulaciones numéricas de canales turbulentos con números de Reynolds lo suficientemente altos como para tener un rango de escalas no trivial y con dominios computacionales lo suficientemente grandes como para representar de forma correcta la dinámica de la capa logarítmica. Nuestros esfuerzos se han desarrollado en cuatro pasos. En primer lugar, hemos realizado una campaña de simulaciones numéricas directas a diferentes números de Reynolds y tamaños de cajas para evaluar el efecto del dominio computacional en las estadísticas de primer orden y el espectro. A partir de los resultados obtenidos, hemos concluido que simulaciones con cajas de longitud 2vr y ancho vr veces la semi-altura del canal son lo suficientemente grandes para reproducir correctamente las interacciones entre estructuras coherentes de la capa logarítmica y el resto de escalas. Estas simulaciones son utilizadas como punto de partida en los siguientes análisis. En segundo lugar, las estructuras coherentes correspondientes a regiones con esfuerzos de Reynolds tangenciales intensos (Qs) en un canal turbulento han sido estudiadas extendiendo a tres dimensiones el análisis de cuadrantes, con especial énfasis en la capa logarítmica y la región exterior. Las estructuras coherentes han sido identificadas como regiones contiguas del espacio donde los esfuerzos de Reynolds tangenciales son más intensos que un cierto nivel. Los resultados muestran que los Qs separados de la pared están orientados de forma isótropa y su contribución neta al esfuerzo de Reynolds medio es nula. La mayor contribución la realiza una familia de estructuras de mayor tamaño y autosemejantes cuya parte inferior está muy cerca de la pared (ligada a la pared), con una geometría compleja y dimensión fractal « 2. Estas estructuras tienen una forma similar a una ‘esponja de placas’, en comparación con los grupos de vorticidad que tienen forma de ‘esponja de cuerdas’. Aunque el número de objetos decae al alejarnos de la pared, la fracción de esfuerzos de Reynolds que contienen es independiente de su altura, y gran parte reside en unas pocas estructuras que se extienden más allá del centro del canal, como en las grandes estructuras propuestas por otros autores. Las estructuras dominantes en la capa logarítmica son parejas de sweeps y eyecciones uno al lado del otro y con grupos de vorticidad asociados que comparten las dimensiones y esfuerzos con los remolinos ligados a la pared propuestos por Townsend. En tercer lugar, hemos estudiado la evolución temporal de Qs y grupos de vorticidad usando las simulaciones numéricas directas presentadas anteriormente hasta números de Reynolds ReT = 4200 (Reynolds de fricción). Las estructuras fueron identificadas siguiendo el proceso descrito en el párrafo anterior y después seguidas en el tiempo. A través de la interseción geométrica de estructuras pertenecientes a instantes de tiempo contiguos, hemos creado gratos de conexiones temporales entre todos los objetos y, a partir de ahí, definido ramas primarias y secundarias, de tal forma que cada rama representa la evolución temporal de una estructura coherente. Una vez que las evoluciones están adecuadamente organizadas, proporcionan toda la información necesaria para caracterizar la historia de las estructuras desde su nacimiento hasta su muerte. Los resultados muestran que las estructuras nacen a todas las distancias de la pared, pero con mayor probabilidad cerca de ella, donde la cortadura es más intensa. La mayoría mantienen tamaños pequeños y no viven mucho tiempo, sin embargo, existe una familia de estructuras que crecen lo suficiente como para ligarse a la pared y extenderse a lo largo de la capa logarítmica convirtiéndose en las estructuras observas anteriormente y descritas por Townsend. Estas estructuras son geométricamente autosemejantes con tiempos de vida proporcionales a su tamaño. La mayoría alcanzan tamaños por encima de la escala de Corrsin, y por ello, su dinámica está controlada por la cortadura media. Los resultados también muestran que las eyecciones se alejan de la pared con velocidad media uT (velocidad de fricción) y su base se liga a la pared muy rápidamente al inicio de sus vidas. Por el contrario, los sweeps se mueven hacia la pared con velocidad -uT y se ligan a ella más tarde. En ambos casos, los objetos permanecen ligados a la pared durante 2/3 de sus vidas. En la dirección de la corriente, las estructuras se desplazan a velocidades cercanas a la convección media del flujo y son deformadas por la cortadura. Finalmente, hemos interpretado la cascada turbulenta, no sólo como una forma conceptual de organizar el flujo, sino como un proceso físico en el cual las estructuras coherentes se unen y se rompen. El volumen de una estructura cambia de forma suave, cuando no se une ni rompe, o lo hace de forma repentina en caso contrario. Los procesos de unión y rotura pueden entenderse como una cascada directa (roturas) o inversa (uniones), siguiendo el concepto de cascada de remolinos ideado por Richardson (1920) y Obukhov (1941). El análisis de los datos muestra que las estructuras con tamaños menores a 30η (unidades de Kolmogorov) nunca se unen ni rompen, es decir, no experimentan el proceso de cascada. Por el contrario, aquellas mayores a 100η siempre se rompen o unen al menos una vez en su vida. En estos casos, el volumen total ganado y perdido es una fracción importante del volumen medio de la estructura implicada, con una tendencia ligeramente mayor a romperse (cascada directa) que a unirse (cascade inversa). La mayor parte de interacciones entre ramas se debe a roturas o uniones de fragmentos muy pequeños en la escala de Kolmogorov con estructuras más grandes, aunque el efecto de fragmentos de mayor tamaño no es despreciable. También hemos encontrado que las roturas tienen a ocurrir al final de la vida de la estructura y las uniones al principio. Aunque los resultados para la cascada directa e inversa no son idénticos, son muy simétricos, lo que sugiere un alto grado de reversibilidad en el proceso de cascada. ABSTRACT The purpose of the present thesis is to study the dynamics of the logarithmic layer of wall-bounded turbulent flows. Specifically, to propose a new structural model based on four different coherent structures: sweeps, ejections, clusters of vortices and velocity streaks. The tool used is the direct numerical simulation of time-resolved turbulent channels. Since the first work by Theodorsen (1952), coherent structures have played an important role in the understanding of turbulence organization and its dynamics. Nowadays, data from individual snapshots of direct numerical simulations allow to study the threedimensional statistical properties of those objects, but their dynamics can only be fully understood by tracking them in time. Although the temporal evolution has already been studied for small structures at moderate Reynolds numbers, e.g., Robinson (1991), a temporal analysis of three-dimensional structures spanning from the smallest to the largest scales across the logarithmic layer has yet to be performed and is the goal of the present thesis. The most interesting problems lie in the logarithmic region, which is the seat of cascades of vorticity, energy, and momentum. Different models involving coherent structures have been proposed to represent the organization of wall-bounded turbulent flows in the logarithmic layer. One of the most extended ones was conceived by Adrian et al. (2000) and built on packets of hairpins that grow from the wall and work cooperatively to gen- ´ erate low-momentum ramps. A different view was presented by del Alamo & Jim´enez (2006), who extracted coherent vortical structures from DNSs and proposed a less organized scenario. Although the two models are kinematically fairly similar, they have important dynamical differences, mostly regarding the relevance of the wall. Another open question is whether such a model can be used to explain the cascade process proposed by Kolmogorov (1941b) in terms of coherent structures. The challenge would be to identify coherent structures undergoing a turbulent cascade that can be quantified. To gain a better insight into the previous questions, we have developed a novel method to track coherent structures in time, and used it to characterize the temporal evolutions of eddies in turbulent channels with Reynolds numbers high enough to include a non-trivial range of length scales, and computational domains sufficiently long and wide to reproduce correctly the dynamics of the logarithmic layer. Our efforts have followed four steps. First, we have conducted a campaign of direct numerical simulations of turbulent channels at different Reynolds numbers and box sizes, and assessed the effect of the computational domain in the one-point statistics and spectra. From the results, we have concluded that computational domains with streamwise and spanwise sizes 2vr and vr times the half-height of the channel, respectively, are large enough to accurately capture the dynamical interactions between structures in the logarithmic layer and the rest of the scales. These simulations are used in the subsequent chapters. Second, the three-dimensional structures of intense tangential Reynolds stress in plane turbulent channels (Qs) have been studied by extending the classical quadrant analysis to three dimensions, with emphasis on the logarithmic and outer layers. The eddies are identified as connected regions of intense tangential Reynolds stress. Qs are then classified according to their streamwise and wall-normal fluctuating velocities as inward interactions, outward interactions, sweeps and ejections. It is found that wall-detached Qs are isotropically oriented background stress fluctuations, common to most turbulent flows, and do not contribute to the mean stress. Most of the stress is carried by a selfsimilar family of larger wall-attached Qs, increasingly complex away from the wall, with fractal dimensions « 2. They have shapes similar to ‘sponges of flakes’, while vortex clusters resemble ‘sponges of strings’. Although their number decays away from the wall, the fraction of the stress that they carry is independent of their heights, and a substantial part resides in a few objects extending beyond the centerline, reminiscent of the very large scale motions of several authors. The predominant logarithmic-layer structures are sideby- side pairs of sweeps and ejections, with an associated vortex cluster, and dimensions and stresses similar to Townsend’s conjectured wall-attached eddies. Third, the temporal evolution of Qs and vortex clusters are studied using time-resolved DNS data up to ReT = 4200 (friction Reynolds number). The eddies are identified following the procedure presented above, and then tracked in time. From the geometric intersection of structures in consecutive fields, we have built temporal connection graphs of all the objects, and defined main and secondary branches in a way that each branch represents the temporal evolution of one coherent structure. Once these evolutions are properly organized, they provide the necessary information to characterize eddies from birth to death. The results show that the eddies are born at all distances from the wall, although with higher probability near it, where the shear is strongest. Most of them stay small and do not last for long times. However, there is a family of eddies that become large enough to attach to the wall while they reach into the logarithmic layer, and become the wall-attached structures previously observed in instantaneous flow fields. They are geometrically self-similar, with sizes and lifetimes proportional to their distance from the wall. Most of them achieve lengths well above the Corrsin’ scale, and hence, their dynamics are controlled by the mean shear. Eddies associated with ejections move away from the wall with an average velocity uT (friction velocity), and their base attaches very fast at the beginning of their lives. Conversely, sweeps move towards the wall at -uT, and attach later. In both cases, they remain attached for 2/3 of their lives. In the streamwise direction, eddies are advected and deformed by the local mean velocity. Finally, we interpret the turbulent cascade not only as a way to conceptualize the flow, but as an actual physical process in which coherent structures merge and split. The volume of an eddy can change either smoothly, when they are not merging or splitting, or through sudden changes. The processes of merging and splitting can be thought of as a direct (when splitting) or an inverse (when merging) cascade, following the ideas envisioned by Richardson (1920) and Obukhov (1941). It is observed that there is a minimum length of 30η (Kolmogorov units) above which mergers and splits begin to be important. Moreover, all eddies above 100η split and merge at least once in their lives. In those cases, the total volume gained and lost is a substantial fraction of the average volume of the structure involved, with slightly more splits (direct cascade) than mergers. Most branch interactions are found to be the shedding or absorption of Kolmogorov-scale fragments by larger structures, but more balanced splits or mergers spanning a wide range of scales are also found to be important. The results show that splits are more probable at the end of the life of the eddy, while mergers take place at the beginning of the life. Although the results for the direct and the inverse cascades are not identical, they are found to be very symmetric, which suggests a high degree of reversibility of the cascade process.

Estimation of Symmetric Channels for Discrete Cosine Transform Type-I Multicarrier Systems: A Compressed Sensing Approach

The problem of channel estimation for multicarrier communications is addressed. We focus on systems employing the Discrete Cosine Transform Type-I (DCT1) even at both the transmitter and the receiver, presenting an algorithm which achieves an accurate estimation of symmetric channel filters using only a small number of training symbols. The solution is obtained by using either matrix inversion or compressed sensing algorithms. We provide the theoretical results which guarantee the validity of the proposed technique for the DCT1. Numerical simulations illustrate the good behaviour of the proposed algorithm.

Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History

Voltage-gated potassium (K+) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K+ channel type segregates into at least two main functional categories—hyperpolarization-activated, inward-rectifying (Kin) and depolarization-activated, outward-rectifying (Kout) channels. Voltage-gated K+ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in Kin and Kout channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K+ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between Kin and Kout channels. Among them is a loop between TMD S5 and the pore that is very short in animal Kout, longer in plant and animal Kin and the longest in plant Kout channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom.

Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: A transmembrane β-subunit homolog

Voltage-dependent and calcium-sensitive K+ (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca2+. In most tissues, their stimulation results in a noninactivating hyperpolarizing K+ current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. Herein, we report a transmembrane β subunit (β2) that yields inactivating MaxiK currents on coexpression with the pore-forming α subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the β2 subunit abolished inactivation of the α subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle β1 subunit leads to an inactivating phenotype of MaxiK channels. Furthermore, addition of a synthetic N-terminal peptide of the β2 subunit causes inactivation of the MaxiK channel α subunit by occluding its K+-conducting pore resembling the inactivation caused by the “ball” peptide in voltage-dependent K+ channels. Thus, the inactivating phenotype of MaxiK channels in native tissues can result from the association with different β subunits.

Calcium block of Na+ channels and its effect on closing rate

Calcium ion transiently blocks Na+ channels, and it shortens the time course for closing of their activation gates. We examined the relation between block and closing kinetics by using the Na+ channels natively expressed in GH3 cells, a clonal line of rat pituitary cells. To simplify analysis, inactivation of the Na+ channels was destroyed by including papain in the internal medium. All divalent cations tested, and trivalent La3+, blocked a progressively larger fraction of the channels as their concentration increased, and they accelerated the closing of the Na+ channel activation gate. For calcium, the most extensively studied cation, there is an approximately linear relation between the fraction of the channels that are calcium-blocked and the closing rate. Extrapolation of the data to very low calcium suggests that closing rate is near zero when there is no block. Analysis shows that, almost with certainty, the channels can close when occupied by calcium. The analysis further suggests that the channels close preferentially or exclusively from the calcium-blocked state.

Distinguishing surface effects of calcium ion from pore-occupancy effects in Na+ channels

The effects of calcium ion on the Na+ activation gate were studied in squid giant axons. Saxitoxin (STX) was used to block ion entry into Na+ channels without hindering access to the membrane surface, making it possible to distinguish surface effects of calcium from pore-occupancy effects. In the presence of STX, gating kinetics were measured from gating current (Ig). The kinetic effects of external calcium concentration changes were small when STX was present. In the absence of STX, lowering the calcium concentration (from 100 to 10 mM) slowed the closing of Na+ channels (measured from INa tails) by more than a factor of 2. Surprisingly, the voltage sensitivity of closing kinetics changed with calcium concentration, and it was modified by STX. Voltage sensitivity apparently depends in part on the ability of calcium to enter and block the channels as voltage is driven negative. In external medium with no added calcium, INa tail current initially increases in amplitude severalfold with the relief of calcium block, then progressively slows and gets smaller, as calcium diffuses out of the layers investing the axon. INa tails seen just before the current disappears suggest that closing in the absence of channel block is very slow or does not occur. INa amplitude and kinetics are completely restored when calcium is returned. The results strongly suggest that calcium occupancy is a requirement for channel closing and that nonoccupied channels fold reversibly into a nonfunctional conformation.

A dimeric crystal structure for the N-terminal two domains of intercellular adhesion molecule-1

The 3.0-Å structure of a 190-residue fragment of intercellular adhesion molecule-1 (ICAM-1, CD54) reveals two tandem Ig-superfamily (IgSF) domains. Each of two independent molecules dimerizes identically with a symmetry-related molecule over a hydrophobic interface on the BED sheet of domain 1, in agreement with dimerization of ICAM-1 on the cell surface. The residues that bind to the integrin LFA-1 are well oriented for bivalent binding in the dimer, with the critical Glu-34 residues pointing away from each other on the periphery. Residues that bind to rhinovirus are in the flexible BC and FG loops at the tip of domain 1, and these and the upper half of domain 1 are well exposed in the dimer for docking to virus. By contrast, a residue important for binding to Plasmodium falciparum-infected erythrocytes is in the dimer interface. The presence of A′ strands in both domains 1 and 2, conserved hydrogen bonds at domain junctions, and elaborate hydrogen bond networks around the key integrin binding residues in domain 1 make these domains suited to resist tensile forces during adhesive interactions. A subdivision of the intermediate (I) set of IgSF domains is proposed in which domain 1 of ICAM-1 and previously described I set domains belong to the I1 set and domain 2 of ICAM-1, ICAM-2, and vascular cell adhesion molecule-1 belong to the I2 set.

Optimal selectin-mediated rolling of leukocytes during inflammation in vivo requires intercellular adhesion molecule-1 expression

Leukocyte interactions with vascular endothelium during inflammation occur through discrete steps involving selectin-mediated leukocyte rolling and subsequent firm adhesion mediated by members of the integrin and Ig families of adhesion molecules. To identify functional synergy between selectin and Ig family members, mice deficient in both L-selectin and intercellular adhesion molecule 1 (ICAM-1) were generated. Leukocyte rolling velocities in cremaster muscle venules were increased significantly in ICAM-1-deficient mice during both trauma- and tumor necrosis factor α-induced inflammation, but rolling leukocyte flux was not reduced. Elimination of ICAM-1 expression in L-selectin-deficient mice resulted in a sharp reduction in the flux of rolling leukocytes during tumor necrosis factor α-induced inflammation. The observed differences in leukocyte rolling behavior demonstrated that ICAM-1 expression was required for optimal P- and L-selectin-mediated rolling. Increased leukocyte rolling velocities presumably translated into decreased tissue emigration because circulating neutrophil, monocyte, and lymphocyte numbers were increased markedly in L-selectin/ICAM-1-deficient mice. Furthermore, neutrophil emigration during acute peritonitis was reduced by 80% in the double-deficient mice compared with either L-selectin or ICAM-1-deficient mice. Thus, members of the selectin and Ig families function synergistically to mediate optimal leukocyte rolling in vivo, which is essential for the generation of effective inflammatory responses.

Factors controlling the competition among rotational and vibrational energy transfer channels in glyoxal

The state-to-state transfer of rotational and vibrational energy has been studied for S1 glyoxal (CHOCHO) in collisions with D2, N2, CO and C2H4 using crossed molecular beams. A laser is used to pump glyoxal seeded in He to its S1 zero point level with zero angular momentum about its top axis (K′ = 0). The inelastic scattering to each of at least 26 S1 glyoxal rotational and rovibrational levels is monitored by dispersed S1–S0 fluorescence. Various collision partners are chosen to investigate the relative influences of reduced mass and the collision pair interaction potential on the competition among the energy transfer channels. When the data are combined with that obtained previously from other collision partners whose masses range from 2 to 84 amu, it is seen that the channel competition is controlled primarily by the kinematics of the collisional interaction. Variations in the intermolecular potential play strictly a secondary role.

The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer

The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a3 (Fe4+ = O2−) to the oxidized form (Fe3+OH−). This redox step requires the delivery of a “chemical” H+ to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly suppressed by mutations in the D channel. The results strongly suggest that the functional roles of the two channels are not the separate delivery of chemical or pumped protons, as proposed recently [Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. (1995) Nature (London) 376, 660–669]. The D channel is likely to be involved in the uptake of both “chemical” and “pumped” protons in the F-to-Ox transition, whereas the K channel is probably idle at this partial reaction and is likely to be used for loading the enzyme with protons at some earlier steps of the catalytic cycle. This conclusion agrees with different redox states of heme a3 in the K362M and E286Q mutants under aerobic steady-state turnover conditions.

RGS proteins reconstitute the rapid gating kinetics of Gβγ-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Gα(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20–40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of “regulators of G protein signaling” (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor → GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent “basal” GIRK current was suppressed by ≈40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of “slow” postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.

hSK4, a member of a novel subfamily of calcium-activated potassium channels

The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca2+ (EC50 of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.

Site-specific, photochemical proteolysis applied to ion channels in vivo

A method for site-specific, nitrobenzyl-induced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K+ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the “signature” Cys128–Cys142 disulfide loop of the nAChR α subunit.

Regulation of the human ether-a-gogo related gene (HERG) K+ channels by reactive oxygen species

Human ether-a-gogo related gene (HERG) K+ channels are key elements in the control of cell excitability in both the cardiovascular and the central nervous systems. For this reason, the possible modulation by reactive oxygen species (ROS) of HERG and other cloned K+ channels expressed in Xenopus oocytes has been explored in the present study. Exposure of Xenopus oocytes to an extracellular solution containing FeSO4 (25–100 μM) and ascorbic acid (50–200 μM) (Fe/Asc) increased both malondialdehyde content and 2′,7′-dichlorofluorescin fluorescence, two indexes of ROS production. Oocyte perfusion with Fe/Asc caused a 50% increase of the outward K+ currents carried by HERG channels, whereas inward currents were not modified. This ROS-induced increase in HERG outward K+ currents was due to a depolarizing shift of the voltage-dependence of channel inactivation, with no change in channel activation. No effect of Fe/Asc was observed on the expressed K+ currents carried by other K+ channels such as bEAG, rDRK1, and mIRK1. Fe/Asc-induced stimulation of HERG outward currents was completely prevented by perfusion of the oocytes with a ROS scavenger mixture (containing 1,000 units/ml catalase, 200 ng/ml superoxide dismutase, and 2 mM mannitol). Furthermore, the scavenger mixture also was able to reduce HERG outward currents in resting conditions by 30%, an effect mimicked by catalase alone. In conclusion, the present results seem to suggest that changes in ROS production can specifically influence K+ currents carried by the HERG channels.

Phentolamine block of KATP channels is mediated by Kir6.2

The ATP-sensitive K+-channel (KATP channel) plays a key role in insulin secretion from pancreatic β cells. It is closed both by glucose metabolism and the sulfonylurea drugs that are used in the treatment of noninsulin-dependent diabetes mellitus, thereby initiating a membrane depolarization that activates voltage-dependent Ca2+ entry and insulin release. The β cell KATP channel is a complex of two proteins: Kir6.2 and SUR1. The former is an ATP-sensitive K+-selective pore, whereas SUR1 is a channel regulator that endows Kir6.2 with sensitivity to sulfonylureas. A number of drugs containing an imidazoline moiety, such as phentolamine, also act as potent stimulators of insulin secretion, but their mechanism of action is unknown. We have used a truncated form of Kir6.2, which expresses independently of SUR1, to show that phentolamine does not inhibit KATP channels by interacting with SUR1. Instead, our results argue that phentolamine may interact directly with Kir6.2 to produce a voltage-independent reduction in channel activity. The single-channel conductance is unaffected. Although the ATP molecule also contains an imidazoline group, the site at which phentolamine blocks is not identical to the ATP-inhibitory site, because phentolamine block of an ATP-insensitive mutant (K185Q) is normal. KATP channels also are found in the heart where they are involved in the response to cardiac ischemia: they also are blocked by phentolamine. Our results suggest that this may be because Kir6.2, which is expressed in the heart, forms the pore of the cardiac KATP channel.