Multidisciplinary Design Optimization Application to Conceptual Design of University-class Microsatellite Projects

Esta tesis se ha realizado en el contexto del proyecto UPMSat-2, que es un microsatélite diseñado, construido y operado por el Instituto Universitario de Microgravedad "Ignacio Da Riva" (IDR / UPM) de la Universidad Politécnica de Madrid. Aplicación de la metodología Ingeniería Concurrente (Concurrent Engineering: CE) en el marco de la aplicación de diseño multidisciplinar (Multidisciplinary Design Optimization: MDO) es uno de los principales objetivos del presente trabajo. En los últimos años, ha habido un interés continuo en la participación de los grupos de investigación de las universidades en los estudios de la tecnología espacial a través de sus propios microsatélites. La participación en este tipo de proyectos tiene algunos desafíos inherentes, tales como presupuestos y servicios limitados. Además, debido al hecho de que el objetivo principal de estos proyectos es fundamentalmente educativo, por lo general hay incertidumbres en cuanto a su misión en órbita y cargas útiles en las primeras fases del proyecto. Por otro lado, existen limitaciones predeterminadas para sus presupuestos de masa, volumen y energía, debido al hecho de que la mayoría de ellos están considerados como una carga útil auxiliar para el lanzamiento. De este modo, el costo de lanzamiento se reduce considerablemente. En este contexto, el subsistema estructural del satélite es uno de los más afectados por las restricciones que impone el lanzador. Esto puede afectar a diferentes aspectos, incluyendo las dimensiones, la resistencia y los requisitos de frecuencia. En la primera parte de esta tesis, la atención se centra en el desarrollo de una herramienta de diseño del subsistema estructural que evalúa, no sólo las propiedades de la estructura primaria como variables, sino también algunas variables de nivel de sistema del satélite, como la masa de la carga útil y la masa y las dimensiones extremas de satélite. Este enfoque permite que el equipo de diseño obtenga una mejor visión del diseño en un espacio de diseño extendido. La herramienta de diseño estructural se basa en las fórmulas y los supuestos apropiados, incluyendo los modelos estáticos y dinámicos del satélite. Un algoritmo genético (Genetic Algorithm: GA) se aplica al espacio de diseño para optimizaciones de objetivo único y también multiobjetivo. El resultado de la optimización multiobjetivo es un Pareto-optimal basado en dos objetivo, la masa total de satélites mínimo y el máximo presupuesto de masa de carga útil. Por otro lado, la aplicación de los microsatélites en misiones espaciales es de interés por su menor coste y tiempo de desarrollo. La gran necesidad de las aplicaciones de teledetección es un fuerte impulsor de su popularidad en este tipo de misiones espaciales. Las misiones de tele-observación por satélite son esenciales para la investigación de los recursos de la tierra y el medio ambiente. En estas misiones existen interrelaciones estrechas entre diferentes requisitos como la altitud orbital, tiempo de revisita, el ciclo de vida y la resolución. Además, todos estos requisitos puede afectar a toda las características de diseño. Durante los últimos años la aplicación de CE en las misiones espaciales ha demostrado una gran ventaja para llegar al diseño óptimo, teniendo en cuenta tanto el rendimiento y el costo del proyecto. Un ejemplo bien conocido de la aplicación de CE es la CDF (Facilidad Diseño Concurrente) de la ESA (Agencia Espacial Europea). Está claro que para los proyectos de microsatélites universitarios tener o desarrollar una instalación de este tipo parece estar más allá de las capacidades del proyecto. Sin embargo, la práctica de la CE a cualquier escala puede ser beneficiosa para los microsatélites universitarios también. En la segunda parte de esta tesis, la atención se centra en el desarrollo de una estructura de optimización de diseño multidisciplinar (Multidisciplinary Design Optimization: MDO) aplicable a la fase de diseño conceptual de microsatélites de teledetección. Este enfoque permite que el equipo de diseño conozca la interacción entre las diferentes variables de diseño. El esquema MDO presentado no sólo incluye variables de nivel de sistema, tales como la masa total del satélite y la potencia total, sino también los requisitos de la misión como la resolución y tiempo de revisita. El proceso de diseño de microsatélites se divide en tres disciplinas; a) diseño de órbita, b) diseño de carga útil y c) diseño de plataforma. En primer lugar, se calculan diferentes parámetros de misión para un rango práctico de órbitas helio-síncronas (sun-synchronous orbits: SS-Os). Luego, según los parámetros orbitales y los datos de un instrumento como referencia, se calcula la masa y la potencia de la carga útil. El diseño de la plataforma del satélite se estima a partir de los datos de la masa y potencia de los diferentes subsistemas utilizando relaciones empíricas de diseño. El diseño del subsistema de potencia se realiza teniendo en cuenta variables de diseño más detalladas, como el escenario de la misión y diferentes tipos de células solares y baterías. El escenario se selecciona, de modo de obtener una banda de cobertura sobre la superficie terrestre paralelo al Ecuador después de cada intervalo de revisita. Con el objetivo de evaluar las interrelaciones entre las diferentes variables en el espacio de diseño, todas las disciplinas de diseño mencionados se combinan en un código unificado. Por último, una forma básica de MDO se ajusta a la herramienta de diseño de sistema de satélite. La optimización del diseño se realiza por medio de un GA con el único objetivo de minimizar la masa total de microsatélite. Según los resultados obtenidos de la aplicación del MDO, existen diferentes puntos de diseños óptimos, pero con diferentes variables de misión. Este análisis demuestra la aplicabilidad de MDO para los estudios de ingeniería de sistema en la fase de diseño conceptual en este tipo de proyectos. La principal conclusión de esta tesis, es que el diseño clásico de los satélites que por lo general comienza con la definición de la misión y la carga útil no es necesariamente la mejor metodología para todos los proyectos de satélites. Un microsatélite universitario, es un ejemplo de este tipo de proyectos. Por eso, se han desarrollado un conjunto de herramientas de diseño para encarar los estudios de la fase inicial de diseño. Este conjunto de herramientas incluye diferentes disciplinas de diseño centrados en el subsistema estructural y teniendo en cuenta una carga útil desconocida a priori. Los resultados demuestran que la mínima masa total del satélite y la máxima masa disponible para una carga útil desconocida a priori, son objetivos conflictivos. En este contexto para encontrar un Pareto-optimal se ha aplicado una optimización multiobjetivo. Según los resultados se concluye que la selección de la masa total por satélite en el rango de 40-60 kg puede considerarse como óptima para un proyecto de microsatélites universitario con carga útil desconocida a priori. También la metodología CE se ha aplicado al proceso de diseño conceptual de microsatélites de teledetección. Los resultados de la aplicación del CE proporcionan una clara comprensión de la interacción entre los requisitos de diseño de sistemas de satélites, tales como la masa total del microsatélite y la potencia y los requisitos de la misión como la resolución y el tiempo de revisita. La aplicación de MDO se hace con la minimización de la masa total de microsatélite. Los resultados de la aplicación de MDO aclaran la relación clara entre los diferentes requisitos de diseño del sistema y de misión, así como que permiten seleccionar las líneas de base para el diseño óptimo con el objetivo seleccionado en las primeras fase de diseño. ABSTRACT This thesis is done in the context of UPMSat-2 project, which is a microsatellite under design and manufacturing at the Instituto Universitario de Microgravedad “Ignacio Da Riva” (IDR/UPM) of the Universidad Politécnica de Madrid. Application of Concurrent Engineering (CE) methodology in the framework of Multidisciplinary Design application (MDO) is one of the main objectives of the present work. In recent years, there has been continuing interest in the participation of university research groups in space technology studies by means of their own microsatellites. The involvement in such projects has some inherent challenges, such as limited budget and facilities. Also, due to the fact that the main objective of these projects is for educational purposes, usually there are uncertainties regarding their in orbit mission and scientific payloads at the early phases of the project. On the other hand, there are predetermined limitations for their mass and volume budgets owing to the fact that most of them are launched as an auxiliary payload in which the launch cost is reduced considerably. The satellite structure subsystem is the one which is most affected by the launcher constraints. This can affect different aspects, including dimensions, strength and frequency requirements. In the first part of this thesis, the main focus is on developing a structural design sizing tool containing not only the primary structures properties as variables but also the satellite system level variables such as payload mass budget and satellite total mass and dimensions. This approach enables the design team to obtain better insight into the design in an extended design envelope. The structural design sizing tool is based on the analytical structural design formulas and appropriate assumptions including both static and dynamic models of the satellite. A Genetic Algorithm (GA) is applied to the design space for both single and multiobejective optimizations. The result of the multiobjective optimization is a Pareto-optimal based on two objectives, minimum satellite total mass and maximum payload mass budget. On the other hand, the application of the microsatellites is of interest for their less cost and response time. The high need for the remote sensing applications is a strong driver of their popularity in space missions. The satellite remote sensing missions are essential for long term research around the condition of the earth resources and environment. In remote sensing missions there are tight interrelations between different requirements such as orbital altitude, revisit time, mission cycle life and spatial resolution. Also, all of these requirements can affect the whole design characteristics. During the last years application of the CE in the space missions has demonstrated a great advantage to reach the optimum design base lines considering both the performance and the cost of the project. A well-known example of CE application is ESA (European Space Agency) CDF (Concurrent Design Facility). It is clear that for the university-class microsatellite projects having or developing such a facility seems beyond the project capabilities. Nevertheless practicing CE at any scale can be beneficiary for the university-class microsatellite projects. In the second part of this thesis, the main focus is on developing a MDO framework applicable to the conceptual design phase of the remote sensing microsatellites. This approach enables the design team to evaluate the interaction between the different system design variables. The presented MDO framework contains not only the system level variables such as the satellite total mass and total power, but also the mission requirements like the spatial resolution and the revisit time. The microsatellite sizing process is divided into the three major design disciplines; a) orbit design, b) payload sizing and c) bus sizing. First, different mission parameters for a practical range of sun-synchronous orbits (SS-Os) are calculated. Then, according to the orbital parameters and a reference remote sensing instrument, mass and power of the payload are calculated. Satellite bus sizing is done based on mass and power calculation of the different subsystems using design estimation relationships. In the satellite bus sizing, the power subsystem design is realized by considering more detailed design variables including a mission scenario and different types of solar cells and batteries. The mission scenario is selected in order to obtain a coverage belt on the earth surface parallel to the earth equatorial after each revisit time. In order to evaluate the interrelations between the different variables inside the design space all the mentioned design disciplines are combined in a unified code. The integrated satellite system sizing tool developed in this section is considered as an application of the CE to the conceptual design of the remote sensing microsatellite projects. Finally, in order to apply the MDO methodology to the design problem, a basic MDO framework is adjusted to the developed satellite system design tool. Design optimization is done by means of a GA single objective algorithm with the objective function as minimizing the microsatellite total mass. According to the results of MDO application, there exist different optimum design points all with the minimum satellite total mass but with different mission variables. This output demonstrates the successful applicability of MDO approach for system engineering trade-off studies at the conceptual design phase of the design in such projects. The main conclusion of this thesis is that the classical design approach for the satellite design which usually starts with the mission and payload definition is not necessarily the best approach for all of the satellite projects. The university-class microsatellite is an example for such projects. Due to this fact an integrated satellite sizing tool including different design disciplines focusing on the structural subsystem and considering unknown payload is developed. According to the results the satellite total mass and available mass for the unknown payload are conflictive objectives. In order to find the Pareto-optimal a multiobjective GA optimization is conducted. Based on the optimization results it is concluded that selecting the satellite total mass in the range of 40-60 kg can be considered as an optimum approach for a university-class microsatellite project with unknown payload(s). Also, the CE methodology is applied to the remote sensing microsatellites conceptual design process. The results of CE application provide a clear understanding of the interaction between satellite system design requirements such as satellite total mass and power and the satellite mission variables such as revisit time and spatial resolution. The MDO application is done with the total mass minimization of a remote sensing satellite. The results from the MDO application clarify the unclear relationship between different system and mission design variables as well as the optimum design base lines according to the selected objective during the initial design phases.

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.

Strychnine activates neuronal α7 nicotinic receptors after mutations in the leucine ring and transmitter binding site domains

Recent work has shown that strychnine, the potent and selective antagonist of glycine receptors, is also an antagonist of nicotinic acetylcholine (AcCho) receptors including neuronal homomeric α7 receptors, and that mutating Leu-247 of the α7 nicotinic AcCho receptor-channel domain (L247Tα7; mut1) converts some nicotinic antagonists into agonists. Therefore, a study was made of the effects of strychnine on Xenopus oocytes expressing the chick wild-type α7 or L247Tα7 receptors. In these oocytes, strychnine itself did not elicit appreciable membrane currents but reduced the currents elicited by AcCho in a reversible and dose-dependent manner. In sharp contrast, in oocytes expressing L247Tα7 receptors with additional mutations at Cys-189 and Cys-190, in the extracellular N-terminal domain (L247T/C189–190Sα7; mut2), micromolar concentrations of strychnine elicited inward currents that were reversibly inhibited by the nicotinic receptor blocker α-bungarotoxin. Single-channel recordings showed that strychnine gated mut2-channels with two conductance levels, 56 pS and 42 pS, and with kinetic properties similar to AcCho-activated channels. We conclude that strychnine is a modulator, as well as an activator, of some homomeric nicotinic α7 receptors. After injecting oocytes with mixtures of cDNAs encoding mut1 and mut2 subunits, the expressed hybrid receptors were activated by strychnine, similar to the mut2, and had a high affinity to AcCho like the mut1. A pentameric symmetrical model yields the striking conclusion that two identical α7 subunits may be sufficient to determine the functional properties of α7 receptors.

Voltage-induced membrane displacement in patch pipettes activates mechanosensitive channels

The patch-clamp technique allows currents to be recorded through single ion channels in patches of cell membrane in the tips of glass pipettes. When recording, voltage is typically applied across the membrane patch to drive ions through open channels and to probe the voltage-sensitivity of channel activity. In this study, we used video microscopy and single-channel recording to show that prolonged depolarization of a membrane patch in borosilicate pipettes results in delayed slow displacement of the membrane into the pipette and that this displacement is associated with the activation of mechanosensitive (MS) channels in the same patch. The membrane displacement, ≈1 μm with each prolonged depolarization, occurs after variable delays ranging from tens of milliseconds to many seconds and is correlated in time with activation of MS channels. Increasing the voltage step shortens both the delay to membrane displacement and the delay to activation. Preventing depolarization-induced membrane displacement by applying positive pressure to the shank of the pipette or by coating the tips of the borosilicate pipettes with soft glass prevents the depolarization-induced activation of MS channels. The correlation between depolarization-induced membrane displacement and activation of MS channels indicates that the membrane displacement is associated with sufficient membrane tension to activate MS channels. Because membrane tension can modulate the activity of various ligand and voltage-activated ion channels as well as some transporters, an apparent voltage dependence of a channel or transporter in a membrane patch in a borosilicate pipette may result from voltage-induced tension rather than from direct modulation by voltage.

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca2+ release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200–500 μM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, Po < 0.005) and brief (<250 μs) gating events and insensitivity to Ca2+. Addition of ryanodine restores Ca2+-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.

Quantal release at a neuronal nicotinic synapse from rat adrenal gland.

The neuronal nicotinic synapse in tissue slices of the adrenal medulla was studied with whole-cell patch-clamp. Excitatory postsynaptic currents (EPSCs) were evoked by local field stimulation or occurred spontaneously especially when external [K+] was increased. EPSCs were carried by channels sharing biophysical and pharmacological properties of neuronal-type nicotinic receptors (nAChRs). A single-channel conductance (gamma) of 43-45 pS was found from nonstationary variance analysis of EPSCs. Spontaneous EPSCs were tetrodotoxin-insensitive and Ca(2+)-dependent and occurred in burst-like clusters. Quantal analysis of spontaneous EPSCs gave a quantal size of 20 pA and amplitude histograms were well described by binomial models with low values of quantal content, consistent with a small number of spontaneously active release sites. However, rare large amplitude EPSCs suggest that the total number of sites is higher and that extrajunctional receptors are involved. Our estimates of quantal content and size at the chromaffin cell neuronal nicotinic synapse may be useful in characterizing central neuronal-type nicotinic receptor-mediated cholinergic synaptic transmission.

Voltage gating and permeation in a gap junction hemichannel.

Gap junction channels are formed by members of the connexin gene family and mediate direct intercellular communication through linked hemichannels (connexons) from each of two adjacent cells. While for most connexins, the hemichannels appear to require an apposing hemichannel to open, macroscopic currents obtained from Xenopus oocytes expressing rat Cx46 suggested that some hemichannels can be readily opened by membrane depolarization [Paul, D. L., Ebihara, L., Takemoto, L. J., Swenson, K. I. & Goodenough, D. A. (1991), J. Cell Biol. 115, 1077-1089]. Here we demonstrate by single channel recording that hemichannels comprised of rat Cx46 exhibit complex voltage gating consistent with there being two distinct gating mechanisms. One mechanism partially closes Cx46 hemichannels from a fully open state, gammaopen, to a substate, gammasub, about one-third of the conductance of gammaopen; these transitions occur when the cell is depolarized to inside positive voltages, consistent with gating by transjunctional voltage in Cx46 gap junctions. The other gating mechanism closes Cx46 hemichannels to a fully closed state, gammaclosed, on hyperpolarization to inside negative voltages and has unusual characteristics; transitions between gammaclosed and gammaopen appear slow (10-20 ms), often involving several transient substates distinct from gammasub. The polarity of activation and kinetics of this latter form of gating indicate that it is the mechanism by which these hemichannels open in the cell surface membrane when unapposed by another hemichannel. Cx46 hemichannels display a substantial preference for cations over anions, yet have a large unitary conductance (approximately 300 pS) and a relatively large pore as inferred from permeability to tetraethylammonium (approximately 8.5 angstroms diameter). These hemichannels open at physiological voltages and could induce substantial cation fluxes in cells expressing Cx46.

Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats.

We investigated the cellular and molecular events associated with the increase in sodium transport across the alveolar epithelium of rats exposed to hyperoxia (85% O2 for 7 days followed by 100% O2 for 4 days). Alveolar type II (ATII) cell RNA was isolated and probed with a cDNA for one of the rat colonic epithelial sodium channel subunits (alpha rENaC). The alpha rENaC mRNA (3.7-kb transcript) increased 3-fold in ATII cell RNA isolated from rats exposed to 85% O2 for 7 days and 6-fold after 4 days of subsequent exposure to 100% O2. In situ hybridization revealed increased expression of alpha rENaC mRNA transcripts in both airway and alveolar epithelial cells of hyperoxic rats. When immunostained with a polyclonal antibody to kidney sodium channel protein, ATII cells from hyperoxic rats exhibited a significant increase in the amount of immunogenic protein present in both the plasma membrane and the cytoplasm. When patched in the whole-cell mode, ATII cells from hyperoxic rats exhibited amiloride and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (EIPA)-sensitive currents that were 100% higher compared with those obtained from air-breathing rats. Single-channel sodium currents (mean conductance of 25 pS) were seen in ATII cells patched in both the inside-out and cell-attached modes. The number and open probability of these channels increased significantly during exposure to hyperoxia. Exposure to sublethal hyperoxia up-regulated both alpha rENaC mRNA and the functional expression of sodium channels in ATII cells.

Hypertonicity activates nonselective cation channels in mouse cortical collecting duct cells.

We investigated the effect of cell shrinkage on whole-cell currents of M-1 mouse cortical collecting duct cells. Addition of 100 mM sucrose to an isotonic NaCl bath solution induced cell shrinkage and increased whole-cell currents within 5-10 min by approximately 12-fold. The effect was reversible upon return to isotonic solution and could also be elicited by adding 100 mM urea or 50 mM NaCl. Replacement of bath Na+ by K+, Cs+, Li+, or Rb+ did not significantly affect the stimulated inward current, but replacement by N-methyl-D-glucamine reduced it by 88.1 +/- 1.3% (n = 34); this demonstrates that hypertonicity activates a nonselective alkali cation conductance. The activation was independent of extra- and intracellular Ca2+, but 1 or 10 mM ATP in the pipette suppressed it in a concentration-dependent manner, indicating that intracellular ATP levels may modulate the degree of channel activation. Flufenamic acid (0.1 mM) and gadolinium (0.1 mM) inhibited the stimulated current by 68.7 +/- 5.9% (n = 9) and 32.4 +/- 11.7% (n = 6), respectively, whereas 0.1 mM amiloride had no significant effect. During the early phase of hypertonic stimulation single-channel transitions could be detected in whole-cell current recordings, and a gradual activation of 30 and more individual channels with a single-channel conductance of 26.7 +/- 0.4 pS (n = 29) could be resolved. Thus, we identified the nonselective cation channel underlying the shrinkage-induced whole-cell conductance that may play a role in volume regulation.

Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors.

Vigilance, anxiety, epileptic activity, and muscle tone can be modulated by drugs acting at the benzodiazepine (BZ) site of gamma-aminobutyric acid type A (GABAA) receptors. In vivo, BZ sites are potential targets for endogenous ligands regulating the corresponding central nervous system states. To assess the physiological relevance of BZ sites, mice were generated containing GABAA receptors devoid of BZ sites. Following targeted disruption of the gamma 2 subunit gene, 94% of the BZ sites were absent in brain of neonatal mice, while the number of GABA sites was only slightly reduced. Except for the gamma 2 subunit, the level of expression and the regional and cellular distribution of the major GABAA receptor subunits were unaltered. The single channel main conductance level and the Hill coefficient were reduced to values consistent with recombinant GABAA receptors composed of alpha and beta subunits. The GABA response was potentiated by pentobarbital but not by flunitrazepam. Diazepam was inactive behaviorally. Thus, the gamma 2 subunit is dispensable for the assembly of functional GABAA receptors but is required for normal channel conductance and the formation of BZ sites in vivo. BZ sites are not essential for embryonic development, as suggested by the normal body weight and histology of newborn mice. Postnatally, however, the reduced GABAA receptor function is associated with retarded growth, sensorimotor dysfunction, and drastically reduced life-span. The lack of postnatal GABAA receptor regulation by endogenous ligands of BZ sites might contribute to this phenotype.

Convergent and parallel activation of low-conductance potassium channels by calcium and cAMP-dependent protein kinase.

K+ channels, which have been linked to regulation of electrogenic solute transport as well as Ca2+ influx, represent a locus in hepatocytes for the concerted actions of hormones that employ Ca2+ and cAMP as intracellular messengers. Despite considerable study, the single-channel basis for synergistic effects of Ca2+ and cAMP on hepatocellular K+ conductance is not well understood. To address this question, patch-clamp recording techniques were applied to a model liver cell line, HTC hepatoma cells. Increasing the cytosolic Ca2+ concentration ([Ca2+]i) in HTC cells, either by activation of purinergic receptors with ATP or by inhibition of intracellular Ca2+ sequestration with thapsigargin, activated low-conductance (9-pS) K+ channels. Studies with excised membrane patches suggested that these channels were directly activated by Ca2+. Exposure of HTC cells to a permeant cAMP analog, 8-(4-chlorophenylthio)-cAMP, also activated 9-pS K+ channels but did not change [Ca2+]i. In excised membrane patches, cAMP-dependent protein kinase (the downstream effector of cAMP) activated K+ channels with conductance and selectivity identical to those of channels activated by Ca2+. In addition, cAMP-dependent protein kinase activated a distinct K+ channel type (5 pS). These data represent the differential regulation of low-conductance K+ channels by signaling pathways mediated by Ca2+ and cAMP. Moreover, since low-conductance Ca(2+)-activated K+ channels have been identified in a variety of cell types, these findings suggest that differential regulation of K+ channels by hormones with distinct signaling pathways may provide a mechanism for hormonal control of solute transport and Ca(2+)-dependent cellular functions in the liver as well as other nonexcitable tissues.

Evidence that neuronal G-protein-gated inwardly rectifying K+ channels are activated by G beta gamma subunits and function as heteromultimers.

Guanine nucleotide-binding proteins (G proteins) activate K+ conductances in cardiac atrial cells to slow heart rate and in neurons to decrease excitability. cDNAs encoding three isoforms of a G-protein-coupled, inwardly rectifying K+ channel (GIRK) have recently been cloned from cardiac (GIRK1/Kir 3.1) and brain cDNA libraries (GIRK2/Kir 3.2 and GIRK3/Kir 3.3). Here we report that GIRK2 but not GIRK3 can be activated by G protein subunits G beta 1 and G gamma 2 in Xenopus oocytes. Furthermore, when either GIRK3 or GIRK2 was coexpressed with GIRK1 and activated either by muscarinic receptors or by G beta gamma subunits, G-protein-mediated inward currents were increased by 5- to 40-fold. The single-channel conductance for GIRK1 plus GIRK2 coexpression was intermediate between those for GIRK1 alone and for GIRK2 alone, and voltage-jump kinetics for the coexpressed channels displayed new kinetic properties. On the other hand, coexpression of GIRK3 with GIRK2 suppressed the GIRK2 alone response. These studies suggest that formation of heteromultimers involving the several GIRKs is an important mechanism for generating diversity in expression level and function of neurotransmitter-coupled, inward rectifier K+ channels.

Structural conservation of ion conduction pathways in K channels and glutamate receptors.

Single channel recordings demonstrate that ion channels switch stochastically between an open and a closed pore conformation. In search of a structural explanation for this universal open/close behavior, we have uncovered a striking degree of amino acid homology across the pore-forming regions of voltage-gated K channels and glutamate receptors. This suggested that the pores of these otherwise unrelated classes of channels could be structurally conserved. Strong experimental evidence supports a hairpin structure for the pore-forming region of K channels. Consequently, we hypothesized the existence of a similar structure for the pore of glutamate receptors. In ligand-gated channels, the pore is formed by M2, the second of four putative transmembrane segments. A hairpin structure for M2 would affect the subsequent membrane topology, inverting the proposed orientation of the next segments, M3. We have tested this idea for the NR1 subunit of the N-methyl-D-aspartate receptor. Mutations that affected the glycosylation pattern of the NR1 subunit localize both extremes of the M3-M4 linker to the extracellular space. Whole cell currents and apparent agonist affinities were not affected by these mutations. Therefore it can be assumed that they represent the native transmembrane topology. The extracellular assignment of the M3-M4 linker challenged the current topology model by inverting M3. Taken together, the amino acid homology and the new topology suggest that the pore-forming M2 segment of glutamate receptors does not transverse the membrane but, rather, forms a hairpin structure, similar to that found in K channels.

Measurement of the conductance of a hydrogen molecule

Recent years have shown steady progress towards molecular electronics, in which molecules form basic components such as switches, diodes and electronic mixers. Often, a scanning tunnelling microscope is used to address an individual molecule, although this arrangement does not provide long-term stability. Therefore, metal–molecule–metal links using break-junction devices have also been explored; however, it is difficult to establish unambiguously that a single molecule forms the contact. Here we show that a single hydrogen molecule can form a stable bridge between platinum electrodes. In contrast to results for organic molecules, the bridge has a nearly perfect conductance of one quantum unit, carried by a single channel. The hydrogen bridge represents a simple test system in which to understand fundamental transport properties of single-molecule devices.

The North Pacific biological pump: Rates, efficiency and influence on ocean carbon uptake

Thesis (Ph.D.)--University of Washington, 2016-05