Convergent and divergent modulatory pathways in {\it Aplysia\/}: Cellular and network studies

Neuromodulation is essential to many functions of the nervous system. In the simple gastropod mollusk Aplysia californica, neuromodulation of the circuits for the defensive withdrawal reflexes has been associated with several forms of learning. In the present work, the neurotransmitters and neural circuitry which contribute to the modulation of the tail-siphon withdrawal reflex were examined.^ A recently-identified neuropeptide transmitter, buccalin A was found to modulate the biophysical properties of the sensory neurons that mediate the reflex. The actions of buccalin A on the sensory neurons were compared with those of the well-characterized modulatory transmitter serotonin, and convergence and divergence in the actions of these two transmitters were evaluated. Buccalin A dramatically increased the excitability of sensory neurons and occluded further enhancement of excitability by serotonin. Buccalin A produced no significant change in spike duration, and it did not block serotonin-induced spike broadening. Voltage-clamp analysis revealed the currents that may be involved in the effects on spike duration and excitability. Buccalin A decreased an outward current similar to the S-K$\sp+$ current (I$\sb{\rm K,S}$). Buccalin A appeared to occlude further modulation of I$\sb{\rm K,S}$ by serotonin, but did not block serotonin-induced modulation of the voltage-dependent delayed rectifier K$\sp+$ current (I$\sb{\rm K,V}$). These results suggest that buccalin A converges on some, but not all, of the same subcellular modulatory pathways as serotonin.^ In order to begin to understand neuromodulation in a more physiological context for the tail-siphon withdrawal reflex, the modulatory circuitry for the tail-withdrawal circuit was examined. Mechanoafferent neurons in the J cluster of the cerebral ganglion were identified as elements of a modulatory circuit for the reflex. Excitatory and inhibitory connections were observed between the J cells and the pleural sensory neurons, the tail motor neurons, and several classes of interneurons for the tail-siphon withdrawal circuit. The J cells produced both fast and slow PSPs in these neurons. Of particular interest was the ability of the J cells to produce slow EPSPs in the pleural sensory neurons. These slow EPSPs were associated with an increase in the excitability of the sensory neurons. The J cells appear to mediate both sensory and modulatory inputs to the circuit for the tail-siphon withdrawal reflex from the anterior part of the animal. ^

Simulación, construcción y medida de un sistema de telealimentación eficiente

Los sistemas de telealimentación han tomado gran importancia en diferentes campos, incluido el de las telecomunicaciones, algunos ejemplos pueden ser: En la red conmutada telefónica junto con la señal de información y llamada existe una alimentación de 48v que se transmite a través de toda la línea de transmisión hasta los terminales. En algunos ferrocarriles eléctricos, se aprovecha la producción de energía eléctrica cuando un tren baja una cuesta y el motor funciona como generador, devolviendo la energía excedente a la propia catenaria por medio de superposición, y siendo esta recuperada en otro lugar y aprovechada por ejemplo por otro tren que requiere energía. Otro uso en ferrocarriles de la telealimentación es la llamada "tecnología del transpondedor magnético", en la que el tren transmite a las balizas una señal en 27MHz además de otras de información propias, que se convierte en energía útil para estas balizas. En este proyecto pretendemos implementar un pequeño ejemplo de sistema de telealimentación trabajando en 5 MHz (RF). Este sistema transforma una señal de CC en una señal de potencia de CA que podría ser, por ejemplo, transmitida a lo largo de una línea de transmisión o radiada por medio de una antena. Después, en el extremo receptor, esta señal RF se transforma finalmente en DC. El objetivo es lograr el mejor rendimiento de conversión de energía, DC a AC y AC a DC. El sistema se divide en dos partes: El inversor, que es la cadena de conversión DC-AC y el rectificador, que es la cadena de conversión AC-DC. Cada parte va a ser calculada, simulada, implementada físicamente y medida aparte. Finalmente el sistema de telealimentación completo se va a medir mediante la interconexión de cada parte por medio de un adaptador o una línea de transmisión. Por último, se mostrarán los resultados obtenidos. ABSTRACT. Remote powering systems have become very important in different fields, including telecommunications, some examples include: In the switched telephone network with the information signal and call there is a 48v supply that is transmitted across the transmission line to the terminals. In some electric railways, the production of electrical energy is used when a train is coming down a hill and the motor acts as a generator, returning the surplus energy to the catenary itself by overlapping, and this being recovered elsewhere and used by other train. Home TV amplifiers that are located in places (storage, remote locations ..) where there is no outlet, remote power allows to carry information and power signal by the same physical medium, for instance a coax. The AC power signal is transformed into DC at the end to feed the amplifier. In medicine, photovoltaic converters and fiber optics can be used as means for feeding devices implanted in patients. Another use of the remote powering systems on railways is the "magnetic transponder technology", in which the station transmits a beacon signal at 27MHz own as well as other information, which is converted into useful energy to these beacons. In this Project we are pretending to implement a little example of remote powering system working in 5 MHz (RF). This system transform DC into an AC-RF power signal which could be, for instance, transmitted throughout a transmission line or radiated by means of an aerial. At the receiving end, this RF signal is then transformed to DC. The objective is to achieve the best power conversion performance, DC to AC and AC to DC. The system is divided in two parts: The inverter, that is the DC-AC conversion chain and the rectifier that is the AC-DC conversion chain. Each part is going to be calculated, simulated, implemented physically and measured apart. Then the complete remote-powering system is to be measured by interconnecting each part by means of a interconnector or a transmission line. Finally, obtained results will be shown.

Development and improvement of protection-system algorithms for ground-fault detection and location in synchronous machines

Uno de los defectos más frecuentes en los generadores síncronos son los defectos a tierra tanto en el devanado estatórico, como de excitación. Se produce un defecto cuando el aislamiento eléctrico entre las partes activas de cualquiera de estos devanados y tierra se reduce considerablemente o desaparece. La detección de los defectos a tierra en ambos devanados es un tema ampliamente estudiado a nivel industrial. Tras la detección y confirmación de la existencia del defecto, dicha falta debe ser localizada a lo largo del devanado para su reparación, para lo que habitualmente el rotor debe ser extraído del estator. Esta operación resulta especialmente compleja y cara. Además, el hecho de limitar la corriente de defecto en ambos devanados provoca que el defecto no sea localizable visualmente, pues apenas existe daño en el generador. Por ello, se deben aplicar técnicas muy laboriosas para localizar exactamente el defecto y poder así reparar el devanado. De cara a reducir el tiempo de reparación, y con ello el tiempo en que el generador esta fuera de servicio, cualquier información por parte del relé de protección acerca de la localización del defecto resultaría de gran utilidad. El principal objetivo de esta tesis doctoral ha sido el desarrollo de nuevos algoritmos que permitan la estimación de la localización de los defectos a tierra tanto en el devanado rotórico como estatórico de máquinas síncronas. Respecto al devanado de excitación, se ha presentado un nuevo método de localización de defectos a tierra para generadores con excitación estática. Este método permite incluso distinguir si el defecto se ha producido en el devanado de excitación, o en cualquiera de los componentes del sistema de excitación, esto es, transformador de excitación, conductores de alimentación del rectificador controlado, etc. En caso de defecto a tierra en del devanado rotórico, este método proporciona una estimación de su localización. Sin embargo, para poder obtener la localización del defecto, se precisa conocer el valor de resistencia de defecto. Por ello, en este trabajo se presenta además un nuevo método para la estimación de este parámetro de forma precisa. Finalmente, se presenta un nuevo método de detección de defectos a tierra, basado en el criterio direccional, que complementa el método de localización, permitiendo tener en cuenta la influencia de las capacidades a tierra del sistema. Estas capacidades resultan determinantes a la hora de localizar el defecto de forma adecuada. En relación con el devanado estatórico, en esta tesis doctoral se presenta un nuevo algoritmo de localización de defectos a tierra para generadores que dispongan de la protección de faltas a tierra basada en la inyección de baja frecuencia. Se ha propuesto un método general, que tiene en cuenta todos los parámetros del sistema, así como una versión simplificada del método para generadores con capacidades a tierra muy reducida, que podría resultar de fácil implementación en relés de protección comercial. Los algoritmos y métodos presentados se han validado mediante ensayos experimentales en un generador de laboratorio de 5 kVA, así como en un generador comercial de 106 MVA con resultados satisfactorios y prometedores. ABSTRACT One of the most common faults in synchronous generators is the ground fault in both the stator winding and the excitation winding. In case of fault, the insulation level between the active part of any of these windings and ground lowers considerably, or even disappears. The detection of ground faults in both windings is a very researched topic. The fault current is typically limited intentionally to a reduced level. This allows to detect easily the ground faults, and therefore to avoid damage in the generator. After the detection and confirmation of the existence of a ground fault, it should be located along the winding in order to repair of the machine. Then, the rotor has to be extracted, which is a very complex and expensive operation. Moreover, the fact of limiting the fault current makes that the insulation failure is not visually detectable, because there is no visible damage in the generator. Therefore, some laborious techniques have to apply to locate accurately the fault. In order to reduce the repair time, and therefore the time that the generator is out of service, any information about the approximate location of the fault would be very useful. The main objective of this doctoral thesis has been the development of new algorithms and methods to estimate the location of ground faults in the stator and in the rotor winding of synchronous generators. Regarding the excitation winding, a new location method of ground faults in excitation winding of synchronous machines with static excitation has been presented. This method allows even to detect if the fault is at the excitation winding, or in any other component of the excitation system: controlled rectifier, excitation transformer, etc. In case of ground fault in the rotor winding, this method provides an estimation of the fault location. However, in order to calculate the location, the value of fault resistance is necessary. Therefore, a new fault-resistance estimation algorithm is presented in this text. Finally, a new fault detection algorithm based on directional criterion is described to complement the fault location method. This algorithm takes into account the influence of the capacitance-to-ground of the system, which has a remarkable impact in the accuracy of the fault location. Regarding the stator winding, a new fault-location algorithm has been presented for stator winding of synchronous generators. This algorithm is applicable to generators with ground-fault protection based in low-frequency injection. A general algorithm, which takes every parameter of the system into account, has been presented. Moreover, a simplified version of the algorithm has been proposed for generators with especially low value of capacitance to ground. This simplified algorithm might be easily implementable in protective relays. The proposed methods and algorithms have been tested in a 5 kVA laboratory generator, as well as in a 106 MVA synchronous generator with satisfactory and promising results.

Transferencia inalámbrica de energía

El trabajo presentado en este documento se centra en la temática de la transferencia inalámbrica de energía, concretamente en aplicaciones de campo lejano, para llevar a cabo dicho trabajo nos centraremos en el diseño, implementación y medición de una rectenna operando en la banda ISM concretamente a una frecuencia de 2.45GHz, el objetivo primordial de este trabajo será analizar que parámetros intervienen en la eficiencia de conversión en la etapa de RF-DC a fin de lograr la máxima eficiencia de conversión posible. Para llevar a cabo dicho análisis se emplearán herramientas informáticas, concretamente se hará uso del software AWR Microwave Office, a través del cual se realizarán simulaciones SourcePull a fin de determinar la impedancia óptima de entrada que se le debe presentar a la etapa rectificadora RF-DC para conseguir la máxima eficiencia de conversión, una vez realizadas dichas pruebas se implementará físicamente un circuito rectenna a través del cual realizar medidas de SourcePull mediante un Wide Matching Range Slide Screw Tuner de MAURY MICROWAVE para cotejar las posibles diferencias con los resultados obtenidos en las simulaciones. Tras la fase de pruebas SourcePull se extrapolará una red de entrada en base a los datos obtenidos en las mediciones anteriores y se diseñará y fabricará un circuito rectenna con máxima eficiencia de conversión para un conjunto de valores de potencia de entrada de RF y carga de DC, tras lo cual se analizará la eficiencia del circuito diseñado para diferentes valores de potencia de RF de entrada y carga de DC. Como elemento rectificador emplearemos en nuestro trabajo el diodo Schottky HSMS-2820, los diodos Schottky se caracterizan por tener tiempos de conmutación relativamente bajos y pérdidas en directa reducidas los cual será fundamental a la hora de trabajar con niveles reducidos de potencia de RF de entrada, para implementar el circuito se empleará un substrato FR4 con espesor de 0.8mm para disminuir en la mayor medida posible las pérdidas introducidas por el dieléctrico, se analizarán diferentes posibilidades a la hora de implementar el filtro de RF a la salida del diodo rectificador y finalmente se optará por el empleo de un stub radial ya que será este el que mejor ancho de banda nos proporcione. Los resultados simulados se compararán con los resultados medidos sobre el circuito rectenna para determinar la similitud entre ambos. ABSTRACT. The work presented in this paper focuses on the issue of wireless transfer of energy, particularly applied to far-field applications, to carry out this work we focus on the design, implementation and measurement of a rectenna operating in the ISM band specifically at a frequency of 2.45GHz, the primary objective of this study is to analyze any parameter involved in the RF-DC conversion efficiency in order to achieve the maximum conversion efficiency as possible. Computer analysis tools will be used, particularly AWR Microwave Office software, in order to carry out SourcePull simulations to determine the optimal input impedance which must be presented to the rectifier stage for maximum conversion efficiency, once obtained, a rectenna circuit will be implemented to compute SourcePull measurements, and finally simulated results will be compared to measured results. Once obtained the result, an input network impedance is extrapolated based on data from previous measurements to design and implement a rectenna circuit with high conversion efficiency for a set of RF input power and DC load values , after that, the designed circuit efficiency will be analyzed for different values of RF input power and DC load. In this work a HSMS-2820 Schottky diode will be used as the rectifier , Schottky diodes are characterized by relatively low switching times and reduced direct losses, that properties will be essential when working with low RF input power levels , to implement the circuit a FR4 substrate with 0.8mm thickness is used to reduce as much as possible the dielectric losses, different possibilities to implement the RF filter to the output of the rectifier diode will be analyzed, finally we will opt for the use of a radial stub as this will provide the best bandwidth possible. The simulated results are compared with the results measured on the rectenna circuit to determine the similarity between them.

A Wireless Charging System Applying Phase-Shift and Amplitude Control to Maximize Efficiency and Extractable Power

Wireless power transfer (WPT) is an emerging technology with an increasing number of potential applications to transfer power from a transmitter to a mobile receiver over a relatively large air gap. However, its widespread application is hampered due to the relatively low efficiency of current Wireless power transfer (WPT) systems. This study presents a concept to maximize the efficiency as well as to increase the amount of extractable power of a WPT system operating in nonresonant operation. The proposed method is based on actively modifying the equivalent secondary-side load impedance by controlling the phase-shift of the active rectifier and its output voltage level. The presented hardware prototype represents a complete wireless charging system, including a dc-dc converter which is used to charge a battery at the output of the system. Experimental results are shown for the proposed concept in comparison to a conventional synchronous rectification approach. The presented optimization method clearly outperforms state-of-the-art solutions in terms of efficiency and extractable power.

A resistance emulation approach to optimize the wave energy harvesting for a direct drive point absorber

In general, a major challenge for the exploitation of renewable energies is to improve their efficiency. In electricity generation from the energy of ocean waves, not unlike other technologies, the converter must be optimized to make the energy harvesting economically feasible. This paper proposes a passive tuning control strategy of a point absorber in which the power captured is maximized by controlling the electromagnetic force of the generator with a resistance emulation approach. The proposed strategy consists of mapping the optimal values for regular waves and applying them to irregular waves. This strategy is tested in a wave energy converter in which the generator is connected to a boost rectifier converter whose controller is designed to emulate a resistance. The power electronics system implemented is validated by comparing its performance with the case in which the generator is directly connected to a resistive load. The simulation results show the effectiveness of the proposed strategy as the maximum captured power is concentrated around the optimal values previously calculated and with the same behavior for both excitations.

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.

pH gating of ROMK (Kir1.1) channels: Control by an Arg-Lys-Arg triad disrupted in antenatal Bartter syndrome

Inward-rectifier K+ channels of the ROMK (Kir1.1) subtype are responsible for K+ secretion and control of NaCl absorption in the kidney. A hallmark of these channels is their gating by intracellular pH in the neutral range. Here we show that a lysine residue close to TM1, identified previously as a structural element required for pH-induced gating, is protonated at neutral pH and that this protonation drives pH gating in ROMK and other Kir channels. Such anomalous titration of this lysine residue (Lys-80 in Kir1.1) is accomplished by the tertiary structure of the Kir protein: two arginines in the distant N and C termini of the same subunit (Arg-41 and Arg-311 in Kir1.1) are located in close spatial proximity to the lysine allowing for electrostatic interactions that shift its pKa into the neutral pH range. Structural disturbance of this triad as a result from a number of point mutations found in patients with antenatal Bartter syndrome shifts the pKa of the lysine residue off the neutral pH range and results in channels permanently inactivated under physiological conditions. Thus, the results provide molecular understanding for normal pH gating of Kir channels as well as for the channel defects found in patients with antenatal Bartter syndrome.

A regenerative link in the ionic fluxes through the weaver potassium channel underlies the pathophysiology of the mutation

The homozygous weaver mouse displays neuronal degeneration in several brain regions. Previous experiments in heterologous expression systems showed that the G protein-gated inward rectifier K+ channel (GIRK2) bearing the weaver pore-region GYG-to-SYG mutation (i) is not activated by Gβγ subunits, but instead shows constitutive activation, and (ii) is no longer a K+-selective channel but conducts Na+ as well. The present experiments on weaverGIRK2 (wvGIRK2) expressed in Xenopus oocytes show that the level of constitutive activation depends on intracellular Na+ concentration. In particular, manipulations that decrease intracellular Na+ produce a component of Na+-permeable current activated via a G protein pathway. Therefore, constitutive activation may not arise because the weaver mutation directly alters the gating transitions of the channel protein. Instead, there may be a regenerative cycle of Na+ influx through the wvGIRK2 channel, leading to additional Na+ activation. We also show that the wvGIRK2 channel is permeable to Ca2+, providing an additional mechanism for the degeneration that characterizes the weaver phenotype. We further demonstrate that the GIRK4 channel bearing the analogous weaver mutation has properties similar to those of the wvGIRK2 channel, providing a glimpse of the selective pressures that have maintained the GYG sequence in nearly all known K+ channels.

γ-Aminobutyric acid type B receptors are expressed and functional in mammalian cardiomyocytes

γ-Hydroxybutyrate (GHB), an anesthetic adjuvant analog of γ-aminobutyrate (GABA), depresses cell excitability in hippocampal neurons by inducing hyperpolarization through the activation of a prominent inwardly rectifying K+ (Kir3) conductance. These GABA type B (GABAB)-like effects are clearly shown at high concentrations of GHB corresponding to blood levels usually reached during anesthesia and are mimicked by the GABAB agonist baclofen. Recent studies of native GABAB receptors (GABABRs) have favored the concept that GHB is also a selective agonist. Furthermore, cloning has demonstrated that GABABRs assemble heteromeric complexes from the GABABR1 and GABABR2 subtypes and that these assemblies are activated by GHB. The surprisingly high tissue content, together with anti-ischemic and protective effects of GHB in the heart, raises the question of a possible influence of GABAB agonists on excitable cardiac cells. In the present study, we provide electrophysiological evidence that GHB activates an inwardly rectifying K+ current in rat ventricular myocytes. This effect is mimicked by baclofen, reversibly inhibited by GABAB antagonists, and prevented by pertussis toxin pretreatment. Both GABABR1 and GABABR2 are detected in cardiomyocytes by Western blotting and are shown to coimmunoprecipitate. Laser scanning confocal microscopy discloses an even distribution of the two receptors in the sarcolemma and along the transverse tubular system. Hence, we conclude that GABABRs are distributed not only in neuronal tissues but also in the heart, where they can be activated and induce electrophysiological alterations through G-protein-coupled inward rectifier potassium channels.

Distinct gene-specific mechanisms of arrhythmia revealed by cardiac gene transfer of two long QT disease genes, HERG and KCNE1

The long QT syndrome (LQTS) is a heritable disorder that predisposes to sudden cardiac death. LQTS is caused by mutations in ion channel genes including HERG and KCNE1, but the precise mechanisms remain unclear. To clarify this situation we injected adenoviral vectors expressing wild-type or LQT mutants of HERG and KCNE1 into guinea pig myocardium. End points at 48–72 h included electrophysiology in isolated myocytes and electrocardiography in vivo. HERG increased the rapid component, IKr, of the delayed rectifier current, thereby accelerating repolarization, increasing refractoriness, and diminishing beat-to-beat action potential variability. Conversely, HERG-G628S suppressed IKr without significantly delaying repolarization. Nevertheless, HERG-G628S abbreviated refractoriness and increased beat-to-beat variability, leading to early afterdepolarizations (EADs). KCNE1 increased the slow component of the delayed rectifier, IKs, without clear phenotypic sequelae. In contrast, KCNE1-D76N suppressed IKs and markedly slowed repolarization, leading to frequent EADs and electrocardiographic QT prolongation. Thus, the two genes predispose to sudden death by distinct mechanisms: the KCNE1 mutant flagrantly undermines cardiac repolarization, and HERG-G628S subtly facilitates the genesis and propagation of premature beats. Our ability to produce electrocardiographic long QT in vivo with a clinical KCNE1 mutation demonstrates the utility of somatic gene transfer in creating genotype-specific disease models.

Spectrum of HERG K+-channel dysfunction in an inherited cardiac arrhythmia.

Long QT syndrome (LQT) is an autosomal dominant disorder that can cause sudden death from cardiac arrhythmias. We recently discovered that mutations in HERG, a K+-channel gene, cause chromosome 7-linked LQT. Heterologous expression of HERG in Xenopus oocytes revealed that HERG current was similar to a well-characterized cardiac delayed rectifier K+ current, IKr, and led to the hypothesis that mutations in HERG reduced IKr, causing prolonged myocellular action potentials. To define the mechanism of LQT, we injected oocytes with mutant HERG complementary RNAs, either singly or in combination with wild-type complementary RNA. Some mutations caused loss of function, whereas others caused dominant negative suppression of HERG function. These mutations are predicted to cause a spectrum of diminished IKr and delayed ventricular repolarization, consistent with the prolonged QT interval observed in individuals with LQT.

Susceptibility of cloned K+ channels to reactive oxygen species.

Free radical-induced oxidant stress has been implicated in a number of physiological and pathophysiological states including ischemia and reperfusion-induced dysrhythmia in the heart, apoptosis of T lymphocytes, phagocytosis, and neurodegeneration. We have studied the effects of oxidant stress on the native K+ channel from T lymphocytes and on K+ channels cloned from cardiac, brain, and T-lymphocyte cells and expressed in Xenopus oocytes. The activity of three Shaker K+ channels (Kv1.3, Kv1.4, and Kv1.5), one Shaw channel (Kv3.4), and one inward rectifier K+ channel (IRK3) was drastically inhibited by photoactivation of rose bengal, a classical generator of reactive oxygen species. Other channel types (such as Shaker K+ channel Kv1.2, Shab channels Kv2.1 and Kv2.2, Shal channel Kv4.1, inward rectifiers IRK1 and ROMK1, and hIsK) were completely resistant to this treatment. On the other hand tert-butyl hydroperoxide, another generator of reactive oxygen species, removed the fast inactivation processes of Kv1.4 and Kv3.4 but did not alter other channels. Xanthine/xanthine oxidase system had no effect on all channels studied. Thus, we show that different types of K+ channels are differently modified by reactive oxygen species, an observation that might be of importance in disease states.

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Characterization of a voltage-gated K+ channel beta subunit expressed in human heart.

Voltage-gated K+ channels are important modulators of the cardiac action potential. However, the correlation of endogenous myocyte currents with K+ channels cloned from human heart is complicated by the possibility that heterotetrameric alpha-subunit combinations and function-altering beta subunits exist in native tissue. Therefore, a variety of subunit interactions may generate cardiac K+ channel diversity. We report here the cloning of a voltage-gated K+ channel beta subunit, hKv beta 3, from adult human left ventricle that shows 84% and 74% amino acid sequence identity with the previously cloned rat Kv beta 1 and Kv beta 2 subunits, respectively. Together these three Kv beta subunits share > 82% identity in the carboxyl-terminal 329 aa and show low identity in the amino-terminal 79 aa. RNA analysis indicated that hKv beta 3 message is 2-fold more abundant in human ventricle than in atrium and is expressed in both healthy and diseased human hearts. Coinjection of hKv beta 3 with a human cardiac delayed rectifier, hKv1.5, in Xenopus oocytes increased inactivation, induced an 18-mV hyperpolarizing shift in the activation curve, and slowed deactivation (tau = 8.0 msec vs. 35.4 msec at -50 mV). hKv beta 3 was localized to human chromosome 3 by using a human/rodent cell hybrid mapping panel. These data confirm the presence of functionally important K+ channel beta subunits in human heart and indicate that beta-subunit composition must be accounted for when comparing cloned channels with endogenous cardiac currents.