We evolved muscarinic receptors in yeast to generate a family of G protein-coupled receptors (GPCRs) that are activated solely by a pharmacologically inert drug-like and bioavailable compound (clozapine-N-oxide). Subsequent screening in human cell lines facilitated the creation of a family of muscarinic acetylcholine GPCRs suitable for in vitro and in situ studies. We subsequently created lines of telomerase-immortalized human pulmonary artery smooth muscle cells stably expressing all five family members and found that each one faithfully recapitulated the signaling phenotype of the parent receptor. We also expressed a G i-coupled designer receptor in hippocampal neurons (hM 4D) and demonstrated its ability to induce membrane hyperpolarization and neuronal silencing. We have thus devised a facile approach for designing families of GPCRs with engineered ligand specificities. Such reverse-engineered GPCRs will prove to be powerful tools for selectively modulating signal-transduction pathways in vitro and in vivo.cell engineering ͉ molecular evolution ͉ receptorome B ecause of the assorted cellular responses directed by them, their number, and the ease of which they are pharmacologically screened, the superfamily of G protein-coupled receptors (GPCRs) is one of the most therapeutically important targets in the proteome (1). However, the potential of this family is restricted by our ability to assess their function, which currently involves transgenic, knockout, and/or in vivo studies with selective drugs. Genetic studies are frequently limited to loss-of-function phenotypes, whereas nonselectiveness of a drug often interferes with interpretation of pharmacological studies. Knowledge of the roles of the individual family members is being bolstered by the ongoing creation of knockout mice for many GPCRs. Selective activation of individual GPCR subtypes in a defined tissue, in either a knockout or wild-type animal, is currently problematic but, if possible, would serve to complement present findings by providing novel insights into disease states resulting from overstimulation of certain signaling pathways.One approach to this problem has been to rationally modify receptors to favor synthetic over natural substrate/ligand recognition, and subsequently, these mutant proteins have been used as bio-tools to study protein function in complex biological environments (2, 3). At the forefront of such modified GPCRs is Ro1, a G i/o -coupled opioid receptor activated by a synthetic but not a native ligand, which has been conditionally expressed in transgenic mice to study cardiac function after its selective activation (4). Such mutant receptors, like Ro1, have been classified as receptors activated solely by synthetic ligands (RASSLs), because they are activated by synthetic ligands but not by their endogenous ligands (5). RASSLs, as in the case of Ro1, have been demonstrated to be valuable tools (4, 6); however, because the synthetic ligand frequently has high affinity and/or potency at the native receptor (5,7,8), this pote...
Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple electrical activity to cellular metabolism through their inhibition by intracellular ATP. ATP inhibition of KATP channels varies among tissues and is affected by the metabolic and regulatory state of individual cells, suggesting involvement of endogenous factors. It is reported here that phosphatidylinositol-4, 5-bisphosphate (PIP2) and phosphatidylinositol-4-phosphate (PIP) controlled ATP inhibition of cloned KATP channels (Kir6.2 and SUR1). These phospholipids acted on the Kir6.2 subunit and shifted ATP sensitivity by several orders of magnitude. Receptor-mediated activation of phospholipase C resulted in inhibition of KATP-mediated currents. These results represent a mechanism for control of excitability through phospholipids.
Techniques for fast noninvasive control of neuronal excitability will be of major importance for analyzing and understanding neuronal networks and animal behavior. To develop these tools we demonstrated that two light-activated signaling proteins, vertebrate rat rhodopsin 4 (RO4) and the green algae channelrhodospin 2 (ChR2), could be used to control neuronal excitability and modulate synaptic transmission. Vertebrate rhodopsin couples to the Gi͞o, pertussis toxin-sensitive pathway to allow modulation of G protein-gated inward rectifying potassium channels and voltagegated Ca 2؉ channels. Light-mediated activation of RO4 in cultured hippocampal neurons reduces neuronal firing within ms by hyperpolarization of the somato-dendritic membrane and when activated at presynaptic sites modulates synaptic transmission and paired-pulse facilitation. In contrast, somato-dendritic activation of ChR2 depolarizes neurons sufficiently to induce immediate action potentials, which precisely follow the ChR2 activation up to light stimulation frequencies of 20 Hz. To demonstrate that these constructs are useful for regulating network behavior in intact organisms, embryonic chick spinal cords were electroporated with either construct, allowing the frequency of episodes of spontaneous bursting activity, known to be important for motor circuit formation, to be precisely controlled. Thus light-activated vertebrate RO4 and green algae ChR2 allow the antagonistic control of neuronal function within ms to s in a precise, reversible, and noninvasive manner in cultured neurons and intact vertebrate spinal cords.A major challenge in understanding the relationship between neural activity and development and between neuronal circuit activity and specific behaviors is to be able to control the activity of large populations of neurons or regions of individual nerve cells simultaneously. Recently, it was demonstrated that neuronal circuits can be manipulated by expressing mutated ion channels or G protein-coupled receptors (GPCRs). For example, the regional expression of a genetically modified K ϩ channel in Drosophila was able to reduce the excitability of targeted cells (i.e., muscle, neurons, photoreceptors) (1). Silencing of cortical neurons was achieved by binding of the peptide allostatin to its exogenously expressed receptor (2). Recently, Zemelman et al. (3) elegantly demonstrated that light activation of the protein complex, encoded by the Drosophila photoreceptor genes (i.e., arrestin-2, rhodopsin, and G protein ␣ subunit), could induce action potential firing of hippocampal neurons. Activation and deactivation of neuronal firing could also be achieved when ligand-gated ion channels, such as the capsaicin receptor, menthol receptor, purinergic receptors, or lightcontrollable K ϩ channel blockers, were used to control firing in hippocampal neurons (4, 5). However, the application of these techniques to control neuronal function especially in neural circuits and living animals is limited by their relatively slow time course, the complex...
containing an RNA helicase can degrade highly structured RNA molecules. It has been reported that the rhlB gene can be deleted without lethal consequences 26. However, the putative deletion strain still has a copy of rhlB, and more recent attempts to delete the gene failed, indicating that rhlB may be essential (our unpublished results; M. Cashel, personal communication). The results reported here provide evidence of a DEAD-box RNA helicase having an active role in bacterial mRNA degradation. D
Calcium ions entering cells through voltage-gated Ca2+ channels initiate rapid release of neurotransmitters and secretion of hormones. Ca2+ currents can be inhibited in many cell types by neurotransmitters acting through G proteins via a membrane-delimited pathway independently of soluble intracellular messengers. Inhibition is typically caused by a positive shift in the voltage dependence and a slowing of channel activation and is relieved by strong depolarization resulting in facilitation of Ca2+ currents. This pathway regulates the activity of N-type and P/Q-type Ca2+ channels, which are localized in presynaptic terminals and participate in neurotransmitter release. Synaptic transmission is inhibited by neurotransmitters through this mechanism. G-protein alpha subunits confer specificity in receptor coupling, but it is not known whether the G alpha or G beta gamma subunits are responsible for modulation of Ca2+ channels. Here we report that G beta gamma subunits can modulate Ca2+ channels. Transfection of G beta gamma into cells expressing P/Q-type Ca2+ channels induces modulation like that caused by activation of G protein-coupled receptors, but G alpha subunits do not. Similarly, injection or expression of G beta gamma subunits in sympathetic ganglion neurons induces facilitation and occludes modulation of N-type channels by noradrenaline, but G alpha subunits do not. In both cases, the G gamma subunit is ineffective by itself, but overexpression of exogenous G beta subunits is sufficient to cause channel modulation.
Serotonin (5HT) is a critical modulator of neural circuits that support diverse behaviors and physiological processes, and multiple lines of evidence implicate abnormal serotonergic signaling in psychiatric pathogenesis. The significance of 5HT underscores the importance of elucidating the molecular pathways involved in serotonergic system development, function, and plasticity. However, these mechanisms remain poorly defined, owing largely to the difficulty of accessing 5HT neurons for experimental manipulation. To address this methodological deficiency, we present a transgenic route to selectively alter 5HT neuron gene expression. This approach is based on the ability of a Pet-1 enhancer region to direct reliable 5HT neuron-specific transgene expression in the CNS. Its versatility is illustrated with several transgenic mouse lines, each of which provides a tool for 5HT neuron studies. Two lines allow Cre-mediated recombination at different stages of 5HT neuron development. A third line in which 5HT neurons are marked with yellow fluorescent protein will have numerous applications, including their electrophysiological characterization. To demonstrate this application, we have characterized active and passive membrane properties of midbrain reticular 5HT neurons, which heretofore have not been reported to our knowledge. A fourth line in which Pet-1 loss of function is rescued by expression of a Pet-1 transgene demonstrates biologically relevant levels of transgene expression and offers a route for investigating serotonergic protein structure and function in a behaving animal. These findings establish a straightforward and reliable approach for developing an array of tools for in vivo and in vitro studies of 5HT neurons.Cre recombinase ͉ pet-1 ͉ transgenic ͉ yellow fluorescent protein ͉ monoamine S erotonin (5HT) is a transmitter of broad relevance to nervous system development and function (1-4). Serotonergic pathways innervate most cytoarchitectonic structures of the CNS, and accordingly they have been implicated in the modulation of circuitry involved in nearly all behaviors and physiological processes (3, 5-7). Additionally, 5HT neurotransmission is modulated through abundant afferent information arising from, for example, other monoaminergic systems (8, 9) and from orexinergic, glutamatergic, and GABAergic pathways (10-12). The remarkably expansive neuromodulatory influence of 5HT is the product of a complex transcriptional cascade that generates 5HT-synthesizing neurons in the ventral hindbrain (13-18). 5HT also figures prominently in mental health disorders as a number of lines of evidence provide strong support for the hypothesis that altered serotonergic signaling contributes to neurological and psychiatric pathogenesis (19)(20)(21)(22). Despite considerable progress in understanding the importance of 5HT neurotransmission, however, the mechanisms governing 5HT neuron development and the precise physiological roles of 5HT in the modulation of CNS circuitry are not yet clear.Studies of 5HT neurons are hind...
Transcriptional cascades are required for specification of 5-HT neurons and 5-HT modulated behaviors. Expression of several cascade factors extends across lifespan suggesting their control of behavior may not be temporally restricted to programming normal numbers of 5-HT neurons. We applied new mouse conditional targeting approaches to investigate ongoing requirements for Pet-1, a cascade factor required for the initiation of 5-HT synthesis but whose expression persists into adulthood. We found that Pet-1 was required after 5-HT neuron generation, for multiple steps in 5-HT neuron maturation including axonal innervation to the somatosensory cortex, firing properties, and 5-HT1A and 5-HT1B autoreceptor expression. Targeting Pet-1 in adult 5-HT neurons showed that it was still needed to preserve normal anxiety-related behaviors through direct autoregulated control of serotonergic gene expression. These findings show that Pet-1 is required across lifespan and therefore behavioral pathogenesis can result from both developmental and adult-onset alterations in serotonergic transcription.
Synaptic transmission is regulated by G protein-coupled receptors whose activation releases G protein ␥ subunits that modulate presynaptic Ca 2؉ channels. The sequence motif QXXER has been proposed to be involved in the interaction between G protein ␥ subunits and target proteins including adenylyl cyclase 2. This motif is present in the intracellular loop connecting domains I and II (L I-II ) of Ca 2؉ channel ␣ 1A subunits, which are modulated by G proteins, but not in ␣ 1C subunits, which are not modulated. Peptides containing the QXXER motif from adenylate cyclase 2 or from ␣ 1A block G protein modulation but a mutant peptide containing the sequence AXXAA does not, suggesting that the QXXER-containing peptide from ␣ 1A can competitively inhibit G␥ modulation. Conversion of the R in the QQIER sequence of ␣ 1A to E as in ␣ 1C slows channel inactivation and shifts the voltage dependence of steady-state inactivation to more positive membrane potentials. Conversion of the final E in the QQLEE sequence of ␣ 1C to R has opposite effects on voltage-dependent inactivation, although the changes are not as large as those for ␣ 1A . Mutation of the QQIER sequence in ␣ 1A to QQIEE enhanced G protein modulation, and mutation to QQLEE as in ␣ 1C greatly reduced G protein modulation and increased the rate of reversal of G protein effects. These results indicate that the QXXER motif in L I-II is an important determinant of both voltage-dependent inactivation and G protein modulation, and that the amino acid in the third position of this motif has an unexpectedly large inf luence on modulation by G␥. Overlap of this motif with the consensus sequence for binding of Ca 2؉ channel  subunits suggests that this region of L I-II is important for three different modulatory inf luences on Ca 2؉ channel activity.Neuronal voltage-gated Ca 2ϩ channels are involved in multiple cellular functions including neurotranstransmitter release, Ca 2ϩ -mediated regulatory processes, and generation of dendritic action potentials. They consist of complexes of a poreforming ␣ 1 subunit of 190-250 kDa in association with ␣ 2 ␦ and  subunits (1, 2). Electrophysiological and pharmacological studies distinguish at least six classes of Ca 2ϩ channel currents designated L-, N-, P-, Q-, R-, and T-type (2, 3). Ca 2ϩ channels containing ␣ 1C or ␣ 1D subunits are thought to be responsible for L-type currents, ␣ 1B for N-type currents, and ␣ 1A for both P-and Q-type calcium currents (2, 4). A major goal of current research is to correlate the observed differences among the properties of these channel types with the structures of their ␣ 1 subunits.P͞Q-type and N-type Ca 2ϩ channels containing ␣ 1A or ␣ 1B subunits differ from L-type Ca 2ϩ channels containing ␣ 1C or ␣ 1D subunits in voltage-dependent inactivation (5-8) and in modulation by G proteins (9). Voltage-dependent inactivation of L-type Ca 2ϩ channels containing cloned ␣ 1C is slower than inactivation of P͞Q-type Ca 2ϩ channels containing cloned ␣ 1A expressed with the same auxiliary subun...
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