Activity of several ion channels is controlled by heterotrimeric GTP-binding proteins (G proteins) via a membrane-delimited pathway that does not involve cytoplasmic intermediates. The best studied example is the K+ channel activated by muscarinic agonists in the atrium, which plays a crucial role in regulating the heartbeat. To enable studies of the molecular mechanisms of activation, this channel, denoted KGA, was cloned from a rat atrium cDNA library by functional coupling to coexpressed serotonin type 1A receptors in Xenopus oocytes. KGA displays regions of sequence homology to other inwardiy rectifying channels as well as unique regions that may govern G-protein interaction. The expressed KGA channel is activated by serotonin 1A, muscarinic m2, and b-opioid receptors via G proteins. KGA is activated by guanosine 5'-[ythio]triphosphate in excised patches, confirming activation by a membrane-delimited pathway, and displays a conductance equal to that of the endogenous channel in atrial cells. The hypothesis that similar channels play a role in neuronal inhibition is supported by the cloning of a nearly identical channel (KGB1) from a rat brain cDNA library.A major signal transduction mechanism in cardiac physiology and neurobiology is the direct coupling of neurotransmitter receptors to ion channels by a membrane-delimited pathway that does not involve cytoplasmic intermediates (for reviews, see refs. 1 and 2). The best studied member of this group is the G protein-activated K+ channel found in atria of all vertebrates. This channel, which we denote KGA, figured in the original discovery of chemical synaptic transmission (3,4) and plays a crucial role in regulating the heartbeat. The KGA channel rectifies at the single-channel level, allowing much larger inward than outward currents (5, 6). It is activated by acetylcholine acting on muscarinic m2 receptors via a pathway that includes a pertussis toxin (PTX)-sensitive G protein, probably of the G, family (1,(7)(8)(9)(10)(11)(12)
A mutant strain of the cyanobacterium Synechocystis 6803, TolE4B, was constructed by genetic deletion of the protein that links phycobilisomes to thylakoid membranes and of the CP43 and CP47 proteins of photosystem II (PSII), leaving the photosystem I (PSI) center as the sole chromophore in the photosynthetic membranes. Both intact membrane and detergent-isolated samples of PSI were characterized by time-resolved and steady-state fluorescence methods. A decay component of -25 ps dominates (99% of the amplitude) the fluorescence of the membrane sample. This result indicates that an intermediate lifetime is not associated with the intact membrane preparation and the charge separation in PSI is irreversible. The decay time of the detergent-isolated sample is similar. The 600-nm excited steady-state fluorescence spectrum displays a red fluorescence peak at "703 nm at room temperature. The 450-nm excited steady-state fluorescence spectrum is dominated by a single peak around 700 nm without 680-nm "bulk" fluorescence. The experimental results were compared with several computer simulations. Assuming an antenna size of 130 chlorophyll molecules, an apparent charge separation time of -1 ps is estimated.Alternatively, the kinetics could be modeled on the basis of a two-domain antenna for PSI, consistent with the available structural data, each containing -65 chlorophyll a molecules. If excitation can migrate freely within each domain and communication between domains occurs only close to the reaction center, a charge separation time of3-4 ps is obtained instead.Understanding of the photochemical reaction center known as photosystem I (PSI) has been greatly increased with the publication of its 6-A x-ray structure (1). However, questions concerning the light-harvesting and trapping mechanisms of this complex remain, particularly since the positions of only half of the chlorophyll (Chl) molecules have been assigned. The PSI center is a membrane-bound, multiprotein complex that catalyzes the lightdriven transport of electrons from reduced plastocyanin or cytochrome C553 to soluble ferredoxin or flavodoxin (2, 3). The complex is known to contain 10 or 11 polypeptides, about 100 Chl molecules, and 10-15 ,B-carotenes. The major reaction center proteins are called PsaA, PsaB, and PsaC, having molecular masses of 83, 83, and 9 kDa, respectively. PsaA and PsaB are disposed symmetrically in the membrane and share bonding to segment P700 itself, the primary electron donor which may be a Chl a dimer, and to Ao, a monomeric Chl-a; A1, a phylloquinone (vitamin K); and F5, a Fe4-S4 center. The two remaining Fe4-S4 centers, FA and FB, are bound to PsaC.We are interested in how energy transfer and trapping occur in PSI. The elementary transfer time (-150-300 fs) in the PSI core antenna of a PSI-only mutant of Chlamydomonas reinhardtii has been determined in a fluorescence up-conversion experiment (4). This single-step transfer time very closely approximates a previous estimate using a simple two-color lattice model (5). Energy tr...
In Xenopus laevis oocytes injected with rat brain poly(A)+ RNA, perfusion with a high-K+ solution (96 mM KCl) generated an inward current (IHK) which was absent in water-injected oocytes. Part of IHK was blocked by low concentrations of Ba2+ (half-maximal inhibitory concentration, IC50: 4.2 +/- 0.5 microM). When serotonin (5-HT) was applied to these oocytes a transient inward oscillating Cl- current arising from activation of Ca2+ -dependent Cl- channels, ICl (Ca), was observed. When this response decayed, a 30% reduction of IHK could be detected. Electrophysiological characterization of the K+ channel down-modulated by 5-HT revealed that it is an inward rectifier. Anti-sense suppression experiments revealed that the 5-HT2C receptor mediates the down-modulatory effect of 5-HT. The nature of the modulatory pathway was investigated by application of phorbol esters and intracellular injection of protein kinase C (PKC) inhibitors, ethylenebis (oxonitrilo)tetraacetate (EGTA) and inositol 1,4,5-trisphosphate. The results demonstrate that PKC is responsible for the down-modulatory effect.
Genes encoding the phycobilisome core subunits allophycocyanin alpha and beta and a small core linker protein in Synechocystis sp. strain PCC 6714 were cloned and sequenced. These genes form an operon, apcABC, with a single transcription start site and two possible termination sites, one following apcB and the other following apcC. The promoter region, like those of the apcABC operons of other cyanobacteria, does not resemble the consensus promoter sequences of Escherichia coli. However, the apcABC promoters identified in four strains of cyanobacteria have conserved sequences centered at -50 and -10 with respect to the start of transcription. The apcE gene, encoding the protein that links the phycobilisome core to the thylakoid membrane, was also cloned from Synechocystis 6714 and sequenced. It is unlinked to the apcABC operon. As in other Synechocystis strains, the LCM polypeptide encoded by the apcE gene contains three repeats of the basic phycobiliprotein linker domain. The apcE gene promoter sequence bears little resemblance to either the E. coli consensus or the apcABC promoter region, but it is similar to the corresponding regions of other cyanobacterial apcE genes. In these cases, there are conserved sequences centered at -40 and -10 with respect to the transcription start site. These conserved promoter elements from the apcABC and apcE genes were also identified in the corresponding 5'-flanking regions of eleven transcript starts for cpc genes encoding phycocyanin subunits in cyanobacteria and algal chloroplasts. These results suggest that a factor yet to be described participates in transcription of phycobiliprotein genes.
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