A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers

Membrane capacitance measurements of depolarization-triggered exocytosis in single nerve terminals from the rat neurohypophysis

Exocytosis of hormones and neurotransmitters is believed to be stimulated by a rise in intracellular calcium concentration. Membrane depolarization and entry of calcium through voltage-gated channels initiate the response, but subsequent steps leading to fusion of secretory vesicles are largely unknown. Investigation of stimulus-secretion coupling mechanisms in neurons is limited by the inaccessibility of most nerve endings to electrophysiological studies. Progress in other secretory cells has been achieved using the technique of cell membrane capacitance measurements, which is a sensitive assay of the increase in membrane area of single cells that accompanies exocytosis. Using patch clamp methods, a sinusoidal voltage stimulation is applied to the cell, and the resulting membrane current is analyzed to determine changes in membrane capacitance. An advancement called phase tracking is described here that automates one aspect of the capacitance technique and that improves the accuracy of the measurement. To investigate the mechanism of exocytosis in excitable cells, we have applied this capacitance technique to single vertebrate nerve endings isolated from the rat neurohypophysis. The nerve endings of this preparation are among the largest found in vertebrates, and are amenable to electrophysiological recording. We find that in response to brief depolarization, the membrane area is rapidly increased by an amount corresponding to the fusion of up to 100 secretory granules. We have investigated the relationship between this exocytotic signal and calcium influx during the voltage pulse. Capacitance responses are sharply reduced by increasing the concentration of intracellular Ca chelator. Also, the dependence of capacitance response amplitude on membrane potential is a bell shaped mirror image of the Ca current-voltage relation. On average, we find a linear relation between Ca influx and exocytosis in this preparation, although there is variability in the Ca sensitivity among terminals. In experiments using higher stimulation rates ($\u3e$1Hz) we observe patterns of facilitation and depression of capacitance responses. These processes occur by a mechanism independent of the amplitude of Ca current, and indicate that steps subsequent to Ca entry are also important in stimulus-secretion coupling

Dense sampling of bird diversity increases power of comparative genomics

Whole-genome sequencing projects are increasingly populating the tree of life and characterizing biodiversity(1-4). Sparse taxon sampling has previously been proposed to confound phylogenetic inference(5), and captures only a fraction of the genomic diversity. Here we report a substantial step towards the dense representation of avian phylogenetic and molecular diversity, by analysing 363 genomes from 92.4% of bird families-including 267 newly sequenced genomes produced for phase II of the Bird 10,000 Genomes (B10K) Project. We use this comparative genome dataset in combination with a pipeline that leverages a reference-free whole-genome alignment to identify orthologous regions in greater numbers than has previously been possible and to recognize genomic novelties in particular bird lineages. The densely sampled alignment provides a single-base-pair map of selection, has more than doubled the fraction of bases that are confidently predicted to be under conservation and reveals extensive patterns of weak selection in predominantly non-coding DNA. Our results demonstrate that increasing the diversity of genomes used in comparative studies can reveal more shared and lineage-specific variation, and improve the investigation of genomic characteristics. We anticipate that this genomic resource will offer new perspectives on evolutionary processes in cross-species comparative analyses and assist in efforts to conserve species. A dataset of the genomes of 363 species from the Bird 10,000 Genomes Project shows increased power to detect shared and lineage-specific variation, demonstrating the importance of phylogenetically diverse taxon sampling in whole-genome sequencing.Peer reviewe