Many neurotransmitters and hormones signal by stimulating G protein-coupled neurotransmitter receptors (GPCRs), which activate G proteins and their downstream effectors. Whether these signalling proteins diffuse freely within the plasma membrane is not well understood. Recent studies have suggested that direct protein-protein interactions exist between GPCRs, G proteins and G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels. Here, we used fluorescence resonance energy transfer (FRET) combined with total internal reflection fluorescence microscopy to investigate whether proteins within this signalling pathway move within 100 A of each other in the plasma membrane of living cells. GABA B R1 and R2 receptors, Kir3 channels, Gαo subunits and regulators of G protein signalling (RGS4) proteins were each fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) and first assessed for functional expression in HEK293 cells. The presence of the fluorophore did not significantly alter the signalling properties of these proteins. Possible FRET was then investigated for different protein pair combinations. As a positive control, FRET was measured between tagged GABA B R1 and R2 subunits (∼12% FRET), which are known to form heterodimers. We measured significant FRET between tagged RGS4 and GABA B R1 or R2 subunits (∼13% FRET), and between Gαo and GABA B R1 or R2 subunits (∼10% FRET). Surprisingly, FRET also occurred between tagged Kir3.2a/Kir3.4 channels and GABA B R1 or R2 subunits (∼10% FRET). FRET was not detected between Kir3.2a and RGS4 nor between Kir3.2a and Gαo. These data are discussed in terms of a model in which GABA B receptors, G proteins, RGS4 proteins and Kir3 channels are closely associated in a signalling complex.

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PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation.

The activity of G protein-activated inwardly rectifying K + channels (GIRK or Kir3) is important for regulating membrane excitability in neuronal, cardiac and endocrine cells. Although G βγ subunits are known to bind the N-and C-termini of GIRK channels, the mechanism underlying G βγ activation of GIRK is not well understood. Here, we used chimeras and point mutants constructed from GIRK2 and IRK1, a G protein-insensitive inward rectifier, to determine the region within GIRK2 important for G βγ binding and activation. An analysis of mutant channels expressed in Xenopus oocytes revealed two amino acid substitutions in the C-terminal domain of GIRK2, GIRK2 L344E and GIRK2 G347H , that exhibited decreased carbacholactivated currents but significantly enhanced basal currents with coexpression of G βγ subunits. Combining the two mutations (GIRK2 EH ) led to a more severe reduction in carbachol-activated and G βγ -stimulated currents. Ethanol-activated currents were normal, however, suggesting that G protein-independent gating was unaffected by the mutations. Both GIRK2 L344E and GIRK2 EH also showed reduced carbachol activation and normal ethanol activation when expressed in HEK-293T cells. Using epitope-tagged channels expressed in HEK-293T cells, immunocytochemistry showed that G βγ -impaired mutants were expressed on the plasma membrane, although to varying extents, and could not account completely for the reduced G βγ activation. In vitro G βγ binding assays revealed an ∼60% decrease in G βγ binding to the C-terminal domain of GIRK2 L344E but no statistical change with GIRK2 EH or GIRK2 G347H , though both mutants exhibited G βγ -impaired activation. Together, these results suggest that L344, and to a lesser extent, G347 play an important functional role in G βγ activation of GIRK2 channels. Based on the 1.8Å structure of GIRK1 cytoplasmic domains, L344 and G347 are positioned in the βL-βM loop, which is situated away from the pore and near the N-terminal domain. The results are discussed in terms of a model for activation in which G βγ alters the interaction between the βL-βM loop and the N-terminal domain.

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Ligand-gated ion channel receptors mediate the response of fast neurotransmitters by opening in less than a millisecond. Here, we investigated the activation mechanism of a serotonin-gated receptor (5-HT 3A ) by systematically introducing cysteine substitutions throughout the pore-lining M1-M2 loop and M2 transmembrane domain. We hypothesized that multiple cysteines in the narrowest region of the pore, which together can form a high affinity binding site for metal cations, would reveal changes in pore structure during gating. Using cadmium (Cd 2؉ ) as a probe, two cysteine substitutions in the cytoplasmic selectivity filter, S2 C and, to a lesser extent, G-2 C, showed high affinity inhibition with Cd 2؉ Ligand-gated ion channel (LGIC) 1 receptors are responsible for rapid chemical transmission between neurons in the nervous system (1). Alterations in the response of ligand-gated receptors have profound effects on neuronal activity in the brain. For example, mutations in nicotinic acetylcholine-gated receptors (nAChRs) and glycine-gated receptors have been linked to certain forms of epilepsy and startle disease, respectively (2-4).LGIC receptors that respond to acetylcholine, ␥-aminobutyric acid, glycine, or serotonin (5-HT) possess a conserved pair of extracellular cysteines in the N-terminal domain ("Cys loop" receptors) and are composed of five subunits, each containing four putative transmembrane domains (5). Of these four domains, the second transmembrane domain (M2) lines the majority of the water-filled pore (Fig. 1A).The rapid response of LGIC receptors is achieved by the energetic coupling of agonist binding in the extracellular Nterminal domain with a "gate" located 60 Å away in the receptor pore (M2 domain) (6). Several studies with LGIC receptors suggest that the gate is situated in the middle of the M2 (6 -12) (but see Ref. 13). The primary determinants of selectivity appear to be located below the gate, near the cytoplasmic membrane surface. Mutations in the cytoplasmic end of the M2 alter ion selectivity (14 -17), and several recent studies show that by mutating sites in this region, cation-conducting LGIC can be converted to anion-conducting channels and vice versa (18 -23). Because permeability of like-charged molecules in LGIC appears to be largely dependent on size, it is believed that the narrowest region of the open LGIC pore coincides with the channel selectivity filter, which is formed by a portion of the M1-M2 loop and the cytoplasmic side of the M2 helix (16,24). Consistent with this region being narrow, site-specific mutations in the filter alter conductivity in a manner that depends upon side-chain volume (8,24). Thus, the pore of the open receptor can be envisaged as a funnel, with a wide extracellular vestibule that tapers to a constriction at the cytoplasmic side of the membrane, where ion selectivity is determined (25).What is known about the extent of movement in the cytoplasmic selectivity filter? The 9-Å three-dimensional structures of open and closed Torpedo nAChRs derived fro...