K+ secretion by the cortical collecting duct (CCD) is stimulated at high flow rates. Patch-clamp analysis has identified a small-conductance secretory K+ (SK) and a high-conductance Ca(2+)-activated K+ (maxi-K) channel in the apical membrane of the CCD. The SK channel, encoded by ROMK, is believed to mediate baseline K+ secretion. The role of the stretch- and Ca2+-activated maxi-K channel is still uncertain. The purpose of this study was to identify the K+ channel mediating flow-dependent K+ secretion in the CCD. Segments isolated from New Zealand White rabbits were microperfused in the absence and presence of luminal tetraethylammonium (TEA) or charybdotoxin, both inhibitors of maxi-K but not SK channels, or apamin, an inhibitor of small-conductance maxi-K+ channels. Net K+ secretion and Na+ absorption were measured at varying flow rates. In the absence of TEA, net K+ secretion increased from 8.3 +/- 1.0 to 23.4 +/- 4.7 pmol. min(-1). mm(-1) (P < 0.03) as the tubular flow rate was increased from 0.5 to 6 nl. min(-1). mm(-1). Flow stimulation of net K+ secretion was blocked by luminal TEA (8.2 +/- 1.2 vs. 9.9 +/- 2.7 pmol. min(-1). mm(-1) at 0.6 and 6 nl. min(-1). mm(-1) flow rates, respectively) or charybdotoxin (6.8 +/- 1.6 vs. 8.3 +/- 1.6 pmol. min(-1). mm(-1) at 1 and 4 nl. min(-1). mm(-1) flow rates, respectively) but not by apamin. These results suggest that flow-dependent K+ secretion is mediated by a maxi-K channel, whereas baseline K+ secretion occurs through a TEA- and charybdotoxin-insensitive SK (ROMK) channel.

Arrestins are members of a superfamily of proteins that arrest the activity of G-protein coupled receptors. Mouse cone photoreceptors express two visual arrestins, Arr1 and Arr4 (Carr). We quantified their expression levels and subcellular distributions in mouse cones: total Arr1 was estimated to be in an ~ 6:1 ratio to cone opsin, about 50-fold higher than Arr4. Recordings from single cones of Arr1−/− and Arr4−/− mice establish that both proteins are competent to arrest the activity of photoactivated S- and M- cone opsins. Recordings from Arr1−/− , Arr4−/− double-knockout mice establish a requirement for at least one of the two visual arrestins for normal cone opsin inactivation at all flash intensities. These recordings also reveal low activity photoproducts of S- and M-opsins that are absent when Grk1 and an arrestin are co-expressed, but which decay 70-fold more rapidly than the comparable photoproducts of rhodopsin in rods.

The topology of most eukaryotic polytopic membrane proteins is established cotranslationally in the endoplasmic reticulum (ER) through a series of coordinated translocation and membrane integration events. For the human aquaporin water channel AQP1, however, the initial four-segment-spanning topology at the ER membrane differs from the mature six-segment-spanning topology at the plasma membrane. Here we use epitope-tagged AQP1 constructs to follow the transmembrane (TM) orientation of key internal peptide loops in Xenopus oocyte and cell-free systems. This analysis revealed that AQP1 maturation in the ER involves a novel topological reorientation of three internal TM segments and two peptide loops. After the synthesis of TMs 4-6, TM3 underwent a 180-degree rotation in which TM3 C-terminal flanking residues were translocated from their initial cytosolic location into the ER lumen and N-terminal flanking residues underwent retrograde translocation from the ER lumen to the cytosol. These events convert TM3 from a type I to a type II topology and reposition TM2 and TM4 into transmembrane conformations consistent with the predicted six-segment-spanning AQP1 topology. AQP1 topological reorientation was also associated with maturation from a protease-sensitive conformation to a protease-resistant structure with water channel function. These studies demonstrate that initial protein topology established via cotranslational translocation events in the ER is dynamic and may be modified by subsequent steps of folding and/or maturation.

Daily, the retinal pigment epithelium (RPE) ingests a bolus of lipid and protein in the form of phagocytized photoreceptor outer segments (OS). The RPE, like the liver, expresses enzymes required for fatty acid oxidation and ketogenesis. This suggests that these pathways play a role in the disposal of lipids from ingested OS, as well as providing a mechanism for recycling metabolic intermediates back to the outer retina. In this study, we examined whether OS phagocytosis was linked to ketogenesis. We found increased levels of β-hydroxybutyrate (β-HB) in the apical medium following ingestion of OS by human fetal RPE and ARPE19 cells cultured on Transwell inserts. No increase in ketogenesis was observed following ingestion of oxidized OS or latex beads. Our studies further defined the connection between OS phagocytosis and ketogenesis in wild-type mice and mice with defects in phagosome maturation using a mouse RPE explant model. In explant studies, the levels of β-HB released were temporally correlated with OS phagocytic burst after light onset. In the mouse where phagosome maturation is delayed, there was a temporal shift in the release of β-HB. An even more pronounced shift in maximal β-HB production was observed in the RPE, in which loss of the ATP-binding cassette A4 transporter results in defective phagosome processing and accumulation of lipid debris. These studies suggest that FAO and ketogenesis are key to supporting the metabolism of the RPE and preventing the accumulation of lipids that lead to oxidative stress and mitochondrial dysfunction.

Background: A repeats-in-toxin (RTX) leukotoxin and its integrin receptor aggregate in cholesterol-rich lipid rafts. Results: The affinity of the toxin to cholesterol is driven by a cholesterol recognition/amino acid consensus (CRAC) motif. Conclusion: Leukotoxin cytotoxicity is regulated by the CRAC motif. Significance: Other RTX toxins contain this CRAC motif, suggesting a role for cholesterol recognition in RTX cytolysis.

Transmembrane topology of most eukaryotic polytopic proteins is established cotranslationally at the endoplasmic reticulum membrane through the action of alternating signal and stop transfer sequences. Here we demonstrate that the cystic fibrosis transmembrane conductance regulator (CFTR) achieves its N terminus topology through a variation of this mechanism that involves both co-and posttranslational translocation events. Using a series of defined chimeric and truncated proteins expressed in a reticulocyte lysate system, we have identified two topogenic determinants encoded within the first (TM1) and second (TM2) membranespanning segments of CFTR. Each sequence independently (i) directed endoplasmic reticulum targeting, (ii) translocated appropriate flanking residues, and (iii) achieved its proper membrane-spanning orientation. Signal sequence activity of TM1, however, was inefficient due to the presence of two charged residues, Glu 92 and Lys 95 , located within its hydrophobic core. As a result, TM1 was able to direct correct topology for less than half of nascent CFTR chains. In contrast to TM1, TM2 signal sequence activity was both efficient and specific. Even in the absence of a functional TM1 signal sequence, TM2 was able to direct CFTR N terminus topology through a ribosome-dependent posttranslational mechanism. Mutating charged residues Glu 92 and Lys 95 to alanine improved TM1 signal sequence activity as well as the ability of TM1 to independently direct CFTR N terminus topology. Thus, a single functional signal sequence in either the first or second TM segment was sufficient for directing proper CFTR topology. These results identify two distinct and redundant translocation pathways for CFTR N terminus transmembrane assembly and support a model in which TM2 functions to ensure correct topology of CFTR chains that fail to translocate via TM1. This novel arrangement of topogenic information provides an alternative to conventional cotranslational pathways of polytopic protein biogenesis.

The hypothesis is tested that enhanced green fluorescent protein (EGFP) can be used to quantify the aqueous spaces of living cells, using as a model transgenic Xenopus rods. Consistent with the hypothesis, regions of rods having structures that exclude EGFP, such as the mitochondrial-rich ellipsoid and the outer segments, have highly reduced EGFP fluorescence. Over a 300-fold range of expression the average EGFP concentration in the outer segment was approximately half that in the most intensely fluorescent regions of the inner segment, in quantitative agreement with prior X-ray diffraction estimates of outer segment cytoplasmic volume. In contrast, the fluorescence of soluble arrestin-EGFP fusion protein in the dark adapted rod outer segment was approximately threefold lower than predicted by the EGFP distribution, establishing that the fusion protein is not equilibrated with the cytoplasm. Arrestin-EGFP mass was conserved during a large-scale, light-driven redistribution in which ∼40% of the protein in the inner segment moved to the outer segment in less than 30 minutes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl− that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl− conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl− conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl >> Br; R347P, Cl >> Br; R347E, Br >> Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl− binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl−. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.