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ABSTRACT: Mutations in the novel serine/threonine WNK (With No lysine [=K]) kinases, WNK1 and WNK4, cause Pseudohypoaldosteronism type II (PHAII or Gordon Syndrome (PHAII)), a rare monogenic syndrome which causes hypertension and hyperkalemia on a background of a normal glomerular filtration rate. Current animal models for PHAII recapitulate some aspects of the disease phenotype, but give no clues to how rapidly the phenotype emerges or whether it is reversible. To this end we have created an inducible PHAII transgenic animal model that expresses a human disease-causing WNK4 mutation, WNK4 Q565E, under the control of the Tet-On system. Several PHAII inducible transgenic mouse lines were created, each with differing transgene (TG) copy numbers and displaying varying degrees of transgene expression (low, medium and high). Each of these transgenic lines demonstrated similar elevations of blood pressure and plasma potassium after 4 weeks of transgene induction. Withdrawal of doxycycline switched off mutant transgene expression and the disappearance of the PHAII phenotype. Western blotting of microdissected kidney nephron segments confirmed that expression of the thiazide-sensitive sodium chloride cotransporter (NCC) was increased, as expected, in the distal convoluted tubule when transgenic mice were induced with doxycycline. The kidneys of these mice also do not show the morphological changes seen in the previous transgenic model expressing the same mutant form of WNK4. This inducible model shows, for the first time, that in vivo expression of a mutant WNK4 protein is sufficient to cause the rapid and reversible appearance of a PHAII disease phenotype in mice.
Clinical Science 01/2013; · 4.61 Impact Factor
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ABSTRACT: Upon illumination several phototransduction proteins translocate between cell body and photosensory compartments. In Drosophila photoreceptors arrestin (Arr2) translocates from cell body to the microvillar rhabdomere down a diffusion gradient created by binding of Arr2 to photo-isomerized metarhodopsin. Translocation is profoundly slowed in mutants of key phototransduction proteins including phospholipase C (PLC) and the Ca(2+)-permeable transient receptor potential channel (TRP), but how the phototransduction cascade accelerates Arr2 translocation is unknown. Using real-time fluorescent imaging of Arr2-green fluorescent protein translocation in dissociated ommatidia, we show that translocation is profoundly slowed in Ca(2+)-free solutions. Conversely, in a blind PLC mutant with ∼100-fold slower translocation, rapid translocation was rescued by the Ca(2+) ionophore, ionomycin. In mutants lacking NINAC (calmodulin [CaM] binding myosin III) in the cell body, translocation remained rapid even in Ca(2+)-free solutions. Immunolabeling revealed that Arr2 in the cell body colocalized with NINAC in the dark. In intact eyes, the impaired translocation found in trp mutants was rescued in ninaC;trp double mutants. Nevertheless, translocation following prolonged dark adaptation was significantly slower in ninaC mutants, than in wild type: a difference that was reflected in the slow decay of the electroretinogram. The results suggest that cytosolic NINAC is a Ca(2+)-dependent binding target for Arr2, which protects Arr2 from immobilization by a second potential sink that sequesters and releases arrestin on a much slower timescale. We propose that rapid Ca(2+)/CaM-dependent release of Arr2 from NINAC upon Ca(2+) influx accounts for the acceleration of translocation by phototransduction.
Journal of Neuroscience 07/2012; 32(27):9205-16. · 7.11 Impact Factor
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ABSTRACT: TRP channels have emerged as key biological sensors in vision, taste, olfaction, hearing, and touch. Despite their importance, virtually nothing is known about the folding and transport of TRP channels during biosynthesis. Here, we identify XPORT (exit protein of rhodopsin and TRP) as a critical chaperone for TRP and its G protein-coupled receptor (GPCR), rhodopsin (Rh1). XPORT is a resident ER and secretory pathway protein that interacts with TRP and Rh1, as well as with Hsp27 and Hsp90. XPORT promotes the targeting of TRP to the membrane in Drosophila S2 cells, a finding that provides a critical first step toward solving a longstanding problem in the successful heterologous expression of TRP. Mutations in xport result in defective transport of TRP and Rh1, leading to retinal degeneration. Our results identify XPORT as a molecular chaperone and provide a mechanistic link between TRP channels and their GPCRs during biosynthesis and transport.
Neuron 11/2011; 72(4):602-15. · 14.74 Impact Factor
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ABSTRACT: INAD is a scaffolding protein that regulates signaling in Drosophila photoreceptors. One of its PDZ domains, PDZ5, cycles between reduced and oxidized forms in response to light, but it is unclear how light affects its redox potential. Through biochemical and structural studies, we show that the redox potential of PDZ5 is allosterically regulated by its interaction with another INAD domain, PDZ4. Whereas isolated PDZ5 is stable in the oxidized state, formation of a PDZ45 "supramodule" locks PDZ5 in the reduced state by raising the redox potential of its Cys606/Cys645 disulfide bond by ∼330 mV. Acidification, potentially mediated via light and PLCβ-mediated hydrolysis of PIP(2), disrupts the interaction between PDZ4 and PDZ5, leading to PDZ5 oxidation and dissociation from the TRP Ca(2+) channel, a key component of fly visual signaling. These results show that scaffolding proteins can actively modulate the intrinsic redox potentials of their disulfide bonds to exert regulatory roles in signaling.
Cell 06/2011; 145(7):1088-101. · 32.40 Impact Factor
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ABSTRACT: A reduced sensitivity to the sedating effects of alcohol is a characteristic associated with alcohol use disorders (AUDs). A genetic screen for ethanol sedation mutants in Drosophila identified arouser (aru), which functions in developing neurons to reduce ethanol sensitivity. Genetic evidence suggests that aru regulates ethanol sensitivity through its activation by Egfr/Erk signaling and its inhibition by PI3K/Akt signaling. The aru mutant also has an increased number of synaptic terminals in the larva and adult fly. Both the increased ethanol sensitivity and synapse number of the aru mutant are restored upon adult social isolation, suggesting a causal relationship between synapse number and ethanol sensitivity. We thus show that a developmental abnormality affecting synapse number and ethanol sensitivity is not permanent and can be reversed by manipulating the environment of the adult fly.
Neuron 06/2011; 70(5):979-90. · 14.74 Impact Factor
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ABSTRACT: Upon illumination, visual arrestin translocates from photoreceptor cell bodies to rhodopsin and membrane-rich photosensory compartments, vertebrate outer segments or invertebrate rhabdomeres, where it quenches activated rhodopsin. Both the mechanism and function of arrestin translocation are unresolved and controversial. In dark-adapted photoreceptors of the fruitfly Drosophila, confocal immunocytochemistry shows arrestin (Arr2) associated with distributed photoreceptor endomembranes. Immunocytochemistry and live imaging of GFP-tagged Arr2 demonstrate rapid reversible translocation to stimulated rhabdomeres in stoichiometric proportion to rhodopsin photoisomerization. Translocation is very rapid in normal photoreceptors (time constant <10 s) and can also be resolved in the time course of electroretinogram recordings. Genetic elimination of key phototransduction proteins, including phospholipase C (PLC), Gq, and the light-sensitive Ca2+-permeable TRP channels, slows translocation by 10- to 100-fold. Our results indicate that Arr2 translocation in Drosophila photoreceptors is driven by diffusion, but profoundly accelerated by phototransduction and Ca2+ influx.
Neuron 09/2010; 67(6):997-1008. · 14.74 Impact Factor
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ABSTRACT: Phototransduction in microvillar photoreceptors is mediated via G protein-coupled phospholipase C (PLC), but how PLC activation leads to the opening of the light-sensitive TRPC channels (TRP and TRPL) remains unresolved. In Drosophila, InsP(3) appears not to be involved, and recent studies have implicated lipid products of PLC activity, e.g., diacylglycerol, its metabolites, or the reduction in PIP(2). The fact that hydrolysis of the phosphodiester bond in PIP(2) by PLC also releases a proton is seldom recognized and has neither been measured in vivo nor implicated previously in a signaling context.
Following depletion of PIP(2) and other phosphoinositides by a variety of experimental manipulations, the light-sensitive channels in Drosophila photoreceptors become remarkably sensitive to rapid and reversible activation by the lipophilic protonophore 2-4 dinitrophenol in a pH-dependent manner. We further show that light induces a rapid (<10 ms) acidification originating in the microvilli, which is eliminated in mutants of PLC, and that heterologously expressed TRPL channels are activated by acidification of the cytosolic surface of inside-out patches.
Our results indicate that a combination of phosphoinositide depletion and acidification of the membrane/boundary layer is sufficient to activate the light-sensitive channels. Together with the demonstration of light-induced, PLC-dependent acidification, this suggests that excitation in Drosophila photoreceptors may be mediated by PLC's dual action of phosphoinositide depletion and proton release.
Current biology: CB 02/2010; 20(3):189-97. · 10.99 Impact Factor
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ABSTRACT: Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this performance is achieved remains unclear. Here, we investigate the mechanism and role of rhodopsin inactivation. We determined the lifetime of activated rhodopsin (metarhodopsin = M( *)) in whole-cell recordings from Drosophila photoreceptors by measuring the time window within which inactivating M( *) by photoreisomerization to rhodopsin could suppress responses to prior illumination. M( *) was inactivated rapidly (tau approximately 20 ms) under control conditions, but approximately 10-fold more slowly in Ca2+-free solutions. This pronounced Ca2+ dependence of M( *) inactivation was unaffected by mutations affecting phosphorylation of rhodopsin or arrestin but was abolished in mutants of calmodulin (CaM) or the CaM-binding myosin III, NINAC. This suggests a mechanism whereby Ca2+ influx acting via CaM and NINAC accelerates the binding of arrestin to M( *). Our results indicate that this strategy promotes quantum efficiency, temporal resolution, and fidelity of visual signaling.
Neuron 10/2008; 59(5):778-89. · 14.74 Impact Factor
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ABSTRACT: The Drosophila phototransduction cascade transforms light into depolarizations that are further shaped by activation of voltage-dependent K+ (Kv) channels. In whole-cell recordings of isolated photoreceptors, we show that light selectively modulated the delayed rectifier (Shab) current. Shab currents were increased by light with similar kinetics to the light-induced current itself (latency approximately 20 ms), recovering to control values with a t(1/2) of approximately 60 s in darkness. Genetic disruption of PLCbeta4, responsible for light-induced PIP(2) hydrolysis, abolished this light-dependent modulation. In mutants of CDP-diaclyglycerol synthase (cds(1)), required for PIP(2) resynthesis, the modulation became irreversible, but exogenously applied PIP(2) restored reversibility. The modulation was accurately and reversibly mimicked by application of PIP(2) to heterologously expressed Shab channels in excised inside-out patches. The results indicate a functionally implemented mechanism of Kv channel modulation by PIP(2) in photoreceptors, which enables light-dependent regulation of signal processing by direct coupling to the phototransduction cascade.
Neuron 09/2008; 59(4):596-607. · 14.74 Impact Factor
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ABSTRACT: Histamine (HA) is the photoreceptor neurotransmitter in arthropods, directly gating chloride channels on large monopolar cells (LMCs), postsynaptic to photoreceptors in the lamina. Two histamine-gated channel genes that could contribute to this channel in Drosophila are hclA (also known as ort) and hclB (also known as hisCl1), both encoding novel members of the Cys-loop receptor superfamily. Drosophila S2 cells transfected with these genes expressed both homomeric and heteromeric histamine-gated chloride channels. The electrophysiological properties of these channels were compared with those from isolated Drosophila LMCs. HCLA homomers had nearly identical HA sensitivity to the native receptors (EC(50) = 25 microM). Single-channel analysis revealed further close similarity in terms of single-channel kinetics and subconductance states ( approximately 25, 40, and 60 pS, the latter strongly voltage dependent). In contrast, HCLB homomers and heteromeric receptors were more sensitive to HA (EC(50) = 14 and 1.2 microM, respectively), with much smaller single-channel conductances ( approximately 4 pS). Null mutations of hclA (ort(US6096)) abolished the synaptic transients in the electroretinograms (ERGs). Surprisingly, the ERG "on" transients in hclB mutants transients were approximately twofold enhanced, whereas intracellular recordings from their LMCs revealed altered responses with slower kinetics. However, HCLB expression within the lamina, assessed by both a GFP (green fluorescent protein) reporter gene strategy and mRNA tagging, was exclusively localized to the glia cells, whereas HCLA expression was confirmed in the LMCs. Our results suggest that the native receptor at the LMC synapse is an HCLA homomer, whereas HCLB signaling via the lamina glia plays a previously unrecognized role in shaping the LMC postsynaptic response.
Journal of Neuroscience 07/2008; 28(29):7250-9. · 7.11 Impact Factor
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ABSTRACT: The novel serine/threonine kinases (with no lysine kinases or WNKs), WNK1 and WNK4, are encoded by the disease genes for Gordon syndrome (PRKWNK1 and PRKWNK4), a rare monogenic syndrome of hypertension and hyperkalemia. These proteins alter the expression of the thiazide-sensitive Na/Cl cotransporter (NCCT) in Xenopus laevis oocytes, although the details are controversial. We describe here our own experience and confirm that kinase-dead WNK4 (318D>A) is unable to affect Na+ fluxes through the thiazide-sensitive Na/Cl transporter (NCCT) or its membrane expression as an ECFP-NCCT fusion protein. However, the kinase domain is not sufficient for a functional WNK4 since deletion of the acidic motif (a motif unique to WNK family members) completely abolishes functional activity. Indeed, the NH2 terminal of WNK4 (1-620) containing the kinase domain and acidic motif retains full activity, but does not interact directly with NCCT in pull-down assays. Coexpression of WNK1 antagonizes the action of WNK4, and kinase-dead WNK1 (368D>A) or WNK1 carrying a WNK4 disease mutation (565Q>E) behaves in the same way as wild-type WNK1. This suggests kinase activity and charge conservation within the acidic motif are not essential for the WNK1-WNK4 interaction. We also report that WNK4 probably reduces surface expression largely through an effect on forward trafficking. Hence, the effect of WNK4 on NCCT expression is mimicked by dynamin, but the dominant-negative K44A dynamin mutant does not block the action of WNK4 itself. These results further highlight important differences in the mechanism by which WNK kinases affect expression of NCCT vs. other membrane proteins such as ROMK.
American journal of physiology. Renal physiology 01/2007; 291(6):F1369-76. · 3.68 Impact Factor
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ABSTRACT: The WNK (with no lysine kinase) kinases are a novel class of serine/threonine kinases that lack a characteristic lysine residue for ATP docking. Both WNK1 and WNK4 are expressed in the mammalian kidney, and mutations in either can cause the rare familial syndrome of hypertension and hyperkalemia (Gordon syndrome, or pseudohypoaldosteronism type 2). The molecular basis for the action of WNK4 is through alteration in the membrane expression of the NaCl co-transporter (NCCT) and the renal outer-medullary K channel KCNJ1 (ROMK). The actions of WNK1 are less well defined, and evidence to date suggests that it can affect NCCT expression but only in the presence of WNK4. The results of co-expressing WNK1 with ROMK in Xenopus oocytes are reported for the first time. These studies show that WNK1 is able to suppress total current directly through ROMK by causing a marked reduction in its surface expression. The effect is mimicked by a kinase-dead mutant of WNK1 (368D > A), suggesting that it is not dependent on its catalytic activity. Study of the time course of ROMK expression further suggests that WNK1 accelerates trafficking of ROMK from the membrane, and this effect seems to be dynamin dependent. Using fragments of full-length WNK1, it also is shown that the effect depends on residues in the middle section of the protein (502 to 1100 WNK1) that contains the acidic motif. Together, these findings emphasize that the molecular mechanisms that underpin WNK1 regulation of ROMK expression are distinct from those that affect NCCT expression.
Journal of the American Society of Nephrology 07/2006; 17(7):1867-74. · 9.66 Impact Factor
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ABSTRACT: We identified a new kindred with the familial syndrome of hypertension and hyperkalemia (pseudohypoaldosteronism type II or Gordon's syndrome) containing an affected father and son. Mutation analysis confirmed a single heterozygous G to C substitution within exon 7 (1690G>C) that causes a missense mutation within the acidic motif of WNK4 (564D>H). We confirmed the function of this novel mutation by coexpressing it in Xenopus oocytes with either the NaCl cotransporter (NCCT) or the inwardly rectifying K-channel (ROMK). Wild-type WNK4 inhibits 22Na+ flux in Xenopus oocytes expressing NCCT by approximately 90% (P<0.001), whereas the 564D>H mutant had no significantly inhibitory effect on flux through NCCT. In oocytes expressing ROMK, wild-type WNK4 produced >50% inhibition of steady-state current through ROMK at a +20-mV holding potential (P<0.001). The 564D>H mutant produced further inhibition with steady-state currents to some 60% to 70% of those seen with the wild-type WNK4. Using fluorescent-tagged NCCT (enhanced cyan fluorescent protein-NCCT) and ROMK (enhanced green fluorescent protein-ROMK) to quantify the expression of the proteins in the oocyte membrane, it appears that the functional effects of the 564D>H mutation can be explained by alteration in the surface expression of NCCT and ROMK. Compared with wild-type WNK4, WNK4 564D>H causes increased cell surface expression of NCCT but reduced expression of ROMK. This work confirms that the novel missense mutation in WNK4, 564D>H, is functionally active and highlights further how switching charge on a single residue in the acid motif of WNK4 affects its interaction with the thiazide-sensitive target NCCT and the potassium channel ROMK.
Hypertension 08/2005; 46(2):295-300. · 6.21 Impact Factor
