The endosomal trafficking factors CORVET and ESCRT suppress plasma membrane residence of the renal outer medullary potassium channel (ROMK)

Mackie, Timothy D.; Kim, Bo Young; Subramanya, Arohan R.; Bain, Daniel J.; O’Donnell, Allyson F.; Welling, Paul A.; Brodsky, Jeffrey L.

doi:10.1074/jbc.m117.819086

Cited by 14 publications

(22 citation statements)

References 104 publications

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“…As shown in Fig 4B, we first determined that yeast expressing WT ROMK1 or Kir2.1, another Kir channel, grew significantly better than yeast containing only the vector control on either low potassium solid medium (top) or liquid medium (bottom), as expected [7,30,32]. We also found that the K80M and V140M alleles exhibited a gain-of-function phenotype, as they grew better than yeast expressing WT ROMK1.…”

Section: Experimental Evaluation Of Single Amino Acid Variants Revealmentioning

confidence: 60%

“…To assay the function of each ROMK variant, we employed a quantitative yeast-based screen in which the relative growth of a yeast strain expressing WT or mutated ROMK can be measured [7,30]. The assay takes advantage of the fact that yeast growth on low potassium medium requires the exogenous expression of a potassium channel.…”

Section: Experimental Evaluation Of Single Amino Acid Variants Revealmentioning

confidence: 99%

“…Plasmids were transformed into yeast via the standard lithium-acetate method [48] and yeast were grown at 30˚C in liquid or solid synthetic complete (SC) medium lacking Leu but supplemented with a final concentration of 2% dextrose. The SC medium contained monosodium glutamate as the main nitrogen source and was buffered to pH 4.5 with MES and supplemented with the indicated amount of KCl, as described [30]. Due to the presence of residual potassium in the agar (VWR, PA), each plate contains 7-10 mM KCl, even in the absence of added potassium [49,50].…”

Section: Plasmid Constructionmentioning

confidence: 99%

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Complementary computational and experimental evaluation of missense variants in the ROMK potassium channel

et al. 2020

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The renal outer medullary potassium (ROMK) channel is essential for potassium transport in the kidney, and its dysfunction is associated with a salt-wasting disorder known as Bartter syndrome. Despite its physiological significance, we lack a mechanistic understanding of the molecular defects in ROMK underlying most Bartter syndrome-associated mutations. To this end, we employed a ROMK-dependent yeast growth assay and tested single amino acid variants selected by a series of computational tools representative of different approaches to predict each variants' pathogenicity. In one approach, we used in silico saturation mutagenesis, i.e. the scanning of all possible single amino acid substitutions at all sequence positions to estimate their impact on function, and then employed a new machine learning classifier known as Rhapsody. We also used two additional tools, EVmutation and Polyphen-2, which permitted us to make consensus predictions on the pathogenicity of single amino acid variants in ROMK. Experimental tests performed for selected mutants in different classes validated the vast majority of our predictions and provided insights into variants implicated in ROMK dysfunction. On a broader scope, our analysis suggests that consolidation of data from complementary computational approaches provides an improved and facile method to predict the severity of an amino acid substitution and may help accelerate the identification of disease-causing mutations in any protein. Author summaryAs the number of sequenced human genomes rises, a major challenge is to identify which single amino acid variations in a protein affect function and predispose individuals to disease. While predictive algorithms are available for this purpose, a comparative analysis of recently developed algorithms has not been adequately performed, nor is it clear whether combining algorithms would improve predictive power. To this end, we compared the efficacy of three publicly available algorithms and applied the results to Bartter syndrome, PLOS COMPUTATIONAL BIOLOGY PLOS Computational Biology | https://doi.Citation: Ponzoni L, Nguyen NH, Bahar I, Brodsky JL (2020) Complementary computational and experimental evaluation of missense variants in the ROMK potassium channel. PLoS Comput Biol 16 (4): e1007749. https://doi.org/10.a human disease for which numerous poorly-characterized single amino acid variants have been identified and for which there is no cure. In silico saturation mutagenesis, i.e., the computational prediction of pathogenesis for every possible amino acid substitution, allowed us to experimentally test predictions by measuring the activity of an ion channel linked to Bartter syndrome. Based on data from blinded experiments, we discovered that Rhapsody and EVmutation successfully predicted deleterious mutations. Moreover, Rhapsody-which takes into account evolutionary as well as structural and dynamic considerations-predicted that >90% of known Bartter syndrome mutations are deleterious. Overall, our data will aid investigators who wish...

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Section: Experimental Evaluation Of Single Amino Acid Variants Revealmentioning

confidence: 60%

Section: Experimental Evaluation Of Single Amino Acid Variants Revealmentioning

confidence: 99%

Section: Plasmid Constructionmentioning

confidence: 99%

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Complementary computational and experimental evaluation of missense variants in the ROMK potassium channel

et al. 2020

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“…K IR 1.1 internalization depends on clathrin-dynamin mediated endocytosis which involved N375 in the K IR 1.1 putative internalization motif NPN [18]. Internalized K IR 1.1 channels depend on CORVET and ESCRT protein complexes for subsequent trafficking to the early endosome and the multivesicular body that eventually fuses with the lysosome, respectively [19]. K IR 2.1 channels are regulated by the ESCRT machinery also [20].…”

Section: Channel Traffickingmentioning

confidence: 99%

Disease Associated Mutations in KIR Proteins Linked to Aberrant Inward Rectifier Channel Trafficking

et al. 2019

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The ubiquitously expressed family of inward rectifier potassium (KIR) channels, encoded by KCNJ genes, is primarily involved in cell excitability and potassium homeostasis. Channel mutations associate with a variety of severe human diseases and syndromes, affecting many organ systems including the central and peripheral neural system, heart, kidney, pancreas, and skeletal muscle. A number of mutations associate with altered ion channel expression at the plasma membrane, which might result from defective channel trafficking. Trafficking involves cellular processes that transport ion channels to and from their place of function. By alignment of all KIR channels, and depicting the trafficking associated mutations, three mutational hotspots were identified. One localized in the transmembrane-domain 1 and immediately adjacent sequences, one was found in the G-loop and Golgi-export domain, and the third one was detected at the immunoglobulin-like domain. Surprisingly, only few mutations were observed in experimentally determined Endoplasmic Reticulum (ER)exit-, export-, or ER-retention motifs. Structural mapping of the trafficking defect causing mutations provided a 3D framework, which indicates that trafficking deficient mutations form clusters. These “mutation clusters” affect trafficking by different mechanisms, including protein stability.

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“…These data indicate that increased trafficking to the membrane is required for this phenomena and are in agreement with a previous study in thick ascending limbs that showed blocking trafficking with tetanus toxin inhibited Cl Ϫ absorption (51). However, our results do not rule out changes in membrane insertion mediated by NKCC2 phosphorylation (2) or activation of other transporters such as renal outer medullary K ϩ channel (ROMK), which is also affected by membrane trafficking (45). We did not directly test whether changes in phosphorylation are also involved.…”

Section: Raising Luminal Flow Enhances Omentioning

confidence: 58%

NADPH oxidase 4-derived superoxide mediates flow-stimulated NKCC2 activity in thick ascending limbs

Sáez

Hong

Garvin

2018

American Journal of Physiology-Renal Physiology

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Luminal flow augments Na reabsorption in the thick ascending limb more than can be explained by increased ion delivery. This segment reabsorbs 30% of the filtered load of Na, playing a key role in its homeostasis. Whether flow elevations enhance Na-K-2Cl cotransporter (NKCC2) activity and the second messenger involved are unknown. We hypothesized that raising luminal flow augments NKCC2 activity by enhancing superoxide ([Formula: see text]) production by NADPH oxidase 4 (NOX4). NKCC2 activity was measured in thick ascending limbs perfused at either 5 or 20 nl/min with and without inhibitors of [Formula: see text] production. Raising luminal flow from 5 to 20 nl/min enhanced NKCC2 activity from 4.8 ± 0.9 to 6.3 ± 1.2 arbitrary fluorescent units (AFU)/s. Maintaining flow at 5 nl/min did not alter NKCC2 activity. The superoxide dismutase mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride blunted NKCC2 activity from 3.5 ± 0.4 to 2.5 ± 0.2 AFU/s when flow was 20 nl/min but not 5 nl/min. When flow was 20 nl/min, NKCC2 activity showed no change with time. The selective NOX1/4 inhibitor GKT-137831 blunted NKCC2 activity when thick ascending limbs were perfused at 20 nl/min from 7.2 ± 1.1 to 4.5 ± 0.8 AFU/s but not at 5 nl/min. The inhibitor also prevented luminal flow from elevating [Formula: see text] production. Allopurinol, a xanthine oxidase inhibitor, had no effect on NKCC2 activity when flow was 20 nl/min. Tetanus toxin prevents flow-induced stimulation of NKCC2 activity. We conclude that elevations in luminal flow enhance NaCl reabsorption in thick ascending limbs by stimulating NKCC2 via NOX4 activation and increased [Formula: see text]. NKCC2 activation is primarily the result of insertion of new transporters in the membrane.

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The endosomal trafficking factors CORVET and ESCRT suppress plasma membrane residence of the renal outer medullary potassium channel (ROMK)

Cited by 14 publications

References 104 publications

Complementary computational and experimental evaluation of missense variants in the ROMK potassium channel

Complementary computational and experimental evaluation of missense variants in the ROMK potassium channel

Disease Associated Mutations in KIR Proteins Linked to Aberrant Inward Rectifier Channel Trafficking

NADPH oxidase 4-derived superoxide mediates flow-stimulated NKCC2 activity in thick ascending limbs

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