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“…As noted above, one of the most significantly altered phosphorylation sites in our study is localized on the BSND protein . This protein is an essential beta subunit of Chloride‐conducting ion channels and is localized to the basolateral membranes of renal tubules . Renal dysfunction is associated with BSND expression , and alterations in the protein have been associated with Bartter syndrome .…”
Multiplexed isobaric tag-based quantitative proteomics and phosphoproteomics strategies can comprehensively analyze drug treatments effects on biological systems. Given the role of MEK signaling in cancer and MAPK-dependent diseases, we sought to determine if this pathway could be inhibited safely by examining the downstream molecular consequences. We used a series of TMT10-plex experiments to analyze the effect of two MEK inhibitors (GSK1120212 and PD0325901) on three tissues (kidney, liver, and pancreas) from nine mice. We quantified ~6000 proteins in each tissue, but significant protein level alterations were minimal with inhibitor treatment. Of particular interest was kidney tissue, as edema is an adverse effect of these inhibitors. From kidney tissue, we enriched phosphopeptides using titanium dioxide (TiO2) and quantified 10,562 phosphorylation events. Further analysis by phosphotyrosine (pY) peptide immunoprecipitation quantified an additional 592 phosphorylation events. Phosphorylation motif analysis revealed that the inhibitors decreased phosphorylation levels of PxSP and SP sites, consistent with ERK inhibition. The MEK inhibitors had the greatest decrease on the phosphorylation of two proteins, Barttin and Slc12a3, which have roles in ion transport and fluid balance. Further studies will provide insight into the effect of these MEK inhibitors with respect to edema and other adverse events in mouse models and human patients.
“…As noted above, one of the most significantly altered phosphorylation sites in our study is localized on the BSND protein . This protein is an essential beta subunit of Chloride‐conducting ion channels and is localized to the basolateral membranes of renal tubules . Renal dysfunction is associated with BSND expression , and alterations in the protein have been associated with Bartter syndrome .…”
Multiplexed isobaric tag-based quantitative proteomics and phosphoproteomics strategies can comprehensively analyze drug treatments effects on biological systems. Given the role of MEK signaling in cancer and MAPK-dependent diseases, we sought to determine if this pathway could be inhibited safely by examining the downstream molecular consequences. We used a series of TMT10-plex experiments to analyze the effect of two MEK inhibitors (GSK1120212 and PD0325901) on three tissues (kidney, liver, and pancreas) from nine mice. We quantified ~6000 proteins in each tissue, but significant protein level alterations were minimal with inhibitor treatment. Of particular interest was kidney tissue, as edema is an adverse effect of these inhibitors. From kidney tissue, we enriched phosphopeptides using titanium dioxide (TiO2) and quantified 10,562 phosphorylation events. Further analysis by phosphotyrosine (pY) peptide immunoprecipitation quantified an additional 592 phosphorylation events. Phosphorylation motif analysis revealed that the inhibitors decreased phosphorylation levels of PxSP and SP sites, consistent with ERK inhibition. The MEK inhibitors had the greatest decrease on the phosphorylation of two proteins, Barttin and Slc12a3, which have roles in ion transport and fluid balance. Further studies will provide insight into the effect of these MEK inhibitors with respect to edema and other adverse events in mouse models and human patients.
“…As said before, similar to other ClC members [ 66 , 67 ], ClC-K channels associate with barttin [ 5 , 6 , 68 , 69 ]. For ClC-7, the structure of the complex formed with its β subunit Ostm1 has been determined [ 70 , 71 ].…”
Section: Structure–function Relationship In Clc-k Channelsmentioning
Given the key role played by ClC-K chloride channels in kidney and inner ear physiology and pathology, they can be considered important targets for drug discovery. Indeed, ClC-Ka and ClC-Kb inhibition would interfere with the urine countercurrent concentration mechanism in Henle’s loop, which is responsible for the reabsorption of water and electrolytes from the collecting duct, producing a diuretic and antihypertensive effect. On the other hand, ClC-K/barttin channel dysfunctions in Bartter Syndrome with or without deafness will require the pharmacological recovery of channel expression and/or activity. In these cases, a channel activator or chaperone would be appealing. Starting from a brief description of the physio-pathological role of ClC-K channels in renal function, this review aims to provide an overview of the recent progress in the discovery of ClC-K channel modulators.
“…Helix L is found in contact with the solvent and might interact with extracellular partners. Interestingly, helices B and J are known to interact with Barttin, a protein that regulates the targeting to the membrane and the ClC-K gating 39 – 41 . Figure 1 Sequence and structural alignment between the bovine ClC-K (template) and the human ClC-Kb (model).…”
Section: Resultsmentioning
confidence: 99%
“…Addition of a proline in these regions of the protein should have important impacts on the local folding, orientation/packing of the helices and stability, and/or channel function, often due to drastic modification of the Phi-Psi angles. Modifications in these regions could also lead to structural modifications of the helix B, known to interact with the barttin protein, required for protein surface expression and function 39 – 41 (L81P), disruption of the channel entrance (L439P), or disruption of a salt-bridge (E136-R182 in the case of L139P).…”
The human ClC-Kb channel plays a key role in exporting chloride ions from the cytosol and is known to be involved in Bartter syndrome type 3 when its permeation capacity is decreased. The ClC-Kb channel has been recently proposed as a potential therapeutic target to treat hypertension. In order to gain new insights into the sequence-structure-function relationships of this channel, to investigate possible impacts of amino-acid substitutions, and to design novel inhibitors, we first built a structural model of the human ClC-Kb channel using comparative modeling strategies. We combined in silico and in vitro techniques to analyze amino acids involved in the chloride ion pathway as well as to rationalize the possible role of several clinically observed mutations leading to the Bartter syndrome type 3. Virtual screening and drug repositioning computations were then carried out. We identified six novel molecules, including 2 approved drugs, diflusinal and loperamide, with Kd values in the low micromolar range, that block the human ClC-Kb channel and that could be used as starting point to design novel chemical probes for this potential therapeutic target.
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