Effect of Known Inhibitors of Ion Transport on Pendrin (SLC26A4) Activity in a Human Kidney Cell Line

Bernardinelli, Emanuele; Costa, Roberta; Nofziger, Charity; Paulmichl, Markus; Dossena, Silvia

doi:10.1159/000445559

Cited by 17 publications

(22 citation statements)

References 93 publications

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“…For testing the function of pendrin variants, the influx of iodide was measured in cells expressing wild type or mutated pendrin and in control cells. The functional test was performed as already described [ 9 , 59 , 72 , 73 ], with adaptations allowing for the use of a multiplate reader [ 27 , 60 , 74 , 75 , 76 ]. Shortly, cells were seeded into black 96-well plates, grown overnight and co-transfected with 0.1 μg of a plasmid encoding for EYFP H148Q;I152L (an EYFP variant with substantially improved sensitivity for iodide [ 77 ]) and 0.1 μg of pTARGET plasmid bearing the cDNA of wild type or mutated pendrin.…”

Section: Methodsmentioning

confidence: 99%

Functional Testing of SLC26A4 Variants—Clinical and Molecular Analysis of a Cohort with Enlarged Vestibular Aqueduct from Austria

Roesch

Bernardinelli

Nofziger³

et al. 2018

IJMS

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Abstract:The prevalence and spectrum of sequence alterations in the SLC26A4 gene, which codes for the anion exchanger pendrin, are population-specific and account for at least 50% of cases of non-syndromic hearing loss associated with an enlarged vestibular aqueduct. A cohort of nineteen patients from Austria with hearing loss and a radiological alteration of the vestibular aqueduct underwent Sanger sequencing of SLC26A4 and GJB2, coding for connexin 26. The pathogenicity of sequence alterations detected was assessed by determining ion transport and molecular features of the corresponding SLC26A4 protein variants. In this group, four uncharacterized sequence alterations within the SLC26A4 coding region were found. Three of these lead to protein variants with abnormal functional and molecular features, while one should be considered with no pathogenic potential. Pathogenic SLC26A4 sequence alterations were only found in 12% of patients. SLC26A4 sequence alterations commonly found in other Caucasian populations were not detected. This survey represents the first study on the prevalence and spectrum of SLC26A4 sequence alterations in an Austrian cohort and further suggests that genetic testing should always be integrated with functional characterization and determination of the molecular features of protein variants in order to unequivocally identify or exclude a causal link between genotype and phenotype.

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Section: Methodsmentioning

confidence: 99%

Functional Testing of SLC26A4 Variants—Clinical and Molecular Analysis of a Cohort with Enlarged Vestibular Aqueduct from Austria

Roesch

Bernardinelli

Nofziger³

et al. 2018

IJMS

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“…To confirm that pendrin was functionally responsible for the increased CFTR current in response to interleukins shown in Figure 2A, we tested the non‐specific pendrin inhibitor niflumic acid 41 on normal HBE cells. Niflumic acid caused a small decrease in CFTR current (Figure 2C‐D).…”

Section: Resultsmentioning

confidence: 99%

Pendrin stimulates a chloride absorption pathway to increase CFTR‐mediated chloride secretion from Cystic Fibrosis airway epithelia

et al. 2020

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Cystic Fibrosis (CF), an inherited multi‐system disease, is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that disrupt its ability to secrete anions from epithelia. Recovery of functional anion secretion may be curative for CF, so different components of the ion transport machinery have become attractive therapeutic targets. Several members of the SLC26 ion transporter family have been linked to epithelial ion flux, some through putative functional interactions with CFTR. Using a small‐scale qPCR screen, we confirmed that the anion transporter SLC26A4 (pendrin) is downregulated in CF. Upregulation of pendrin using interleukins IL‐4 or IL‐13 increased Cl− secretion through CFTR in human bronchial epithelial cell (HBEC) derived epithelia differentiated in vitro and measured in the Ussing Chamber. Inhibition or knockdown of pendrin prevented this increased secretion. Increased CFTR activity was not driven by increases in CFTR protein or upstream regulatory pathway components. When basolateral Cl− absorption through NKCC1 was inhibited, a pendrin‐dependent Cl− absorption pathway allowing CFTR to continue secreting Cl− from the epithelium was revealed. Although CFTR is often considered the bottleneck in the transepithelial Cl− transport pathway, these studies indicate that basolateral Cl− permeability becomes limiting as CFTR activity increases. Therefore, an increase of epithelial Cl− absorption via pendrin might have additional therapeutic benefit in combination with CFTR modulators.

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“…The increasing inhibition of the peak current observed with DIDS concentrations higher than 0.01 mM (>20% with 1 mM DIDS, Table 2, Fig. 1A and [28] masked the effect on the current inactivation (Table 2). …”

Section: Resultsmentioning

confidence: 99%

“…This drug and its derivatives are widely used in clinics for the relief of chronic and acute pain conditions [56]. Niflumic acid is also known as an inhibitor of several anion channels, including Ca ++ -activated [57-59] and swelling-activated [12] Cl - channels, and was reported to variably affect different membrane ion transport systems, such as anion exchangers [28, 60], the glycine [61], GABA(A) [62] and N-methyl-D-aspartate [63] receptors and T-type calcium channels [64]. The findings presented here show that 0.01-1 mM niflumic acid, despite being more potent on the swelling-activated Cl - current, also significantly blocked the K + current (Table 2 and Fig.…”

Section: Discussionmentioning

confidence: 99%

A Potassium-Selective Current Affected by Micromolar Concentrations of Anion Transport Inhibitors

Costa

Civello

Bernardinelli

et al. 2018

Cell Physiol Biochem

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Background/Aims: In the human genome, more than 400 genes encode ion channels, which are ubiquitously expressed and often coexist and participate in almost all physiological processes. Therefore, ion channel blockers represent fundamental tools in discriminating the contribution of individual channel types to a physiological phenomenon. However, unspecific effects of these compounds may represent a confounding factor. Three commonly used chloride channel inhibitors, i.e. 4,4′-diisothiocyano-2,2′-stilbene-disulfonic acid (DIDS), 5-nitro-2-[(3-phenylpropyl) amino]benzoic acid (NPPB) and the anti-inflammatory drug niflumic acid were tested to identify the lowest concentration effective on Cl^- channels and ineffective on K⁺ channels. Methods: The activity of the above mentioned compounds was tested by whole cell patch-clamp on the swelling-activated Cl^- current ICl,swell and on the endogenous voltage-dependent, outwardly rectifying K⁺ selective current in human kidney cell lines (HEK 293/HEK 293 Phoenix). Results: Micromolar (1-10 µM) concentrations of DIDS and NPPB could not discriminate between the Cl^- and K⁺ selective currents. Specifically, 1 µM DIDS only affected the K⁺ current and 10 µM NPPB equally affected the Cl^- and K⁺ currents. Only relatively high (0.1-1 mM) concentrations of DIDS and prolonged (5 minutes) exposure to 0.1-1 mM NPPB preferentially suppressed the Cl^- current. Niflumic acid preferentially inhibited the Cl^- current, but also significantly affected the K⁺ current. The endogenous voltage-dependent, outwardly rectifying K⁺ selective current in HEK 293/HEK 293 Phoenix cells was shown to arise from the Kv 3.1 channel, which is extensively expressed in brain and is involved in neurological diseases. Conclusion: The results of the present study underscore that sensitivity of a given physiological phenomenon to the Cl^- channel inhibitors NPPB, DIDS and niflumic acid may actually arise from an inhibition of Cl^- channels but can also result from an inhibition of voltage-dependent K⁺ channels, including the Kv 3.1 channel. The use of niflumic acid as anti-inflammatory drug in patients with concomitant Kv 3.1 dysfunction may result contraindicated.

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Effect of Known Inhibitors of Ion Transport on Pendrin (SLC26A4) Activity in a Human Kidney Cell Line

Cited by 17 publications

References 93 publications

Functional Testing of SLC26A4 Variants—Clinical and Molecular Analysis of a Cohort with Enlarged Vestibular Aqueduct from Austria

Functional Testing of SLC26A4 Variants—Clinical and Molecular Analysis of a Cohort with Enlarged Vestibular Aqueduct from Austria

Pendrin stimulates a chloride absorption pathway to increase CFTR‐mediated chloride secretion from Cystic Fibrosis airway epithelia

A Potassium-Selective Current Affected by Micromolar Concentrations of Anion Transport Inhibitors

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