Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking

Stölting, Gabriel; Bungert-Plümke, Stefanie; Franzen, Arne; Fahlke, Christoph

doi:10.1074/jbc.m115.675827

Cited by 17 publications

(24 citation statements)

References 65 publications

(107 reference statements)

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“…As depicted in Figure a, ClC‐Kb expression produced two immune‐reactive bands at about 90–100 kDa and a fainter signal around 100–130 kDa, all absent in NI oocytes. This pattern is qualitatively similar to the one previously described by the group of Fahlke (Janssen et al, ; Stolting et al, ), suggesting that the upper bands correspond to complex‐glycosylated ClC‐Kb meanwhile the lower bands correspond to core‐ and nonglycosylated ClC‐Kb. Quantitatively, the total amount of G437R mutant, whose 90–100 kDa bands as well as 100–130 kDa signal are weakly visible, shows a significant 65% reduction in total protein abundance as compared with WT EGFP‐ClC‐Kb (Figure b).…”

Section: Resultssupporting

confidence: 89%

“…As depicted in Figure 4a, ClC-Kb expression produced two immune-reactive bands at about 90-100 kDa and a fainter signal around 100-130 kDa, all absent in NI oocytes. This pattern is qualitatively similar to the one previously described by the group of Fahlke (Janssen et al, 2009;Stolting et al, 2015), suggesting that the upper bands correspond to complex-glycosylated ClC-Kb meanwhile the lower bands correspond to core-and nonglycosylated ClC-Kb.…”

Section: Surface Expression and Protein Expression Of Clc-kb Mutantsupporting

confidence: 89%

“…A relatively large number of pathological CLCNKB mutations positioned throughout the whole length of the protein and affecting particularly the pore and dimer interface has been identified (Andrini et al, 2014;Brochard et al, 2009;Keck et al, 2013;Konrad et al, 2000;Lee et al, 2012;Seys et al, 2017;Stolting, Bungert-Plumke, Franzen, & Fahlke, 2015;Yu et al, 2009;see Andrini et al, 2015 for review). Functional studies began to assess the currents generated by several mutants in Xenopus laevis oocytes immediately after the discovery of Barttin (Estevez et al, 2001;Waldegger et al, 2002), an effort that was continued by attempts to describe the functional consequences of CLCNKB mutations (Andrini et al, 2014;Andrini et al, 2015;Cheng, Lo, Chen, Huang, & Lin, 2017;Martinez & Maduke, 2008;Stolting et al, 2015;Yu et al, 2009). However, due to technical difficulties encountered in expressing ClC-Kb/barttin, no general view of how CLCNKB mutations cause the disease is yet available.…”

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confidence: 99%

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Analysis ofCLCNKBmutations at dimer‐interface, calcium‐binding site, and pore reveals a variety of functional alterations in ClC‐Kb channel leading to Bartter syndrome

et al. 2019

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Pathological missense mutations in CLCNKB gene give a wide spectrum of clinical phenotypes in Bartter syndrome type III patients. Molecular analysis of the mutated ClC‐Kb channels can be helpful to classify the mutations according to their functional alteration. We investigated the functional consequences of nine mutations in the CLCNKB gene causing Bartter syndrome. We first established that all tested mutations lead to decreased ClC‐Kb currents. Combining electrophysiological and biochemical methods in Xenopus laevis oocytes and in MDCKII cells, we identified three classes of mutations. One class is characterized by altered channel trafficking. p.A210V, p.P216L, p.G424R, and p.G437R are totally or partially retained in the endoplasmic reticulum. p.S218N is characterized by reduced channel insertion at the plasma membrane and altered pH‐sensitivity; thus, it falls in the second class of mutations. Finally, we found a novel class of functionally inactivated mutants normally present at the plasma membrane. Indeed, we found that p.A204T alters the pH‐sensitivity, p.A254V abolishes the calcium‐sensitivity. p.G219C and p.G465R are probably partially inactive at the plasma membrane. In conclusion, most pathogenic mutants accumulate partly or totally in intracellular compartments, but some mutants are normally present at the membrane surface and simultaneously show a large range of altered channel gating properties.

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Section: Resultssupporting

confidence: 89%

Section: Surface Expression and Protein Expression Of Clc-kb Mutantsupporting

confidence: 89%

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confidence: 99%

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Analysis ofCLCNKBmutations at dimer‐interface, calcium‐binding site, and pore reveals a variety of functional alterations in ClC‐Kb channel leading to Bartter syndrome

et al. 2019

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“…The homologous ClC-K2 channel appears to exhibit properties that are incompatible with whole-cell recordings of ClC-Kb (Pinelli et al, 2016) and may not be used for comparison. We therefore estimated unitary properties using a modified noise analysis according to a method described in Stölting et al (2015). For a regular CLC-type channel with two conduction pathways exhibiting protopore as well as common gating mechanisms (Fischer et al, 2010; Stölting et al, 2014b), combining Equations (1) and (2) results in

\begin{matrix} eqnarrayleft \\ \frac{σ^{2}}{I} = i \cdot false(1 - P_{p} false(2 P_{c} - 1 false) false) \end{matrix}

The ratio of variance to the mean macroscopic current depends on the product of single channel amplitudes and open probabilities of protopore and common gates, but is indifferent to variations in the number of channels.…”

Section: Methodsmentioning

confidence: 99%

Reduced Membrane Insertion of CLC-K by V33L Barttin Results in Loss of Hearing, but Leaves Kidney Function Intact

et al. 2017

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In the mammalian ear, transduction of sound stimuli is initiated by K+ entry through mechano-sensitive channels into inner hair cells. K+ entry is driven by a positive endocochlear potential that is maintained by the marginal cell layer of the stria vascularis. This process requires basolateral K+ import by NKCC1 Na+−2Cl−−K+ co-transporters as well as Cl− efflux through ClC-Ka/barttin or ClC-Kb/barttin channels. Multiple mutations in the gene encoding the obligatory CLC-K subunit barttin, BSND, have been identified in patients with Bartter syndrome type IV. These mutations reduce the endocochlear potential and cause deafness. As CLC-K/barttin channels are also expressed in the kidney, patients with Bartter syndrome IV typically also suffer from salt-wasting hyperuria and electrolyte imbalances. However, there was a single report on a BSND mutation that resulted only in deafness, but not kidney disease. We herein studied the functional consequences of another recently discovered BSND mutation that predicts exchange of valine at position 33 by leucine. We combined whole-cell patch clamp, confocal microscopy and protein biochemistry to analyze how V33L affects distinct functions of barttin. We found that V33L reduced membrane insertion of CLC-K/barttin complexes without altering unitary CLC-K channel function. Our findings support the hypothesis of a common pathophysiology for the selective loss of hearing due to an attenuation of the total chloride conductance in the stria vascularis while providing enough residual function to maintain normal kidney function.

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“…They bind nucleotides and are required for nucleotide-dependent regulation of various CLCs (7,17,18,27,28). They have also been suggested to play a role in CLC trafficking (29,30), in the regulation of epithelial Na þ channels by CLC-2 (31), and in the regulation of CLC-Kb by the accessory protein barttin (32). CBS domains also participate, in a poorly defined way, in CLC common gating (33,34).…”

Section: Introductionmentioning

confidence: 99%

Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel

Yamada

Krzeminski

Bozóky

et al. 2016

Biophysical Journal

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Eukaryotic CLC anion channels and transporters are homodimeric proteins composed of multiple a-helical membrane domains and large cytoplasmic C-termini containing two cystathionine-b-synthase domains (CBS1 and CBS2) that dimerize to form a Bateman domain. The Bateman domains of adjacent CLC subunits interact to form a Bateman domain dimer. The functions of CLC CBS and Bateman domains are poorly understood. We utilized the Caenorhabditis elegans CLC-1/2/Ka/ Kb anion channel homolog CLH-3b to characterize the regulatory roles of CLC cytoplasmic domains. CLH-3b activity is reduced by phosphorylation or deletion of a 14-amino-acid activation domain (AD) located on the linker connecting CBS1 and CBS2. We demonstrate here that phosphorylation-dependent reductions in channel activity require an intact Bateman domain dimer and concomitant phosphorylation or deletion of both ADs. Regulation of a CLH-3b AD deletion mutant is reconstituted by intracellular perfusion with recombinant 14-amino-acid AD peptides. The sulfhydryl reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) alters in a phosphorylation-dependent manner the activity of channels containing single cysteine residues that are engineered into the short intracellular loop connecting membrane a-helices H and I (H-I loop), the AD, CBS1, and CBS2. In contrast, MTSET has no effect on channels in which cysteine residues are engineered into intracellular regions that are dispensable for regulation. These studies together with our previous work suggest that binding and unbinding of the AD to the Bateman domain dimer induces conformational changes that are transduced to channel membrane domains via the H-I loop. Our findings provide new, to our knowledge, insights into the roles of CLC Bateman domains and the structurefunction relationships that govern the regulation of CLC protein activity by diverse ligands and signaling pathways.

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Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking

Cited by 17 publications

References 65 publications

Analysis ofCLCNKBmutations at dimer‐interface, calcium‐binding site, and pore reveals a variety of functional alterations in ClC‐Kb channel leading to Bartter syndrome

Analysis ofCLCNKBmutations at dimer‐interface, calcium‐binding site, and pore reveals a variety of functional alterations in ClC‐Kb channel leading to Bartter syndrome

Reduced Membrane Insertion of CLC-K by V33L Barttin Results in Loss of Hearing, but Leaves Kidney Function Intact

Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel

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