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

2019 ◽  
Vol 41 (4) ◽  
pp. 774-785 ◽  
Author(s):  
Yohan Bignon ◽  
Imene Sakhi ◽  
Sara Bitam ◽  
Naziha Bakouh ◽  
Mathilde Keck ◽  
...  
Keyword(s):  
Binding Site ◽  
Calcium Binding ◽  
Bartter Syndrome ◽  
Calcium Binding Site ◽  
Dimer Interface

scholarly journals A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels

2010 ◽  
Vol 136 (3) ◽  
pp. 311-323 ◽  
Author(s):  
Antonella Gradogna ◽  
Elena Babini ◽  
Alessandra Picollo ◽  
Michael Pusch
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Calcium Binding ◽  
Chloride Channels ◽  
Single Channel ◽  
Bartter Syndrome ◽  
K Channel ◽  
Response Analysis ◽  
Triple Mutant ◽  
Calcium Binding Site

The two human CLC Cl− channels, ClC-Ka and ClC-Kb, are almost exclusively expressed in kidney and inner ear epithelia. Mutations in the genes coding for ClC-Kb and barttin, an essential CLC-K channel β subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca2+ and H+ on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca2+]ext without full saturation up to 50 mM. However, in the absence of Ca2+, ClC-Ka currents were still 20% of currents in 10 mM [Ca2+]ext, demonstrating that Ca2+ is not strictly essential for opening. Vice versa, ClC-Ka and ClC-Kb were blocked by increasing [H+]ext with a practically complete block at pH 6. Ca2+ and H+ act as gating modifiers without changing the single-channel conductance. Dose–response analysis suggested that two protons are necessary to induce block with an apparent pK of ∼7.1. A simple four-state allosteric model described the modulation by Ca2+ assuming a 13-fold higher Ca2+ affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca2+ and H+. A mutagenic screen of a large number of extracellularly accessible amino acids identified a pair of acidic residues (E261 and D278 on the loop connecting helices I and J), which are close to each other but positioned on different subunits of the channel, as a likely candidate for forming an intersubunit Ca2+-binding site. Single mutants E261Q and D278N greatly diminished and the double mutant E261Q/D278N completely abolished modulation by Ca2+. Several mutations of a histidine residue (H497) that is homologous to a histidine that is responsible for H+ block in ClC-2 did not yield functional channels. However, the triple mutant E261Q/D278N/H497M completely eliminated H+ -induced current block. We have thus identified a protein region that is involved in binding these physiologically important ligands and that is likely undergoing conformational changes underlying the complex gating of CLC-K channels.

scholarly journals A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate

FEBS Open Bio ◽  
2016 ◽  
Vol 6 (5) ◽  
pp. 425-432 ◽  
Author(s):  
Johannes Then ◽  
Ren Wei ◽  
Thorsten Oeser ◽  
André Gerdts ◽  
Juliane Schmidt ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Binding Site ◽  
Polyethylene Terephthalate ◽  
Calcium Binding ◽  
Disulfide Bridge ◽  
Calcium Binding Site

Calcium-Binding Site β2, Adjacent to the “b” Polymerization Site, Modulates Lateral Aggregation of Protofibrils during Fibrin Polymerization†,‡

Biochemistry ◽  
2004 ◽  
Vol 43 (9) ◽  
pp. 2475-2483 ◽  
Author(s):  
Michael S. Kostelansky ◽  
Karim C. Lounes ◽  
Li Fang Ping ◽  
Sarah K. Dickerson ◽  
Oleg V. Gorkun ◽  
...  
Keyword(s):  
Binding Site ◽  
Calcium Binding ◽  
Calcium Binding Site ◽  
Fibrin Polymerization

scholarly journals The calcium-binding site of human glutamate carboxypeptidase II is critical for dimerization, thermal stability, and enzymatic activity

2018 ◽  
Vol 27 (9) ◽  
pp. 1575-1584 ◽  
Author(s):  
Jakub Ptacek ◽  
Jana Nedvedova ◽  
Michal Navratil ◽  
Barbora Havlinova ◽  
Jan Konvalinka ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Enzymatic Activity ◽  
Binding Site ◽  
Calcium Binding ◽  
Calcium Binding Site ◽  
Glutamate Carboxypeptidase Ii ◽  
Glutamate Carboxypeptidase
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