Gradogna A et al. (2010), A regulatory calcium-binding site at the subuni...

XB-ART-42014

J Gen Physiol 2010 Sep 01;1363:311-23. doi: 10.1085/jgp.201010455.

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

Gradogna A , Babini E , Picollo A , Pusch M .

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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 beta subunit, lead to Bartter syndrome. We performed a biophysical analysis of the modulatory effect of extracellular Ca(2+) and H(+) on ClC-Ka and ClC-Kb in Xenopus oocytes. Currents increased with increasing [Ca(2+)](ext) without full saturation up to 50 mM. However, in the absence of Ca(2+), ClC-Ka currents were still 20% of currents in 10 mM [Ca(2+)](ext), demonstrating that Ca(2+) 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. Ca(2+) 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 approximately 7.1. A simple four-state allosteric model described the modulation by Ca(2+) assuming a 13-fold higher Ca(2+) affinity of the open state compared with the closed state. The quantitative analysis suggested separate binding sites for Ca(2+) 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 Ca(2+)-binding site. Single mutants E261Q and D278N greatly diminished and the double mutant E261Q/D278N completely abolished modulation by Ca(2+). 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.

???displayArticle.pubmedLink??? 20805576
???displayArticle.pmcLink??? PMC2931146
???displayArticle.link??? J Gen Physiol
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Species referenced: Xenopus
Genes referenced: bsnd clcn2 clcnkb pkm
GO keywords: chloride channel activity [+]

???displayArticle.disOnts??? Bartter disease
???displayArticle.omims??? BARTTER SYNDROME, TYPE 3; BARTS3

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	Figure 1. Ca2+ext dependence of ClC-Ka and ClC-Kb. (A) Typical currents of ClC-Ka evoked by the standard IV-pulse protocol at different Ca2+ concentrations as indicated, at pH 7.3. (B) Effect of [Ca2+]ext on ClC-Ka (circles; n = 9). Current at 60 mV acquired at different conditions was normalized to values measured in standard solution (I/I10 mM Ca2+, pH 7.3) and plotted versus [Ca2+]ext (used concentrations: 0.1, 0.5, 2, 5, 10, 20, 30, and 50 mM). The line represents the fit obtained using Eq. 2 as described in Results. (C) Superimposed traces measured at 60 mV at 5 mM [Ca2+]ext (black line) and at 20 mM [Ca2+]ext (gray line) measured from the same oocyte. Currents were scaled to the steady-state current at 60 mV. Horizontal bar, 200 ms. (D) Typical currents of ClC-Kb measured at different Ca2+ concentrations as indicated. (E) Effect of [Ca2+]ext (concentrations as in B) on ClC-Kb (squares; n = 5). Horizontal bars, 200 ms. Vertical bars, 5 ÂµA.
	Figure 2. pHext dependence of ClC-Ka and ClC-Kb. (A) Voltage clamp traces of ClC-Ka measured at different pH as indicated, in 10 mM [Ca2+]ext. (B) Effect of pHext on ClC-Ka (circles; n = 7). Normalized currents were calculated as for Fig. 1. I/I10 mM Ca2+, pH 7.3, is plotted versus pH. The line represents the fit obtained using Eq. 1 as described in Results. (C) Superimposed traces measured at pH 7.0 (black line) and pH 8.0 (gray line) measured from the same oocyte. Currents were scaled to the steady-state current at 60 mV. Horizontal bar, 200 ms. (D) Traces of ClC-Kb measured at different pH as indicated, in 10 mM [Ca2+]ext. (E) Effect of pHext on ClC-Kb (squares; n = 4). Horizontal bars, 200 ms. Vertical bars, 5 ÂµA.
	Figure 3. Relaxation kinetics upon fast concentration jumps in outside-out patches. (A) Example recording of a patch held at 60 mV in which [Ca2+]ext was switched from 10 to 1.8 mM, and then back to 10 mM. Finally, the patch was exposed to a solution without Clâ (and without Ca2+). The three insets below show the transition regions on an expanded time scale (bar, 500 ms), superimposed with a single-exponential fit (red line). Time constants for the solution exchange (inset c) were on the order of 10 ms. (B) Average time constants (n > 3 patches) for the indicated transitions.
	Figure 4. Ca2+ and pH effects on ClC-Ka at mixed conditions. (A and B) Effect of [Ca2+]ext on ClC-Ka measured at pHext 8 (A, n = 6) and pHext 6.5 (B, n = 4). Currents at 60 mV were normalized to values measured in 10 mM [Ca2+]ext and plotted versus [Ca2+]ext. (C) Effect of pHext on ClC-Ka in 0.5 mM Ca2+ (n = 3). Currents at 60 mV were normalized to values measured at pH 7.3 and plotted versus pHext. Lines represent the theoretical predictions obtained using Eqs. 1 and 2 as described in Results using the same parameters obtained from the fit in the standard conditions (i.e., pK = 7.11; KC = 19.6 mM, KO = 1.5 mM, and r0 = 434).
	Figure 5. Location of mutants mapped on the structure of ecClC-1. (A) A surface representation of the bacterial ecClC-1 (Protein Databank accession no. 1OTS) viewed from the extracellular side is shown. The two subunits that compose ecClC-1 are colored in gray and light gray, respectively. The residues corresponding to those selected for mutation are shown in pink and light blue in the two different subunits, respectively. The transparent surface also allows a glimpse of the internal mutated residues. The residues responsible for Ca2+ and H+ sensitivity are shown in different colors: yellow, E261 (corresponding to E235 of ecClC-1); red, D278 (corresponding to N250 of ecClC-1); green, H497 (corresponding to L421 of ecClC-1); the numbers of residues indicated in the figure correspond to those of ClC-Ka. (B) A zoom of a selected region is shown in cartoon representation. The three residues E235, N250, and L421 are highlighted as sticks and colored as in A. A hypothetical Ca2+ ion is shown as a light blue sphere between E261 and D278. (C) Alignments around the three residues responsible for Ca2+ and proton sensitivity are shown. E261, D278, H497, and the corresponding residues in other CLCs are in bold.
	Figure 6. Effect of [Ca2+]ext on ClC-Ka WT and its mutants E261Q, D278N, and E261Q/D278N. (AâD) The mean current, shown in color, at 60 mV plotted as a function of time. The type of solution applied is color coded as indicated in the middle inset. Breaks during the experiment are indicated with short dashed lines. Insets display representative current traces evoked by the standard IV-pulse protocol in standard solution. Horizontal bars, 100 ms; vertical bars, 3 ÂµA. (E) Doseâresponse relationship of the modulation by Ca2+ of ClC-Ka WT (black circles; n = 16) and the mutants E261Q (yellow squares; n = 4), D278N (red triangles; n = 5), and E261Q/D278N (light blue rhombi; n = 5). Currents at 60 mV were normalized to values measured in standard solution and plotted versus [Ca2+]ext. Data for WT are different compared with Fig. 1 because they were obtained from different oocytes as control measurements in the mutagenic screen. The currents shown for the mutants D278N and E261Q/D278N are from oocytes with exceptionally large expression. On average, the current expression level measured at 60 mV were (in ÂµA Â± SD [no. of oocytes]): WT (2 d), 2.5 Â± 1.4 (7); WT (3 d), 5.3 Â± 1.9 (8); E261Q (1â2 d), 4.3 Â± 0.9 (4); D278N (>3 d), 1.1 Â± 0.9 (8); E261Q/D278N (>3 d), 2.2 Â± 0.9 (7); E261Q/D278N/H497M (>3 d), 0.8 Â± 0.3 (15); not injected, 0.17 Â± 0.07 (8).
	Figure 7. Effect of [H+]ext on ClC-Ka WT and its mutants E261Q, D278N, E261Q/D278N, and E261Q/D278N/H497M. (A) The current at 60 mV, shown in color, of the triple mutant E261Q/D278N/H497M as a function of time (color code as in Fig. 6). Inset in A shows representative current traces of the mutant in standard solution. Horizontal bars, 100 ms; vertical bars, 1 ÂµA. Capacitive transients have been blanked for clarity. (B) Doseâresponse relationship of the modulation by protons of ClC-Ka WT (black circles; n = 14) and the mutants E261Q (yellow squares; n = 4), D278N (red triangles; n = 5), E261Q/D278N (light blue rhombi; n = 5), and E261Q/D278N/E261Q (green hexagons; n = 5). Currents at 60 mV were normalized to values measured in standard solution and plotted versus pH. Data for WT are different compared with Fig. 2 because they were obtained from different oocytes as control measurements in the mutagenic screen.

References [+] :

Accardi, Conformational changes in the pore of CLC-0. 2003, Pubmed, Xenbase