Martinez GQ and Maduke M (2008), A cytoplasmic domain mutation in ClC-Kb affects...

XB-ART-38447

PLoS One 2008 Jul 23;37:e2746. doi: 10.1371/journal.pone.0002746.

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A cytoplasmic domain mutation in ClC-Kb affects long-distance communication across the membrane.

Martinez GQ , Maduke M .

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BACKGROUND: ClC-Kb and ClC-Ka are homologous chloride channels that facilitate chloride homeostasis in the kidney and inner ear. Disruption of ClC-Kb leads to Bartter's Syndrome, a kidney disease. A point mutation in ClC-Kb, R538P, linked to Bartter's Syndrome and located in the C-terminal cytoplasmic domain was hypothesized to alter electrophysiological properties due to its proximity to an important membrane-embedded helix. METHODOLOGY/PRINCIPAL FINDINGS: Two-electrode voltage clamp experiments were used to examine the electrophysiological properties of the mutation R538P in both ClC-Kb and ClC-Ka. R538P selectively abolishes extracellular calcium activation of ClC-Kb but not ClC-Ka. In attempting to determine the reason for this specificity, we hypothesized that the ClC-Kb C-terminal domain had either a different oligomeric status or dimerization interface than that of ClC-Ka, for which a crystal structure has been published. We purified a recombinant protein corresponding to the ClC-Kb C-terminal domain and used multi-angle light scattering together with a cysteine-crosslinking approach to show that the dimerization interface is conserved between the ClC-Kb and ClC-Ka C-terminal domains, despite the fact that there are several differences in the amino acids that occur at this interface. CONCLUSIONS: The R538P mutation in ClC-Kb, which leads to Bartter's Syndrome, abolishes calcium activation of the channel. This suggests that a significant conformational change--ranging from the cytoplasmic side of the protein to the extracellular side of the protein--is involved in the Ca(2+)-activation process for ClC-Kb, and shows that the cytoplasmic domain is important for the channel's electrophysiological properties. In the highly similar ClC-Ka (90% identical), the R538P mutation does not affect activation by extracellular Ca(2+). This selective outcome indicates that ClC-Ka and ClC-Kb differ in how conformational changes are translated to the extracellular domain, despite the fact that the cytoplasmic domains share the same quaternary structure.

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Species referenced: Xenopus
Genes referenced: clcnkb

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	Figure 1. CLC domain architecture and the location of R538.(A) Homology model of ClC-Kb using ClC-ec1 (1OTS) as a template for the membrane domain and the ClC-Ka C-terminal domain structure (2PFI) as a template for the cytoplasmic domain. Subunit A (grey) has several amino acids removed to show the ion coordination of chloride by Y520 (in spacefill), located on helix-R. R538 is located in the cytoplasmic-domain loop (red) that connects helix-R to CBSD1. (B) Structure of the ClC-Ka C-terminal domain (2PFI) highlighting the different CBSDs. Subunit 1 has CBSD1 in blue and CBSD2 in cyan; subunit 2 has CBSD1 in red and CBSD2 in violet. (C) Structure of TM0935 (1OTS) with the color scheme as in B. All cartoons were generated in PyMol (http://www.pymol.org).
	Figure 2. Gating and selectivity of ClC-Kb-R538P.Two-electrode voltage clamp recordings of (A) wild type and (B) R538P ClC-Kb channels. Currents are in response to a 200-ms prepulse to +60 mV followed by 500-ms test pulses ranging from +60 mV to −140 mV in −20 mV decrements. (C) Steady-state currents at the different voltages were normalized to the current value at +60 mV for wild type (filled squares) and R538P (open squares). Error bars are s.e.m. with n = 8 (WT) and n = 6 (R538P). Error bars smaller than the symbols are not shown. Representative steady state I–V curves for (D) wild type and (E) R538P channels in the presence of chloride (filled squares), bromide (open squares), or iodide (open diamonds). (F) Permeability ratios relative to chloride for wild type (filled bars) and R538P (open bars). The bromide/chloride permeability ratio of R538P is statistically different from that of wild type (p<0.0005, n = 5).
	Figure 3. R538P abolishes calcium activation in hClC-Kb.Current recordings for (A) wild type and (B) R538P ClC-Kb channels at 0.1 mM calcium (left) and 10.0 mM calcium (right). (C) Calcium dependent activation of wild type (filled squares) and R538P (open squares) channels. Steady-state current levels at +60 mV plotted as a function of [Ca2+], normalized to the value at 0.1 mM calcium. Each data point is the average of several (n = 4–6) oocytes and error bars show the s.e.m. Only oocytes that showed reversibility were included in the analysis.
	Figure 4. Expression of hClC-Ka-R538P.Two-electrode voltage clamp current recordings of wild type (A) and R538P (B) expressing channels. Currents are in response to a 100-ms prepulse to +60 mV follow by 500-ms test pulses ranging from +60 mV to −140 mV in −20 mV steps. (C) Relative permeability ratios for ClC-Ka wild type (filled bars) and R538P (open bars) channels. The iodide/chloride permeability ratio of R538P is statistically different from that of wild type (p<0.02, n = 3 (WT) and n = 5 (R538P)). (D) Calcium dependence at +60 mV of wild type (open squares) and R538P (open triangles) normalized to the current at +60 mV in the presence of 0.1 mM calcium, as in Figure 3. Error bars for WT (n = 4) and R538P (n = 3) are the s.e.m.
	Figure 5. Dimerization of the ClC-Kb C-terminal domain.(A) Stereo view of the ClC-Ka cytoplasmic domain structure (2PFI) with amino acid differences between ClC-Ka and ClC-Kb shown in space fill. Amino acid F636 (shown in black) is on the dimer interface in ClC-Ka and when mutated to aspartic acid removes ClC-Ka dimerization. In ClC-Kb this residue is the non-polar residue serine. (B) Gel filtration combined with multi-angle light scattering shows that the C-terminal domain of ClC-Kb forms a dimer in solution. The solid line represents the absorbance at 280 nm as a function of elution volume. Filled circles represent the calculated mass at the different elution volumes. The molar mass was determined to be 37 kDa, consistent with dimerization.
	Figure 6. Conserved dimerization interface in the ClC-Kb C-terminal domain.(A) Stereo view of TM0935 structure (1O50), a CBSD-containing protein that adopts an alternate dimer interface. Amino acids corresponding to residues that differ between ClC-Ka and ClC-Kb are shown in spacefill. The residue corresponding to 636 in ClC-Kb (see Figure 5A) is shown in black. (B) Residues chosen for cross-linking studies mapped onto the ClC-Ka C-terminal domain structure: L650 (black), 603 (cyan), 604 (green), 644 (olive), 645 (magenta). For this dimer interface, the L650C mutant is expected to form an intersubunit cross-link. (C) Residues chosen for cross-linking studies mapped onto the TM0935 structure; color scheme as in B. For this dimer interface, the 603–645 and the 604–644 cysteine double mutants are expected to form intersubunit cross-links. (D) SDS-PAGE of purified ClC-Kb C-terminal domains with different cysteine mutations in reducing (−) and oxidizing (+) conditions.

References [+] :

Bateman, The structure of a domain common to archaebacteria and the homocystinuria disease protein. 1997, Pubmed