title: A regulatory calcium-binding site at the subunit interface of CLC-K kidney chloride channels.
abstract: 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.
authors.affiliation: Istituto di Biofisica, Consiglio Nazionale delle Ricerche, 16149 Genoa, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Gradogna
authors.forename: Elena
authors.initials: E
authors.lastname: Babini
authors.forename: Alessandra
authors.initials: A
authors.lastname: Picollo
authors.forename: Michael
authors.initials: M
authors.lastname: Pusch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D021122:Protein Subunits; D013329:Structure-Activity Relationship; D014981:Xenopus
chemical_list: C489932:CLCNKA protein, human; C412733:CLCNKB protein, human; D018118:Chloride Channels; D021122:Protein Subunits; D001224:Aspartic Acid; D018698:Glutamic Acid; D002118:Calcium
references.citation: J Gen Physiol. 2003 Sep;122(3):277-93
references.pmid: 12913090
references.citation: J Gen Physiol. 1999 Mar;113(3):457-68
references.pmid: 10051520
references.citation: EMBO Rep. 2004 Jun;5(6):584-9
references.pmid: 15167890
references.citation: J Physiol. 2009 Apr 1;587(Pt 7):1387-400
references.pmid: 19153159
references.citation: EMBO J. 2008 Nov 5;27(21):2907-17
references.pmid: 18833191
references.citation: J Gen Physiol. 2001 Jul;118(1):23-32
references.pmid: 11429442
references.citation: Nature. 2005 Jul 21;436(7049):420-3
references.pmid: 16034421
references.citation: J Gen Physiol. 2008 Jul;132(1):185-98
references.pmid: 18591423
references.citation: J Biol Chem. 1993 Feb 25;268(6):3821-4
references.pmid: 7680033
references.citation: J Biol Chem. 1999 Apr 23;274(17):11687-92
references.pmid: 10206982
references.citation: Br J Pharmacol. 2010 Aug;160(7):1652-61
references.pmid: 20649569
references.citation: Nature. 2001 Nov 29;414(6863):558-61
references.pmid: 11734858
references.citation: Proc Natl Acad Sci U S A. 2008 Jan 29;105(4):1369-73
references.pmid: 18216243
references.citation: PLoS One. 2008 Jul 23;3(7):e2746
references.pmid: 18648499
references.citation: Methods Enzymol. 1998;293:280-300
references.pmid: 9711614
references.citation: J Physiol. 1996 Dec 1;497 ( Pt 2):423-35
references.pmid: 8961185
references.citation: N Engl J Med. 2004 Mar 25;350(13):1314-9
references.pmid: 15044642
references.citation: J Biol Chem. 1999 Jan 8;274(2):896-902
references.pmid: 9873029
references.citation: J Am Soc Nephrol. 2003 Apr;14(4):1082-95
references.pmid: 12660344
references.citation: Nat Genet. 1999 Jan;21(1):95-8
references.pmid: 9916798
references.citation: Nature. 2002 Jan 17;415(6869):287-94
references.pmid: 11796999
references.citation: Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6943-7
references.pmid: 8041726
references.citation: Nat Genet. 1997 Oct;17(2):171-8
references.pmid: 9326936
references.citation: EMBO J. 1997 Apr 1;16(7):1582-92
references.pmid: 9130703
references.citation: Eur Biophys J. 2010 May;39(6):869-75
references.pmid: 19132363
references.citation: Clin Genet. 2010 Feb;77(2):155-62
references.pmid: 19807735
references.citation: Nature. 2004 Feb 26;427(6977):803-7
references.pmid: 14985752
references.citation: Nephrol Dial Transplant. 2006 Jan;21(1):29-32
references.pmid: 16287914
references.citation: Pflugers Arch. 2002 Jun;444(3):411-8
references.pmid: 12111250
references.citation: Science. 2003 Apr 4;300(5616):108-12
references.pmid: 12649487
references.citation: Nat Genet. 2001 Nov;29(3):310-4
references.pmid: 11687798
references.citation: J Clin Invest. 2005 Aug;115(8):2039-46
references.pmid: 16075045
references.citation: Mol Pharmacol. 2006 Jan;69(1):165-73
references.pmid: 16244177
references.citation: J Gen Physiol. 1983 Jul;82(1):25-45
references.pmid: 6310023
references.citation: J Gen Physiol. 2006 Jan;127(1):51-65
references.pmid: 16380443
references.citation: Pflugers Arch. 1981 Aug;391(2):85-100
references.pmid: 6270629
references.citation: Am J Physiol Regul Integr Comp Physiol. 2005 Apr;288(4):R782-95
references.pmid: 15793031
references.citation: EMBO J. 2009 Feb 4;28(3):175-82
references.pmid: 19131966
references.citation: J Am Soc Nephrol. 2002 Sep;13(9):2259-66
references.pmid: 12191970
references.citation: Methods Enzymol. 1992;207:131-48
references.pmid: 1326701
references.citation: J Am Soc Nephrol. 2005 Jun;16(6):1549-61
references.pmid: 15829707
references.citation: J Physiol. 2002 May 15;541(Pt 1):103-12
references.pmid: 12015423
references.citation: Biophys J. 1994 Jan;66(1):149-52
references.pmid: 8130334
references.citation: J Clin Invest. 1995 Jan;95(1):104-13
references.pmid: 7814604
references.citation: Annu Rev Biophys Biomol Struct. 2000;29:411-38
references.pmid: 10940254