Residues Lining the Inner Pore Vestibule of Human Muscle Chloride Channels

Abstract

Chloride channels belonging to the ClC family are ubiquitous and participate in a wide variety of physiological and pathophysiological processes. To define sequence segments in ClC channels that contribute to the formation of their ion conduction pathway, we employed a combination of site-directed mutagenesis, heterologous expression, patch clamp recordings, and chemical modification of the human muscle ClC isoform, hClC-1. We demonstrate that a highly conserved 8-amino acid motif (P3) located in the linker between transmembrane domains D2 and D3 contributes to the formation of a wide pore vestibule facing the cell interior. Similar to a previously defined pore region (P1 region), this segment functionally interacts with the corresponding segment of the contralateral subunit. The use of cysteine-specific reagents of different size revealed marked differences in the diameter of pore-forming regions implying that ClC channels exhibit a pore architecture quite similar to that of certain cation channels, in which a narrow constriction containing major structural determinants of ion selectivity is neighbored by wide vestibules on both sides of the membrane. The ClC family of voltage-gated ClϪ channels represents the largest known gene family coding for anion channels (1-3). At least nine human isoforms (ClC-1 to ClC-7, ClC-Ka, and ClCKb) are expressed in various tissues and play important roles in the function of various organs. Mutations in the genes coding for three ClC isoforms cause inherited human diseases. CLCN1 represents the genetic locus for myotonia congenita (4, 5), a muscle disease characterized by stiffness upon sudden forceful movement. Mutations in CLCN5 cause Dent's disease, an inherited renal disorder associated with hypercalciuria, nephrolithiasis, and low molecular weight proteinuria (6). Genetic alterations of CLCNKB are responsible for type III Bartter's syndrome, a salt-wasting renal tubular disorder causing hypovolemia, hyponatremia, and hypotension (7).Heterologous expression of many cloned ClC isoforms have revealed highly anion-selective ion channels, and it is therefore reasonable to predict that evolutionarily conserved structures confer anion selectivity to their ion conduction pathways. Elucidating the molecular mechanisms responsible for ion selectivity and conduction in ClC channels is key to understanding the function and dysfunction of this important family of ion channels. Moreover, primary sequence information about the ionic pore represents the first step for a rationally designing compounds to block or open these ion channels that play important roles in human diseases.We recently probed the ion conduction pathway of human ClC-1 in the presence of several different permeant anions (8), and we demonstrated an unusual ion selectivity mechanism that depends upon differential ion binding to discriminate among anions (8, 9). Human ClC-1 (hClC-1) 1 exhibits at least two functionally distinct ion binding sites within the pore (8, 10), both preferring large and polyatomic anions over chl...

Proton-coupled gating in chloride channels

“…Over the past two decades, ClC-1 and ClC-0 have been extensively mutated-nearly all of the predicted protonatable residues in the transmembrane region have been mutated-yet no other residue exhibits this predicted phenotype (Pusch et al 1995;Ludewig et al 1997;Kurz et al 1999;Fahlke et al 2001;Engh & Maduke 2005;Zhang et al 2006). Therefore, our results are in agreement with the hypothesis that this conserved glutamate is a part of the proton pathway in the CLC channels.…”

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

“…For biochemical experiments with hClC-Kb, HEK293T cells were co-transfected with 1 g of pcDNA3.1 eGFP-or mCherryhClC-Kb and 4 g of pcDNA3.1 barttin-mCherry or -mYFP using the calcium phosphate technique (33). For all other experiments on hClC-Kb a combination of 1 g of pcDNA3.1 eGFP-hClC-Kb and 2 g pcDNA3.1 barttin-mCherry was used.…”

Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations, but Mediate Identical Transport Functions

“…ClC-3 splice variants were transiently expressed in HEK293T or MDCK II cells alone or in combination with fluorescent markers such as LAMP1 (which was a gift from Walther Mothes (Addgene plasmid 1817) (22), RAB7, RAB11 (a gift from Richard Pagano (Addgene plasmid 12605) (23), TfR (a gift from Gary Banker (Addgene plasmid 45060) (24), or the membrane marker farnesylated eGFP (provided by Dr. M. Filippov, Nizhny Novgorod, Russia) and examined typically 24 h or 36 h after transfection of 2 to 5 g of cDNA using Lipofectamine 2000 (Invitrogen) or calcium phosphate transfection methods (25).…”