Idiopathic generalized epilepsy (IGE) is an inherited neurologi-

Barttin is an accessory subunit of a subgroup of ClC-type chloride channels expressed in renal and inner ear epithelia. In this study, we examined the effects of barttin on two ClC-K channel isoforms, rat ClC-K1 and human ClC-Kb, using heterologous expression, patch clamping, confocal imaging, and flow cytometry. In the absence of barttin, only a small percentage of rClC-K1 and hClC-Kb channels are inserted into the plasma membrane. Coexpression of barttin enhances surface membrane insertion and furthermore modifies permeation and gating of ClC-K channels. hClC-Kb channels are nonfunctional without barttin and require the coexpressed accessory subunit to become anion conducting. In contrast, rClC-K1 channels are active without barttin, but at the cost of reduced unitary conductance as well as altered voltage dependence of activation. We mapped the separate functions of barttin to structural domains by a deletion analysis. Whereas the transmembrane core is necessary and sufficient to promote ClC-K channel exit from the endoplasmic reticulum, a short cytoplasmic segment following the second transmembrane helix modifies the unitary conductance. The entire cytoplasmic carboxyl terminus affects the open probability of ClC-K channels. The multiple functions of barttin might be necessary for a tight adjustment of epithelial Cl ؊ conductances to ensure a precise regulation of body salt content and endocochlear potential.chloride channels ͉ gating ͉ kidney ͉ ClC family ͉ accessory subunit

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The human muscle chloride channel ClC-1 has a 398-amino acid carboxyl-terminal domain that resides in the cytoplasm and contains two CBS (cystathionine-␤-synthase) domains. To examine the role of this region, we studied various carboxyl-terminal truncations by heterologous expression in mammalian cells, whole-cell patch clamp recording, and confocal imaging. Channel constructs lacking parts of the distal CBS domain, CBS2, did not produce functional channels, whereas deletion of CBS1 was tolerated. ClC channels are dimeric proteins with two ion conduction pathways (protopores). In heterodimeric channels consisting of one wild type subunit and one subunit in which the carboxyl terminus was completely deleted, only the wild type protopore was functional, indicating that the carboxyl terminus supports the function of the protopore. All carboxylterminal-truncated mutant channels fused to yellow fluorescent protein were translated and the majority inserted into the plasma membrane as revealed by confocal microscopy. Fusion proteins of cyan fluorescent protein linked to various fragments of the carboxyl terminus formed soluble proteins that could be redistributed to the surface membrane through binding to certain truncated channel subunits. Stable binding only occurs between carboxyl-terminal fragments of a single subunit, not between carboxyl termini of different subunits and not between carboxyl-terminal and transmembrane domains. However, an interaction with transmembrane domains can modify the binding properties of particular carboxyl-terminal proteins. Our results demonstrate that the carboxyl terminus of ClC-1 is not necessary for intracellular trafficking but is critical for channel function. Carboxyl termini fold independently and modify individual protopores of the double-barreled channel.ClC channels are found in almost all prokaryotic and in eukaryotic cells. Nine isoforms (ClC-1 to ClC-7, ClC-Ka and ClC-Kb) were shown to be expressed in human tissues and to fulfil a variety of functional roles. ClC-1 is the major muscle chloride channel responsible for the regulation of muscle excitability (1, 2), and ClC-2 is crucial for neuronal chloride homeostasis (3). ClC-Kb is involved in transepithelial NaCl movement in the thick ascending limb of Henle (4), and ClC-3, -5, and -7 are necessary for the pH adjustment of several cell compartments (5-7).Eukaryotic and prokaryotic ClC channels exhibit 18 transmembrane domains (8) followed by cytoplasmic carboxyl termini of variable sequences. The carboxyl-terminal tails of mammalian isoforms contain between 146 and 404 amino acids and exhibit two structurally defined domains, so-called CBS domains (9, 10). The functional importance of the carboxyl terminus is illustrated by disease-causing mutations in ClC-1, -2, -5 and ClC- Kb (3,6,11,12). Moreover, truncations removing parts of the distal cystathionine-␤-synthase (CBS) 1 domain of ClC channels were shown to abolish functional expression in heterologous systems (6,(13)(14)(15)(16). Although co-expression of the complemen...