The Role of the Carboxyl Terminus in ClC Chloride Channel Function

Abstract

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...

A Two-Holed Story: Structural Secrets About ClC Proteins Become Unraveled?

“…Moreover, they observed that mutations in the COOH terminus did not alter the gating properties of the channel but cause a decrease in the maximal current. Surprisingly, Hebeisen et al (18) found that the deletion of CBS1 but not that of CBS2 was tolerated, in complete contrast to the results of Estévez et al (12) (FIGURE 4). These two studies underline the difficulties involved in investigating the role of CBS domains.…”

“…The conclusion of Hebeisen et al was mainly based on the use of concatameric constructs in which one of the subunits bore a point mutation (S537F) that renders the channel less sensitive to block by 9-AC (13). The authors assumed that 9-AC block represents a property of a single pore not influenced by the neighboring subunit (18). However, this assumption is probably not true, because results with ClC-0 indicate a dependence of 9-AC and CPA…”

“…These two studies underline the difficulties involved in investigating the role of CBS domains. The use of different methods and constructs used in these two studies (12,18) is probably one of the reasons for such discordant results. For example, the employment of concatameric construct could force an assembly and a targeting different from that found in coexpression studies.…”

“…Two recent studies employed different techniques to explore in more detail the function of CBS domains in ClC channels, in particular in ClC-1 (12,18). Previously, it had been shown that truncating ClC-1 after CBS1 does not give rise to any current (39).…”

Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies

“…Although the physiological role of CBS domains is still unknown, truncations removing parts of the distal CBS domain of ClC channels abolished functional expression in heterologous systems (20,36).…”