Gating and trafficking of ClC-2 chloride channel without cystathionine β-synthase domains

ClC-2 is an inwardly rectifying Cl⁻ channel that belongs to the double pore ClC Cl⁻ channel family. It is widely expressed but its physiological function remains unsettled. ClC channels are homodimers, assembled from monomers that comprise 18 α-helices (A-R) located in the plasma membrane and a large, cytoplasmic carboxy terminus containing two cystathionine β-synthase (CBS1 and CBS2) domains (Jentsch et al. 2002). Channel gating is controlled by protopore gates (fast acting) that close each pore separately and a common gate (slow acting) which simultaneously closes both pores (Richard & Miller, 1990). A conserved glutamic acid located in a loop connecting the α-helices E and F (E⇔F loop) forms the protopore gate of ClC-0, ClC-1 and ClC-2 (Dutzler et al. 2003) but displays different properties. In ClC-0, this gate opens by depolarizations and is controlled by external and internal [Cl⁻] and [H⁺], whereas in ClC-2 it is open by hyperpolarizations and controlled by the intracellular but not extracellular [Cl⁻]. Defining the exact molecular domains that outline the common gate has been an elusive task, although two regions have already been identified (Lin et al. 1999; Estevez et al. 2004). Mutational analysis revealed that a cysteine residue in loop I⇔J of ClC-0 and ClC-1 participate in slow gating; furthermore, in ClC-0 closure of this gate is facilitated by Zn²⁺ application. Similarly, histidine and glutamic acid residues located in the CBS2 domain of either ClC-0 or ClC-1 participate in slow gating and Zn²⁺ sensitivity. By contrast, mutating cysteine 258 (or cysteine 256) in loop I⇔J or histidine 811 in CBS2 domain of the ClC-2 channel neither completely locked the slow gate in the open state nor eliminated Cd²⁺ sensitivity. In these mutants, however, the protopore gate voltage dependence was shifted to positive voltages (de Santiago et al. 2005; Yusef et al. 2006). Interestingly, in ClC-0 movements of the carboxy terminus have been measured during slow gating (Bykova et al. 2006). So, it is clear that the carboxy terminus is important for gating yet the picture is blurred.

An additional but lesser studied role in trafficking ClC channels to the plasma membrane has also been suggested for the carboxy terminus (Estevez et al. 2004). For example, ClC-0 without its carboxy terminus is not functional when expressed in Xenopus oocytes, although this channel was functional when expressed in HEK cells. Another example are ClC-1 channels lacking the CBS2 domain that do not reach the plasma membrane of the oocyte but can be rescued after co-expressing them with a fragment containing CBS2. Also, changes in slow gating are observed in some ClC-linked hereditary diseases associated with point mutations or truncations of the CBS domains. Consequently, determining the role of CBS domains in gating and trafficking is an important issue.

The work by Garcia-Olivares et al. (2008) published in this issue of The Journal of Physiology further explores the role of the carboxy terminus in ClC-2 gating and trafficking. Surprisingly, channels without both CBS domains were functional and reached the plasma membrane as well as WT ClC-2. Moreover, the protopore gate of these channels shows normal voltage dependence but the normalized open probability of the common gate increased (> 0.8 at all voltages). These results support the idea that the carboxy terminus is essential for slow gating but are contrary to previous findings showing that CBS2 domain is essential for ClC-1 trafficking. In addition, ClC-2 channels with only CBS1 reached the plasma membrane but were not functional. Apparently, without CBS2 these channels were locked in the close state because the normal conductance was restored only after mutating the glutamic acid that comprises the protopore gate. Thus, not all ClC channels use the carboxy terminus domains to traffic to the plasma membrane but need the CBS domains to coordinate their gating.

How does the carboxy terminus then alter ClC gating? A missing piece of the puzzle may be the coupling between the protopore and common gates. Here the work by Garcia-Olivares et al. (2008) already suggests a coupling mechanism. They observed that the removal of the carboxy terminus of ClC-2 accelerated the opening and closing of the protopore gate and concluded that the carboxy terminus controls ClC-2 activation by hindering the protopore gate. It has already been suggested that additional conformational rearrangements take place on the intracellular side of the ClC-0 pore during fast gating (Accardi & Pusch, 2003). Moreover, the model of the channel structure shows that a carboxy terminus is connected to the intracellular side of the protopore via the α-helix R. This helix contributes a tyrosine residue to the conduction pathway and this residue coordinates a Cl⁻ ion sitting in the central binding site near the glutamate COO⁻ group forming the protopore gate. When this tyrosine (512) was mutated in ClC-0 the result was a protopore gate with slower kinetics. Therefore, assuming that the carboxy terminus of ClC-2 also moves during slow gating as it does in ClC-0, it is conceivably that such movement may be transmitted to the pore through the R helix, thus coupling the gates.

Garcia-Olivares's work provides important results that add to the mounting evidence which suggests that the main molecular domain outlining the common gate in ClC channels is located in the CBS2 domain.