Transmembrane topology of a CLC chloride channel

Abstract

CLC chloride channels form a large and conserved gene family unrelated to other channel proteins. Knowledge of the transmembrane topology of these channels is important for understanding the effects of mutations found in human myotonia and inherited hypercalciuric kidney stone diseases and for the interpretation of structure-function studies. We now systematically study the topology of human ClC-1, a prototype CLC channel that is defective in human myotonia. Using a combination of in vitro glycosylation scanning and protease protection assays, we show that both N and C termini face the cytoplasm and demonstrate the presence of 10 (or less likely 12) transmembrane spans. Difficult regions were additionally tested by inserting cysteines and probing the effect of cysteine-modifying reagents on ClC-1 currents. The results show that D3 crosses the membrane and D4 does not, and that L549 between D11 and D12 is accessible from the outside. Further, since the modification of cysteines introduced between D11 and D12 and at the extracellular end of D3 strongly affect ClC-1 currents, these regions are suggested to be important for ion permeation.Voltage-gated chloride channels of the CLC family are highly conserved during evolution and are expressed in organisms ranging from bacteria (1) and yeast (2) to plants (3) and animals (4). Their physiological functions in higher organisms include the regulation of cell volume, control of electrical excitability, and transepithelial transport.The CLC proteins were identified by expression cloning of the Torpedo chloride channel ClC-0 (5). At present, nine mammalian members are known (for review, see ref. 6). ClC-1 is nearly specific for skeletal muscle (4). It ensures its electrical stability, which is evident from ClC-1 mutations leading to myotonia congenita, a disease characterized by defective muscle relaxation. Many different ClC-1 mutations were found in human myotonia and in animal models (7-11). The importance of CLC channels is underscored by another, recently identified disease. Inactivating mutations of ClC-5 cause a syndrome that is characterized by low molecular weight proteinuria, hypercalciuria, and kidney stones (12). ClC-2 may play a role in cell volume control (13) and in setting the intracellular chloride concentration, which in turn is important for synaptic transmission in certain neurons (14). However, the function of most CLC proteins is still unclear.CLC proteins are structurally unrelated to other ion channel classes. Topological models are still mainly based on hydropathy analysis, suggesting the presence of about 12-13 transmembrane domains, originally termed D1 to D13 (5). This model has already had to be revised to accommodate new experimental findings (15,16).CLC channels function as multimers of identical (11) or homologous subunits (17). Dominant negative mutations suggested that ClC-1 channels have more than three subunits (11).However, ClC-0 is a homodimeric channel with one pore per subunit (16,18,19); this may also apply for .In structure...

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

“…However, their gating is strongly dependent on pH, indicating a kind of memory of the bacterial transporter. This notion is also supported by the conserved crucial role of E148 for ClC gating in all ClC channels studied (10,13,14,16,32,40,47). The new findings could be also helpful in future studies on some human channels, such as ClC-6 and ClC-7, that are still poorly understood.…”

Identification of Three Cysteines as Targets for the Zn2+ Blockade of the Human Skeletal Muscle Chloride Channel

“…Strong evidence suggests regions between transmembrane segments D3 and the end of D5 to participate in the pore-forming structure (11). However, these results are not in agreement with the most recently postulated topology model of ClC-1 (5). Furthermore, studies of ClC-0 (reviewed in Ref.…”

“…The first membrane topology model of ClC proteins was derived from hydropathy analysis (2); several times it had to be revised on the grounds of firmer experimental evidence (3)(4)(5). Certain mutations in the human ClC-1 gene (reviewed in Ref.…”

Molecular determinants of common gating of a ClC chloride channel

“…The importance of residue E232 for ClC-1 common gating has been established elsewhere 27,28,52 . The data presented here expound a novel role for Y578 in ClC-1 common gating.…”