Chloride Ion Channels: Structure, Functions, and Blockers - Ion Channels and Their Inhibitors - page 314

only one of the two subunits, has been revealed in the crystal structure coming close
to the cytoplasmic end of the helix R of the other subunit. However, the two
monomers of the CLC channel are tilted by 45 towards each other. With all such
observations, however, the biochemical topology studies of CLC channels remain
inconclusive, because of which sometimes complications arise in interpreting the
effects of mutations [ 26 ].
2.1.2 The Pore, Gating, and Cl Permeation of CLC Channels
Although it is stated that the dimeric structure of CLC channels have two pores, but
which protein segments line the pores have not yet been fully identified. The CLC
pore is assumed to be formed by a single subunit [ 20 ], where it must be lined by
different, nonhomologous parts of a single protein. This is in contrast to K +
channels, where four identical or homologous “P loops” contribute to the perme-
ation pathway. From the crystal structure study, it has been found that the various
regions of the protein come together to form the pore. Four anti-parallel helices
extend from the inside and the outside into the center plane of the membrane and the
Cl is coordinated by residues at the ends of these residues, which contain highly
conserved regions from the amino terminals of helix D (GSGIP), helix F (GREGP),
helix N (GIFAP), and an amino acid from helix R (Ty445, St ClC numbering) [ 25 ].
From some transplantation experiments, it was however hypothesized that the
region between helices E and F directly lines the pore [ 27 ], but its role in perme-
ation and gating is poorly understood.
Regarding the gating of CLC channels, it is said that most CLC proteins that can be
expressed functionally show voltage-dependent gating [ 9 ]. The charged amino acids
in CLC trans-membrane domains are supposed to act as voltage sensors, and it
hasbeenindeedproposedthatanasparticacidattheextracellularendofhelixB
acts as a voltage sensor in ClC-1 [ 28 ]. However, the most thorough study on gating has
been made for ClC-0 because of its relatively high channel conductance (~10 ps) and
relatively simple gating [ 9 ]. It has fast gating that is two-state process with
monoexponential kinetics. The ClC-0 opening is promoted by its substrate Cl .
An unusual gating model for ClC-0 has also been proposed by Pusch et al. [ 29 ]in
which the binding of Cl to a site deep within the pore promotes the opening (voltage-
independent) of the channel. This results in voltage-dependent gating, as chloride ions
have to travel along the electric field to reach this site. The voltage-dependent gating
of many CLCs is strongly modulated by extracellular anions and pH [ 29 - 35 ].
The binding of Cl to the channels involves the nitrogen and oxygen atoms of the
protein. The ions are coordinated with nitrogen atoms of main chain amino acid amide
groups of Ile 356, Phe 357 (helix N) and with the oxygen atoms of the side chain of
Ser107 (helix D) and Tyr 445 (helix R). The oxygen atomof Ser107 is also involved in
the formation of a hydrogen bond with the accompanying Ile109 of the same
conserved region, allowing a higher degree of polarization. The Cl permeation,
however, can be blocked when the side chain of a glutamate residue at the start of
helix F projects into the pore on the extracellular side from the chloride binding site.