From the Department of Physiology and Biophysics, Wright State University, Dayton, Ohio 45435
The molecular identity of the channels producing
swelling-activated Cl conductance (gswell) is of great interest since this physiologic response to swelling is common to many cells. As presented in the May Perspectives (Clapham, 1998 Clearly, permeation by Cl is a characteristic of gswell,
but organic osmolytes also flow through these channels
at significant rates. Taurine is an important component
of intracellular osmolarity in many cell types and can
exit through these channels, which indicates a pore
large enough for navigation by ions bulkier than Cl.
Determination of pore size, to delimit the class of permeating molecules, has been a goal of ion selectivity
studies; as pioneered by Hille (1971); Strange, 1998
), the question of
whether the observed gswell represents a family of channel proteins is presently unanswered, but to be included in this family a channel must be activated by cell
swelling and conduct Cl. Determining if a candidate Cl
channel should be included based on swelling sensitivity may be problematic since the volume-sensing element could reside in an auxiliary protein that associates with the channel protein and, through allosteric
modulation, creates a swelling-activated complex. If the
associated sensor is removed inadvertently by experimental manipulation or incomplete reconstitution, then
a legitimate family member would be excluded. Permeation selectivity is intrinsic to the pore of the channel
protein and thus provides a less easily lost characteristic
to aid in identification.
, the method involves calculating relative permeabilities (PX/PCl) from
reversal potential measurements combined with knowledge of ion dimensions (using space-filling models)
and affinities. A plot of relative permeability versus ion
diameter provides insight into pore geometry; PX/PCl
reported for gswell (Jackson et al., 1994
, 1996
) are shown
in Fig. 1, together with values for CLC-1 (Rychkov et al.,
1998
), a member of a large Cl channel family. Anion diameter often has been estimated using Stokes' Law for spherical particles (Bormann et al., 1987
; Rychkov et
al., 1998
); unfortunately, Stokes' diameters often severely underestimate ion size for diameters <1.0 nm
(Robinson and Stokes, 1959
), which includes most anions and osmolytes of interest during swelling activation. For this reason, molecular sizes obtained from
space-filling models are the most appropriate, which
suggests that the pore sizes of the GABA and glycine receptor Cl channel family and the CLC family need to
be reevaluated.
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Fig. 1.
Relative permeabilities and diameters of anions. Relative anion permeabilities for gswell
( ; Jackson et al., 1994
, 1996
),
CLC-1(
; Rychkov et al., 1998
),
and mutant (G230E)hCLC-1 (
,
PSCN = 7.2 PCl; Fahlke et al.,
1997a
) are plotted versus diameters determined from space-filling
models (Halm and Frizzell, 1992
).
Geometric mean diameters were
used for the nonspherical ions
NO3, SCN, HCO3, and ClO4. Cylindrical diameters were used for
the elongated ions methanesulfonate (MeSO3), isethionate,
taurine, and gluconate.
The permeability patterns for gswell and CLC-1 (Fig.
1) illustrate two distinct classes of anion selectivity. For
gswell, the selectivity sequence is lyotropic, SCN > I NO3 > Br > Cl > F, similar to that reported for many
anion channels (Frizzell and Halm, 1990
) in which
larger anions having lower hydration energies pass
through the channels more easily than small anions (up to a point set by steric factors). The low field
strength sites expected for a pore exhibiting a lyotropic
sequence could be formed from the positive ends of
the dipoles contributed by the imino groups of the peptide backbone, analogous to the carbonyl oxygen interaction sites of potassium channels (Doyle et al., 1998
).
Many organic ions also permeate through gswell; the
zwitterionic taurine (2-aminoethanesulfonate) is shown
in Fig. 1. Passage of organic ions together with a lyotropic sequence for halide anions is practically identical to
the permeation pattern of ions in the so called outward-rectifying Cl channel (ORCC; Halm and Frizzell, 1992
). In comparison, CLC-1 has reduced permeabilities for larger anions, SCN > Cl > Br > I
NO3 > F,
that would suggest steric restrictions limiting flow of
bulkier anions such as methanesulfonate (MeSO3),
except that the large polyatomic anion perchlorate
(ClO4) has permeability greater than Cl. Hydrogen
bonding of ClO4 and sulfonates (including taurine)
with imino groups could reduce the effective ionic dimensions by up to 0.1 nm, making these ions behave as
small as iodide in the pore of such a channel. Low PI/ PCl is considered a hallmark of the CLC family, particularly CLC-1, and has been used to suggest that this family of channels does not contribute to gswell.
High PX/PCl for ClO4 and SCN supports a lyotropic
sequence (Wright and Diamond, 1977) for CLC-1, similar to gswell. Anomalously low PI/PCl and PBr/PCl reported for CLC-1 probably result from the current
block reported for these ions (Fahlke et al., 1997b
; Rychkov et al., 1998
); blocking action by permeant ions
reduces current, making reversal potential determinations difficult and the resulting PX/PCl unreliable. Erroneously low PI/PCl has been recognized in the cystic fibrosis transmembrane conductance regulator Cl channel to result from current block by iodide (Tabcharani et al., 1997
). A report on hCLC-1 (Fahlke et al., 1997a
)
suggests that the taurine mimic MeSO3 may have a
small, nonzero permeability in this channel. Even more
dramatic, the anion selectivity of hCLC-1 can be converted by a single amino acid substitution (G230E, a
mutation found in myotonia congenita) to a lyotropic
sequence, SCN > NO3
I > Br > Cl > MeSO3 (Fig.
1), suggesting that this mutation relieves iodide block
(Fahlke et al., 1997a
) such that the resulting larger currents allow a more reliable measure of permeability. In
addition, pore dimensions should permit flow of organic osmolytes such as taurine. Interestingly, the glycine at position 230 in CLC-1 is conserved throughout
the CLC family, yet wild-type CLC-3 has a lyotropic sequence for halide permeabilities (Fahlke et al., 1997c
).
Knowledge of permeability for taurine and its mimics
in CLC-3 would greatly aid an assessment of the physiologic functions for this member of the CLC family.
Based on what ion selectivity can illuminate about pore dimensions and ion interactions with the pore, the outward-rectifying Cl channel, and members of the CLC family are likely candidates for inclusion in a family of channels producing gswell. Useful probes of permeation by osmolytes such as taurine are ethanesulfonate, isethionate (2-hydroxyethanesulfonate), and ClO4; currents can be measured directly with these probes since each is a monovalent anion at physiologic pH, and all have similar size and electronic configuration for the anionic part of the molecule. At this time, CLC-3 appears the most likely member of the gswell family, but the allosteric modifications that would occur during physiologic activation (possibly cell swelling) seemingly also could reduce iodide block in other members of the CLC family such that ion permeation measures would be consistent with that of gswell.
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FOOTNOTES |
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Address correspondence to Dan R. Halm, Department of Physiology and Biophysics, Wright State University, Dayton, OH 45435. Fax: 937-775-3769; E-mail: dhalm{at}wright.edu
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