From the Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109
In the 8 yr since the gene encoding the cystic fibrosis
transmembrane conductance regulator (CFTR) was
identified by means of positional cloning (Kerem et al.,
1989 The amino acid sequence of the CFTR protein and
its predicted topology accurately foreshadowed the regulation of CFTR channel function by phosphorylation
and ATP binding/hydrolysis (Kerem et al., 1989 Previous studies of CFTR's anion conduction properties suggested a pattern that is common to a number of
anion channels. Relative halide permeabilities, determined from shifts in reversal potential, appear to be ordered more or less as would be predicted for a so-called
"weak site" ion-channel interaction, so that permeability ratios are highly dependent on differences in anion-water interactions (Eisenman and Horn, 1983 In the first paper of this series, Tabcharani et al.
(1997) In the second paper, Linsdell et al. (1997a) In the final paper, Linsdell et al. (1997b) It is important to note the limitations of any modeling effort, and the authors are appropriately circumspect about their results. Rate theory models provide
an approach to describing the process of ion conduction, which is, in a sense, removed from any direct consideration of structure. As such, it provides a very useful
way of integrating an ever increasing database of conduction measurements, which will ultimately place
some constraints on structure. It is interesting to note
that in this case the authors, despite their conservative
approach to interpretation, could not resist the temptation to assign the three binding sites in the model to
three residues found in TM6, where mutations have been found to alter anion conduction.
The structural basis for anion conduction by CFTR
remains largely a mystery, but research such as that presented here represents the sort of systematic approach
to the biophysics of the permeation process that will be
central to any understanding of the physical basis for
anion entry and translocation. It will be of interest to
compare conduction across anion channel types and
separate out the general properties of an anion-selective pore from those that may be specific to an individual channel such as CFTR. A detailed analysis of the
conduction mechanism will also establish an important
standard that can be used to compare anion conduction by channels that are covalently modified (Akabas
et al., 1994; Riordan et al., 1989
; Rommens et al., 1989
), the
Cl channel function of CFTR has been studied in a
wide variety of expression systems. Macroscopic and single-channel currents have been measured and a
large number of mutant constructs containing either
single amino acid substitutions or major deletions have
been examined. It is ironic, however, that despite this
intense activity, there is little available in the published
literature in the way of a systematic characterization of
the conduction properties of this unique Cl channel.
In this issue of The Journal of General Physiology, John
Hanrahan and his colleagues make an important contribution to this question in the form of three papers in
which they report experimental and modeling results
pertaining to anion permeation. Although it seems unlikely that these papers will attract the same attention as
the cloning of farm animals or new approaches to gene
therapy, they contain information that is crucial to developing an understanding of the Cl permeation mechanism of CFTR; and the results extend the functional
fingerprint of the channel in a way that will be useful to
other investigators seeking to identify CFTR channels (e.g., in reconstituted systems).
; Riordan et al., 1989
; Rommens et al., 1989
; Anderson and
Welsh, 1992
; Gadsby and Nairn, 1994
), but the primary
structure provided no hint as to the nature of the anion
conduction path. There is no homology or internal
symmetry to suggest how the 12 membrane-spanning
segments might be arranged to form a pore, although the abundance of patient mutations provides clues as
to specific residues that might be important for pore
function (Anderson et al., 1991
; Sheppard et al., 1993
;
Nasr et al., 1996
). The difficulty in probing pore structure is compounded by the fact that CFTR, expressed
predominantly in epithelial cells, does not appear to be
a target for the sorts of highly specific toxins that have been so useful in deciphering the conduction properties of voltage-gated channels. Although several compounds have been advanced as blockers of the CFTR
pore (Sheppard and Welsh, 1992
; McCarty et al., 1993
; Linsdell and Hanrahan, 1996
), it nevertheless appears
that the best probes of the pore are, in fact, permeant
anions that must, by definition, enter, traverse, and interact with the conduction path. In this regard, the
study of CFTR conduction is well endowed as this channel shares with other anion channels the property of
permitting the permeation of a wide variety of inorganic and organic anions; e.g., Cl, Br, NO3, I, SCN, formate, and acetate. It seems likely that important keys to
understanding the conduction path are likely to
emerge from studies like those presented in the three
subject papers, in which behavior of permeating ions is
used to draw inferences about the physical nature of
the interaction of anions with the CFTR pore.
;
Bormann et al., 1987
; Anderson et al., 1991
). Some anions, however, most notably SCN
, tend to stick tightly
in anion channels such that ionic throughput is slowed
and conductance is reduced. Substitution of external Cl
by SCN
tends to produce a result that seems at
first to be anomalous. The reversal potential shifts to
more negative values consistent with PSCN/PCl of between 3 and 4, but the conductance is reduced to
~15% of that seen with Cl
. This behavior is perfectly
consistent, however, with that predicted for channels in
which the conduction pathway contains binding sites
with which anions associate transiently as they traverse the pore. It is useful (although not completely accurate) to think of permeability ratios determined from
shifts in reversal potential as measuring how easy it is
for an ion to leave some or all of its waters of hydration
and enter the pore. Conductance ratios, in contrast,
measure relative rates of anion throughput. As a consequence, the latter are very sensitive to block by a sticky anion, whereas permeability ratios, derived from zero
current measurements, are relatively insensitive to ion
binding. In a previous report from the Hanrahan laboratory (Tabcharani et al., 1993
), it was shown that
SCN
was apparently so sticky that at least two of these
anions could bind in the channel simultaneously, giving rise to the so-called "anomalous mole fraction effect" in which interionic repulsion due to multiple anion occupancy increases the throughput of sticky ions.
examine the permeability ratios for Cl
, Br
,
F
, and I
. The results are consistent with a "weak field
strength" sequence indicative of a major role for anion-water interactions in determining how readily halides may enter the channel. This conclusion seems simple enough in itself, but reaching it required that
the authors confront the rather puzzling behavior of
I
, which appears to be either more or less permeant
than Cl
, depending upon how the measurements are
made. The authors conclude that it is possible to distinguish two states of the channel, one in which conduction is partially blocked by I
, and another in which it is
not. The conversion between these two apparent states
exhibits an intriguing dependence on the polarity of
the membrane potential and the flow of Cl
. If I
is
present on one side of the membrane and Cl
on the
other, the rate of conversion is enhanced at potentials that drive Cl
into and I
out of the channel! Importantly, the conversion is associated with a dramatic
change in PI/PCl from 1.8 to <0.4, an observation that
may be relevant to the range of values of PI/PCl that
have been reported for CFTR. Conversion was not seen
at high ionic strength. Nor was it seen in R347D CFTR,
a construct previously shown to lack the anomalous dependence of channel conductance on the mole fraction of SCN characteristic of wild-type CFTR (Tabcharani et al., 1993
). Interestingly, however, the R347D
construct was characterized by an intermediate value of
PI/PCl (~1.0) and exhibited a reduced conductance in
the presence of I
as expected if this ion binds more
tightly than Cl
in the pore of the mutant protein. The
authors conclude that it may be possible for I
to occupy the pore by binding to a site in such a way as to
only partially obstruct Cl
flow. Clearly, a mechanistic
understanding of the behavior of I
will be an important clue to pore structure.
examine
permeability ratios for a series of polyatomic anions in
an attempt to estimate the size of the narrowest part of
the pore. By comparing permeability ratios for nitrate,
formate, pyruvate, propionate, gluconate, methane sulfate, ethane sulfate, and acetate, they arrive at an estimated diameter of ~5.3 Å, larger than the unhydrated diameter of Cl
, ~3.60 Å. The behavior of these ions is
consistent with a Hofmeister series; i.e., one governed
by hydration energy. Of particular interest in this paper
is the behavior of a mutant CFTR, T338A, T339A, in
which two threonines in TM6 were substituted with alanines. This construct exhibited markedly increased
permeability ratios for iodide and nitrate and apparent
permeation of propionate and pyruvate not seen in wild-type CFTR, leading the authors to speculate that these
two residues may be near a "selectivity filter." This result
is of interest because, in another study in which 11 mutants were compared, permeability ratios were found to
be relatively insensitive to point mutations in TM1, TM5,
and TM6 (Mansoura, M.K., S.S. Smith, A.D. Choi, N.W.
Richards, T.V. Strong, M.L. Drumm, F.S. Collins, and
D.C. Dawson, manuscript submitted for publication).
present a
simulation of some of the CFTR multi-ion behavior using a rate theory model. The model is derived primarily
from experiments in which block of CFTR channels by
gluconate applied to the cytoplasmic side was studied.
A strong interaction between gluconate and the trans
concentration of Cl
is modeled by allowing the pore
to be occupied simultaneously by gluconate and Cl
.
The I-V curves can be described by models that allow
for either two or three intrachannel, anion binding
sites. The two-site model was also used to simulate the
block of CFTR by SCN
, but a three-site model was
used to generate the anomalous mole fraction effect
previously reported, as well as the properties of the mutant, R347D, in which the anomalous mole fraction effect is lost. It will be interesting to extend these first
modeling efforts and determine the minimal requirements for single and multiple occupancy models that
are necessary to simulate permeation properties. For
example, can relief of gluconate block be simulated by
a single-site model and will alternate placement of sites
allow the two-site model to simulate anomalous mole
fraction effects?
), that result from the expression of truncated CFTRs (Sheppard et al., 1994
; Carroll et al.,
1995
), or that are observed when the channel, or parts
of it, are reconstituted in planar bilayers (Oblatt-Montal et al., 1994
; Ma et al., 1996
). Where there is a pore,
permeant ions will find it and their behavior will constitute its fingerprint.
.
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