From the Department of Neurobiology, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts 02114
Ion channels don't like having their permeant ions
taken away. Particularly when they are in the mood to
let ions through, this sort of deprivation makes them
feel downright unstable. Like jilted lovers, they shut the
door and become listless, inactive(-ated), and sometimes
even immobilized. Channel physiologists have long tried
to ignore this dejected behavior on the part of the channels, even though for many years it has been clear that
channels often refuse to gate normally in unfriendly
ionic conditions. The best known such effect is the slowed
closing of channels in the presence of high concentrations of certain permeant ions, often called the "foot in
the door" effect (Yeh and Armstrong, 1978 Three recent papers in this journal highlight the different possible manifestations of such ion effects on
gating and emphasize their importance for a good biophysical understanding of the channel mechanisms.
Cloned voltage-activated Na+ channels show two distinct changes in gating when extracellular Na+ ions are
replaced by impermeant ions (Townsend et al., 1997 The second effect of reduced Na+ concentration is to
enhance Na+ channel slow inactivation (Townsend and
Horn, 1997 Both of these effects on Na+ channels have analogs
in the history of ion effects on K+ channels. For a number of K+ channels, reducing external [K+] can markedly reduce the open probability (Pardo et al., 1992 Townsend et al. (1997) To understand their results, it is necessary first to
know what the previous gating current measurements
have shown, in the absence of permeant ion interactions. Depolarizing voltage steps produce outward gating current ("on" currents) and the return voltage step
produces a restoring "off" gating current; the net charge
(integral of current × dt) for on and off charge movement is generally equal and opposite. For small or brief
depolarizations (too small to produce much channel
opening), the rate of off charge movement is quite rapid.
However, for larger and longer depolarizations that
would open the channels, the off charge movement becomes much slower. This has previously been interpreted to mean that the rate-determining opening step
involves a concerted motion of the channel subunits to
a particularly stable open state, which slows down the
return of the off charge (Bezanilla et al., 1994 Chen et al. (1997) Is there any physiological importance to these permeant ion effects on the gating of voltage-dependent
channels? With rare exceptions, such as the taste buds,
the concentrations of permeant ions around voltage-dependent channels in animal cells are carefully regulated by homeostatic mechanisms. This precludes most
of the ion effects that are provoked by the dramatic manipulations of channel biophysicists; e.g., when they
measure gating currents. However, this regulation can
fail to some extent, particularly in the case of extracellular [K+]: local accumulation during neuronal activity
can produce increases of [K+] up to 6-10 mM (Sykova,
1983 What can we learn from these effects about ion channel structure and mechanisms? The theme that pervades all three of the effects described here and almost
all the ion effects in the literature is that permeant ions
stabilize the open channel structure. Why is it so common for open pore stability to depend on permeant ions? Perhaps the most obvious explanation is that
these channels evolved to work under particular ionic
conditions; because charged permeant ions have substantial energetic interactions with the open channel
structure, these were included in the structural and energetic "design" of the channel proteins in the open
state. But why aren't the open channel structures "overstabilized"? Why don't they have a design safety factor
that allows pore stability to be maintained even under
stress? One can propose two teleological answers to this
question. First, the inherent instability or metastability of the open state produces a variety of inactivation
mechanisms, which apparently prove to be useful at
times. Second, if some of the interaction energy between the permeant ion and the channel is used to stabilize the open channel structure (à la Jencks, 1975). This type
of effect was first noticed for synaptic channels (Ascher et al., 1978
; Marchais and Marty, 1979
) and has been
studied in detail for a variety of K+ channels (Stanfield
et al., 1981
; Swenson and Armstrong, 1981
; Matteson
and Swenson, 1986
; Shapiro and DeCoursey, 1991
; Neyton and Pelleschi, 1992; Demo and Yellen, 1992
).
; Townsend and Horn, 1997
). The first change is that
their open probability is reduced, particularly for very
large depolarizations. This decrease is not seen with
normal concentrations of extracellular Na+ and is likely
to result from a voltage-dependent depletion of ions
from the pore. Only the highly permeant ions Na+ and
Li+ can prevent this effect, which does not depend on
the normal fast inactivation mechanism. In fact, it is
more prominent in a mutant with reduced fast inactivation. Townsend et al. (1997)
show that the ion-sensitive
steps are very rapid, and they argue that the most likely
explanation of the effect is that, in the absence of extracellular permeant ions, there is a rapid inactivation that
competes with the opening process.
). In low [Na+], the onset of inactivation is
faster and recovery is slower. In contrast with the rapid
ion effects on open probability, all of the alkali cations
tested can oppose slow inactivation, even the weakly
permeant K+ and Cs+ ions.
; López-Barneo et al., 1993
). It is not clear that these effects have the same rapid time course as the Na+ channel effects, but they too are proposed to result from
closed-channel inactivation. External [K+] can also modulate both onset and recovery from slow (C-type) inactivation (López-Barneo et al., 1993
; Levy and Deutsch,
1996
); the effects on the onset of inactivation are particularly marked under conditions where K+ efflux through
the channel is reduced by N-type inactivation or blockade (Baukrowitz and Yellen, 1995
, 1996
).
predict that the ion effects on
open probability may have significant effects on the
gating currents, since inactivated states often produce a
dramatic slowing or "immobilization" of gating charge
movement. Their prediction appears to have been borne out in the third recent paper on Shaker-family
K+ channels (Chen et al., 1997
). The problem with
studying ion effects on gating charge movement is that
to measure gating currents it usually is necessary to
eliminate ionic currents. This is generally done either
by removing permeant ions, adding blockers (which often act at the same sites as permeant ions), or using
"nonconducting" mutants (which by definition disrupt
some of the normal interactions of permeant ions with
the channel). Chen et al. (1997)
overcome this problem, partially, by measuring gating currents at or near the reversal potential for ionic currents in the presence
of low concentrations of K+ or the weakly permeant
ion Cs+.
; Zagotta
et al., 1994
).
find that in the presence of permeant ions this slowing of the off gating current is not
seen, even though it is clear that the channels indeed
are opening. This could be explained by supposing
that permeant ions just speed up the closing of the
open state, but it seems more natural to explain it by revising the original interpretation of the slow off currents. Chen et al. (1997)
propose that the original observation of slowing actually is due to a rapid inactivation process, which predominates when permeant ions
are absent (much like the rapid inactivation inferred by
Townsend et al., 1997
) in the absence of extracellular Na+. K+ ions cannot prevent the development of slow
off currents in the nonconducting W472F mutant of
Kv1.5 channels, which may be explained simply by the
inability of ions to interact with the nonconducting channel. If these interpretations are correct, they argue for considerable caution in combining information obtained
under widely varying experimental conditions (e.g., gating current measurements and ionic current measurements) to construct coherent models of channel gating.
), and the changes might be even more pronounced in ischemic conditions. These [K+] changes
may be enough to produce changes in open probability of certain K+ channels (Pardo et al., 1992
), particularly
in the rate and extent of C-type inactivation (López-Barneo et al., 1993
; Baukrowitz and Yellen, 1995
, 1996
).
),
the net binding energy of the ion will be weaker. Perhaps, like multi-ion occupancy, this allows the channel
protein to have strong and selective interactions with its
favorite ion without binding the ion so strongly as to
prevent rapid permeation.