From the Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6085
This is a wildly exciting period in the study of ion channels. Since the sequencing of Na+ and K+ channels was
accomplished (Noda et al., 1984 Missing from the list of identified functional parts is
the activation gate. The paper by Holmgren, Smith,
and Yellen in this issue of The Journal of General Physiology (Holmgren et al., 1997 Where should one begin to look for the gate? As
Holmgren et al. generously note, old experiments using TEA+ and analogues to block K channels (Armstrong, 1971 Fig. 1.
C9+ has a "head" group (the charged nitrogen and
three surrounding ethyl groups) and a hydrophobic
tail (nine carbons with their hydrogens). The head is
similar in size to a K+ ion with one hydration shell,
roughly 8 Å, and this is thought to make C9+ acceptable
to the inner end of the pore. C9+ blocks because, unlike K+, which is thought to dehydrate partially as it
moves into the narrow part of the pore (Hille, 1973 What happens when the gate tries to shut on repolarization? As might be expected, C9+ acts like a foot in
the door, and hinders closing of the gate. At The gate thus seems to be inside, but where is it
within the sequence? And where is the TEA+ binding
site? Choi et al. showed that C10+ binding is affected
not only by mutations in the P region but also by S6
(sixth transmembrane segment) mutations. The S6 mutation T469I, for example, greatly increases the affinity
of the channel for C8+ and C10+, almost certainly by improving interaction between the channel and the hydrophobic tail. Interestingly, the affinity increase is
largest with C8+, suggesting that C10+ is too large to fit
well in the mutated channel.
The wild-type Shaker channel, when exposed to a TEA+
analogue, shows all of the states seen in the cartoon of
Fig. 1 except the trapped state: the gate cannot close
until C10+ leaves the channel. Holmgren et al. have now
discovered that I470C, an S6 mutation, confers the ability to trap C10+. Presumably replacing isoleucine by a
smaller cysteine residue makes it sterically possible for
C10+ to reside in the inner vestibule of the closed channel.
Significantly, it is relatively easy (and easier than in
squid) to close the gate of this mutant even with C10+
trapped inside: the closed state was destabilized by only
~1.2 kcal/mol, less than the energy of a hydrogen bond.
This implies that C10+ occupies a cavity that changes little when the gate closes. The smaller TEA+ ion actually
stabilizes the closed state. Since TEA+ is similar in size
to a hydrated K+ ion, does this mean that the channel
normally closes with a K+ ion in the inner vestibule?
Overall, these fascinating findings make it almost irresistible to think that one or more flaps fold over the inner end of a commodious vestibule that can accommodate C10+ without much strain.
What are the flaps? Since the channel is a tetramer, it
is easiest to imagine there are four flaps. Where are
they, and how are they linked to S4, the voltage sensor?
One candidate is the S4-5 linker, an intracellular loop
of 13 amino acids. This region was tested for this role in
elegant fashion (Holmgren et al., 1996, Tempel et al., 1987
),
many of the functional parts of the channels have been located within the sequence. These include the pore
through which ions travel (P region: Guy and Conti,
1990
; Hartmann et al., 1991
; Yellen et al., 1991
), the
voltage sensor (S4, the positively charged fourth transmembrane segment: Stühmer et al., 1989
; Larsson et al., 1996
; Yang et al., 1996
) and the inactivation gate of
both K and Na channels (NH2 terminus of Shaker B:
Hoshi et al., 1990
; for the Na channel, the linker between domains III and IV: Stühmer et al., 1989
; West et
al., 1992
).
) and a preceding paper
(Holmgren et al., 1996
) give fascinating evidence about
the location of this gate if not yet a complete answer.
) suggested that the activation gate is inside and that the search should be in the intracellular
segments of the sequence. These experiments are summarized in Fig. 1. TEA+ and analogues block squid K
channels only when applied inside. Further, they are
"open channel blockers," meaning they have access to
their binding site in the pore only when the channel
gate is open. During a voltage step the number of open
K channels (measured as IK) normally rises to a steady
level (Fig. 1, upper trace). If C9+, an analogue of TEA+, is
present inside the axon, the channels then "inactivate"
as C9+ ions diffuse in and block them.
),
C9+ cannot shed its covalently linked ethyl groups. The
hydrophobic tail increases binding of the blocker in
the pore, the longer the tail the better, up to about 10 carbons (C10+; Choi et al., 1993
).
60 mV
few squid K channels close until C9+ has dissociated. At
100 mV, however, the closing pressure is very strong,
and the gates of about one-third of the channels apparently close at least partially before C9+ has dissociated.
The result is that C9+ is trapped in the channel and remains there for (relatively) a very long time. This trapping effect is easy to picture in terms of a gate at the
inner end of the channel, as shown in the cartoon. Altogether, the experiments favor the idea of a stable
channel with a gate at the inner end. The gate is the
major moving part.
) by labeling cysteine mutants with MTS reagents (Akabas et al., 1992
).
They found that labeling rates at some positions differed 4- to 10-fold between the open and the closed
state, showing that there is movement in this region
during opening and closing. Perhaps the S4-5 linkers
are the flaps, but conviction must await further experiments. We eagerly await the next chapter.