From the * Research Unit Molecular and Cellular Biophysics, Max Planck Society, D-07747 Jena, Germany; C-type inactivation of Shaker potassium channels involves entry into a state (or states) in which the inactivated channels appear nonconducting in physiological solutions. However, when Shaker channels, from which
fast N-type inactivation has been removed by NH2-terminal deletions, are expressed in Xenopus oocytes and evaluated in inside-out patches, complete removal of K+ ions from the internal solution exposes conduction of Na+ and
Li+ in C-type inactivated conformational states. The present paper uses this observation to investigate the properties of ion conduction through C-type inactivated channel states, and demonstrates that both activation and deactivation can occur in C-type states, although with slower than normal kinetics. Channels in the C-type states appear
"inactivated" (i.e., nonconducting) in physiological solutions due to the summation of two separate effects: first,
internal K+ ions prevent Na+ ions from permeating through the channel; second, C-type inactivation greatly reduces the permeability of K+ relative to the permeability of Na+, thus altering the ion selectivity of the channel.
Inactivation in both sodium and A-type potassium channels (K+ channels) can be separated into two forms with
different putative mechanisms. Thus, in sodium channels, fast inactivation is sensitive to pronase (Armstrong
et al., 1973 On the other hand, previous studies have reported
conduction of normally impermeant monovalent cations (e.g., Na+ and Li+) through delayed rectifier K+
channels (Callahan and Korn, 1994 We propose here that conduction through Shaker
channels is determined by summation of two different
regulatory effects: first, there is the internal K+ ion effect that appears similar in both normal and C-type inactivated channels. This effect prevents significant permeation by Na+ or Li+ ions when K+ ions are present in
the internal solution. Second, there is an external site
that changes its conformation on entry into C-type inactivated states. This outer site is normally highly selective for K+ over Na+, but, during C-type inactivation,
the structurally modified outer site prevents permeation of K+ but not of Na+ ions. Thus, in normal physiological solutions, C-type inactivation results in Shaker
channels becoming functionally impermeable to all
available ion species, since the outer site becomes impermeable to K+ ions while internal K+ ions prevent
Na+ permeation.
Channel Expression
All studies reported here were carried out using the Shaker D 29-4 construct (Iverson and Rudy, 1990 Electrophysiology
All data reported here were obtained in inside-out macropatch
recordings (Hamill et al., 1981 Where appropriate, estimates of the effective voltage sensitivity
of transitions in the deactivation or reactivation pathways were
obtained by fitting time constant-voltage curves to the following relationship: As all experiments were done in the inside-out configuration,
only the intracellular bath solution could be changed in individual patches. Solution changes were carried out either (a) by exchange of the entire bath volume (taking ~1 min), or (b) via a
valve-operated quartz-capillary manifold where the pipette tip
was positioned in the manifold outlet. This second method permitted solution changes within ~100 ms. Great care had to be
taken in carrying out solution exchanges in order to ensure effective removal of residual K+ from earlier bath solutions, from
close proximity of the patch to the oocyte surface, or from cytoplasm adhering to a freshly excised inside-out patch.
Solutions
In addition to monovalent chloride salt, external solutions always
contained 1.8 mM CaCl2 and 10 mM HEPES, pH 7.2. Internal solutions contained 1.8 mM EGTA and 10 mM HEPES, pH 7.2. The
solutions were named according to the content of monovalent
cations. External (mM): Normal Frog Ringer (NFR),1 containing
115 NaCl and 2.5 KCl; K-Ringer, containing 115 KCl; Na-Ringer,
containing 115 NaCl; Li-Ringer, containing 115 LiCl; Tris-Ringer,
containing 115 TrisCl. Internal (mM): K-EGTA, containing 115 KCl; Na-EGTA, containing 115 NaCl; Tris-EGTA, containing 115 TrisCl.
Combinations of these monovalent cations were obtained by
appropriate mixing of these solutions. In the text and figures, the
solutions are only specified by the concentrations of the monovalent cations: external/internal solution.
Deletion of the residues that constitute the NH2-terminal "ball" domain has made it possible to generate
Shaker channel constructs with otherwise normal properties, but in which fast, N-type inactivation has been
completely eliminated (Hoshi et al., 1990
Fig. 1, A-C presents the previously characterized effects of extracellular K+ in modulation of the onset rate
of C-type inactivation. Fig. 1 A demonstrates the slow
onset of inactivation in symmetric K+ solutions as compared with the faster onset in more physiological media
(Fig. 1 B). Onset rate is further increased when K+ is removed from the external medium by Tris+ substitution
(Fig. 1C). The trace shown in Fig. 1C further suggests that steady state is approached after ~3 s at +40 mV.
Although the time course of C-type inactivation is voltage independent in physiological solutions, this inactivation rate cannot be fully characterized by single exponentials (López-Barneo et al., 1993 Fig. 1, D-F shows the effects of reducing internal K+
concentration on the onset rate of C-type inactivation
(note the expanded time base for these records). With
external cations substituted by Tris+ and internal K+
concentration reduced from 115 to 10 mM (Fig. 1 D)
and to 2.5 mM (Fig. 1 E), the inactivation time constant
further decreases. Complete substitution of internal K+
ions using symmetric 115 mM internal and external
Na+ solutions (Fig. 1 F) again decreases the inactivation time constant to ~30 ms, and outward INa inactivates to a steady state level of ~50% of peak INa at +40
mV. This steady state Na+ current is larger relative to
peak INa at less positive test potentials, although the observed current increases monotonically with increasing
test potential (data not shown).
Since these records were obtained using leak subtraction, the outward steady state INa must be occurring either through residual noninactivated channels or through
inactivated channels or some mix of normal and inactivated channels. Although this result shows that Na+ can
pass through Sh Effects of Test Pulse Duration on Na+ Tail Currents
Fig. 2 compares the development of C-type inactivation
during the test pulse with the changing time course of
inward Na+ tail currents using records obtained in
K+-free solutions. In Fig. 2 A, using symmetric 115 mM
Na+ solutions where the test pulse potential was +40
mV for direct comparability with Fig. 1 F, tail currents
show fast kinetics at the shortest test pulse duration (2 ms) when the repolarizing step is initiated on the rising
phase of the outward Na+ current. However, with increasing test pulse duration, and consequently increasing C-type inactivation, this initial fast tail current component is progressively reduced as a slower component
develops in the tail current decay. Thus, after a 64-ms
test pulse (the longest duration shown here), the fast
component entirely disappears and a pronounced rising phase (or "hook") becomes apparent in this tail
current.
In Fig. 2 B, the internal Na+ concentration was adjusted to 38 mM (plus 77 mM Tris) such that the test
potential (here +20 mV) would be close to the Na+ reversal potential. Thus, the outward current is primarily
gating current (IgON), permitting clear determination
that ionic conductance is the major contribution to
these tail currents (i.e., the ratio of the OFF/ON integrals is significantly greater than unity). As in Fig. 2 A,
at the shortest test pulse duration there is a fast initial
decay followed by a long slow relaxation component. As the pulse duration increases, the initial fast decay
decreases in magnitude while there is a corresponding
increase in the slow component. Disappearance of the
fast component at the longest test-pulse duration
shown here (64 ms) again reveals a substantial slow rising phase in the Na+ tail current. Noting that the loss
of the fast component corresponds to the time course
of C-type inactivation (Figs. 1 F and 2 A), the slow tail
current component appears likely to represent deactivation of a Na+ current that is occurring through C-type
inactivated channels.
However, it is also possible that noninactivated channels may change their deactivation rate for reasons unrelated to the coincident inactivation process, or that
inactivated channels become conducting during recovery from C-type inactivation. Finally, we have already
raised the concern that this very fast, non-N-type inactivation might be different in its mechanism from the
dominant C-type inactivation seen in more physiological solutions, despite the lack of any pronounced discontinuity in the rates seen in Fig. 1 between the slow
inactivation rate in symmetric K+ solutions (Fig. 1 A)
and records with reduced K+ concentration (Fig. 1, B-F ).
Effects of Test Pulse Duration on Tail Currents in the Presence
of External K+
To address the questions raised above, we repeated a
pulse-duration study using external solutions containing either 2.5 mM K+ plus 115 mM Tris+ (Fig. 3, A and
B) or 2.5 mM K+ plus 115 mM Na+ (Fig. 3, C and D). In
both cases, the internal solution was Tris-EGTA (see
MATERIALS AND METHODS). Fig. 3, A and C shows superimposed current traces yielding various degrees of inactivation at the end of the depolarization. Fig. 3, B and D
shows the tail currents on an expanded time base and
aligned in time to the end of the depolarizing pulse. In
Fig. 3 A, with no external Na+, it is clear that the tail
currents are fast at all prepulse durations and are markedly reduced coincident with the onset of C-type inactivation (visible from the decay of the inward K+ current
at
One hypothesis would be that the entire slow tail currents of Fig. 3, B and C are being generated by K+ permeation through channels that recover rapidly from
C-type inactivation at negative potentials in the presence of external Na+ ions, but which do not recover in
the absence of external Na+ (Fig. 3 A). This hypothesis
predicts that the slow tail currents should return to
baseline only when channels fully recover from C-type
inactivation. Thus, recovery should be complete within 0.5 s at It might seem reasonable, therefore, to presume that
these tail currents represent Na+ permeating through
C-type inactivated channels, in the absence of blocking
effects that would normally be generated by intracellular K+ ions (Callahan and Korn, 1994 Ion Permeation through C-Type Inactivated Channels in
K+-free Internal Solutions
The central hypothesis to be evaluated in this section is
that Shaker channels change their relative permeabilities for K+ and Na+ as they enter into the C-type inactivated state(s). Thus, we question whether they progress
from their normal K+-selective state into a C-type state
in which they are no longer permeant to K+ ions, although they remain permeable to Na+ ions (as long as
the blocking action of intracellular K+ can be prevented, see below). If this hypothesis is correct, and if
channels are provided with an external solution that
contains both K+ and Na+ ions, then noninactivated
channels would be primarily K+ conductors, whereas
inactivated channels should be either nonconducting or Na+ conducting, depending on the presence or absence, respectively, of K+ in the internal solution. This
prediction could be tested if it were possible to report
which ion species were the primary current carriers at
different times during a long, inactivating test pulse.
We have explored this possibility by evaluating the instantaneous current-voltage relationship in carefully
chosen internal and external solutions. With an external solution containing 2.5 mM K+ and 115 mM Na+
and a K+-free internal solution (also containing 115 mM Na+), the reversal potential for K+-conducting
channels should be >+50 mV, whereas the reversal potential for Na+-conducting channels should be close to
0 mV. Thus, after a test pulse to The standard instantaneous current-voltage (I-V) procedure is demonstrated in Fig. 4 A, in which "tail" steps
to both +40 and
Unfortunately, problems occur in using the simple
approach described above for careful, quantitative studies. Long interpulse intervals are required to prevent
accumulation of C-type inactivation and it is not easy to
complete the number of pulses required to obtain accurate measures of reversal potentials at both short and
long times in the same patch. This method was, therefore, modified by inserting voltage ramps (between Fig. 4 B, a and b indicates the results obtained in the
solutions described above, after a 20-ms prepulse to
Quantitative errors in this data set could arise from
two principal sources. First, in view of the low external
K+ concentration (2.5 mM), any significant accumulation of internal K+ ions during the long prepulse to
Finally, it may seem surprising, initially, that the reversal potential at long times does not move closer to 0 mV than ~+25 mV. However, the relative permeability
of Na+ versus K+ (PNa/PK) is normally ~0.02 in Shaker
channels; moreover, PNa falls rather than increases during C-type inactivation (see Fig. 1 F). Thus, even if 98%
of the channels become inactivated, Na+ flux through
those channels would be expected to be not greater than the K+ flux occurring through the remaining 2%
of noninactivated channels. Hence, the reversal potential data suggest that, at long test pulse durations, an
equilibrium becomes established between normal and
C-type inactivated channels such that some very small
fraction of residual noninactivated channels remains,
even after 10 s at Conduction through C-Type Inactivated Channels Is Blocked
by Internal K+
We next demonstrate the blocking of ion conduction
through C-type inactivated channels by internal K+
ions. Fig. 5 explores the effects of washout of K+ ions
from the internal medium. Fig. 5, i was obtained using
Li-Ringer (see MATERIALS AND METHODS) as the external solution and with 5 mM of K+ in the Tris+-substituted internal solution. Test pulse duration was 40 ms, which is long enough to produce considerable C-type
inactivation in K+-free solutions (see Fig. 1 F ). However, with 5 mM of K+ in the internal solution, it is clear
that little C-type inactivation takes place during this
short pulse and IgOFF is clearly seen here as part of the
tail currents (see arrow). The remaining traces (Fig. 5,
ii-iv) show successive records obtained during slow
washout of the internal solution with K+-free Tris-EGTA solution. As the internal K+ concentration falls,
the peak outward current also falls and the fraction of
channels that become C-type inactivated within the 40-ms duration of the test pulse increases (as assessed
from [Ipeak
Activation and Deactivation in C-Type Inactivated Channels
We have seen that Sh This question is addressed in Fig. 6. In Figs. 6 A and,
particularly, B, where the same experiments are presented on a faster time base, it is apparent that the rising phase (or hook) follows an initial increase in inward current resulting from a change of driving force
before the settling of the voltage clamp. After the
clamp has settled (in <200 µs), we see a slower increase in tail current amplitude that we presume reflects either some process specific to the C-type inactivated state, relief of divalent ion block, or, alternatively,
a rapid initial component of recovery from C-type inactivation. Further work will be required to distinguish between these possibilities.
The rising and falling phases of these tail currents
were analyzed by fitting double exponential functions
to leak-subtracted data. In Fig. 6 C, the rising and falling phase time constants, Recovery from C-type inactivation is not complete for
at least 5 s at
The voltage sensitivity of reactivation in C-type inactivated channels is addressed in Figs. 7, B and C. In Fig. 7
B, the depolarization was varied from Fig. 7 A has shown effects of changes in external solutions on rates of recovery from C-type inactivation. We
next explore the more complex effects that can arise after changes in internal solutions as a result of combinations of internal K+ block and changes in the onset and
recovery rates of C-type inactivation. Fig. 8 shows six
consecutive traces obtained from the same patch, separated at 15-s intervals. In all cases, the external solution was Tris-Ringer. Fig. 8 A, i and ii was obtained using 114 mM Na+ and 1 mM K+ in the bath, which was then
changed to a K+-free, 115 mM Na+ solution for Fig. 8 B,
i and ii. Finally, the internal solution was switched back
to 114 mM Na+ and 1 mM K+ for Fig. 8 C, i and ii. In
Fig. 8 A, i, in the presence of 1 mM K+, a fast C-type inactivating outward K+ current is visible, followed by a
small but detectable steady state current. Recovery
from C-type inactivation is fast enough, in the presence
of internal K+ ions, for Fig. 8 A, ii to show almost complete recovery of the initial outward K+ current, although some accumulation of inactivation is noticed despite the 15-s interpulse interval. After washout of internal K+, Fig. 8 B, i shows an initial fast inactivating
Na+ current followed by a substantial steady state current. However, in Fig. 8 B, ii, we see that recovery from
C-type inactivation has been markedly slowed in the absence of internal K+, and there is no evidence of any recovery of the fast decaying initial current in this trace.
By contrast, Fig. 8 B, ii now shows the slow rising phase
of activation characteristic of C-type inactivated channels (see Fig. 7 B). During reapplication of 1 mM internal K+, recovery from C-type inactivation is accelerated
but still appears incomplete in Fig. 8 C, ii, taken ~30 s
after the solution exchange.
Thus, Shaker channels appear capable of both opening
and closing while remaining C-type inactivated, although
with altered kinetics and perhaps through different pathways than those used by normal, noninactivated channels.
Previous studies of C-type inactivation in Shaker channels have clarified the functional differences between
this slow inactivation mechanism and the mechanism
of fast, N-type inactivation (Hoshi et al., 1990 The critical evidence required to reach this conclusion has involved demonstrating, first, that the rapid inactivation found using K+-free, Na+-substituted internal
and external solutions has the characteristic properties
of the C-type inactivation found in normal physiological solutions and, second, that the Na+ permeation
found under these conditions is occurring through inactivated channels rather than through a population of
normal channels in equilibrium with the inactivated
population. A residual population of noninactivated
channels must be present at negative test potentials, although C-type inactivation appears to become almost
complete at potentials >+20 mV. However, previous
work has shown that the same impermeant ions will
also pass through normal, noninactivated K+ channels
provided that K+ is first removed from the internal solution (Callahan and Korn, 1994 Nevertheless, the rapid non-N-type inactivation occurring in K+-free solutions could be associated with
some alternative inactivated state, rather than with the
classic, slow onset C-type states that have been characterized in physiological internal and external solutions.
Therefore, we demonstrate that equivalent tail current waveform changes occur during entry into C-type states
in more physiological solutions (Fig. 3) and that these
changes in tail current kinetics are correlated with coincident changes in PK, such that C-type inactivated
channels become effectively impermeant to K+ ions (in
all solutions). However, in K+-free internal solutions,
channels remain permeable to Na+ and Li+ as they enter into C-type inactivated states (Fig. 4), indicating that
the lack of permeation by these ions in physiological solutions arises from internal K+ block (Fig. 5). We note
that large inward Na+ tail currents remain unblocked
by 2.5 mM external K+ (see Fig. 3, C and D) and suspect
that access to this blocking site may prove to be substantially asymmetrical, although further and more quantitative studies will be required to confirm this point.
The Na+ tail currents that follow inactivating prepulses indicate that channels must close within ~500
ms even at In the nonstandard solutions used here, deactivation
and reactivation of channels that have entered C-type
states generates the expected changes in macroscopic
currents, albeit with modified kinetics. By contrast, in
physiological solutions C-type deactivation and reactivation will be characterized only by gating currents. Similarly, single-channel records obtained in physiological
solutions would be expected to show only entry into or
escape from long "closed" states, although such closing
would be a consequence of changed permeation properties rather than of true channel closure. Unfortunately, the very low permeation for Na+ and Li+ during
C-type inactivation will make it difficult to provide a direct test of this conclusion from single-channel records.
It seems mechanistically significant that C-type inactivation develops with time constants that vary from seconds (in symmetric 115-mM K+ solutions) to tens of
milliseconds (in symmetric 115-mM Na+ solutions). In
view of this wide range of rates, we speculate that C-type
inactivation is an intrinsically fast process (with a time
constant in the range of microseconds) that is modulated by binding of K+ ions (and to a lesser extent by
Na+ ions). For example, activation of the charge-carrying S4 segments could initiate both channel opening
and a cooperative process, which, if unopposed, will
trigger conformational changes in the region of the external "filter," thus reducing the permeability of the
channel to K+ ions. Presuming this conformational
change is prevented by the presence of a K+ ion at this
external site, the rate of C-type inactivation would be
determined by the availability of K+ ions in this region,
becoming faster as K+ concentration is reduced. Since
this site lies in the permeation path, it should be accessible from both external and internal sides of the membrane, in open, conducting channels, and internal K+
might be expected to substitute for external K+, as shown
in Fig. 1. Furthermore, this hypothesis predicts that C-type
inactivation will be fast in symmetric Na+ solutions, faster
in Tris/Na solutions (see Fig. 7), and presumably even
faster (or not measurable) in Tris/Tris solutions.
The reduced efficacy of other ion species in slowing
C-type inactivation could be due either to the reduced
dwell time of these ions or to their reduced ability to
slow the external conformational change. The occupancy hypothesis (Matteson and Swenson, 1986 In conclusion, we restate the hypothesis that seems to
provide the most elegant explanation of the results obtained in the present study. Permeation, in Shaker channels, is primarily determined by the properties of the
externally located "selectivity filter," which is normally
highly selective for K+ over Na+ ions. However, during
C-type inactivation, this external filter changes its properties; i.e., greatly reducing K+ permeability while leaving Na+ permeability relatively unchanged. We presume that this change in relative permeabilities is the
primary mechanism of the "inactivation" seen in C-type
inactivated states. Nevertheless, Na+ permeation does
not occur in C-type inactivated states in physiological solutions, due to blocking actions of internal K+ ions.
Removal of K+ from the internal solution thus permits
limited permeation of the channel by ions such as Na+
and Li+ and, in a more general sense, permits direct
evaluation of the selectivity of the external filter.
Address correspondence to Stefan H. Heinemann, Research Unit
Molecular and Cellular Biophysics, Max Planck Society, Drackendorfer Str. 1, D-07747 Jena, Germany. Fax: 49 3641 304 542; E-mail:
ite{at}rz.uni-jena.de Received for publication 11 July 1997 and accepted in revised form 5 September 1997.
We thank K. McCormack for providing us with Sh
Békésy Laboratory of Neurobiology,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
ABSTRACT
INTRODUCTION
), although slow inactivation is not (Rudy,
1978
). Further, the simple kinetics and low independent voltage sensitivity of fast inactivation in Na+ channels lead Armstrong and Bezanilla (1977)
to suggest
their "ball and chain" hypothesis. This hypothesis has
since been confirmed as the mechanism of fast (N-type)
inactivation in Shaker K+ channels at the structural level
(Hoshi et al., 1990
; Zagotta et al., 1990
). However, the
mechanisms underlying the slower, pronase-resistant inactivation processes (Hoshi et al., 1991
) are less well understood, although recent evidence has clarified that
structural changes occur in the outer mouth of the
Shaker channel during this slow (C-type) inactivation
(Liu et al., 1996
; Schlief et al., 1996
). These changes
were interpreted as indicating a collapse of the external channel mouth, which occludes the ion permeation
pathway.
; Ikeda and Korn,
1995
) after removal of K+ ions from both internal and
external solutions. Similarly, Na+ permeation has been
reported through noninactivated Shaker channels (Ogielska and Aldrich, 1997
) after K+ ions have been removed from the internal solution. The present study
extends these observations to include permeation of
Na+ and Li+ ions through C-type inactivated Shaker
channels. Thus, we demonstrate that even channels
that have become impermeant to K+ ions are capable
of conducting normally impermeant monovalent cations after the careful removal of internal K+. If C-type
inactivated channels can conduct these ions in K+-free
internal solutions, then the mechanism of C-type inactivation is unlikely to involve a structural collapse affecting the conduction pathway. Moreover, the finding
that permeation of Na+ or Li+ can be blocked by submillimolar concentrations of internal K+ ions suggests
the presence of a high affinity K+ binding site at the internal surface of the permeation path, where K+ binding contributes to the blockade of "impermeant" ion
species.
MATERIALS AND METHODS
) in which fast, N-type inactivation has been removed by deletion of the residues 2-29 (McCormack et al., 1994
; this construct will be referred to as Sh
). Oocytes of Xenopus laevis were surgically obtained under tricaine/ice
water anesthesia. Follicular layers were removed according to
standard methods. mRNA was synthesized in vitro and was injected into Xenopus oocytes at a concentration ranging from 0.005 to 0.1 µg/µl (total volume ~50 nl per oocyte). Oocytes
were incubated at 18°C for 1-7 d before electrophysiological recordings.
) using either an EPC-7 or EPC-9 patch clamp amplifier (HEKA Elektronik, Lambrecht, Germany). Patch pipettes were fabricated from aluminum silicate
or borosilicate glass, yielding resistances in standard solutions
between 0.5 and 2 M
. Data acquisition was controlled with
the Pulse+PulseFit software package (HEKA Elektronik). Experiments were carried out at room temperature ranging between 20 and 22°C. Data analysis was performed with PulseFit,
PulseTools (HEKA Elektronik) and IgorPro (Wave Metrics, Lake
Oswego, OR) software. Leak and capacitive transients were compensated using a variable P/n correction (Heinemann et al.,
1992
) with a typical leak holding potential of
120 mV. In
several cases, leak and capacitive transients were eliminated by
off-line leak correction methods. Unless otherwise stated, data
values are specified as mean ± SD (n = number of independent experiments).
(V) =
(0) exp (±Vq/kT), where
(V) is the observed time constant at the applied transmembrane voltage, V; k
is Boltzmann's constant, T is the absolute temperature, and q is
the apparent charge moved between the appropriate thermodynamic energy well and the energy barrier for the transition in
question.
RESULTS
). However, although the onset of C-type inactivation is extremely
slow in high K+ extracellular media, this onset rate is
accelerated by reducing extracellular K+ concentration
or by substitution with other extracellular monovalent cations following the order K+ < Rb+ << Na+, Cs+,
NH4+ (López-Barneo et al., 1993
; Levy and Deutsch,
1996
). Onset of inactivation is further accelerated
when internal as well as external K+ concentration is reduced by impermeant ion substitutions. Thus, even
channels in which N-type inactivation has been removed by deletion of the NH2-terminal ball domain
can inactivate in K+-free media with a time course in
the tens of milliseconds range in symmetric Na+ solutions (see Fig. 1 F ). This rate is approximately equivalent to the rate of normal fast N-type inactivation in
physiological solutions and is almost three orders of
magnitude faster than the rates of C-type inactivation
in high external K+ media. Clearly, a question that
must be addressed here is whether this very fast inactivation process shares a common mechanism with the
slow C-type inactivation previously characterized in more physiological solutions.
Fig. 1.
C-type inactivation rate is affected by
changes in external (A-C) and internal (D-F) cation concentrations, as shown using inside-out
macro-patch recordings from Sh channels. The
potential profile is indicated at the top and the
current traces were digitally low pass filtered at 1 kHz. (A) K-Ringer versus K-EGTA. (B) NFR versus
K-EGTA. (C) Tris-Ringer versus K-EGTA. (D) Tris-Ringer versus 10 mM KCl, 105 mM TrisCl, 1.8 mM
EGTA. (E) Tris-Ringer versus 2.5 mM KCl, 112.5 mM TrisCl, 1.8 mM EGTA. (F ) Na-Ringer versus
Na-EGTA. Single-exponential fits (see text) resulted in the following time constants: 9.3 s, 1.6 s,
550 ms, 520 ms, 120 ms, and 31 ms in A-F, respectively. In F, the current reached a steady state level
that was 50% of the peak current.
[View Larger Version of this Image (19K GIF file)]
; Meyer and Heinemann, 1997
). An initial, faster component of inactivation is particularly noticeable in Fig. 1, A-C. Nevertheless,
after the decay of this initial component, single time
constants well fit the remaining inactivation time course
and indicate the sensitivity of this slow inactivation mechanism to extracellular K+ ions (for time constants, see
figure legend).
channels (after removal of internal
K+), it is not yet clear that inactivated channels are involved in generation of these steady state Na+ currents.
We have addressed this important question through
several different approaches.
Fig. 2.
Tail current kinetics change as C-type inactivation develops (A and B). (A) Recordings obtained in symmetric (115 mM)
Na+ solutions, using test pulses (to +40 mV) of differing durations demonstrate that development of C-type inactivation parallels significant changes in tail current waveform. After short depolarizations, they are dominated by a rapid component associated with
deactivation of noninactivated channels, followed by a small slow
ionic component. This slow ionic current component increases
with increasing depolarization duration. Finally, a rising phase
(hook) in the onset of the slow ionic current becomes apparent
while the initial, fast peak disappears as C-type inactivation approaches steady state. (B) Recordings obtained under ionic conditions where the estimated reversal potential for Na+ (115 mM versus 38 mM) nearly matches the potential of depolarization (+20
mV) such that IgON becomes clearly visible. Upon increasing duration of the depolarizing pulse segment, the tail currents undergo
kinetic changes similar to those seen in A. Data traces were low
pass filtered at 4 kHz.
[View Larger Version of this Image (31K GIF file)]
20 mV). These tail currents are reduced to little more than the expected magnitude of IgOFF by the time
C-type inactivation reaches a steady state. Clearly, channels do not conduct K+ ions to any significant extent in
the C-type inactivated state. By contrast, if 115 mM Na+
is added to the external solution, as in Fig. 3 C, two effects are seen. First, fast tail currents are visible at
shorter prepulse durations, but are gradually replaced
during the course of C-type inactivation by slow tail currents that no longer diminish in size. In Fig. 3 D we see
that these tail currents develop the characteristic rising
phase with a time course similar to that of C-type inactivation. This relationship between the development of
C-type inactivation and the increase in the slow tail current peak is shown to be linear in Fig. 3 E. Second, Fig.
3 C shows a substantial increase in the inward steady
state current in the presence of external Na+ ions.
Fig. 3.
Dependence of inward tail current
waveforms on the development of C-type inactivation, without (A and B) and with (C and D) addition of external Na+ ions. Inward currents were
measured in the absence of intracellular permeating ions (Tris-EGTA in the bath), during pulses to
20 mV in external 2.5 mM K+ plus 115 mM Tris
(A and B) or in NFR containing 2.5 mM K+ plus
115 mM Na+ (C and D). In these solutions, channels inactivate with a time constant of ~1 s. In the
absence of Na+ (A), the tail currents upon repolarization to
120 mV are rapid and decrease in
magnitude as the degree of inactivation increases (see B). In the presence of external Na+ (C), however, tail currents are only rapid after very brief
depolarizations. After longer depolarizations (i.e.,
as C-type inactivation occurs), tail currents slow
down and do not decrease in size. D shows the tail
current sections of C, aligned in time to the end
of the test depolarization. Note the 10-fold
change in time scale compared with B. E shows an
analysis of data from C and D, demonstrating a
linear relationship between the fraction of channels that have entered into C-type inactivation
and the peak magnitude of the slow component
of Na+ tail currents.
[View Larger Version of this Image (28K GIF file)]
100 mV return potential. By contrast, we find
that recovery from C-type inactivation is still incomplete after a 3-s return to
100 mV, even in 115 mM external Na+ solution (data not shown).
; Ikeda and Korn,
1995
). In the next section, we address the question as to
whether or not any change in the permeating ion species
occurs coincident with the onset of C-type inactivation.
20 mV, either steps to
appropriate return potentials or voltage ramps can be
used to evaluate the reversal potential seen at different
test pulse durations. In either case, it should be possible to demonstrate whether or not the hypothesized
change in relative permeabilities occurs, as C-type inactivation develops during the test pulse. The predicted
result will be a progressive left shifting of the reversal
potential, from ~+50 mV towards less positive potentials, coincident with development of C-type inactivation.
40 mV were applied after the patches
were depolarized to
20 mV for either 200 ms or 3 s.
After the short (200-ms) test pulse in which ~10% of
peak inward current has become C-type inactivated, the
step to
40 mV generates a composite tail current with
both fast and slow components, whereas the step to
+40 mV reaches the zero current baseline, as if the effective reversal potential is ~+40 mV at this early stage
in the onset of C-type inactivation. However, after the
long (3-s) test pulse, in which some 90% of peak current has been eliminated by C-type inactivation, the tail current in the +40-mV step is now clearly outwardly directed, whereas the tail current is still inward at
40
mV. As a first approach, linear interpolation between
these outward and inward tail currents suggests a reversal potential of ~+15 mV at this time. Comparing
these tail currents imposed at different points in the
development of C-type inactivation suggests that the reversal potential changes in the direction predicted by
our hypothesis.
Fig. 4.
Shaker channels become less permeable to K+ ions
and relatively more permeable to
Na+ ions when C-type inactivated. (A) Superposition of four
current traces recorded in NFR
versus Na-EGTA; i.e., in symmetrical 115 mM Na+ but with 2.5 mM K+ added to the external solution. Pairs of traces were recorded with a depolarization to
20 mV for 200 ms and 3 s leading to quite different degrees of
inactivation at the end of the depolarization. Subsequently, the
potential was stepped to either
40 or +40 mV. After the short
depolarization, the subsequent
step to +40 mV shows only marginal outward current, the same
step after almost complete inactivation results in outward currents that are ~50% of the size of the inward currents obtained at
40 mV,
indicating an enhanced relative Na+ permeation after development of C-type inactivation. (B) Demonstration of the protocol used for collection of instantaneous I-V data at two different durations within a
20-mV test depolarization. Ramp potential changes of 40-ms duration
were imposed after 20-ms (B, a and b) and 10.04-s (B, c and d) test pulses. Ramps were upward (from
20 to +70 mV) in B, a and c, and
downward (from +70 to
20 mV) in B, b and d. See text for further description of this protocol. (C) Reversal potentials obtained from instantaneous I-V data are plotted as functions of the degree of C-type inactivation that had occurred before starting the ramps. Results are
shown for eight patches from which full data sets were collected. Straight lines connect data points from the same patch. Upward ramp
data points are shown by open symbols and connected by solid lines, downward ramp points are shown by filled symbols and connected by
dashed lines.
[View Larger Version of this Image (19K GIF file)]
20
and +70 mV) in which the instantaneous I-V data could
be directly visualized within the 40-ms time course of
each ramp. Additionally, both increasing (
20 to +70
mV) and decreasing (+70 to
20 mV) ramps were
used to evaluate any possible directional artifacts in this method. These voltage-ramp protocols are illustrated
in Fig. 4 B, which shows four instantaneous I-V plots that
were obtained from a single patch.
20 mV and (Fig. 4 B, c and d) after a 10.04-s prepulse.
Note that traces c and d do not show the complete time
course of the current record, a 10-s interval was inserted in the pulse paradigm, where indicated, in which
data traces were not digitized, yielding a more faithful P/n leak correction (Steffan et al., 1997
). Upward ramps
from
20 to +70 mV are shown in Fig. 4 B, a and c,
while downward ramps from +70 to
20 mV are shown
in traces b and d (after a depolarizing step from the
20-mV test potential to +70 mV, and a brief preramp delay of 10 ms). Voltage protocols are shown above the
current records in each panel. Thus, for each test pulse
duration, the observed reversal potential could be evaluated from the leak-subtracted currents seen during
both upwardly and downwardly directed voltage ramps.
In the patch shown here, after the 20-ms prepulse, reversal potential was +52.2 or +43.5 mV depending on
ramp direction (Fig. 4 B, a and b), whereas after 10.04 s,
the corresponding values were +27.1 and +22.6 mV
(Fig. 4 B, c and d). Fig. 4 C shows the cumulative data
obtained from eight different patches using ramps in
both directions, plotted against the degree of C-type inactivation obtained by the depolarization to
20 mV
before the start of each ramp protocol. In this figure,
the mean reversal potential shifts to the left by ~26 mV
with increasing C-type inactivation, as predicted by the
hypothesis stated above.
20 mV might be expected to lower the reversal potential at long times. However, such K+ accumulation
would also block Na+ permeation (see below), thus obscuring the major shift of reversal potential predicted
by the data shown in Fig. 4. To address this problem,
inside-out patches were positioned in the direct path of
a pipette perfusing the Na-EGTA internal solution so as
to minimize any possible accumulation of internal K+.
In addition, any effect due to K+ accumulation should
be much smaller in protocols using downward ramps.
Second, changes in the extent of steady state C-type inactivation could occur during voltage ramps between
20 and +70 mV, altering the fraction of noninactivated channels remaining after long prepulse duration.
This problem was minimized by using short (40 ms)
ramps with only a short delay of 10 ms at +70 mV before the start of the down ramp. The estimated reversal
potentials in the absence of inactivation at the start of
the ramp protocol were 51.4 ± 1.5 mV for upward
ramps and 44.8 ± 1.1 mV for downward ramps, indicating that this difference is indeed caused by additional inactivation induced by the preramp delay of 10 ms at
+70 ms used for the downward ramps. Nevertheless,
the measured shifts of the reversal potential as a function of inactivation are highly significant for both protocols. The inactivation-induced reduction in reversal potential between partially and completely inactivated
channels was 26.2 ± 2.0 mV for downward ramps (Fig.
4 C, closed symbols) and 25.8 ± 2.5 mV for upward ramps
(Fig. 4 C, open symbols), providing strong confirmation
for the hypothesis presented above.
20 mV.
I40 ms]/Ipeak). When the outward K+ current has been reduced by approximately one order of
magnitude (see Fig. 5, iii) compared with the original
record in 5 mM K+, the Li+ tail current starts to show
the slow rising phase as seen in Fig. 2, which is characteristic of tail currents through C-type inactivated channels. A further increase in the slow tail peak occurs in
trace iv, associated with additional loss of outward K+
current and presumably a further reduction in internal
K+ concentration.
Fig. 5.
Influence of intracellular K+ on the tail currents. The
first trace (i) was recorded with external Li-Ringer (same results would be obtained with Na+ instead of Li+ as permeating ion, not
shown) and an internal solution containing 5 mM K+ plus 110 mM
Tris+. The arrow points to an inflection in the tail currents indicating two components. The following traces (ii-iv) were recorded
while the internal K+ ions were slowly washed out by Tris-EGTA solution. As the outward currents disappear, due to removal of internal K+ ions, the tail currents become slower and larger, indicating increased Li+ permeation.
[View Larger Version of this Image (16K GIF file)]
channels can show steady state
conductance after entering into C-type inactivated states
(Figs. 1 F, 3 C, and 4) and that they can generate slow
tail currents while apparently deactivating in C-type inactivated states (Figs. 2 and 3 D). At these negative return
potentials, Na+ current decays to zero. Since the duration of the hooked tail currents (<500 ms) is at least
one order of magnitude less than the time required for
full recovery from C-type inactivation (>5 s), we presume that these channels are deactivating to a closed C-type state. On the other hand, the "closed C-type state"
reached in this apparent deactivation may not be the
same as the normal closed state, and the hook in these
tail currents implies an additional process occurring
before the channel closing step. Furthermore, the fast
tail currents of noninactivated channels fail to provide any kinetic evidence to indicate a corresponding "preclosing" transition. Is the deactivation of C-type inactivated channels in any way comparable with the deactivation of noninactivated channels?
Fig. 6.
Deactivation of
C-type inactivated channels. (A
and B) Tail currents were recorded after depolarizations to
+20 mV for 100 ms at tail potentials between 170 and
80
mV using 10-mV steps. Representative traces are shown in A
and B using different time scales to demonstrate the voltage sensitivity of deactivation in
A and of the hooked rising
phase in B. The recording
bandwidth was 4 kHz. The time
constants of both, the hooks
(
Cd1) and the deactivation
(
Cd2), were estimated by fitting double-exponential functions to the data. The mean
time constants resulting from six experiments are plotted in C as a function of the tail potential. The filled symbols indicate those data
points that were considered for the determination of the slopes (straight lines) that yielded a voltage dependence for both processes corresponding to effective gating valences of 0.51 (
Cd1) and 1.05 e0 (
Cd2).
[View Larger Version of this Image (19K GIF file)]
Cd1 and
Cd2, respectively, are
plotted on a logarithmic scale against return potential.
At the most negative return potentials, the voltage sensitivities of the two time constants in Na/Na solutions correspond to an apparent charge movement of 0.51 ± 0.05 and 1.05 ± 0.03 e0 for
Cd1 and
Cd2, respectively
(see MATERIALS AND METHODS). The estimated time
constants at 0 mV were 6.1 ± 1.5 s and 884 ± 134 ms
for
Cd1 and
Cd2, respectively (n = 6). Under asymmetrical conditions (Na/Tris), the voltage sensitivities were
0.53 ± 0.08 and 1.13 ± 0.11 e0, and the estimated time
constants at 0 mV were 13.5 ± 6.2 s and 5.2 ± 2.7 s
(n = 5). Interestingly, the slopes for
Cd2 are similar to
reported values for normal tail currents (Zagotta et al.,
1994
a), although the time constants are considerably slower than is normal for Shaker tail currents at similar
potentials. These results suggest that both rising (
Cd1)
and falling (
Cd2) transitions are driven by voltage-sensitive processes.
100 mV in K+-free solutions, although
tail current deactivation closes all channels within 500 ms. Therefore, it seems reasonable that these channels
might reactivate, although to the reduced permeability characteristic of depolarized C-type states, when a second test depolarization is applied before significant recovery from C-type inactivation has occurred. As demonstrated in Fig. 7 A, recovery from C-type inactivation
is affected by the ionic content of the external solution
(the internal solution was 115 mM Na+ for both traces).
For the upper trace, where the external solution also
contained 115 mM Na+, partial recovery is visible after
a 500-ms return to holding potential. By contrast, in the
lower trace obtained with Tris-Ringer as the external
solution, there was no evident recovery from C-type inactivation during this same interpulse interval. Note
that in this lower trace, the second depolarization produces an apparently monoexponential approach to the
same steady state current level as seen in the first pulse.
We conclude that this second depolarizing step induces
reactivation of channels that are still C-type inactivated and, since they activate to the same level as the steady
state current, it appears that these channels remain
C-type inactivated despite this activation process. Thus,
in appropriate conditions, channels can both close (Fig.
6) and reopen (Fig. 7) without necessarily recovering
from C-type inactivation.
Fig. 7.
Activation of ionic
current through C-type inactivated channels. Recovery from
C-type inactivation is affected
by external cations (A). Double-pulse experiments were
performed in the indicated solutions. The duration of the interpulse interval at 100 mV was chosen such that the slow
tail currents in Na/Na solutions reach the baseline. In
Na/Na solutions (top) depolarization after complete tail current deactivation shows only
partial recovery from inactivation. In Tris/Na solutions (bottom), no significant recovery
from inactivation is visible. Instead, slow, mono-exponential reactivation to the same steady state level as in the first pulse is observed (dashed lines). (B) Activation of current through C-type inactivated
channels is shown in Tris/Na solutions in response to depolarizations from
100 mV to voltages between
30 and +80 mV in steps of 10 mV. The depolarizations were given in rapid succession such that no marked recovery from inactivation occurred (see A). The activation
kinetics (under these conditions of steady state inactivation) were estimated by fitting single-exponentials functions to the data. The resulting time constants are plotted in C as a function of the test potential. The straight line corresponds to a single exponential function in this
semilogarithmic plot. The slope of this line yields an estimate for the apparent gating charge associated with the process of reactivation of
1.13 e0.
[View Larger Version of this Image (21K GIF file)]
30 to +80 mV
to explore the voltage sensitivity of this reactivation
process. Fig. 7 C shows the activation time constants
(
Ca) obtained from monoexponential fits to currents at test potentials sufficient to generate measurable reactivation under these conditions. From this semilogarithmic plot we note an e-fold change in time constant
in 22.3 mV, indicating an effective reactivation valence
of ~1 e0. This value is substantially greater than the ~0.3
e0 reported for reactivation of noninactivated Shaker channels by Zagotta et al. (1994
a), suggesting that there
is some underlying difference between the mechanisms
of reactivation in normal and C-type inactivated channels.
Fig. 8.
Effects of internal K+ ions on recovery from C-type inactivation. Currents were recorded in the absence of extracellular
permeating ions (Tris-Ringer), individual traces being separated
by 15-s intervals. Between A, B, and C, bath solutions were altered
as indicated. In A, in the presence of 1 mM internal K+, channels
undergo rapid and complete inactivation (A, i and ii). Switching to
K+-free 115 mM internal Na+ solutions results in a similarly fast inactivation (B, i) that falls to a steady state value that indicates continued Na+ permeation through C-type inactivated channels (see
also Fig. 7 A). The next pulse in Na+-solution (B, ii) shows no recovery from inactivation, thus this trace shows only activation of
C-type inactivated channels (as in Fig. 7 B). Switching back to
K+-containing internal solutions reveals almost complete inactivation (C, i). However, the final pulse (C, ii) shows partial recovery of
"normal" inactivation.
[View Larger Version of this Image (15K GIF file)]
DISCUSSION
, 1991
).
Additionally, this inactivation mechanism has been
found to be affected by site-directed mutations in the S6 segment (Hoshi et al., 1991
; Holmgren et al., 1997
),
as well as in the pore domain (López-Barneo et al.,
1993
; Schlief et al., 1996
). Specifically, Schlief et al.
(1996)
concluded that the externally facing residue at
position 448 becomes more accessible during C-type inactivation, while evidence favoring a cooperative action
between monomers in the initiation of C-type inactivation has been provided by several groups (Ogielska et
al., 1995
; Panyi et al., 1995
). Finally, a cysteine cross-linking study has demonstrated that C-type inactivation
involves conformational changes in the outer mouth of
the pore indicating a structural constriction of the
outer mouth, potentially sufficient to produce long-term closure of the ion-conducting pathway (Liu et al.,
1996
). In contrast to the hypothesis that C-type inactivation is accompanied by a "collapse" of the pore leading
to a virtually closed channel, we show here that channels remain capable of conducting impermeant Na+
and Li+ ions in the C-type inactivated state, provided
that internal blocking effects of K+ are first removed by
careful washout of K+ from the internal medium.
; Ikeda and Korn,
1995
; Ogielska and Aldrich, 1997
). Since both noninactivated and C-type inactivated channels appear to be
Na+ permeable in the absence of internal K+, we show
here (see Fig. 2) that C-type inactivation can be detected from marked and characteristic changes in tail
current waveform that far exceed the rate changes previously reported to result from substitutions in permeating ion species (Zagotta et al., 1994
a). Thus, Fig. 2
shows fast initial Na+ tail currents through noninactivated channels converting to slowly deactivating Na+
tail currents through C-type inactivated channels.
100 mV, although complete recovery from
C-type inactivation takes ~15 s even at these negative
return potentials. It seems clear, therefore, that these
tail currents represent the closing (deactivation) of
channels that remain predominantly in a C-type inactivated state (see Fig. 6). Similarly, we have demonstrated reactivation of C-type inactivated channels from
a closed to a Na+-conducting C-type state (see Fig. 7)
on subsequent depolarization. These deactivation and
reactivation rates are considerably slower than the corresponding rates for normal, noninactivated channels. However, the voltage sensitivity of deactivation seems
similar to the apparent gating valence of ~1 e0 reported for Shaker channels by Zagotta et al. (1994
a).
Thus, the deactivation process characterized by the falling phase of these Na+ tail currents may be similar to
that which occurs in noninactivated channels. By contrast (see Fig. 7 C), reactivation seems to be substantially more voltage sensitive in these C-type inactivated channels (~1 e0) than in noninactivated Shaker channels (Zagotta et al., 1994
a, 1994b) over the potential
range of +20 to +100 mV. It seems possible that reactivation of C-type inactivated channels involves additional transitions that are not resolved as separate kinetic components in the ionic current records. Finally, we demonstrate (see Fig. 8) some of the complex effects of internal solution changes on conduction through
C-type inactivated channels.
; Sala
and Matteson, 1991
; Zagotta et al., 1994
a) suggests that changes in dwell time within the channel are important
determinants of the deactivation rate. We note that K+
has the shortest dwell time (according to this hypothesis) but has the highest efficacy in preventing/delaying
C-type inactivation. Since the order of efficacy is opposite to that which would be predicted on the basis of
the occupancy hypothesis, we conclude that efficacy is
determined through ion specificity (i.e., by closeness of
interaction with critical residues in the outer selectivity filter) rather than by a nonspecific dwell time effect on
channel gating.
FOOTNOTES
Preliminary results of this work have been presented in abstract form
(Heinemann, S.H., J.G. Starkus, and M.D. Rayner. 1997. Biophys. J. 72:A29; Starkus, J.G., M.D. Rayner, and S.H. Heinemann. 1997. Biophys. J. 72:A232).
1
Abbreviations used in this paper: I-V, current-voltage; NFR, Normal
Frog Ringer.
. The valuable technical assistance of A. Grimm, A. Rossner, A. Hakeem, and M. Henteleff is appreciated.
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