From the * Hopkins Marine Station, Department of Biological Sciences, Stanford University, Pacific Grove, California 93950; and Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Inactivation of delayed rectifier K conductance (gK) was studied in squid giant axons and in the somata of giant fiber lobe (GFL) neurons. Axon measurements were made with an axial wire voltage clamp by pulsing to VK (~10 mV in 50-70 mM external K) for a variable time and then assaying available gK with a strong, brief test pulse. GFL cells were studied with whole-cell patch clamp using the same prepulse procedure as well as
with long depolarizations. Under our experimental conditions (12-18°C, 4 mM internal MgATP) a large fraction
of gK inactivates within 250 ms at
10 mV in both cell bodies and axons, although inactivation tends to be more
complete in cell bodies. Inactivation in both preparations shows two kinetic components. The faster component is
more temperature-sensitive and becomes very prominent above 12°C. Contribution of the fast component to inactivation shows a similar voltage dependence to that of gK, suggesting a strong coupling of this inactivation path to
the open state. Omission of internal MgATP or application of internal protease reduces the amount of fast inactivation. High external K decreases the amount of rapidly inactivating IK but does not greatly alter inactivation kinetics. Neither external nor internal tetraethylammonium has a marked effect on inactivation kinetics. Squid delayed rectifier K channels in GFL cell bodies and giant axons thus share complex fast inactivation properties that
do not closely resemble those associated with either C-type or N-type inactivation of cloned Kv1 channels studied
in heterologous expression systems.
Functional properties of the diverse array of voltage-dependent K channels are important determinants of
electrical excitability in many cells (Rudy, 1988). Numerous K channel
-subunits have now been molecularly identified, and their functional properties have
been extensively studied in heterologous expression
systems (Jan and Jan, 1992
; Pongs, 1992
). It remains
problematic, however, to correlate molecular, biochemical, and physiological properties of specific K channels
expressed in native cells or in specific cellular domains,
such as the cell body and axon of individual neurons.
Squid giant axon and its cell bodies in the giant fiber
lobe (GFL)1 of the stellate ganglion provide an attractive system for carrying out such work. Cell bodies and
giant axons can be selectively studied physiologically
and biochemically, and the complement of K channel
subtypes is relatively simple. The major portion of macroscopic K conductance (gK) in the GFL/giant axon
system is due to a 20-pS channel (Llano and Bookman,
1986; Perozo et al., 1991
; Nealey et al., 1993
), and this
delayed rectifier gK has been extensively studied. Only
two other minor K channel species (10 pS and 40 pS)
have been reported (Llano et al., 1988
; Nealey et al.,
1993
).
Activation properties of the 20-pS channels are similar in axons and cell bodies (Llano and Bookman,
1986; Nealey et al., 1993
), but the picture is less clear for
inactivation. Macroscopic gK in GFL cell bodies partially
inactivates with a time constant of <100 ms at 10-12°C,
and this property is shared by the somatic 20-pS channel (Llano and Bookman, 1986
). Delayed rectifier gK in
giant axon, on the other hand, is generally thought to
show no fast inactivation (Keynes, 1994
), and single
channel recordings of axonal 20-pS channels have not
been focused on this issue (Llano et al., 1988
; Perozo et
al., 1991
).
In reality, few studies have specifically looked for fast
inactivation in giant axons, and such experiments in
perfused axons are complicated by technical difficulties. Depolarizations that produce outward K current
(IK) result in K accumulation in the extracellular space
and a change in driving force (Frankenhaeuser and
Hodgkin, 1956). In addition, the axial wire polarizes
during long pulses and can lead to inadequate voltage
control (Hodgkin and Huxley, 1952a
). Both problems
produce a decline of IK during a pulse that superficially
resembles inactivation. These errors can be circumvented by studying inactivation in high external K
(Ehrenstein and Gilbert, 1966
) and at a voltage equal
to the K channel reversal potential (VK). This approach
showed that axonal gK inactivates with a complex time-course, but no inactivation was detected before 100 ms
at voltages up to
10 mV (Clay, 1989
; see also Chabala,
1984
).
Recently we described similar experiments which revealed that axonal and somatic gK inactivate in a comparable manner and proposed that a single type of 20-pS
channel constitutes the delayed rectifier in axons and
in cell bodies (Rosenthal et al., 1996). In this paper we
present a more complete analysis of gK inactivation in
the GFL/giant axon system. A fast phase of inactivation
is very dependent on temperature and the presence of
internal ATP. A prominent slower component of inactivation is relatively insensitive to these agents. Complex
inactivation kinetics that are susceptible to modification are thus characteristic of squid delayed rectifier gK.
Recordings from GFL Neurons
Adult Loligo opalescens were collected from Monterey Bay and
maintained in flow-through seawater at ambient temperature
(~15°C). Somata of GFL neurons were isolated from the posterior tip of the stellate ganglion and cultured at 16°C as described
elsewhere (Gilly et al., 1990). Cells were normally used for recording within 2 d of isolation.
Conventional, whole-cell patch clamp was employed (Gilly et
al., 1990). Effective series resistance after electronic compensation was estimated by analyzing capacity current transients for a
10-mV step (Armstrong and Gilly, 1992
) and ranged from 0.2 to
0.8 M
. Holding potential was
80 mV throughout, and linear
capacity and ionic currents were removed on-line with a standard
P/
4 technique. Filtering was at 10 kHz; sampling was carried
out at either 20 or 2 kHz.
The internal solution (150 Ki) was similar to the perfusate used
for giant axons (see below) and contained (in mM): 20 KCl, 80 K
glutamate, 50 KF, 10 lysine (titrated to pH 7.0 with HEPES), 1 EGTA, 1 EDTA, 381 glycine, 291 sucrose, 4 Mg ATP, and 5 TMA OH (pH 7; 966 mosm). The standard external solution (20 Ko)
contained (in mM): 20 KCl, 480 NaCl, 10 MgCl2, 10 CaCl2, 10 MgSO4, 10 HEPES, and 0.0002 TTX (pH 7.7; 980 mosm). In
some cases, higher external K was used, and Na was reduced on
an equimolar basis (see appropriate figure legends). Experiments described in conjunction with Fig. 5 used slightly different
external and internal solutions whose compositions are given in
the figure legend. Junction potentials of ~10 mV exist between
the internal and external solutions and have been ignored. Tityustoxin-K
was a gift from Drs. M.P. Blaustein and D.R. Matteson,
University of Maryland (Baltimore, MD).
Recordings from Giant Axons
Adult squid (L. pealei) were supplied by the Marine Biological
Laboratory, Woods Hole, MA during July 1995 and maintained at
14°C. Cleaned giant axons were studied with a conventional axial-wire voltage clamp (Gilly and Armstrong, 1982) following internal perfusion with (in mM): 50 or 70 KF, 10 HEPES, 1 EGTA, 1 EDTA, 450 glycine, 450 sucrose, 3 lysine, 4 Mg ATP, and 4 TMA OH (pH 7.2; 980 mosm). The standard external solution contained (in mM): 50 or 70 KCl, 380 or 360 NaCl, 38 CaCl2, 14 MgSO4, 10 HEPES, and 0.0002 TTX (pH 7.2; 980 mosm). Junction
potentials were ignored. Unfiltered data were sampled at 50 kHz.
Properties of Inactivating and Noninactivating K Conductance in GFL Neurons
Results in this paper are developed around the idea
that a single type of K channel underlies delayed rectifier gK in both squid giant axons and cell bodies of GFL
neurons. Logic behind this assertion is summarized
above, and additional arguments are presented elsewhere (Horrigan and Gilly, 1996). Identification of the
cDNA SqKv1A also supports this idea (Rosenthal et al., 1996
). SqKv1A mRNA is expressed in GFL neurons and
appears to encode both somatic and axonal K channel
protein, and injection of cRNA into Xenopus oocytes
produces K channels with appropriate functional properties. Regardless of whether SqKv1A
subunits are expressed in frog oocytes, squid axons, or GFL cell bodies, inactivation is incomplete.
Results in Fig.1 indicate that the K channels underlying inactivating and noninactivating IK in GFL neurons
share important biophysical properties. A 500-ms prepulse
to 0 mV produces a decrease in peak IK of ~50% but has
almost no effect on steady-state IK (Fig. 1 A). We consider
the prepulse-resistant IK () to be essentially noninactivating on this time-scale. Fig. 1 B illustrates corresponding IK
traces obtained at higher sampling rate. Scaling the
prepulse-resistant trace (
) by a factor of 2.4 shows that
noninactivating IK and total IK have indistinguishable activation and deactivation kinetics. This comparison between total and noninactivating IK was carried out over a
range of activation (ON) and deactivation (OFF) voltages (Fig. 1 C), and single exponentials were fit to determine
ON and
OFF as indicated in the insets. Fig. 1 D shows that
the normalized gK-voltage relationships are also indistinguishable. Thus, fundamental activation properties of inactivating and noninactivating K channels in GFL neurons appear to be identical, consistent with a single population of channels that inactivate incompletely.
Fig. 2 provides pharmacological support for this idea.
Tityustoxin-K is a scorpion polypeptide that blocks certain mammalian K channels (Werkman et al., 1993
), and its effect on IK in a GFL neuron is illustrated in Fig. 2, A and B. 300 nM tityustoxin reversibly reduces peak and steady-state
IK by a comparable factor with little effect on kinetics. Tityustoxin also blocks IK in giant axons during brief pulses
(data not illustrated), but long pulses were not studied.
Similar results in GFL cells were produced by high concentrations of external TEA (Fig. 2, C and D). Internal TEA blocks IK at much lower concentrations, but also produces no major kinetic alteration. Records in Fig. 2, E and F were obtained 1 min and 18 min after achieving the whole-cell configuration with 1.6 mM TEA in the pipette. Results with all three ways of blocking IK are thus consistent with a single type of channel underlying the delayed rectifier gK in GFL cells.
The fact that neither internal nor external TEA produces a strong effect on inactivation kinetics in GFL
cells is unusual, because TEA is known to compete with
both N-type (intracellular) and C-type (extracellular)
inactivation in cloned Kv1 channels (Choi et al., 1991).
More subtle effects of external TEA on inactivation kinetics in GFL cells are described below.
Inactivation Kinetics in GFL Neurons and in Giant Axons
Fig. 3 A illustrates the procedure used to study inactivation kinetics in this study and raw data from a GFL neuron. Each prepulse to 10 mV of varying duration (
t)
was followed by a strong test pulse to determine the
amount of available IK (I(
t)). Maximal IK occurs with
a short prepulse (
t = 1.6 ms), and longer prepulses
produce progressively smaller IK due to developing inactivation. Values of I(
t)/I(1.6 ms) from this procedure are compared in Fig. 3 B (
) to a record of IK directly recorded at
10 mV after scaling so that I (1.6 ms) = peak IK (
10 mV).
Fig. 3 C establishes the time course at which the inactivating portion of IK declined at 10 mV in the
prepulse experiment (noninactivating IK was subtracted
out, see legend). Inactivation kinetics can be described
by the sum of two exponentials (solid curve) whose amplitudes and time constants were obtained by simultaneous fitting. Fast (
F) and slow (
S) time constants are
55 and 277 ms; the corresponding fractional amplitudes are 0.77 (AF) and 0.25 (AS). Inactivation thus has
fast and slow components operating on time scales of
tens and hundreds of ms, respectively.
Application of the same prepulse procedure to a giant
axon reveals similar fast and slow components of inactivation. Fig. 3 D shows I(t) records for three different
prepulses to
7 mV, which was identified as the reversal potential (VK) for IK tails following a brief pulse (0.5 ms to +60 mV). Normalized peak amplitude of I(
t)
from this experiment is plotted versus prepulse duration in Fig. 3 E (
), along with the value of current at
the end of each prepulse (
). Zero current flow during
the prepulse indicates that VK did not change during
the measurements, and deviation of VK from its original
value was estimated to be <
2 mV in this experiment.
These data demonstrate that the large decline in
I(t) in Fig. 3 E is an inactivation process analogous to
that in GFL neurons. Kinetics of inactivation in this
axon are further analyzed in Fig. 3 F. Fast and slow
components are evident, and the solid curve is the sum
of two exponentials with time constants (
F = 62 ms,
S = 447 ms) and amplitudes (AF = 0.67, AS = 0.34) comparable to those derived from GFL data (dashed curve is
replotted fit from Fig. 3 C).
Results thus obtained from a number of GFL neurons and giant axons are compared in Table I. Although significant inactivation exists in both cases, the
total fraction of IK inactivated within 250 ms in giant axons is smaller than that in cell bodies (P < 0.05 by t
test). Values for fast (F) and slow (
S) time constants are comparable in cell bodies and axons, and the fast
component accounts for the majority of inactivating IK
in both cases.
Table I. Comparison of Inactivation Properties in GFL Neurons and Giant Axons |
Voltage Dependence of Inactivation Kinetics
Although measurements on giant axons are complicated by the requirement that inactivation can be studied only at voltages equal to VK, experiments on GFL
neurons are not thus limited. Prepulses to negative
(25 mV) or positive (+40 mV) voltages yield very different overall rates of inactivation, with either the slow
or fast component predominating (Fig. 4 A). At
5
mV, a more even balance of fast and slow components
exists. Bi-exponential fits to such data from a number
of cells indicate that values of
F and
S are essentially
voltage independent (Fig. 4 B), whereas the fractional
amplitude of each component depends strongly on voltage. Fig. 4 C shows that the amplitude of the fast
component (
= AF) increases as voltage becomes
more positive, and the steep voltage dependence of AF
bears strong similarity to that of gK measured in the
same cell (
). As decreases over the same voltage range, because AF + As = 1.
Correspondence between the voltage dependencies of AF and gK suggests that the process underlying fast inactivation is strongly coupled to channel opening. Conversely, predominance of the slow phase at negative voltages where gK is small suggests that inactivation may also occur from closed states independently of channel opening or through a less directly coupled pathway.
Selective Modulation of Fast Inactivation
Fast inactivation in GFL neurons and axons can be reduced by withdrawal of internal MgATP. Consequences of omitting ATP from the pipette solution in whole-cell recordings from GFL neurons are illustrated in Fig. 5. After achieving the whole-cell configuration at time 0, a slow decline in both peak IK (Fig. 5 A, filled symbols) and in the fraction of IK inactivated during a 250 ms pulse to +40 mV (Fig. 5 B, filled symbols) typically occurs over 10-15 min. However, with 4 mM MgATP included in the pipette solution, both parameters are much more stable (open symbols in Fig. 5, A and B). Representative IK traces taken at time 0 and after whole-cell dialysis are shown in Fig. 5, C (0 ATP) and D (4 mM ATP). At this voltage, most inactivation occurring on this time scale occurs via the fast pathway (Fig. 4 C). Although the pulses used for Fig. 5, C and D, were too short to allow reliable fitting of two exponentials to the falling phase of IK, fitting single exponentials in Fig. 5 C yields time constants of 90 ms at time 0 and 200 ms at 620 s, consistent with a selective decrease in the fast component. A time constant of 85 ms adequately describes both traces in Fig. 5 D. Similar results were obtained for the other cells included in Fig. 5, A and B.
ATP was also necessary to maintain fast inactivation in
perfused axons. The time course of inactivation with ATP
in the internal perfusate is indicated in Fig. 6 (), and
ATP withdrawal for 30 min produced a decrease in both
peak IK and the amount of fast inactivation (
), comparable to the situation in GFL cells as described above.
At the end of the experiment, internal ATP was replaced, but neither peak IK nor the degree of inactivation showed significant recovery within 15 min (not illustrated). Papain (1 mg/ml) was then introduced into the axon for 90 s and then washed out. This treatment produced an immediate decrease in peak IK of ~50% and eliminated the fast component of inactivation (× in Fig. 6). A second application of papain produced no additional effect (not illustrated). ATP withdrawal and papain treatment were carried out in this manner in only one axon, but measurements made on two other axons which were both ATP deprived and papain treated failed to reveal significant inactivation occurring over 500 ms (not illustrated).
Preferential Effect of Temperature on Fast Inactivation Kinetics
Inactivation in both GFL cell bodies and giant axons is
highly temperature dependent. Fig. 7 A displays IK
traces recorded at +60 mV from a GFL neuron at several temperatures between 5.5 and 26°C. Low temperature produces a decrease in peak IK and a loss of fast inactivation similar to that seen with ATP withdrawal. Fig.
7 B shows inactivation kinetics at 12°C () and 18°C
(
) determined with the prepulse method at 0 mV in a
GFL cell. Solid curves are bi-exponential fits. The major effect of the temperature decrease is to reduce the
rate of fast inactivation, with
F decreasing from 40 to
93 ms. The amplitude of the rapidly inactivating component is basically unchanged (AF = 0.83 versus 0.76 at
18°C and 12°C, respectively), as is the slower time constant of inactivation (
S = 313 ms at 18°C and 265 ms at
12°C). Similar results were obtained in giant axons at
these two temperatures (not illustrated).
Recordings made in a number (n) of GFL cells with
prepulses to +40 mV yield mean values (± SEM) of F = 82.4 ± 5.4 ms (n = 19) at 12°C and 37 ± 1.4 ms (n = 12) at 18°C. In these same experiments
S only changed
from 365 ± 31 ms at 12°C to 282 ± 24 ms at 18°C. These figures correspond to Q10 values of 3.9 for
F and
1.5 for
S.
At temperatures below ~8°C, fast inactivation becomes undetectable. Fig. 7 C gives results from a
prepulse-experiment at +40 mV in a GFL neuron at 18 and 8°C. Inactivation kinetics at 8°C () are adequately
described by a single time constant (
= 306 ms) comparable to that of the slow component at 18°C (
;
S = 295 ms, AS = 0.21). In the same cell at
25 mV, a single
time constant of 440 ms adequately described inactivation kinetics at both 18 and 8°C (not illustrated). These
data reinforce the idea that the effect of temperature
on inactivation kinetics is almost entirely due to
changes in behavior of the fast component.
Effect of Temperature on "Steady-state" Inactivation
Temperature also exerts an effect on steady-state properties of inactivation. Fig. 8, Ai and ii, show IK recorded
at +40 mV following 1.5-s prepulses to the indicated
voltages in a GFL neuron at 18 () and 8°C (
), respectively. Figure 8 B plots normalized peak IK versus
prepulse voltage from this experiment. Low temperature decreases the maximum fraction of steady-state inactivation and shifts the inactivation-voltage relation by
about
15 mV but does not significantly change its
shape, i.e., steepness.
Recovery from Inactivation in GFL Neurons and Giant Axons
Recovery from inactivation was studied in giant axons
and GFL cells under similar conditions using a standard protocol. Fig. 9 A shows results from a GFL experiment at 18°C. The inactivating prepulse to +40 mV
lasted 250 ms, and the amount of inactivation is indicated by the decrease of IK from its peak value to the final value at the end of the prepulse (ISS). Sampling was
turned off during most of the prepulse and also for
most of the recovery period, t. The increase of I(
t)
above ISS represents recovery after a time (
t) at
80
mV, and the fraction of IK recovered was computed as
[I(
t)
I0]/[I
I0], where I0 = I (3.5 ms) (no recovery) and I
= I (2,000 ms) (nearly complete recovery).
The time course of recovery thus defined is shown in Fig. 9 B (
). A similar recovery time course was obtained in a giant axon with an inactivating prepulse to
VK =
7 mV (
). Open symbols in Fig. 9 B give results
from the same GFL neuron (
) and axon (
) at 12°C.
In no case does recovery follow an exponential time
course.
Because inactivation can proceed by fast and slow
pathways, recovery kinetics in GFL cells were compared
for inactivating pulses that primarily produced either
fast or slow inactivation. In order to preferentially inactivate channels slowly, a prepulse was made to 25 mV
for 500 ms (Fig. 9 C, upper records). Nearly pure fast inactivation was obtained with a prepulse to +40 mV for
61 ms (Fig. 9 C, lower records). If inactivation produced by these pulse protocols put channels into distinct inactivated states, recovery kinetics might be different in
the two cases. This expectation was not born out, however. Results in Fig. 9 D indicate that the slow (
) and
fast (
) inactivation protocols yielded the same recovery time course, as did a prepulse to 0 mV for 500 ms
which produced mixed slow and fast inactivation (
).
Cumulative Inactivation
Application of brief, repetitive pulses produces an accumulating decrease in the amplitude of IK in GFL neurons, and this decrease is more prominent if the inter-pulse voltage is made more positive. Fig. 10 A illustrates
these effects for 5-ms pulses applied every 30 ms with
inter-pulse voltages of 40,
80, and
120 mV. These
results are characteristic of cumulative inactivation (Aldrich et al., 1979
). Development of cumulative inactivation continues as long as channels are open, including
the time when tail currents are flowing, and the largest
decrease of IK thus occurs with an inter-pulse voltage of
40 mV where channel closing is slow. Although cumulative inactivation is generally associated with C-type
inactivation in mammalian (Lee and Deutsch, 1990
; Marom and Levitan, 1994
) and Shaker (Baukrowitz and
Yellen, 1995
) Kv1 channels, it is not clear at this stage if
this phenomenon occurs only with C-type mechanisms.
Very little "ordinary" fast (or slow) inactivation of
squid gK occurs in 5 ms, so repetitive pulsing provides a
third way to selectively inactivate channels. Recovery
from the cumulative-inactivated condition was therefore studied to shed light on the relationship of this inactivation pathway to the fast and slow processes. Fig. 10 B shows recovery at 80 mV from the effect of four
brief pulses for recovery periods of (
t) = 3.5 ms (no
recovery), 250 ms, and 4,000 ms (full recovery). Analysis of recovery kinetics in this experiment is presented
in Fig. 10 C. Recovery from cumulative inactivation (
)
proceeds with the same time course as recovery from
fast inactivation (
) or from mixed fast and slow inactivation (
) produced as described above. These results
indicate that the cumulative-inactivated state is not
readily distinguishable from the inactivated state (or
states) reached via fast or slow inactivation.
Two observations suggest that cumulative inactivation in squid K channels may be associated with processes underlying fast inactivation. First, the amount of cumulative inactivation induced by a single 5-10-ms pulse is greater at 18°C (19 ± 2%, n = 5) than at 12°C (11 ± 2%, n = 14). This temperature-sensitivity (Q10 = 2.5) is less than that shown by the rate of fast inactivation (Q10 = 3.7; 38 ± 2 ms at 18°C versus 82 ± 7 ms at 12°C, determined in the same cells by bi-exponential fitting), but it is considerably greater than that of the slow rate of inactivation (Q10 = 1.5). Second, a fair correlation exists between the degree of cumulative inactivation and the fraction of IK inactivated during a 250-ms pulse at 12 or 18°C (data not illustrated).
Dependence of Inactivation on External Potassium
External potassium ions are known to exert a strong influence on the kinetics of C-type inactivation of Kv1
channels, including Shaker B (Lopez-Barneo et al.,
1993; Baukrowitz and Yellen, 1995
) and mammalian Kv1.3
(Marom and Levitan, 1994
; Levy and Deutsch, 1996
)
and Kv1.4 (Pardo et al., 1992
; Rasmusson et al., 1995
). In light of the results on cumulative inactivation, which
suggest some similarity between C-type inactivation and
the fast process in squid K channels, experiments were
carried out on GFL neurons in which the effects of elevated external K on inactivation were assessed.
Fig. 11 A shows IK traces recorded at +60 mV in the
presence of 5-500 mM external K. Although the amount
of inactivation clearly depends on external K, very little
effect of high K on inactivation kinetics is evident. This
impression is supported by the kinetic analysis of
prepulse-procedure data obtained in 5 mM () and
200 mM (
) external K (Fig. 11 B). Fig. 11 C presents
the steady-state relation between inactivation and voltage
measured with 1.5-s prepulses in 5 mM (
) and 200 mM
(
) K . High K primarily decreases the maximum degree
of inactivation.
Recovery from inactivation in GFL neurons is accelerated in high external K. Fig. 11 D shows the recovery
time course in the presence of 5 mM () and 200 mM
(
) external K. The time course in high K remains
nonexponential. Accelerated recovery from inactivation in high external K occurs with both C- and N-type
mechanisms in Kv1 channels (Demo and Yellen, 1991
;
Gomez-Lagunas and Armstrong, 1994
; Rasmusson et
al., 1995
; Levy and Deutsch, 1996
).
Effect of External TEA on Inactivation
Competition with internal and external TEA is known
to slow the rates of N- and C-type inactivation, respectively (Choi et al., 1991), and sensitivity to external TEA
has been used as a diagnostic feature to identify C-type
inactivation (Rasmusson, et al., 1995). Although results
in Fig. 2 show that neither external nor internal TEA
markedly alter inactivation kinetics of squid delayed
rectifier gK, the effects of external TEA were examined more carefully. Fig. 12 Ai shows the reduction in IK at
+40 mV (
) by 500 mM external TEA (
). To facilitate comparison of inactivation kinetics, the traces are
replotted in Fig. 12 Aii after fitting the baseline to the
IK level at the end of the pulse along with a version of
the record in TEA (arrow) scaled to the control peak IK.
Although the control and scaled TEA traces show a similar early time course, they deviate at later times, with
the TEA trace decaying more slowly. These traces are
plotted with superimposed bi-exponential fits in Fig. 12
Aiii (see legend). Neither the fast nor slow time constant appears to be increased by TEA, and the major effect of external TEA is a decrease in AF. This result is
quite different from that seen in studies of cloned Kv1
channels.
Results of the prepulse method for determining inactivation kinetics in the same cell are shown in Fig. 12 B. Although the effect at +20 mV (, control;
, TEA) is
similar to that described above, there is no comparable
effect at
30 mV (
, control;
, TEA), consistent with
the fact that little inactivation occurs by the fast pathway at
such negative voltages. Although the slowing of inactivation by external TEA in this manner is subtle, this effect was seen in each of four cells studied in the presence of TEA at concentrations sufficient to block IK by
50%.
Internal TEA does not appear to alter the kinetics of
inactivation at either 12 or 18°C. IK at +40 mV was periodically monitored with 250-500-ms pulses during the
initial period of intracellular dialysis with the standard
internal solution plus 1.6 mM TEA-Cl as described in
conjunction with Fig. 2, E and F. Although this procedure regularly led to a decrease in peak IK of 50%
over 5-10 min, there was no consistent effect of the kinetics of the decaying portion of IK when traces were
compared as described for Fig. 12 A (not illustrated).
This paper describes inactivation properties of delayed rectifier gK in squid giant axons and in the cell bodies of GFL neurons from which these axons arise. Inactivation of gK shows similar complex properties in both parts of this system and proceeds with two kinetic components, the faster of which has a time constant of ~50 ms at 18°C. The fast component is very sensitive to temperature, becoming undetectable below 8°C, and requires internal ATP. The slower component (time constant of ~300 ms) is not much affected by either temperature or ATP.
Similarities between fast inactivation in GFL neuron
cell bodies and giant axons as described in this paper
are consistent with the conjecture that the relevant K
channels are encoded by the same mRNA, SqKv1A
(Rosenthal et al., 1996). We have not yet carried out
detailed studies of inactivation in the cloned channel, however. The present work provides a more complete
picture of inactivation of the native channels which will
guide analysis of SqKv1A.
Complex Inactivation of Squid Delayed Rectifier gK
Fast and slow inactivation processes of squid K channels interact in important and seemingly novel ways. Although the rates of fast and slow inactivation individually show little voltage dependence, the overall time
course of inactivation is quite voltage dependent. This
comes about by a shift in the balance between the fraction of channels inactivating by the complementary
pathways. Growth of the fast-inactivating fraction with
voltage matches the voltage dependence of gK, suggesting
that the fast-inactivating pathway is very accessible from
the open state. At voltages negative to 25 mV, the probability of channel opening is relatively low, and most channels that inactivate in this voltage range do so slowly.
Steady-state inactivation is fairly prominent over this voltage range, however, suggesting that the slow inactivation processes may also be important in determining steady-state properties. Low temperature (8 vs. 18°C) does not significantly alter the gK-V relation (not illustrated), but it does shift the voltage dependence of steady-state inactivation. This would be consistent with the idea that slow inactivation can occur from closed states in the activation pathway, the kinetics of which are quite temperature sensitive.
Recovery from inactivation shows a nonexponential time course, but there are not two kinetic components corresponding to the fast and slow inactivation processes. Regardless of whether inactivation occurs via the fast or slow pathway, or by a cumulative-type process, recovery proceeds with the same time course. Multiple inactivation pathways thus appear to either converge on a single inactivated state or lead to a pool of inactivated states that are likely to be in rapid equilibrium at voltages where recovery is measured.
Inactivation of the squid delayed rectifier appears to
be unusually complex, and it is very likely that this complexity is responsible for variability in the amount of
fast inactivation seen between different cells and between
cell bodies and axons. At present a specific kinetic
scheme for inactivation cannot be uniquely identified.
In any such model the rates and amounts of inactivation
from closed versus open states depend not only on the
rates of the inactivation steps themselves but also on details of the activation pathway leading into and out of the
open state (Hoshi et al., 1994; Zagotta et al., 1994
a, b).
Such data are not yet available for squid channels.
Reduction of Fast Inactivation by Withdrawal of Internal ATP
Functional modification of squid axon K channels by
internal ATP has been extensively studied at the macroscopic (Perozo et al., 1989; Augustine and Bezanilla,
1990) and single channel (Perozo et al., 1991
) levels.
All effects of the putative phosphorylation event were
accounted for by a shift of activation and steady-state inactivation properties along the voltage axis by ~+20
mV (Perozo and Bezanilla, 1990
, 1991
).
In this paper we show that internal ATP is necessary
to maintain fast inactivation in both axons and cell bodies. ATP withdrawal produces a decrease in peak IK and
a reduction in the amount of fast inactivation. Both of
these effects appear to be independent of the effects on
voltage-dependent properties expected to accompany ATP withdrawal (i.e., a 20-mV hyperpolarizing shift),
because our experiments were carried out with very
negative holding potential (80 mV) and positive test
potentials (+40 mV or higher).
Presumably, the fast inactivation mechanism is operative only in the phosphorylated condition, but we have
no direct evidence for this. The site(s) of phosphorylation need not be the same as those responsible for effects
on voltage-dependent gating, and it is likely that reported effects of phosphorylation on steady-state inactivation indirectly reflect the effects on activation gating (Perozo and Bezanilla, 1991). A role for phosphorylation in determining the rate of C-type inactivation in
mammalian Kv1.3 channels has been proposed, but the
precise nature of this regulation remains to be determined (Kupper et al., 1995
).
Is Fast Inactivation in Squid N-type or C-type?
Delayed rectifier K channels in squid are quite sensitive
to internal TEA, but slowing of inactivation was not observed in the presence of internal TEA. This suggests
that neither fast nor slow inactivation in is mediated by
an N-type mechanism involving intracellular pore-block
(Choi et al., 1991). Although elimination of fast inactivation by internal papain (Fig. 6) might suggest an intracellular, N-type mechanism, we do not regard irreversible protease sensitivity as definitive.
Dependence of inactivation kinetics on external K
and TEA, along with the presence of significant cumulative inactivation are associated with C-type inactivation mechanisms that involve the extracellular mouth
of the channel (Liu et al., 1996). Several features of fast
inactivation of squid gK are to some extent similar to results obtained in cloned channels showing C-type inactivation, but some clear differences deserve comment.
In the case of external TEA and high K, the time course
of inactivation in squid is not altered very much, and the
primary effect we describe is a selective decrease in the
amount of fast inactivation. These results are clearly not
equivalent to those in other systems where the actual
rate of inactivation is slowed (Lopez-Barneo et al., 1993;
Marom and Levitan, 1994
; Baukrowitz and Yellen, 1995
;
Rasmusson et al., 1995
; Levy and Deutsch, 1996
).
On the other hand, changes in the amount of inactivation produced by these modifiers of C-type processes
may not be unique to squid K channels. C-type inactivation in Shaker (Oglieska et al., 1995) and mammalian
(Panyi et al., 1995
; Rasmusson et al., 1995
) Kv1 channels is incomplete, even in low external K, and a decrease in the amount of inactivation in high external K
appears to accompany the decreased inactivation rate
(Lopez-Barneo et al., 1993
; Marom and Levitan, 1994
;
Baukrowitz and Yellen, 1995
).
We have proposed elsewhere that the delayed rectifier
gK in GFL neurons and giant axons corresponds to protein encoded by the cDNA SqKv1A (Rosenthal et al.,
1996). The predicted NH2 terminus of this protein shows
little similarity to features known to be important to N-type
inactivation in Shaker variants (Hoshi et al., 1990
; Murrel-Lagnado and Aldrich, 1993). Structural determinants of
N-type inactivation in rat Kv1.4 (Tseng-Crank et al., 1993
) are not identical to those in Shaker, however, and some
form of N-type inactivation for the SqKv1A
subunit cannot be ruled out based on sequence data alone.
Presently there is no molecular structure that defines
C-type inactivation, but specific residues in Shaker B (e.g.,
T449) are critically important to the rate of inactivation
and its dependence on external K (Lopez-Barneo et al.,
1993; Baukrowitz and Yellen, 1995
). SqKv1A has a serine
at the position equivalent to Shaker T449 (Rosenthal et
al., 1996
), and the T449S mutation in Shaker produces
extremely fast C-type inactivation (Schlief et al., 1996
).
Although a plausible case can be made for fast inactivation of squid K channels being related to a C-type process, the relationship would appear to be rather distant.
It is even less clear how the slower process in squid fits in.
The lack of a simple correspondence between N- or C-type processes may not, in fact, be that surprising, since
both processes have been carefully studied in only a few channel types (mainly Shaker), and their properties depend not only on the inactivation mechanisms themselves, but on the molecular and kinetic coupling to
other aspects of channel function (Baukrowitz and
Yellen, 1995). Future work on the cloned squid delayed
rectifier in heterologous expression systems will reveal
how inactivation compares to that described here for
the native channels in giant axons and GFL neurons.
Original version received 25 October 1996 and accepted version received 9 January 1997.
Address correspondence to Dr. William F. Gilly, Hopkins Marine Station, Stanford University, Pacific Grove, CA 93950. Fax: 408-655-6220; E-mail: Lignje{at}leland.stanford.edu
This work was supported by grants from the National Institutes of Health (NS-12547 to C.M. Armstrong and NS-17510 to W.F. Gilly) and by an ONR AASERT award to J.J.C. Rosenthal.
GFL, giant fiber lobe.