Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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Yao, Wei-Dong and
Chun-Fang Wu.
Auxiliary Hyperkinetic subunit of K+
channels: regulation of firing properties and K+ currents
in Drosophila neurons. Molecular analysis and
heterologous expression have shown that K+ channel
subunits regulate the properties of the pore-forming
subunits,
although how they influence neuronal K+ currents and
excitability remains to be explored. We studied cultured
Drosophila "giant" neurons derived from mutants of the Hyperkinetic (Hk) gene, which codes for a
K+ channel
subunit. Whole cell patch-clamp recording
revealed broadened action potentials and, more strikingly, persistent
rhythmic spontaneous activities in a portion of mutant neurons.
Voltage-clamp analysis demonstrated extensive alterations in the
kinetics and voltage dependence of K+ current activation
and inactivation, especially at subthreshold membrane potentials,
suggesting a role in regulating the quiescent state of neurons that are
capable of tonic firing. Altered sensitivity of Hk currents
to classical K+ channel blockers (4-aminopyridine,
-dendrotoxin, and TEA) indicated that Hk mutations modify
interactions between voltage-activated K+ channels and
these pharmacological probes, apparently by changing both the intra-
and extracellular regions of the channel pore. Correlation of voltage-
and current-clamp data from the same cells indicated that Hk
mutations affect not only the persistently active neurons, but also
other neuronal categories. Shaker (Sh) mutations, which alter K+ channel
subunits, increased neuronal
excitability but did not cause the robust spontaneous activity
characteristic of some Hk neurons. Significantly, Hk
Sh double mutants were indistinguishable from Sh single
mutants, implying that the rhythmic Hk firing pattern is
conferred by intact Sh
subunits in a distinct neuronal
subpopulation. Our results suggest that alterations in
subunit
regulation, rather than elimination or addition of
subunits, may
cause striking modifications in the excitability state of neurons,
which may be important for complex neuronal function and plasticity.
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INTRODUCTION |
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The proper functioning of the nervous system
depends on a large variety of neurons displaying distinct excitability
patterns. The firing properties of neurons are regulated by potassium
channels (Hille 1992; Rudy 1988
), which
represent one of the most diverse groups of ion channels.
K+ channels have been implicated in activity-dependent
neural plasticity, including learning in Aplysia
(Byrne and Kandel 1996
) and habituation in a defined
neural circuit in Drosophila (Engel and Wu
1998
). Screening for leg-shaking mutants in
Drosophila has led to the identification of a number of
behavioral genes (Kaplan and Trout 1969
), such as
Shaker (Sh) and ether a go-go
(eag), which encode different membrane-spanning,
pore-forming
subunits of K+ channels (Kamb et
al. 1988
; Pongs et al. 1988
; Schwarz et
al. 1988
; Warmke et al. 1991
), and
Hyperkinetic (Hk), which recently was identified
as a cytoplasmic auxiliary
subunit (Chouinard et al.
1995
). Homo- and hetero-multimeric assembly among Sh
splicing variants provides one mechanism for generating diverse
K+ channels (Christie et al. 1990
;
Haugland and Wu 1990
; Isacoff et al.
1990
; McComack et al. 1990
; Ruppersberg
et al. 1990
; Wu and Ganetzky 1986
, 1988
).
Heteromultimeric interactions of K+ channel
subunits
across different families, e.g., Sh, Slo, and Eag, could contribute
further to structural and functional diversity (Chen et al.
1996
; Schopperle et al. 1998
; Wu and Chen 1995
; Zhong and Wu 1991
, 1993
).
Conceivably, association of subunits with different
subunits
can contribute further to channel functional diversity. The structural
complexity of voltage-gated Na+, Ca2+, and
K+ channels is enhanced by the auxiliary or
subunits,
which are cytoplasmic proteins in tight association with
subunits
(Catterall 1991
, 1992
; Isom et al. 1994
).
For K+ channels, at least four subtypes of
subunit
polypeptides, Kv
1, Kv
2, Kv
3, and Kv
4, have been identified
biochemically and molecularly in mammalian species, which are
associated with specific
subunits (England et al.
1995
; Fink et al. 1996
; Majumder et al.
1995
; McCormack et al. 1995
; Morales et
al. 1995
; Nakahira et al. 1996
; Parcej
and Dolly 1989
; Scott et al. 1994
; Sewing
et al. 1996
; Yu et al. 1996
). Extensive in vitro
studies using heterologous coexpression with
subunits have
demonstrated that
subunits regulate surface expression of
subunits (Accili et al. 1997b
; Shi et al.
1996
) and their inactivation kinetics and voltage sensitivity (Chouinard et al. 1995
; Rettig et al.
1994
). Furthermore, interaction among different types of
subunits potentially generates additional complexity in K+
channel properties (Accili et al. 1997a
; Xu and
Li 1997
).
Although the regulatory role of K+ channel subunits has
been well established in heterologous expression systems, the
physiological consequences of
-subunit regulation have not been
determined in neurons. How are K+ currents in native
neurons modulated by the
subunit? What are the functional
consequences of such modulation as reflected in excitability and firing
patterns? Are neurons of different functional categories differentially
regulated by the
subunits? Such questions are largely unanswered,
and exploring these issues can provide insights into the underlying
molecular and cellular basis.
Defects in non-pore-forming auxiliary subunits of Na+ and
Ca2+ channels are responsible for certain types of
mammalian epileptic disorders (Burgess et al. 1997;
Letts et al. 1998
; Wallace et al. 1998
).
Mutations of the K+ channel
subunit may further our
understanding of certain neurological conditions and behavioral
defects. Drosophila Hk mutants provide a unique opportunity
for defining the in vivo function of a K+ channel
subunit in neurons to bridge the gap between the molecular interactions
(Chouinard et al. 1995
) and altered neuronal activity (Ikeda and Kaplan 1970
; Stern and Ganetzky
1989
; Yao and Wu 1995
) responsible for
behavioral abnormalities including leg shaking (Kaplan and Trout
1969
) and abnormal locomotion in larvae (Wang et al.
1997
). Moreover, K+ channel mutations have been
shown to affect the habituation process of the jump-and-flight escape
response in a defined circuit, and interestingly the effect of altering
Hk
subunits is far more extreme than altering the
subunits such
as Sh and Slo (Engel and Wu 1998
). In
Drosophila, the in vivo function of the K+
channel
subunits encoded by the Sh, eag, and
slo genes has been well characterized in neuromuscular
junctions (Ganetzky and Wu 1982
; Jan et al.
1977
), muscle fibers (Elkins et al. 1986
; Salkoff 1983
; Salkoff and Wyman 1981
;
Singh and Wu 1989
; Wu and Haugland 1985
;
Wu et al. 1983
; Zhong and Wu 1993
), and
neurons (Baker and Salkoff 1990
; Saito and Wu
1991
, 1993
; Tanouye et al. 1981
). Therefore it
is desirable and also feasible to study the interaction between Hk
and specific
subunits in excitable cells of the corresponding
double mutants, such as Hk Sh.
We employed cultured Drosophila giant neurons (Wu et
al. 1990), which enabled voltage- and current-clamp studies on
the same cells (Saito and Wu 1991
; Zhao and Wu
1997
), to delineate the Hk phenotype in terms of
neuronal excitability and firing patterns and to characterize the
modulation of K+ current properties through the
interactions of Hk
with
subunits. Some of the results have
previously been presented in abstract form (Yao and Wu
1995
).
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METHODS |
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Fly stocks
All fly stocks were maintained at 20-22°C on standard
Drosophila media. The Canton S strain was used as wild-type
control in all experiments. Three mutant alleles,
Hk0, Hk1, and
Hk4 were used. Among them,
HkIE18, a deletion mutant designated
as Hk0 in this paper, was kindly provided by Dr.
B. Ganetzky; Hk1, induced by ethyl
methanesulfonate, is likely a point mutation. Hk4 was isolated in an X-ray-irradiation screen
(by R. Kreber in Ganetzky's lab) and has been characterized in
behavioral studies (Wang et al. 1997). The Sh
mutants, ShM and ShKS133
(Sh133), were from the collection of Dr. S. Benzer. The double mutants Hk1ShM and
Hk1Sh133 were both
generated in the authors' laboratory.
Cell culture
The procedure for culturing Drosophila giant neurons
has been described previously (Saito and Wu 1991;
Wu et al. 1990
). Briefly, embryos at the early
gastrulation stage were homogenized in modified Schneider medium
(GIBCO, Grand Island, NJ) containing 200 ng/ml insulin (Sigma, St.
Louis, MO), 20% fetal bovine serum (FBS), 50 µg/ml streptomycin, and
50 U/ml penicillin. Cells were washed two times in the above medium and
resuspended in medium containing 2 µg/ml cytochalasin B (Sigma) and
then plated on glass coverslips. Cultures were maintained in humidified
chambers at room temperature (20-25°C) for 2-5 days before recordings.
Electrophysiology
Whole cell patch-clamp recording was described previously
(Saito and Wu 1991; Zhao and Wu 1997
).
Recording electrodes were prepared from 75 µl glass micropipettes
(VWR Scientific, Chicago, IL). The tip opening of the electrodes had a
diameter of ~1.2 µm and an input resistance of 3-5 M
in bath
solutions. The bath solution contained (in mM) 128 NaCl, 2 KCl, 4 MgCl2, 1.8 CaCl2, and 35.5 sucrose, buffered
with 5 mM HEPES at pH 7.1-7.2. Patch pipettes were filled with a
solution containing (in mM) 144 KCl, 1.0 MgCl2, 0.5 CaCl2, and 5.0 EGTA, buffered with 10 mM HEPES, pH
7.1-7.2. Recordings were performed primarily on the neuronal soma with
diameters ranging from 10 to 25 µm, using a patch-clamp amplifier
(Axopatch 1B, Axon Instruments, Foster City, CA). The seal resistance
was usually >10 G
. Junction potentials were nulled before the
establishment of the whole cell configuration. All recordings were made
at room temperature.
An IBM 386 PC computer in conjunction with an A/D and a D/A converter
and pClamp software (version 5.5.1, Axon Instruments) was used to
generate the current and voltage commands and data acquisition. During
current-clamp experiments, the membrane potential was maintained at or
near the resting level through constant current injection. The majority
of cells did not require current injection, whereas in some cells, the
resting membrane potential varied slowly and required readjustments of
the amplitude of current injected within a period of several minutes.
Cells that showed significant drift (>15% in 2 min) were discarded.
In voltage-clamp studies, the membrane was held at 80 mV. Voltage and
current data were filtered at 1-2 kHz (4-pole Bessel filter) and
digitized at 1-10 kHz. Recordings of spontaneous neuronal activity
were stored in an FM tape recorder (Store 4, Lockheed Electronics,
Plainfield, NJ) for later analysis. A series of four pulses from
80
to
75 mV were applied and the resultant currents were used to
determine, and to compensate for, the series resistance and whole cell
capacitance. Membrane currents as a result of leakage were subtracted
digitally off-line. All data analysis was performed using pClamp 5.5, Excel 4.0 (Microsoft, Redmond, WA), AxoGraph 2.0 (Axon Instruments), Systat 5.2 (SYSTAT, Evanston, IL), and Superpaint 2.0 (Aldus, San
Diego, CA) software.
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RESULTS |
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Spontaneous firing in Hk neurons
We performed whole cell current-clamp recordings from isolated
giant neurons in culture. These multinucleated neurons, derived from
cytokinesis-arrested embryonic neuroblasts, develop a variety of
neuronal phenotypes, including characteristic arborization patterns
(Wu et al. 1990), neurotransmitters (Huff et al.
1989
), and ionic currents and firing properties (Saito
and Wu 1991
, 1993
; Zhao and Wu 1997
). The
preserved morphological and functional diversity allowed an assessment
of
subunit function in isolated CNS neurons of different functional
categories. We studied neurons isolated from wild type and three
alleles of Hk (Hk0, a null
mutation, and Hk1 and
Hk4, two hypomorph mutations, see
METHODS).
Figure 1 compares the spontaneous
activities for wild-type and Hk neurons. An immediate
hallmark of the Hk cells was a significant increase in the
sustained activities of spontaneous spikes (maintained for minutes and
often for the entire experiment period), which were rarely seen in
wild-type neurons. Other spontaneous activities, including sporadic
firing and bursts of spikes terminated by a plateau potential, were
found in both wild-type and Hk cultures. The majority of
wild-type neurons were quiescent, showing no activity at resting
levels. The proportion of quiescent cells was reduced in Hk
cultures, primarily due to an increase in persistent rhythmic firing.
Among the three categories of spontaneous activity, nonrhythmic "sporadic" activity was observed in 6.3% of wild-type neurons versus 7.5, 8.6, and 6.6% of Hk0,
Hk1, and Hk4
neurons, respectively; "plateau" potentials, developed from
occasional bursts of spikes, were observed in 2.1% of wild-type
neurons versus 2.5, 0.0, and 4.9% of Hk0,
Hk1, and Hk4
neurons, respectively. Significantly, only 3.6% of wild-type cells, in
contrast to 20.0, 15.7 and 13.4% of Hk0,
Hk1, and Hk4
neurons, displayed persistent rhythmic firing (Fig. 1). Such cells in
wild-type cultures might correspond to the endogenous pace-making cells
expected to be present in the normal CNS. In addition, the variety of
spontaneous activity patterns observed in the heterogeneous giant
neuron culture system might reflect activities of the diverse cell
types previously described in the nervous system of
Drosophila (Fischbach and Dittrich 1989;
Heisenberg and Wolf 1984
) and may involve differential
expression of and interplay between multiple channel proteins (see
DISCUSSION).
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On the basis of the spike waveform, two patterns of persistent spontaneous firing can be recognized in Hk neurons (Fig. 2). Class I neurons (16.3, 12.8, and 9.7% in Hk0, Hk1, and Hk4, respectively) showed regular all-or-none action potentials with varying prepotential, spike duration, and hyperpolarizing afterpotential. Class II neurons (3.8, 2.9, and 3.7% in Hk0, Hk1, and Hk4, respectively), a smaller population of cells, were characterized by repetitive bursts of spikes riding on depolarizing potentials. It should be noted that Class II firing appears to be Hk-specific because it was not seen in wild type and in other mutant genotypes (see Sh and Hk Sh in the following text). Notably, the two classes of firing patterns could not be interconverted by simple current injections but in certain cases could be converted between each other by blocking Na+ channel or Ca2+ channels (data not shown), suggesting that Class I and II neurons are indeed physiologically different.
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The results suggest that the Hk subunit is important in regulating
the quiescent state of CNS neurons. Intriguingly, among the various
cell types found in wild-type cultures, only the persistent rhythmic
activity was enhanced by Hk mutations (Fig. 1), apparently at the expense of converting a subpopulation of quiescent cells. A
further question would be whether the mutational effect, indicative of
the expression of Hk
subunits, is restricted to this category of
neurons only. Additional observations suggested that, although the
robust spontaneous firing clearly identifies a category of neurons in
which Hk
subunits play a distinct functional role, it is not the
sole category affected by Hk mutations (see following text).
Increased action potential duration in Hk neurons
The functional role of subunits in neuronal firing properties
was examined further by step current injection in neurons with and
without spontaneous activities. Three types of voltage responses,
all-or-none, graded, and nonregenerative (Saito and Wu
1991
), were observed for both wild-type and Hk
neurons. Action potential duration was determined in neurons displaying
all-or-none spikes. As shown in Fig. 3
and Table 1, action potentials were broadened in some Hk neurons. The broadening of action
potentials was variable, to ~10-fold in duration, as exemplified in
Fig. 3A. It is worth noting that the percentage of
Hk neurons displaying spontaneous activities was not
significantly different from the ensemble population of neurons for
each Hk allele examined (Fig. 3B). Furthermore,
some quiescent Hk cells also showed prolonged action
potentials in response to current injection.
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Alterations in K+ currents in Hk neurons
The effect of the subunit on neuronal excitability as revealed
in different alleles of Hk mutants suggests modifications of
the underlying K+ currents in mutant neurons.
Voltage-activated K+ currents were examined in saline
containing TTX and Cd2+ to eliminate the inward
Na+ and Ca2+ currents as well as outward
Ca2+-activated K+ currents (Saito and Wu
1991
). We studied K+ current properties in large
samples of neurons from wild type and three alleles of Hk.
The different mutant alleles showed consistent defects in
K+ currents, although the deletion mutant,
Hk0, was the most extreme. For
simplicity, only Hk0 is illustrated in this
section while results on the other two alleles are summarized in Table
1.
As shown in Fig. 4, the voltage-activated
outward K+ currents were affected substantially in
Hk0. Apparently both the peak and
sustained currents were reduced by Hk mutations as indicated
by current density measured at the peak and at 700 ms after the onset
of the depolarization step (the end of the pulse). More significantly,
the reduction in K+ currents was not uniform over all
membrane potentials but was proportionally more pronounced at lower
potentials near the spike threshold (Fig. 4, inset). In
addition, the kinetics of K+ current activation also were
affected in Hk neurons. The rise time of K+
currents, measured from the onset of the depolarization steps to the
peak, was slower in Hk (Fig.
5, A and B).
Notably, the effect was again more pronounced at lower voltages (Fig.
5A, compare between 20 and +20 mV; and Fig. 5B,
top). The results suggest that Hk
subunits modulate
multiple aspects of K+ channel activation especially at
lower levels of depolarization, where spike initiation is critically
controlled by K+ currents.
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Interestingly, the effects of subunit mutations on K+
current inactivation again varied with membrane potential. The decay phase of the K+ currents in these cultured neurons could be
fitted with one or two exponential plus a baseline current (Zhao
et al. 1995
). The time constant of the exponential decay (or of
the faster component if double exponential approximation was required
to fit the decay) was examined for indications of altered rates of
inactivation. We found that the decay of K+ currents was
slower in Hk neurons (Fig. 5).
Distribution of the time constant in Fig. 5C indicated
that Hk mutations affect K+ currents with faster
decay (1 < 100 ms, region I). There was a decrease in
the population of cells showing faster decay (region I) and a
corresponding increase in the number of neurons displaying slower decay
(region II but not in region III). The proportions of cells in regions
I, II, and III are 49, 38, and 13% for wild type and 28, 57, and 15%
for Hk0 [Fig. 5C; the percentage
differences for regions I and II, but not III, are significant
(P < 0.0001) under 1-sample binomial tests]. The
preferential effects of Hk mutations on faster decaying currents are consistent with the idea that Hk
interacts mainly with
the
subunits of certain fast-inactivating channels. Among the
K+ channel
subunits expressed in Drosophila
neurons, Sh subunits are thought to be associated preferentially with
channels showing faster inactivation (Baker and Salkoff
1990
). The slower decay of K+ currents observed at
the whole cell level in Hk neurons may not simply represent
a slower inactivation process because a convolution of a delay in
channel opening, which has been demonstrated at the single-channel
level in some Sh mutants (Zagotta et al.
1989
), and a slowed transition from the open to inactivated
state (Wang and Wu 1996
) could lead to similar results.
The parameters for steady-state inactivation and recovery from
inactivation also were determined in wild-type and mutant neurons. The
voltage for 50% inactivation (Vh1/2) of the
transient component was slightly shifted to positive potentials in
Hk mutants (Table 1). Figure 6
shows that recovery of the transient component from inactivation was
significantly slower in Hk0. The
recovery process could be approximated by two exponential processes
(cf. Wang and Wu 1996; Wu and Haugland
1985
; Zhao et al. 1995
) with time constants of
90 and 300 ms for wild type versus 170 and 700 ms for
Hk0. The time required for 50%
recovery from inactivation was 112.8 ms in wild-type neurons versus
206.7, 353.0, and 212.1 ms for Hk0,
Hk1, and Hk4
neurons, respectively.
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Reduced sensitivity of Hk K+ channel to blockade by neurotoxins
It can be concluded that the cytoplasmic Hk subunit is
important in the regulation of the amplitude, voltage dependence, and
kinetics of voltage-activated K+ currents. Such regulation
may involve modulation of the channel conformation. We examined how
mutations of the Hk
subunit would affect the interaction of
pharmacological agents with membrane-spanning
subunits for evidence
of conformation changes. Sensitivity to 4-aminopyridine (4-AP), a
routinely used A-type channel blocker (Hille 1992
;
Rudy 1988
), was examined in both wild-type and
Hk neurons. As in other Drosophila preparations,
4-AP inhibits inactivating currents in both muscle (Haugland
1987
; Wang and Wu 1996
) and neurons (Zhao
et al. 1995
). We used the difference between the peak and
sustained components to indicate the amplitude of inactivating current.
We found that Hk currents were less sensitive to 4-AP, as
0.5 mM 4-AP blocked ~52% of inactivating currents in wild type but
only 21% in Hk0 and 11% in
Hk1 (Fig. 7,
A and D). The altered inhibition implies that
functional
subunits are necessary to confer normal 4-AP sensitivity
and supports the idea that Hk
subunits influence the conformation in
the cytoplasmic pore region of certain
subunits (Fig. 7D, inset) (cf. Wang and Wu 1996
), in which the 4-AP
binding site is thought to reside (Kirsch et al. 1993
;
McCormack et al. 1991
; Yao and Tseng
1994
).
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The K+ channel subunit initially was identified by
copurification with the
subunits by using dendrotoxin (DTX)
(Pacej and Dolly 1989
; Scott et al.
1994
). We examined how Hk mutations alter K+ current sensitivity to
-DTX, which inhibits certain
fast-inactivating voltage-dependent K+ channels in
vertebrate neurons (Dolly et al. 1984
; Halliwell et al. 1986
). We found that in Drosophila neurons
the toxin inhibited preferentially the transient component, leaving the
steady-state current unchanged (Fig. 7, B and D).
At 100 nM, the suppression was 21% for wild type, 1% for
Hk0 and Hk1,
and 10% for Hk4. Unlike the 4-AP
effect (Fig. 7A), the suppression of K+ currents
by
-DTX was not reversible after washing, consistent with a tight
association of
-DTX with the K+ channels. Because the
binding sites of the toxin have been mapped to a region on the
subunit near the outer mouth of the channel pore (Hurst et al.
1991
) (Fig. 7D, inset), the data suggested that the
cytoplasmic
subunit exerts transmembrane influence on channel
conformation in the extracellular pore region as well.
We found that Hk mutations also affect the sensitivity of
K+ currents to a broad-spectrum blocker, tetraethylammonium
(TEA). A variety of K+ channels, both inactivating and
noninactivating, have been shown to be inhibited by TEA (Hille
1992; Rudy 1988
). Externally applied TEA blocks
K+ channels by binding to the extracellular mouth region of
the pore (Fig. 7D, inset) (Choi et al. 1991
;
Grissmer and Cahalan 1989
). Our results showed that TEA
significantly reduced both the peak and sustained currents in
Drosophila neurons. At 5 mM, TEA inhibited 22% of the peak
and 24% of the sustained currents in wild type. In contrast, the same
concentration of TEA did not affect peak currents in Hk
neurons but blocked sustained currents to an extent similar to that in
wild type (19% for Hk0 and 15% for
Hk1). Thus the net effect of TEA on
Hk current was an apparent increase, rather than a decrease,
of the inactivating current component (operationally defined as the
difference between the peak and sustained currents). Apparently, the
sensitivity of slower decaying currents to TEA was not significantly
altered whereas the fast decaying components became clearly less
sensitive in Hk mutant neurons.
It should be noted that TEA, as well as 4-AP and -DTX, did not seem
to alter either the fast or the slow decay time constant required for
fitting the decay process but merely changed the relative proportion of
the two components (i.e., the same 2 time constants could be used to
fit the current decay before and after drug treatment; data not shown).
The altered pharmacological profiles of Hk mutant channels
support the idea that Hk
subunits influence the function of
K+ channels by modulating channel conformation, which is
detectable by pharmacological probes to specific regions of the
subunits.
Correlation of K+ current properties with firing patterns: effects of Hk on functionally distinct cell types
As stated earlier, the robust phenotype of persistent spontaneous
activity can be used as an indicator for identifying neurons that are
affected by Hk mutations. However, the extensive effects of
Hk on K+ currents demonstrated by voltage-clamp
analysis of the entire neuronal population imply that not only the
cells capable of spontaneous firing are affected. To examine how Hk
subunits function in various cell types, we performed both current-and
voltage-clamp experiments on the same neuron to correlate the
underlying K+ current with different excitability
properties. Current clamping was performed first in normal
physiological saline and followed by voltage-clamp recording in
solution containing TTX and Cd2+. As shown in Fig.
8, a great majority of Hk
neurons examined in this manner could be divided into three types:
cells with persistent rhythmic spontaneous activity (Per. spont.; cf.
Fig. 1, persistent), cells that were quiescent but exhibited
all-or-none action potentials in response to current injection
(All-or-none), and quiescent cells unable to fire all-or-none action
potentials (Graded). Because of the rare occurrence (<4%) of
wild-type cells displaying rhythmic spontaneous firing, a satisfactory
sample was not obtained to determine the current properties of this
cell category in wild-type cultures. Furthermore, cells with
spontaneous activities of sporadic spikes or plateau potentials
constituted a small population in both Hk and wild-type
cultures (Fig. 1B) and were excluded from the analysis.
|
As shown in Fig. 9A, a reduction in current density was observed unexpectedly in all three types of Hk neurons. Furthermore the density for either the peak or the sustained K+ current was not significantly different among the three types of Hk neurons, with or without spontaneous activities (1-way ANOVA tests, see Fig. 9). Therefore Hk mutations appear to affect different cell types with distinct excitability properties. This result also suggests that simple reduction in K+ current amplitude alone is not sufficient to account for the abnormal spontaneous firing: additional explanations may include altered kinetic properties of K+ currents (see following text). Furthermore a complete account for the effect of Hk mutations on the firing pattern in a given neuron also should include voltage-clamp examinations of the underlying inward (e.g., Ca2+ and Na+) and nonvoltage-dependent K+ (e.g., Ca2+-activated K+) currents.
|
As shown in the preceding text, spontaneously active and quiescent
Hk neurons could not be distinguished by the extent of reduction in current density (Fig. 9A) nor by action
potential broadening (Fig. 3). Furthermore, the slowed recovery process of K+ currents in Hk cells with (confirmed)
spontaneous activity was no more severe than those of the overall
Hk0 population (Fig. 6). Nevertheless, the decay
time constant appeared to show more extreme alterations in
Hk cells with persistent spontaneous activities. Figure
9B compares the distribution of decay time constant in the
three cell types between wild type and Hk. There appear to
be no obvious differences among cells of the graded and all-or-none
types in both Hk and wild type. However, Hk
neurons that displayed persistent spontaneous activity deviated from
other types by showing an increased proportion of cells with
slower K+ current decay ( > 100 ms, Fig.
9B). Even though Hk mutations affect several
aspects of K+ currents in multiple cell categories, the
demonstrable enhancement in deceleration of the current decay process
associated with persistently active Hk cells suggests
interaction between Hk
and a particular type of
subunit,
rendering robust rhythmic activities in a subpopulation of neurons.
Role of Sh in mediating Hk mutant phenotypes
Hk has been shown to interact with Sh
, which confers a
fast-inactivating K+ current IA in
Drosophila larval muscle (Wang and Wu 1996
)
as well as in a heterologous expression system (Chouinard et al. 1995
). We analyzed the functional consequences of interaction between Hk
subunits and Sh
subunits in cultured central neurons.
Spontaneous nerve activities were compared for wild type,
Hk (Hk0 and
Hk1) and Sh
(ShM) single mutants and Hk Sh
(Hk1 ShM and
Hk1 Sh133) double
mutants. ShM is a null allele (Iverson
and Rudy 1990; Zhao et al. 1995
) and Sh133 an antimorphic allele (Haugland and
Wu 1987
; Tanouye et al. 1986
). Each single
mutant and double mutant exhibited higher excitability with
characteristic spontaneous activities as described earlier (Fig. 1). As
shown in Fig. 10A, the total
percentage of spontaneously active ShM neurons
was similar to that seen in Hk1 and
Hk0 cultures. However, the proportion of
ShM neurons with persistent rhythmic firing was
significantly lower than that of Hk neurons, attaining only
a level comparable to that of wild-type neurons. Correspondingly,
sporadic or plateau activities were increased in
ShM cultures.
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We then examined Hk Sh double-mutant neurons to see if mutating Hk in a Sh mutant background would result in additional abnormalities attributable to Hk. Interestingly, double-mutant neurons showed defects similar to ShM, but distinct from Hk, single-mutant neurons. Mostly transient clusters of spontaneous spikes and plateau potentials, but rarely prolonged rhythmic spike trains, were seen in Hk Sh neurons (Fig. 10A).
The data also raised the possibility that mutations of the
Sh gene in Hk Sh double-mutant neurons convert a
major portion of those cells expected to display persistent rhythmic
firing (when only Hk is mutated) into those displaying
sporadic or plateau activities (see Hk data in Fig.
10A, replotted from Fig. 1). In other words, intact
Sh products seem to be required to enable Hk
mutations to induce persistent rhythmic activities in this population
of neurons. It is also interesting to note that, as shown in Fig.
10A, a small percentage of Sh and Hk
Sh neurons, in which the Sh gene is disrupted, exhibited
persistent rhythmic firing, suggesting a mechanism independent of Sh
subunits. Significantly, a similar proportion of such cells also was
observed in wild-type cultures (Figs. 1 and 10A). Therefore
these cells are likely the endogenous pace-making neurons in the fly
nervous system, which may involve non-Sh channels such as Shal, Shab,
and Shaw (Butler et al. 1989
).
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DISCUSSION |
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This paper presents a comprehensive study of the function of ion
channel auxiliary subunits in native neurons, in which we examined the
role of Hk subunits in regulation of neuronal K+
currents and activity patterns, which may be important in determining neuronal circuit function, such as activity-dependent plasticity (Byrne and Kandel 1996
; Engel and Wu
1998
; Getting 1989
; Turrigiano et al.
1994
, 1995
). Discrete patterns of spontaneous activity were
categorized in cultured Drosophila neurons.
Hk mutations enhanced neuronal excitability by lengthening
action potential duration and increasing the proportion of cells firing
persistent rhythmic spikes. Voltage-clamp analysis suggested extensive
alterations in properties of voltage-gated K+ currents,
including density, voltage dependence, and kinetics. Alterations in
both activation and inactivation processes were more pronounced at
subthreshold voltage levels, suggesting a role in the regulation of the
quiescent state in certain categories of neurons. Altered
pharmacological profiles in Hk neurons suggested that the
principal effector domain of
subunits may include both the intra-
and extracellular pore regions of K+ channels, the
previously proposed acceptor sites of 4-AP,
-DTX, and TEA.
A combination of genetic and neurophysiological analyses enabled us to
identify a distinct category of neurons in which the effects of
Hk and Sh mutations were not additive, and the
mutational consequence of Hk is conferred through intact Sh
subunits to generate persistent rhythmic firing in this category of
neurons. Our results also suggested that Hk mutations affect
additional categories of neurons as well, possibly through interaction
with non-Sh
subunits. Recent molecular and genetic studies have
linked mammalian epileptic diseases to several Na+ and
Ca2+ channel auxiliary subunits (Burgess et al.
1997
; Letts et al. 1998
; Wallace et al.
1998
). It will be of interest to explore whether K+
channel
subunits are also candidates for neurological disorders, such as epilepsy.
Regulation of firing patterns by Hk subunits
A general picture appears to emerge from the analysis of
spontaneous activities in Sh, Hk, and Hk Sh
mutant neurons. It suggests a model that according to the differential
roles of Sh and Hk
subunits, Drosophila neurons can be
subdivided into four or more categories (Fig. 10). In this model based
on mutant phenotypes, "category a" represents a small population of
neurons (~4%, Fig. 10) that display pace making-like activity (Class
I, Fig. 2) in wild-type, Sh and Hk Sh cultures.
As mentioned in RESULTS, these presumably are associated
with native pace-making cells, similar to those documented in other
preparations (Adams and Benson 1985
; Kramer and
Zucker 1985
; McCormick and Prince 1988
;
Smith and Thompson 1987
). Because a similar proportion
of cells was observed in wild-type as in Sh and Hk
Sh cultures, rhythmic activities in this category of cells do not
depend on intact Sh
subunits. "Category b" consists of a
slightly larger population in wild-type neurons (~8%, Fig. 10) in
which sporadic and plateau activities can be seen. Note that since a
similar percentage of Hk neurons also showed the same
spontaneous activities, Hk
may not be required for generating such
activities. Our preliminary results indicated that K+
current properties were not significantly different in this category of
cells between wild-type and Hk cultures.
"Category c," as schematized in Fig. 10, represents a larger
population of neurons (~15%, Fig. 10), which are normally quiescent in wild-type cultures. The suggestion for this category is based on the
interesting observation that the persistent rhythmic neurons in
Hk0 and Hk1 cultures in
excess of those seen in wild-type cultures were roughly the same as the
increased proportion of neurons with sporadic and plateau activities in
Sh and Hk Sh cultures as compared with wild type
(Fig. 10, A and B). It can be hypothesized that
this category of quiescent cells in wild-type cultures is converted to
display distinct firing patterns by either Sh or
Hk mutations. Significantly, the phenotypes of Hk
Sh double-mutant neurons resembled those of Sh neurons
rather than those of Hk neurons, suggesting that the effects
of Hk and Sh mutations are not additive and that Sh is epistatic to Hk (see Stern and
Ganetzky 1989 for a similar epistatic effect at larval
neuromuscular junctions). The notion that the consequences of Hk
subunits are mediated by Sh-related
subunits in this
category is supported by the lack of detectable differences in either
voltage dependence, inactivation, or recovery kinetics of
K+ currents between Sh and Sh Hk
neurons (data not shown). It is interesting to note that roughly the
same percentage of neurons was proposed previously to express
Sh products in dissociated pupal (15%) (Baker and
Salkoff 1990
) and embryonic (13%) (Tsunoda and Salkoff
1995
) neuronal preparations.
"Category d" includes the remaining cell population. Cells in
category d are quiescent in all genotypes (~73%, Fig. 10). At the
present time, we cannot determine how the Sh and
Hk products function in these cells, although the reduction
in K+ currents in quiescent Hk neurons (Figs. 8
and 9) suggests that the Hk subunit does play a role in at least
some of the category d cells.
Our results are consistent with earlier in situ intracellular studies
that showed that neurons in the motor regions of adult Hk
thoracic ganglia spontaneously fire persistent rhythmic action potentials not seen in wild-type preparations (Ikeda and Kaplan 1970). Most interestingly, a major portion of these neurons
were reported to display typical pace-making potentials (type II)
(Ikeda and Kaplan 1970
, 1974
) and our Class I firing
pattern (Fig. 2) appears remarkably similar to such spontaneous
activity revealed by intracellular recording. This agreement lends
support to the idea that basic in vivo neuronal properties may be
preserved in the giant neuron culture system, which provides a more
accessible preparation for genetic analysis of physiological functions
in different categories of neurons.
In addition to analyses based on spontaneous activity presented above,
the firing patterns in response to current injection can provide
further information about the functional diversity of these cell
categories. Previous work has demonstrated that cultured giant neurons
can be characterized functionally by distinct firing patterns,
including adaptive, tonic, delayed, and interrupted spike trains, as
observed in current-clamp experiments (Zhao and Wu
1997). We noticed a suggestive correlation between the
categories of neurons in wild-type cultures described here and the
previously characterized firing patterns (Fig. 10C).
Category a neurons were exclusively of the tonic type at low levels of
current injection. Category b consisted mostly of neurons firing tonic
and adaptive spike trains during current clamp. The population of
"quiescent" neurons in wild-type cultures, which includes
categories c and d, was a mixture of subpopulations exhibiting the
different firing patterns previously described, i.e., adaptive, tonic,
delayed and interrupted spike trains, plus graded membrane oscillations (cf. Zhao and Wu 1997
).
Regulation of K+ channels by the Hk subunit
Our data suggest that the primary role of the Hk subunit is to
modulate voltage-gated K+ channels. A preliminary
voltage-clamp survey has not detected any appreciable differences in
voltage-activated Ca2+ and Na+ currents between
Hk and WT neurons (unpublished results). Moreover, a
previous study (Wang and Wu 1996
) on
Drosophila muscle also detected no differences in either
inward Ca2+ currents or outward Ca2+-activated
K+ currents in Hk alleles. Nevertheless subtle
developmental adjustments of Ca2+ or Na+
currents in some Hk neurons cannot be ruled out; this may
influence the expression of mutant firing patterns.
Because the giant neuron culture system potentially contains a variety
of cell types expressing different K+ channel subunits
that are encoded by Sh and other related genes (Butler et al. 1989
), as mentioned in
INTRODUCTION, multimeric assemblies of these subunits may
generate channels with considerable overlap in voltage dependence and
kinetic properties. Further diversity is possible because additional
K+ channel
subunits may be encoded by genes other than
Hk. Although our studies provide some insight into the in
vivo role of
subunits, it will be technically more difficult to
pinpoint the molecular mechanisms and specific subunit interactions in
native neurons than in simple heterologous expression systems.
Nevertheless, most of our results on
-subunit modulation of
K+ current properties are consistent with specific
information obtained in heterologous expression studies using defined
molecular lesions and subunit interactions.
The reduction in both peak and sustained voltage-activated
K+ currents in Hk neurons is consistent with the
role of Hk in enhancing the magnitude of the Sh currents
heterologously expressed (Chouinard et al. 1995
). The
Sh mutations are likely to affect transient as well as
sustained K+ currents in cultured Drosophila
neurons (Saito et al. 1993
) and both types of currents
can be mediated by different Sh splicing variants in the
Xenopus oocyte (Iverson and Rudy 1990
;
Timple et al. 1988
). The reduced current density may be
due to a decrease in either single-channel conductance or surface
expression, as
subunits have been shown to regulate the surface
expression of
subunits in mammalian cell lines (Shi et al.
1996
). Further work at the single-channel level will be
required to distinguish these two possibilities in Hk neurons.
Besides Sh subunits, K+ channel
subunits of other
subfamilies, encoded by the Shal, Shab, and
Shaw genes, exist in Drosophila (Butler et
al. 1989
; Tsunoda and Salkoff 1995
). Our results
showed that Hk mutations affect K+ currents not
only in cells that show the persistent spontaneous activity conferred
by Sh channels but also in quiescent cells of other functional
categories (Figs. 8-10). Thus potential interactions between Hk
and
non-Sh
subunits also may exist in certain native neurons.
Our results confirmed that the normal role of subunits is to
accelerate the time course of activation (Chouinard et al. 1995
) and inactivation, as demonstrated in heterologous
expression studies (Chouinard et al. 1995
;
England et al. 1995
; Majumder et al.
1995
; McCormack et al. 1995
; Morales et
al. 1995
; Rettig et al. 1994
; Uebele et
al. 1996
). In one such study (Chouinard et al.
1995
), a more pronounced effect was found at low voltage levels, similar to our findings in native neurons. Results obtained from the oocyte expression system also suggest that
subunits influence both the N- and C-type inactivation of Sh channels
(Chouinard et al. 1995
; Morales et al.
1996
). In the present study, complete isolation of identified
inactivating current is difficult, preventing an analysis of C-type
inactivation in separation of N-type inactivation. In addition to
comparable effects on kinetic properties, altered voltage dependence of
activation and inactivation of K+ currents in Hk
neurons is in general agreement with the results in expression systems
(Chouinard et al. 1995
; England et al.
1995
; Uebele et al. 1996
), although more
pronounced defects at lower voltages were seen in native neurons (this
report) and muscles (Wang and Wu 1996
).
Although the binding of the subunit has been shown to involve
N-terminal domains of the
subunit (Rettig et al.
1994
), the putative acceptor domain for
subunit action has
been suggested to reside in the internal pore region of
subunits
(Chouinard et al. 1995
; Wang and Wu 1996
;
Yu et al. 1996
). The altered pharmacological profile in
Hk neurons demonstrated by the use of TEA (cf.
Chouinard et al. 1995
), and 4-AP and
-DTX (which have
not been used in heterologous expression studies) lends further support
to this idea. Whereas 4-AP, an A-channel blocker, is known to bind to the cytoplasmic face (Kirsch et al. 1993
;
McCormack et al. 1991
; Yao and Tseng
1994
),
-DTX and TEA are thought to bind to the outer mouth
of the pore region (Choi et al. 1991
; Grissmer
and Cahalan 1989
; Hurst et al. 1991
) of the
subunits. Therefore the interaction of
and
subunits on the
cytoplasmic side can lead to a transmembrane change in the external
mouth region.
Despite the consistency detailed in the preceding text, one critical
difference between observations derived from native neurons and
heterologous expression systems is the rate of recovery of transient
K+ currents after inactivation. Coexpression of normal
Hk with Sh
in Xenopus oocytes slowed down recovery of
Sh currents (Chouinard et al. 1995
), whereas
in native neurons (this report) and muscles (Wang and Wu
1996
), data indicate that the wild-type Hk
subunits accelerate recovery from inactivation (Fig. 6). Previous experiments on
neuromuscular junctions of Hk mutant larvae have shown that high-frequency nerve stimulation results in repetitive nerve firing and
increased postsynaptic response (Stern and Ganetzky
1989
). This can be explained more readily by our data from
native neurons, as slower recovery of K+ currents from
inactivation would accumulate depolarization over time and cause
repetitive firing at Hk nerve terminals. Thus functional Hk
subunits might be important in activity-dependent processes, such
as those previously described in identified neurons of other species
(Marcus and Carew 1991
; Turrigiano et al. 1994
,
1995
; Wilson and Kaczmarek 1993
) during
development or physiological activity.
In conclusion, Hk subunits may be involved in the regulation of the
quiescent state and the fine tuning of firing patterns in neurons. Our
results reveal the intricacy in
-subunit regulation of nerve
excitability: this auxiliary subunit may function as a switch to
determine the excitability state of neurons and as a consequence,
complex repertoires in neuronal firing can be achieved by
subunit-mediated modulation, rather than through elimination or
addition, of specific types of K+ channels. Such regulation
may contribute critically to the behavior of neuronal circuits
(Getting 1989
), and it may not be surprising to see that
mutations affecting Hk
subunits can impose effects on habituation of
an identified neural circuit more severely than mutations affecting
Sh
subunits (Engel and Wu 1998
).
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ACKNOWLEDGMENTS |
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We thank Dr. B. Ganetzky for providing the HkIE18 stock, Drs. M. Daily, D. Eberl, S. England, and C. Rodesch and P. Taft for comments on the manuscript, and Drs. T. Hoshi, A. Ueda, and J. W. Wang for helpful discussions.
This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-18500 and NS-26528.
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FOOTNOTES |
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Address reprint requests to C.-F. Wu.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 September 1998; accepted in final form 25 January 1999.
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REFERENCES |
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