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The Journal of Neuroscience, August 15, 1999, 19(16):6838-6843
Unmasking of a Novel Potassium Current in Drosophila
by a Mutation and Drugs
Amandeep
Singh1, 2 and
Satpal
Singh1
1 Department of Biochemical Pharmacology, State
University of New York at Buffalo, Buffalo, New York 14260, and
2 Williamsville North High School, Williamsville, New
York 14221
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ABSTRACT |
The delayed rectifier potassium current plays a critical role in
cellular physiology. This current
(IK) in
Drosophila larvae is believed to be a single current.
However, a likely null mutation in the Shab
K+ channel gene
(Shab3) reduces
IK but does not eliminate it. This raises a
question as to whether or not the entire IK
passes through channels encoded by one gene. Similarly, an incomplete
blockade of IK by high concentrations of
quinidine, a selective IK blocker, raises a
question as to whether IK consists of two
components that are differentially sensitive to quinidine. We have
addressed these questions by a combined use of genetics, pharmacology,
and physiology. The current component removed by the
Shab3 mutation differed from the
remaining component in activation kinetics, inactivation kinetics,
threshold of activation, and voltage dependence. The two components
showed strong differences in sensitivity to quinidine. Physiological
properties of the current component removed by the
Shab3 mutation were similar to those
of the quinidine-sensitive fraction of IK.
Complementary to this, properties of the current component remaining in
the Shab3 mutant muscles were similar
to those of the quinidine-resistant fraction of
IK. These observations strongly suggest
that, in contrast to the current belief, IK
consists of two components in Drosophila, which are
genetically, pharmacologically, and physiologically distinct. These
components are being called IKS and
IKF. IKS is carried via Shab-encoded channels.
IKF defines a new voltage-activated K+ current in Drosophila.
Key words:
Drosophila; K+
channels; Shab; delayed rectifier; larval muscles; quinidine
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INTRODUCTION |
Diversity of
K+ channels provides a basis for a wide
spectrum of physiological properties among excitable and nonexcitable cells. For example, K+ channels with
diverse characteristics play a vital role in several phenomena,
including repolarization of membrane potential, cardiac and neuronal
pacemaker activity, repetitive firing, sensory receptor potentials,
secretion, fertilization, and learning (Rudy, 1988 ; Colatsky, 1990;
Cook, 1990; Hille, 1992; Wu and Ganetzky, 1992; Jan and Jan, 1997;
Armstrong and Hille, 1998). Our understanding of diversity of
K+ channels, as well as of properties and
function of a variety of K+ channels, has
been advanced greatly by single gene mutations of
Drosophila. Combining mutations that selectively disrupt
channels with drugs that block specific channels has helped in
resolving various ionic currents and in determining the role of
specific currents in excitability of nerve and muscle cell membranes
(Salkoff, 1983; Wu et al., 1983; Gho and Mallart, 1986; Elkins and
Ganetzky, 1988; Singh and Wu, 1989; Singh and Wu, 1990; Gho and
Ganetzky, 1992). Voltage-activated K+
current in the larval muscles of Drosophila has been
resolved into two components, a fast transient current
(IA) and a delayed sustained current
(IK) (Salkoff, 1983; Wu et al., 1983;
Wu and Haugland, 1985; Singh and Wu, 1989).
Among the two voltage-activated K+
currents, IA is disrupted by mutations
in the Shaker gene, which codes for the structure of the
IA channels (Kaplan and Trout, 1969;
Kamb et al., 1987; Papazian et al., 1987; Pongs et al., 1988). This
current is blocked by 4-aminopyridine (4-AP) (Wu and Haugland,
1985; Wu and Ganetzky, 1992). Whereas it has been possible to partially
block IK with quinidine, a cinchona
alkaloid used as an antiarrhythmic agent in humans (Singh and Wu, 1989;
Kraliz et al., 1998), a mutational analysis of this current has not
been possible because of absence of mutations that disrupt this
current. However, a recently identified mutation
(Shab1) in the Shab gene
selectively reduces IK without
affecting other known ionic currents in the larval muscles (Chopra,
1994; M. Chopra, G.-G. Gu, and S. Singh, unpublished
observations). Mutations at the Shab locus, including a
likely null allele (Shab3), enable
us to ask questions about this current that have not been possible before.
Until now, IK in Drosophila
has been believed to be one homogenous current (Wu and Haugland, 1985;
Singh and Wu, 1989, 1999; Wu and Ganetzky, 1992; Tsunoda and Salkoff,
1995b). However, the current is not completely removed by
Shab3, which appears to be a
genetically null allele of the gene that codes for the structure of the
channels (Hegde et al., 1999). This raises a question as to whether
IK is indeed a single current or
whether it consists of two components, only one of which is carried by
the Shab-encoded channels. The current is also not blocked
completely by high concentrations of quinidine and its analogs, which
selectively block IK. The data
presented here strongly argue that the delayed sustained current in the
larval muscles of Drosophila may consist of two distinct
components. Identification of a new current component
(IKF) in these experiments raises
important questions on the identity of the gene that codes for the
channels carrying IKF, in
vivo physiological role of IKF in
muscle excitability, and pharmacological specificity of the current.
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MATERIALS AND METHODS |
Flies were grown on a standard cornmeal medium at 21°C (Chopra
and Singh, 1994). K+ currents were
recorded from body-wall muscles 12 and 13 (Gu and Singh, 1997) of
wandering third instar larvae by two-microelectrode voltage clamping
(Wu and Haugland, 1985). Larvae were dissected from dorsal side, and
internal organs were removed. All recordings were completed within 30 min from the start of the dissection (Gielow et al., 1995). Electrodes
were made from thin-walled borosilicate glass capillaries with an
outside diameter of 1.0 mm (World Precision Instruments, Sarasota, FL).
The voltage electrode was filled with 2.5 M KCl and the
current electrode with a 3:1 mixture of KCl and potassium citrate (Wu
and Haugland, 1985). Resistances of both electrodes were in the range
of 10-15 M .
All recordings were made in a Ca2+-free
bath solution. This prevents the activation of the two
Ca2+ currents and the two
Ca2+-activated
K+ currents (Wu and Haugland, 1985; Singh
et al., 1989). These recording conditions produce only the
voltage-activated fast transient (IA) and the delayed sustained (IK)
currents. Currents were elicited by 500 msec voltage steps from a
holding potential of  80 mV to potentials between -40 and +40 mV, in
10 mV increments. In some experiments, as mentioned in the figure
legends, a prepulse of 2 sec duration, to 20 mV, was used to
inactivate IA (Wu and Haugland, 1985).
The recording solution contained (in mM): NaCl 77.5, KCl 5, MgCl2 20, NaHCO3 2.5, trehalose 5, sucrose 115, EGTA 0.5, and HEPES 5 (Stewart et al.,
1994; Gu and Singh, 1997). In addition, the recording solution in some
experiments, as explained in Results, also contained quinidine, 4-AP,
and tetraethylammonium (TEA). The pH was adjusted to 7.1 with NaOH.
Voltage stimuli for eliciting the currents were generated with the help
of a Macintosh IISi computer through a 12-bit digital-to-analog converter (MacADIOS II/16 board; GW Instruments, Somerville, MA). The
resulting current was recorded with the help of an amplifier (TEC
01C/02/03; NPI Electronic GmbH, Haeldenstrasse, Germany) connected to
the computer. Data were converted from analog to digital form with the
help of a 16-bit analog to digital converter (MacADIOS II/16 board; GW
Instruments). Currents were sampled every 500 µs for digital
conversion, except during capacitance transients (every 100 µs),
which were used for measuring cell capacitance. The digitized data were
analyzed off-line with the help of a program written in "C" language.
Control recordings were performed independently for each set of
experiments, and digital subtraction was performed between data
obtained during the same set of experiments. The current measurements
are given as current density (nanoampere per nanofaraday) to
avoid differences attributable to fiber size. The values are given as mean ± SE.
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RESULTS |
The voltage-activated delayed sustained current
(IK) recorded from the normal and the
Shab3 mutant muscles is shown in
Figure 1, A and B,
respectively. Under the recording conditions used in this experiment
(see Materials and Methods), the voltage-clamp traces show a fast
transient peak (IA) and the delayed
sustained current (IK). Figure
1C shows the current-voltage
(I-V) relationships for
IK recorded from the normal and the
mutant muscles. The Shab3 mutation
has two small deletions in the N-terminal region of the channel protein
upstream of the first transmembrane domain (Hegde et al., 1999). The
first deletion of 24 base pairs removes nucleotides 508-531. The
second deletion removes nucleotides 656-1011, in turn shifting the
reading frame and introducing a stop codon 74 bases downstream of the
mutation. The Shab3 protein is thus
expected to be truncated before the S1 segment, which starts at amino
acid 436. The truncated protein is also expected to lack the N-terminal
tetramerization region before the S1 segment (Li et al., 1992; Shen et
al., 1993) and is thus not likely to act in a dominant-negative manner.
Shab3 is thus most likely a null
mutation in the gene. If the entire IK
current passes through Shab-encoded channels, the
Shab3 mutation is expected to
completely eliminate IK. However,
Shab3 removes the current only
partially (Fig. 1), reducing it by ~65% of the total current. One
possibility raised by these data are that the sustained
K+ current may consist of two distinct
current components, only one of which is carried by channels encoded by
the Shab gene.
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Figure 1.
The Shab3
mutation reduces IK only partly. Membrane
currents recorded from the normal (A,  ) and the
mutant (B,  ) muscles are shown. Under the recording
conditions used (see Materials and Methods), only
IA (the fast transient peak in
A and B) and
IK (the sustained current) are seen.
C, Current-voltage relationships for the sustained
current, as measured at the end of the 500 msec pulse. For
A, number of larvae (L) = 9; number of fibers
(F) = 19. For B, L = 10; F = 33.
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To examine whether properties of the current component eliminated by
Shab3 differed from those of the
remaining component, the two components were compared for their
activation and inactivation kinetics (Fig. 2). To enable this comparison, the fast
transient current (IA), which masks
the rise of IK, needs to be removed.
IA was inactivated by using a 2 sec
prepulse to 20 mV. Under these conditions, only IK is observed (Wu and Haugland,
1985). IK was recorded from wild-type [Canton-S (CS)] and Shab3
muscles. The component removed by the
Shab3 mutation (Fig. 2C)
was obtained by digitally subtracting the mutant current (Fig.
2B) from the wild-type current (Fig.
2A). Figure 2D compares the
kinetics of the two current components. In this figure, the
Shab-independent component was scaled vertically to bring
its maximal value to the level of the maximal value of the
Shab-affected component. Traces are shown for voltage steps to +20 and +40 mV. Activation kinetics of the
Shab-independent component were faster than those of the
Shab-affected component. In addition, the
Shab-independent component showed slight inactivation after
reaching the maximum value, whereas the Shab-affected
component did not show inactivation until the end of the pulse. Kinetic differences between the Shab-affected and the
Shab-independent components lend support to the possibility
of IK consisting of two distinct
components. For the following discussion, the current component
eliminated by Shab3 (presumably
representing channels encoded by the Shab gene) is designated as IKS (for
"slow" activation). The current component unaffected by the
Shab3 mutation (presumably
representing channels not encoded by the Shab gene) is
designated as IKF (for "fast"
activation).
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Figure 2.
Properties of the two components of
IK. IA was
inactivated by a prepulse (2 sec, 20 mV). A, Total
current (IKF + IKS) as recorded from the normal (CS)
muscles. B, Current remaining in the
Shab3 mutant muscles
(IKF). C, Current
component (IKS) affected by the
Shab3 mutation as obtained by
subtracting the current shown in B from that shown in
A. D, IKF
digitally scaled up for comparison, so as to bring its maximum value to
the maximum value of IKS. To avoid clutter,
current traces are shown for voltage pulses to +20 and +40 mV only.
E, I-V relationships for
IKF () and IKS
(). D shows relative currents, and the current scale
does not apply to it. For A, L = 3; F = 7. For
B, L = 3; F = 10.
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Figure 2E shows that voltage-dependence of the
IKF component is different from that
of the IKS component. Activation threshold of the
two components is also different, with
IKF activating at approximately 30
mV and IKS at approximately 10 mV.
This further strengthens arguments for the distinct nature of the two
current components.
The possibility that IK may consist of
two distinct components is also raised by another independent set of
experiments. Quinidine and its analogs, which selectively block
IK in larval muscles (Singh and Wu,
1989, 1990) do not block IK
completely, even at high concentrations (Kraliz and Singh, 1997; Kraliz
et al., 1998). One possibility raised by these data is that
IK may consist of two components, only
one of which is sensitive to blockade by quinidine. To determine
whether there was any correlation between the two likely current
components resolved by quinidine and the two likely components resolved
by the Shab3 mutation, we examined
the blockade of IKF and
IKS by quinidine (Fig.
3). As in the experiments mentioned
above, IKF was recorded from
Shab3, and
IKS was obtained by digitally
subtracting IKF from the total current
obtained in the wild-type (CS). Figure 3, A and
B, respectively, show recordings from the CS and the
Shab3 muscles in the presence of 100 µM quinidine. Currents recorded from CS and
Shab3 in the absence of quinidine
were similar to those shown in Figure 2, A and B,
respectively, and are not shown here. Figure 3A represents residual IK (i.e.,
IKF + IKS) not affected by quinidine. Figure 3B represents residual IKF
not affected by quinidine. Data shown in Figure 3B were
digitally subtracted from data shown in Figure 3A to obtain
residual IKS not affected by quinidine
(Fig. 3C). Figure 3D shows I-V plots
for IKF and
IKS in the presence of 100 µM quinidine, with I-V plots for
IKF and
IKS in quinidine-free solution shown
for comparison as dotted and dashed lines,
respectively. The bar graph in Figure 3E shows percentages
of IKF and
IKS obtained during a pulse to +40 mV,
remaining in the presence of quinidine. Quinidine reduced
IKF by ~35% and
IKS by ~89%. Thus, the two current components showed a strong difference in their blockade by quinidine. The data indicate that the Shab3
mutation and quinidine affect the same component of the total current,
whereas the component left unaffected by the
Shab3 mutation is the one that is
less sensitive to quinidine.
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Figure 3.
Differential sensitivity of
IKF and IKS to
blockade by quinidine. A prepulse was used to inactivate
IA. Currents recorded from CS (representing
IKF + IKS in
quinidine-free solution) and Shab3
(representing IKF in quinidine-free
solution) were similar to those shown in Figure 2, A and
B, respectively, and are not shown here.
A, Currents recorded from CS in the presence of 100 µM quinidine. This represents fraction of
(IKF + IKS) not blocked by quinidine.
B, Currents recorded from the
Shab3 muscles in the presence of
quinidine. This represents the fraction of
IKF not blocked by quinidine.
C, The current obtained by digitally subtracting the
current seen in B from that seen in A.
This provides the fraction of IKS not
blocked by quinidine. D, I-V plots for
IKS without quinidine (dashed
line), IKF without quinidine
(dotted line), IKF with
quinidine (), and IKS with quinidine
( ). E, Bar graph shows percentages of
IKF and IKS
remaining in the presence of quinidine for a voltage step to +40 mV.
For A, L = 8; F = 22. For B,
L = 4; F = 13.
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According to the above interpretation, properties of the current
carried by the Shab-encoded channels
(IKS) are expected to be similar to
those of the quinidine-sensitive fraction of the current, and
properties of the Shab-independent current
(IKF) are expected to be similar to
those of the quinidine-resistant fraction of the current, with only a
minor deviation attributable to some blockade of
IKF by quinidine. Figure
4 compares the properties of
IKF and
IKS with those of the
quinidine-resistant and the quinidine-sensitive fractions,
respectively. As described for Figure 2 above,
IKF was measured in
Shab3 muscles, and
IKS was obtained by subtracting
IKF from the CS current. Similarly,
quinidine-resistant fraction was obtained by measuring current from CS
in the presence of 100 µM quinidine, and the
quinidine-sensitive fraction was obtained by subtracting quinidine-resistant fraction from the total CS current.
IKF,
IKS, and the quinidine-resistant
fraction of total IK were as shown in
Figures 2, B and C, and 3A,
respectively, and are not shown in Figure 4. The quinidine-sensitive
fraction of total IK is shown in
Figure 4A. Figure 4, B and C,
compares the activation and inactivation kinetics of the two current
components as resolved by the two methods. To avoid clutter, current
traces are shown for voltage pulses to +20 and +40 mV only. Traces for
quinidine-resistant fraction were scaled up to traces for
IKF for comparison of kinetics. Similarly, traces for IKS were scaled
up to those for the quinidine-sensitive fraction for comparison.
Activation and inactivation kinetics of
IKF were similar to those of the
quinidine-resistant fraction (Fig. 4B). Similarly,
kinetics of IKS were similar to those
of the quinidine-sensitive fraction (Fig. 4C). Figure
4D shows that voltage dependence of
IKS and
IKF (as seen in Fig.
2E and shown here for comparison as dashed
and dotted lines, respectively) were similar to those of
quinidine-sensitive and quinidine-resistant fractions, respectively.
Partial blockage of IKF by quinidine is reflected in the quinidine-resistant fraction being slightly less
than IKF and the quinidine-sensitive
fraction being slightly more than IKS
(Fig. 4D).
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Figure 4.
Comparison of the properties of different current
components. IA was inactivated by a
prepulse. IKF,
IKS, and the quinidine-resistant fraction of
IK (i.e., IKF + IKS) were as shown in Figures 2,
B and C, and 3A,
respectively, and are not shown here. A, This panel
shows IK recorded in the presence of
quinidine subtracted from IK recorded in
quinidine-free solution. This gives quinidine-sensitive fraction of the
total current (IKF + IKS). B, Kinetics of
the quinidine-resistant fraction compared with those of
IKF. For comparison of kinetics, the
quinidine-resistant fraction was digitally scaled up so as to bring its
maximum value to the maximum value of IKF.
Each of the two traces seen here (and in C) are a near
overlap of two currents, which annot be distinguished in the traces.
C, Kinetics of the quinidine-sensitive current compared
with those of IKS.
IKS was digitally scaled up to bring its
maximum value to the maximum value of the quinidine-sensitive current.
D, Comparison of I-V plots for
IKF (dotted line) and
IKS (dashed line) with those of
quinidine-resistant () and quinidine-sensitive () fractions,
respectively. B and C show relative
currents, and the current scale does not apply to them. For
A, L = 8; F = 22.
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Shab3 and quinidine affect the
current by very different mechanisms.
Shab3 is expected to eliminate the
channel protein itself, whereas quinidine blocks the channels present
in the membrane. The above data showing similar effects of quinidine
and the Shab3 mutation on the
amplitude, activation kinetics, inactivation kinetics, threshold of
activation, and voltage dependence of two current components suggest
that the delayed sustained current consists of two distinct components,
one carried by Shab-encoded channels (which are blocked by
quinidine) and the other carried by a different set of channels (which
are relatively less sensitive to blockade by quinidine).
To examine the sensitivity of IKF and
IKS to other
K+ channel blockers, we tested the two
currents for blockade by 4-AP and TEA. Figures
5 and 6
show the effect of these drugs on the two currents. 4-AP (5 mM) blocked both IKF and
IKS to a similar extent, to ~56 and
~60% of the control current, respectively. Thus,
IKF and
IKS are much less sensitive to
blockade by 4-AP than IA, which is
almost completely blocked by 50 µM 4-AP (Wu and
Ganetzky, 1988). This is consistent with the inability of 1 mM 4-AP to affect Drosophila Shab
channels expressed in Xenopus oocytes (Covarrubias et al., 1991). The effect of 10 mM TEA is shown in Figure
6 with IKF and IKS being ~75 and 84% of the
control current, respectively. This compares with ~100
mM TEA nearly eliminating
IA and
ICF in the larval muscles (Wu and
Ganetzky, 1988). We are currently testing other
K+ channel blockers to identify drugs and
toxins that can selectively block IKF.
Pharmacological agents and mutations that eliminate IKF selectively, and in general a
pharmacological profile of the two currents, will be very helpful in
analyzing the properties of the two currents, in determining their
individual roles in membrane excitability, and in studying the
mechanisms underlying their regulation.
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Figure 5.
Blockade of the two current components by
4-AP. Fractions of IKF and
IKS blocked by 4-AP were calculated in the
same way as for quinidine in Figure 3. Currents recorded from CS
(representing IKF + IKS in 4-AP-free solution) and from
Shab3 (representing
IKF in 4-AP-free solution) were similar to
those shown in Figure 1, A and B,
respectively, and are not shown here. A, Currents
recorded from CS in the presence of 5 mM 4-AP. This
represents fraction of (IKF + IKS) not blocked by 4-AP. No
IA (the fast transient current) is seen in
this figure because 5 mM 4-AP blocks
IA. B, Currents recorded from
the Shab3 muscles in the presence of
5 mM 4-AP. This represents the fraction of
IKF not blocked by 4-AP. C,
The current obtained by digitally subtracting the current seen in
B from the current seen in A. This
provides the 4-AP-resistant fraction of IKS.
D, I-V plots for
IKS without 4-AP (dashed
line), IKF without 4-AP
(dotted line), IKF with 4-AP
(), and IKS with 4-AP ().
E, Bar graph showing percentages of
IKF and IKS
remaining in the presence of 4-AP for a voltage step to +40 mV. For
A, L = 2; F = 5. For B, L = 6; F = 20.
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Figure 6.
Effect of TEA on IKF
and IKS. Fractions of
IKF and IKS
blocked by TEA were calculated in the same way as for quinidine in
Figure 3. Currents recorded from CS and
Shab3 were similar to those shown in
Figure 1, A and B, respectively.
A, Currents from CS in saline containing 10 mM TEA. B, Currents from
Shab3 in saline with 10 mM TEA. C, The current obtained by digitally
subtracting the current seen in B from the current seen
in A. This provides the TEA-resistant fraction of
IKS. D, I-V
plots for IKS without TEA (dashed
line), IKF without TEA
(dotted line), IKS with TEA
(), and IKF with TEA ().
E, Bar graph showing percentages of
IKF and IKS
remaining in the presence of TEA for a voltage step to +40 mV. For
A, L = 4; F = 11. For B, L = 4; F = 17.
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With the resolution of IK into two
components, the total voltage-activated K+
current in the larval muscles of Drosophila, as shown in
Figure 1A, can be now resolved into three distinct
components (Fig. 7) in several ways.
These include the Shaker and the Shab mutations, which eliminate IA and
IKS, respectively, and 4-AP and
quinidine, which block IA and
IKS, respectively. Differences in
physiological properties between the three currents further help in
resolving these currents. In combination with a similar resolution of
the two Ca2+-activated
K+ currents
(ICF and
ICS) (Gho and Mallart, 1986; Singh and
Wu, 1989, 1990) and two Ca2+ currents (the
1,4-dihydropyridine-sensitive and the amiloride-sensitive current)
(Gielow et al., 1995), the larval muscles of Drosophila provide an excellent preparation in which all known specific current components can now be resolved and studied individually.
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Figure 7.
A schematic representation of the three
voltage-activated K+ currents in the larval muscles.
Gene refers to the gene that codes for the channels
carrying a particular current. Blocker refers to the
drug that selectively blocks the current.
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DISCUSSION |
Experiments described in this report provide a strong argument for
the existence of two distinct current components
(IKF and IKS) in the slow sustained
voltage-activated K+ current
(IK) in the larval muscles of
Drosophila. Voltage-activated K+ current in the larval muscles of
Drosophila has been previously resolved into two distinct
currents. With the data presented here, we can now resolve the total
voltage-activated K+ current into three
components. Resolution of IK into
IKS and IKF will be particularly helpful in
analyzing the properties of these two currents, deciphering the
functional role of each current in muscle excitability, and studying
mechanisms underlying their function and regulation.
Channels carrying IKS are encoded by
the Shab gene. IKS shares
properties with the current generated by expressing Shab
channels in Xenopus oocytes. These properties include
relative resistance to blockade by 4-AP and a relatively slow
activation (Covarrubias et al., 1991; Tsunoda and Salkoff, 1995a).
However, in contrast to a slight inactivation of the delayed rectifier
current recorded from Xenopus oocytes expressing
Shab channels (Salkoff et al., 1992),
IKS shows no observable inactivation
of IKS during the 500 msec pulse.
The gene that encodes the channels carrying the new current
(IKF) identified in this study remains
to be identified. Among the channels that give rise to
voltage-activated K+ currents in in
vitro expression systems, the Shaker and the
Shal channels give rise to fast transient currents, whereas
the Shab and the Shaw channels give rise to slow
sustained currents (Iverson et al., 1988; Timpe et al., 1988; Salkoff
et al., 1992). One of the two sustained currents
(IKS) resolved in this study is
carried by the Shab-encoded channels. These data raise the
possibility that the second channel may be encoded by the
Shaw gene (Tsunoda and Salkoff, 1995a). However,
physiological and pharmacological properties of
IKF seen in our recordings differ from
those of Drosophila and mammalian Shaw current
observed in Xenopus oocyte system (Salkoff et al., 1992;
Kirsch and Drewe, 1993; Kanemasa et al., 1995). For example, Kv3.1
channels (a mammalian representative of Shaw) are ~150
times more sensitive to 4-AP than Kv2.1 (Shab) channels
(Kirsch and Drewe, 1993). This contrasts to almost similar blockade of
the IKF channels to that of the
Shab-encoded IKS channels (Fig. 5). Similarly, in contrast to
IKF (Fig. 6E), Kv3.1
channels are blocked by TEA with a half-blocking dose of ~220
µM (Iverson et al., 1988; Timpe et al., 1988;
Salkoff et al., 1992). One possibility is that the observed differences
arise from an in vitro expression of the channels. On the
other hand, IKF may be carried via
channels encoded by other genes, such as seizure
(sei) or ether-a-go-go (eag) (Warmke
et al., 1991; Titus et al., 1997; Wang et al., 1997). Mutations at the
eag locus have been shown to reduce
IK (Zhong and Wu, 1991), although the
exact mechanism for this effect is not yet clear. It is not clear at
this stage whether IKF is a Shaw current with novel properties, whether it is carried
via sei- or eag-encoded channels, or whether it
represents an as yet unidentified gene for a voltage-activated
K+ channel in Drosophila. It
will be very instructive to examine the nature of
IKF and identify the gene that codes
for the channels carrying this current. Availability of a
pharmacological agent that selectively blocks these channels can
provide a valuable tool for this purpose. Similarly, single gene
mutations that affect the current can also help greatly. Such mutations
and pharmacological agents will also be very valuable for a molecular
analysis of the IKF channels and their
function in Drosophila.
The delayed rectifier current is a ubiquitous current present in a
large variety of cells in most species. In human cardiac cells, it
consists of two components that show differential sensitivity to
various antiarrhythmic agents (Sanguinetti and Jurkiewicz, 1990; Singh,
1998). It will be interesting to analyze correlations, if any, between
the two components of IK in
Drosophila and the two components of the delayed rectifier
current in human cardiac cells. There is already some indication of a
pharmacological overlap between Drosophila and human cardiac
K+ currents. Quinidine, which affects the
delayed rectifier K+ channels in human
heart (Roden, 1996), affects heartbeat in Drosophila (Gu and
Singh, 1995), as well as blocks IKS in
Drosophila larval muscles (Kraliz et al., 1998). Any
correspondence between components of Drosophila and
mammalian delayed rectifier currents will be very useful in undertaking
a genetic analysis of cardiac excitability, particularly with the help
of mutations that affect IKF and
IKS in Drosophila.
| |
FOOTNOTES |
Received March 17, 1999; revised May 28, 1999; accepted June 1, 1999.
This work was supported by National Science Foundation Grant
MCB-9604457 and National Institutes of Health Grant GM-50779. A.S.
would like to thank Karen Snyder of Williamsville North High School for her support during the conduct of these experiments.
Correspondence should be addressed to Dr. Satpal Singh, Department of
Biochemical Pharmacology, 308 Hochstetter Hall, State University of New
York at Buffalo, Buffalo, NY, 14260-1200.
Dr. A. Singh's present address: College of Arts and
Sciences, Cornell University, Ithaca, NY 14853.
| |
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INTRODUCTION
