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The Journal of Neuroscience, January 15, 2000, 20(2):511-520
Ultrafast Inactivation Causes Inward Rectification in a
Voltage-Gated K+ Channel from Caenorhabditis
elegans
Richard
Fleischhauer1,
M. Wayne
Davis2,
Igor
Dzhura3,
Alan
Neely3,
Leon
Avery2, and
Rolf H.
Joho1
1 Center for Basic Neuroscience and
2 Department of Molecular Biology, The University of Texas
Southwestern Medical Center at Dallas, Dallas, Texas 75390-9111, and
3 Department of Physiology, Texas Tech University Health
Science Center, Lubbock, Texas 79430
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ABSTRACT |
The exp-2 gene in the nematode Caenorhabditis
elegans influences the shape and duration of the action
potential of pharyngeal muscle cells. Several loss-of-function
mutations in exp-2 lead to broadening of the action
potential and to a concomitant slowing of the pumping action of the
pharynx. In contrast, a gain-of-function mutation leads to narrow
action potentials and shallow pumping. We cloned and functionally
characterized the exp-2 gene. The exp-2 gene is homologous to genes of the family of voltage-gated
K+ channels (Kv type). The Xenopus
oocyte-expressed EXP-2 channel, although structurally closely related
to Kv-type channels, is functionally distinct and very similar to the
human ether-à-gogo-related gene (HERG) K+
channel. In response to depolarization, EXP-2 activates slowly and
inactivates very rapidly. On repolarization, recovery from inactivation
is also rapid and strongly voltage-dependent. These kinetic properties
make the Kv-type EXP-2 channel an inward rectifier that resembles the
structurally unrelated HERG channel. Apart from many similarities to
HERG, however, the molecular mechanism of fast inactivation appears to
be different. Moreover, the single-channel conductance is 5- to 10-fold
larger than that of HERG and most Kv-type K+
channels. It appears that the inward rectification mechanism by rapid
inactivation has evolved independently in two distinct classes of
structurally unrelated, voltage-gated K+ channels.
Key words:
potassium channel; Kv channel; inward rectifier; C.
elegans; pharyngeal muscle; Xenopus oocyte
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INTRODUCTION |
To maintain long-lasting action
potentials (APs), voltage-gated K+
channels are required that do not conduct at high levels during the
plateau phase. Inward-rectifying K+
channels have been shown to be important for the proper repolarization of APs with long plateaus (Hille, 1992 ). So far, two structurally different classes of inward rectifiers have been identified: tetrameric channels consisting of subunits with either two transmembrane (2-TM) or
six transmembrane (6-TM) segments. The 2-TM-type channels may be
activated by hyperpolarization without previous depolarization and
derive the property of inward rectification from block of outward
K+ current by intracellular polyamines or
Mg2+ ions (Dascal et al., 1993; Ho et al.,
1993; Kubo et al., 1993). Two functionally different 6-TM-type inward
rectifiers are known: the human ether-à-gogo-related gene (HERG)
channel (Sanguinetti et al., 1995; Trudeau et al., 1995) and the plant
inward rectifiers KAT1 and AKT1 (Anderson et al., 1992;
Sentenac et al., 1992). HERG channels are activated by depolarization
but inactivate rapidly, leading to relatively small outward currents
(Trudeau et al., 1995; Schönherr and Heinemann, 1996; Smith et
al., 1996; Spector et al., 1996; Wang et al., 1996). In contrast to
HERG, KAT1 channels are activated by hyperpolarization but not
depolarization. Unlike 2-TM-type inward rectifiers, KAT1 activation
reflects a channel-intrinsic mechanism and not simply the relief of
block from intracellular Mg2+ ions (Hoshi,
1995; Schroeder, 1995). 2-TM and 6-TM inward rectifiers show sequence
similarities to voltage-gated (Kv) K+
channels in the ion conduction pathway. Outside this pore region, there
is very limited, if any, similarity between HERG and Kv-type channels,
except for the fact that each K+ channel
subunit contains six transmembrane segments.
Here, we present evidence for a novel type of inward rectifier. The
exp-2 gene of the nematode Caenorhabditis elegans
has been found to determine the shape and duration of the AP of
pharyngeal muscle cells (Davis et al., 1995, 1999; Davis, 1999).
Several loss-of-function mutations in exp-2 lead to dramatic
broadening of the muscle APs with a concomitant slowing of the pumping
action of the pharynx. In contrast, a gain-of-function mutation leads to narrow APs with fast and shallow pumping. Based on the physiological role in rapid repolarization of the pharyngeal muscle, which was reminiscent of the function of HERG in the human heart, we surmised that EXP-2 might be closely related to HERG. When the exp-2
gene was isolated, however, it became clear that it was homologous to
genes from the Kv-type family of voltage-gated
K+ channels (Davis, 1999; Davis et al.,
1999).
Below, we show that the Xenopus oocyte-expressed EXP-2
channel is functionally very similar to the HERG channel, although it
is structurally closely related to Kv-type channels. EXP-2 activates
slowly in response to depolarization and inactivates very rapidly. On
repolarization, EXP-2 channels recover rapidly from inactivation in a
strongly voltage-dependent manner. In contrast to HERG and most
voltage-gated K+ channels, EXP-2 has a
relatively large unit conductance.
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MATERIALS AND METHODS |
Synthesis of EXP-2 cRNA
The identification and isolation of cDNA encoding EXP-2 is
described in detail elsewhere (Davis, 1999). Briefly, a fully-spliced EXP-2 cDNA (1595 bp) was inserted into the pT7 expression vector (a
derivative of pSP64; Cary et al., 1994) to allow in vitro
transcription of cRNA that could be expressed in Xenopus
oocytes. This vector contains a T7 promoter, 44 bases of the
Xenopus  -globin 5' untranslated region (UTR), and 143 bases of the Xenopus -globin 3' UTR followed by a poly(A)
tract. The construct was linearized with BamHI, and in
vitro transcription was done using the Ribomax T7 kit (Promega, Madison, WI) to generate a cRNA 2134 nucleotides long. Capped transcripts were produced by the addition of 3 mM
m7G(5')ppp(5')G (New England Biolabs,
Beverly, MA) to the reaction.
Electrophysiology
Approximately 1-3 ng cRNA were injected into Xenopus
laevis oocytes. After 1-4 d at 16°C, cells were subjected to a
standard two-electrode voltage-clamp protocol (VanDongen et al., 1990) using an OC-725A amplifier (Warner Instruments, Hamden, CT).
Experiments were done at room temperature (22 ± 1°C) in ND96
(96 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2 in 5 mM HEPES, pH 7.4) or in a solution in which KCl
replaced NaCl. Both voltage-sensing and current-passing electrodes were
filled with 3 M KCl and had resistances of
0.2-1.0 M . The pCLAMP 6.0 software (Axon Instruments, Foster City,
CA) was used to generate voltage pulse protocols and for data
acquisition. Signals were filtered at 0.5-2.0 kHz and digitized at
1-10 kHz. Capacitive and leakage currents were not subtracted, and
membrane potentials were not corrected for series resistance errors.
Macroscopic currents were also recorded using the cut-open oocyte
voltage-clamp technique (Taglialatela et al., 1992) with a CA-1
amplifier (Dagan, Minneapolis, MN). The oocyte membrane exposed to the
bottom chamber was permeabilized by a brief treatment with 0.1%
saponin. The voltage electrodes contained 3 M KCl and had
tip resistances from 0.6 to 1.2 M. Data acquisition and analysis were performed using the pCLAMP system. External and internal solutions
were the same as for the two-electrode voltage-clamp recording. Signals
were filtered at 10 kHz and sampled at 20 kHz.
For single-channel recordings, experiments were essentially done as
described previously (Moorman et al., 1990; Liu and Joho, 1998).
Oocytes were kept in (in mM): 100 KCl, 1.0 MgCl2, and 5.0 HEPES, pH 7.4. The pipette
contained (in mM): 100 KCl, 1.0 MgCl2, 1.8 CaCl2, and 5.0 HEPES, pH 7.4, unless otherwise indicated. Analog signals were filtered
at 2 kHz (Axopatch 200A and pCLAMP version 6.0), digitized at 86.6 kHz, and stored for off-line analysis. Passive leak and
capacitance currents were subtracted using the mean of traces without
channel openings. Single-channel data were analyzed using Fetchan and
pSTAT (pCLAMP 6.0).
Data acquisition and analysis
The pCLAMP 6.0 software was used for data acquisition and to fit
current traces to exponential functions.
Activation. Oocytes were depolarized for 1 sec to different
potentials (from  80 to 60 mV), and the peaks of the inward tail currents (at 120 mV) were normalized to the maximal current. A
conductance-voltage (g-V) relationship was
fit to a Boltzmann equation, and the midpoint of activation
(V0.5) and the slope factor k were calculated.
To obtain the instantaneous I-V relationship, oocytes were
depolarized to 20 mV for 1 sec to activate and inactivate EXP-2 channels. The channels were then allowed to recover for 20-40 msec at
80 or 120 mV before 200 msec test pulses in 10 mV increments were
applied from 120 to 60 mV. For experiments with the cut-open oocyte,
the instantaneous current amplitude was determined by extrapolating the
tail current to the beginning of the test pulse. In cases of
experiments with whole oocytes, the peak inward current was used, and
the outward current was used after the capacitive component had settled
(mean current from 5 to 10 msec after the onset of the test pulse). The
latter approach underestimates the outward current at positive
potentials and is probably the reason why almost no outward current is
visible in Figure 3C.
The time dependence of activation was determined by varying the
duration (1-4000 msec) of the depolarizing pulses at different voltages (20 to 40 mV). The peaks of the tail currents (at 120 mV)
were normalized to the maximum tail current, fit by a monoexponential function, and used to calculate the time constant for activation ( act).
Inactivation and deactivation. Deactivation and inactivation
could be separated from each other because they differed greatly in
their time and voltage dependence. After a 1 sec prepulse to 20 mV,
channels were allowed to recover from inactivation for 20-40 msec at
80 or 120 mV. One second test pulses were applied in 10 mV
increments to potentials in the range from 120 to 60 mV. To measure
inactivation in the range from 60 to 0 mV (to +60 mV in the cut-open
oocyte), the current representing the first 20 msec was fit with a
monoexponential equation, which was then used to calculate the time
constants of inactivation (inact) at different
voltages. In the voltage range in which the two-electrode voltage-clamp
and cut-open oocyte techniques can be compared, the two approaches
yielded similar results (see Fig. 5).
Deactivation could be separated from inactivation at test potentials
more negative than 80 mV. The slow current decrease in the range from
80 to 120 mV was fit with a monoexponential function, which was
used to calculate the time constants of deactivation (deact).
Recovery from inactivation (deinactivation). Oocytes were
depolarized for 1 sec to 20 mV to activate and inactivate EXP-2 channels. Channels were then allowed to recover from inactivation for
200 msec at different potentials from 60 to 120 mV. The rising
phase of the tail current was well fit by a monoexponential function,
which was used to calculate the time constants of recovery from
inactivation (rec) at different voltages.
Effects of external and internal tetraethylammonium.
Inactivation and recovery from inactivation were studied in the
presence of external or internal tetraethylammonium (TEA). A
frog Ringer's solution containing 50 mM KCl and 50 mM TEA as monovalent ions was used to measure the effect of
external TEA. To measure the effect of internally applied TEA, a 24 nl
aliquot of a 1.0 M TEA solution was directly injected in
the oocyte during the experiment. The volume of an oocyte was assumed
to be 0.5 × 10 6 l (1 mm diameter).
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RESULTS |
The C. elegans gene exp-2 is a member of
the Kv family of voltage-gated K+ channels
The exp-2 gene of the nematode C. elegans is
involved in the shape and duration of the pharyngeal muscle action
potential (Davis, 1999). The action potential of wild-type pharyngeal
muscle shows a characteristic plateau phase lasting 150-250 msec
before rapid repolarization takes place, driving the membrane potential toward EK. In loss-of-function
mutations of exp-2, this plateau phase is dramatically
prolonged and may last up to 6 sec (Davis et al., 1995; Davis, 1999).
In contrast, an exp-2 gain-of-function mutation
(sa26) results in much briefer than normal action
potentials (~50 msec). The action potential properties affected by
these exp-2 mutations led us to believe that the
exp-2 gene might encode a K+
channel with properties similar to those of the HERG
K+ channel, which is involved in the rapid
repolarization of the cardiac action potential. Indeed, the cloning of
genomic DNA (and cDNA) encoding exp-2 revealed an amino acid
sequence of a putative K+ channel (Davis,
1999; Davis et al., 1999). Contrary to expectation, EXP-2 showed no
similarity to HERG except for its putative transmembrane topology. The
EXP-2 sequence was, however, clearly related to the Kv family of
voltage-gated K+ channels represented by
the subfamilies Shaker, Shab, Shaw,
and Shal in Drosophila and Kv1-Kv9 in mammals
(Fig. 1, Table
1).
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Figure 1.
The EXP-2 channel is closely related to
voltage-sensitive K+ channels of the Kv type. The
amino acid sequence of the C. elegans channel EXP-2 is
shown in single-letter code. The tetramerization domain T1 is
underlined, and the transmembrane segments S1-S6 are
highlighted by a wavy line. The pore region between S5
and S6 is doubly underlined. Three consensus sites for
phosphorylation by protein kinase C and two glycosylation sites are
shown above the sequence by a black dot and a
diamond, respectively. There are no consensus sites for
phosphorylation by cAMP-dependent protein kinase. The amino acid
sequence of EXP-2 is from Davis (1999).
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The exp-2 gene encodes a protein of 528 amino acid residues
with a calculated molecular weight of 59,575 daltons. The EXP-2 protein
has all the characteristics of Kv-type K+
channels. It contains a hydrophobic core region with six putative transmembrane segments (S1-S6). The S4 segment contains six positively charged residues, and the linker region between S5 and S6 has the
typical sequence of a potassium-selective ion channel (Hartmann et al.,
1991; Yellen et al., 1991; Yool and Schwarz, 1991; Heginbotham et al.,
1994). The hydrophobic core of EXP-2 is preceded by the conserved
tetramerization domain T1, consisting of the subdomains A and B (Drewe
et al., 1992; Li et al., 1992; Shen et al., 1993). The EXP-2 channel
does not contain any consensus sites for cAMP-dependent protein kinase;
it contains, however, three consensus sites for phosphorylation by
protein kinase C (T131, T309, and S407). The extracellular S1-S2
linker region shows two consensus sites for N-glycosylation. Two such
consensus sites at similar positions in Shaker are indeed
glycosylated in insect cells and in oocytes (Santacruz-Toloza et al.,
1994).
When the EXP-2 amino acid sequence was compared with the sequences of
several other Kv-type channels, we found 35-43% identity, a value
that is typical for K+ channels that are
members of different Kv subfamilies (Table 1). In contrast,
K+ channels from the same subfamily are
61-84% identical. EXP-2 had previously been placed in the
Shab subfamily of K+ channels
(Wei et al., 1996); however, our analysis presented in Table 1 does not
allow an assignment of EXP-2 to any of the known Kv subfamilies. It is
possible that EXP-2 is not the C. elegans ortholog of a
Drosophila or mammalian channel gene from one of the known
Kv subfamilies but represents the prototype member of a novel subfamily
of Kv channels (in this case Kv10).
The Kv-type channel EXP-2 acts as an inward rectifier
We injected Xenopus oocytes with cDNA-derived RNA
(cRNA) to study the biophysical properties of EXP-2 channels. Oocytes
were subjected to a two-electrode voltage-clamp protocol to determine voltage-dependent channel kinetics and ion selectivity. Initial experiments were done in frog Ringer's solution containing 100 mM KCl. To determine the voltage dependence of
activation, oocytes were held at 80 mV, and depolarizing 1 sec test
pulses were applied in 10 mV increments to 60 mV. The depolarizing test
pulses were followed by 200 msec pulses to 120 mV (Fig.
2). During the 1 sec pulses, we could not
detect any substantial steady-state outward current, even at potentials
as positive as 60 mV; however, we consistently saw large
inward-directed tail currents during the subsequent 200 msec pulses to
120 mV. These tail currents were visible only when the preceding 1 sec test pulses were to potentials more positive than 40 mV (Fig.
2A). Apparently, EXP-2 channels become activated
during depolarization but inactivate rapidly, so little steady-state
outward current can be observed during the 1 sec depolarization, a
behavior reminiscent of HERG channels (Trudeau et al., 1995; Smith et
al., 1996; Spector et al., 1996; Wang et al., 1996). On repolarization
to 120 mV, again similar to HERG, EXP-2 channels recover rapidly from
inactivation and generate large tail currents. We used the peak values
of the tail currents to determine the g-V relationship of
EXP-2 (Fig. 2A, inset). The midpoint of activation
was V0.5 = 17 ± 2 mV (n = 5) with slope k = 6.5 ± 0.3 mV per e-fold
change in conductance (n = 5).
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Figure 2.
Voltage and time dependence of EXP-2 activation
and recovery from inactivation. A, Oocytes were held at
80 mV, and 1 sec test pulses to potentials from 60 to 60 mV were
applied in 10 mV increments, followed by 200 msec pulses to 120 mV.
The first inward tail current could be detected at 30 mV, indicating
the onset of activation of EXP-2 channels. Inset,
g-V curve with a midpoint of activation
V0.5 = 17 ± 2 mV and slope
k = 6.5 ± 0.3 mV per e-fold change in
conductance (n = 5 oocytes). B,
Oocytes were held at 80 mV, and test pulses to potentials from 20
to 40 mV were applied lasting from 1 to 4000 msec. The time dependence
of EXP-2 activation was determined from the peak tail currents during
the subsequent pulses to 120 mV. Inset, Actual
recording at 20 mV. In A and B, oocytes
were bathed in 100 mM KCl in frog Ringer's solution, and
current traces were not leak-subtracted. C, EXP-2
channels were activated by 1 sec prepulses to 20 mV. Two hundred
millisecond test pulses to different potentials from 120 to 60 mV
were applied to initiate recovery from inactivation. Recovery from
inactivation is faster at more negative potentials, indicating its
steep voltage dependence.
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To obtain the time constants for channel activation at different
voltages, the duration of the depolarizing pulse was varied from 1 to
4000 msec in the range from 20 to 40 mV, and the fraction of
activated channels was measured as the relative amplitude of the tail
current evoked during the subsequent 200 msec pulse to 120 mV (Fig.
2B). Time constants were obtained from a single exponential function fit to the experimental data obtained at each
voltage. Like the structurally unrelated HERG channel and unlike most
other Kv-type channels, EXP-2 activates very slowly even at positive
potentials. At 0 mV, the time constant for activation was 191 ± 23 msec (n = 5). Activation of EXP-2 showed a strong voltage dependence with time constants of 788 ± 117 msec at 20 mV (n = 5) to 35 ± 4 msec at 40 mV
(n = 10) (see Fig. 5). Given the function of EXP-2 in
repolarization after long action potential plateaus (150-200 msec),
relatively slow activation is consistent with its putative
physiological role.
Because the voltage and time dependence of activation and the apparent
fast inactivation of EXP-2 were reminiscent of the structurally
unrelated HERG channel, we compared these two channels directly (Fig.
3). Oocyte-expressed EXP-2 or HERG
channels were activated by 1 sec prepulses to 20 mV, followed by 40 msec pulses to 80 mV. Channels activated and inactivated during the 1 sec prepulses to 20 mV. During the 40 msec pulses to 80 mV, tail currents rise, indicating that EXP-2 channels have begun to recover from inactivation (rec = ~4 msec) before
they begin to deactivate slowly under these conditions (Fig.
3A). When the voltage is changed to 120 mV at the end of
the 40 msec pulse, there is further time-dependent current increase
before the onset of deactivation, which proceeds relatively slowly even
at 120 mV. This additional current probably reflects channels that
have not yet recovered from inactivation after 40 msec at 80 mV but
recover under more hyperpolarized conditions. When the voltage was
changed to less negative potentials, we could detect an initial rapid
component of current decay (Fig. 3A, arrow at 60 mV). This
decline in current was faster than deactivation, was strongly voltage
dependent, and probably results from EXP-2 channels entering the
inactivated state again. Hence, it appears that EXP-2 channels recover
rapidly from inactivation at 80 mV but begin to inactivate again at
less negative potentials, explaining the apparent lack of a
steady-state outward current at positive test potentials. HERG channels
also recover from inactivation at 80 mV, although more slowly than
EXP-2 (Fig. 3B). Inactivation of HERG is, however, much
slower than that of EXP-2, leading to detectable outward
K+ currents at potentials >0 mV (Fig.
3B, arrow), as has been shown previously (Schönherr
and Heinemann, 1996; Smith et al., 1996; Spector et al., 1996; Wang et
al., 1997). It seems that inactivation of EXP-2 is too fast for outward
K+ currents to be measured using the
two-electrode voltage-clamp protocol in Xenopus oocytes.
Uninjected control oocytes never showed any currents like the ones
described for EXP-2 (Fig. 3D).
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Figure 3.
Instantaneous I-V relationship of
EXP-2. A, EXP-2 channels were activated and inactivated
by 1 sec prepulses to 20 mV and then allowed to recover from
inactivation at 80 mV for 40 msec. After recovery from inactivation,
the instantaneous current through open channels was determined for the
beginning of 1 sec test pulses applied in 10 mV increments ranging from
120 to 60 mV. The arrow points to current decline
attributable to rapid channel inactivation at 60 mV. No outward
currents could be detected at positive potentials. B,
HERG channels show little inactivation at negative potentials and
outward current at positive potentials (arrow at 60 mV).
C, I-V relationship for EXP-2 at
different extracellular KCl concentrations, ranging from 2 to 100 mM (same oocyte). Inward currents are only detectable at
voltages more negative than EK,
indicating the potassium selectivity of EXP-2. D,
Uninjected control oocytes show no EXP-2-like currents. The same pulse
protocol was used for experiments presented in A,
B, and D.
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We determined the instantaneous I-V relationship and the
selectivity for K+ ions. At the end of the
40 msec recovery period (after the 1 sec depolarizing prepulse to 20 mV), test pulses to different potentials from 120 to 60 mV were
applied to measure the instantaneous current through open EXP-2
channels before the onset of inactivation or deactivation. We used
different extracellular K+ concentrations
to measure inward and, possibly, outward currents as a function of
voltage and EK (Fig. 3C).
As expected for a K+-selective channel,
inward currents could only be seen in voltage ranges more negative than
EK. It was, however, difficult to
determine the reversal potential for EXP-2 channels. Outward currents
were very small even under conditions of relatively slow inactivation because of the concomitant small driving forces in the negative potential range (Fig. 3C). EXP-2 channels are more permeable
for K+ than
Rb+ ions. With 100 mM RbCl in the bath solution, tail currents were ~30% compared with currents in the presence of 100 mM KCl (data not shown).
Voltage dependence of inactivation and recovery
from inactivation
The experiments described above suggested that EXP-2 may
inactivate too rapidly for outward currents to be detected
using the two-electrode voltage clamp on whole oocytes. We
therefore used the cut-open oocyte technique, which provides much
faster voltage control and time resolution (Taglialatela et al., 1992). Using a similar pulse protocol as above, we could clearly detect outward K+ currents, both in 2.5 and 100 mM extracellular K+ ions (Fig.
4). With 100 mM KCl in the
bath, the currents reversed direction at approximately 5 mV, close to
the expected value of EK. In 2.5 mM extracellular KCl, outward
K+ currents could be detected at
potentials positive to approximately 60 mV, again indicating that
EXP-2 channels are selective for K+ over
Na+ ions. An increase in the extracellular
potassium concentration increases EXP-2 channel conductance despite a
decrease in driving force, a phenomenon observed for several
voltage-gated K+ channels and inward
rectifiers (Hille, 1992; Pardo et al., 1992; López-Barneo et al.,
1993).
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Figure 4.
Inward rectification is caused by ultrafast
inactivation. The cut-open oocyte technique was used to study fast
inactivation. EXP-2 channels were activated and inactivated by 1 sec
prepulses to 20 mV from a holding potential at 80 mV and then allowed
to recover from inactivation for 20 msec at 120 mV. To determine the
instantaneous current, the voltage was then stepped in 20 mV increments
to different potentials from 120 to 60 mV. The arrow
points to current decline attributable to rapid channel inactivation.
A, With 2.5 mM KCl in the bath, the current
reversed direction at approximately 60 mV. B, In 100 mM KCl, outward and inward currents are larger, and the
current reversed direction at ~0 mV. C,
I-V relationship in 2.5 and 100 mM
extracellular KCl solution (same oocyte).
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We also determined the voltage dependence of deactivation,
inactivation, and recovery from inactivation. To study the voltage dependence of inactivation and deactivation, EXP-2 channels were activated and inactivated by a 1 sec prepulse to 20 mV, followed by a
20 msec pulse to 120 mV during which all channels recovered from
inactivation (Fig. 4). At this point, 1 sec test pulses were applied in
10 mV increments between 120 and 60 mV. To determine the time
constant of inactivation, the first 20 msec of the current traces in
the voltage range from 60 to 0 mV were fitted with a single
exponential. This initial process is mainly governed by inactivation
that becomes faster at less negative potentials (see Figs.
3A, 4B, arrows). Deactivation time
constants were determined in the potential range from 80 to 120 mV
in which inactivation is relatively slow and can be neglected. To
determine the time and voltage dependence of recovery from
inactivation, EXP-2 channels were activated by 1 sec prepulses to 20 mV, followed by 200 msec test pulses to different potentials from 60
to 120 mV (Fig. 2C). The rising phase of the currents was
fit to a monoexponential function and used to determine time constants
for the recovery from inactivation at different voltages.
The time constants for activation (act),
deactivation (deact), inactivation
(inact), and recovery from inactivation
(rec) were used to calculate the corresponding
apparent transition rates as a function of voltage (Fig.
5). As expected, the rates for activation
are strongly voltage-dependent and nearly semilogarithmic from 20 to
20 mV. In contrast, deactivation rates show no voltage dependence and
are relatively small (~13/sec) between 120 and 80 mV. Both the
rates for inactivation and recovery from inactivation are strongly
voltage-dependent and up to ~100 times greater than the rates for
activation or deactivation at similar voltages. Hence, it appears that
inactivation and recovery from inactivation are strongly
voltage-dependent like the corresponding processes in HERG, although
EXP-2 forms a Kv-type K+ channel unrelated
in sequence to HERG.
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Figure 5.
Apparent transition rate constants of EXP-2. The
apparent voltage-dependent rate constants (per second) for activation,
inactivation, recovery from inactivation (deinactivation), and
deactivation were calculated as the reciprocal values of the
corresponding time constants act,
inact, rec, and
deact. It is apparent that inactivation and recovery
from inactivation is up to ~100 times faster than activation.
Deactivation is very slow and not voltage-dependent between 80 and
120 mV. Open inverted triangles (inactivation)
indicate data points obtained with the cut-open oocyte technique; all
other data were obtained using the two-electrode voltage clamp on whole
oocytes.
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Internal TEA blocks EXP-2 channels and slows fast inactivation
Kv-type K+ channels have been shown
to undergo two distinct types of inactivation (Choi et al., 1991; Hoshi
et al., 1991). The relatively fast N-type inactivation involves the
binding of an N-terminal particle of a single-channel subunit to the
cytoplasmic face or inner vestibule of the channel, thereby occluding
the pore and preventing ion flow (Hoshi et al., 1990). The inactivation particle corresponds to a segment of ~20 amino acids at the N terminus of each channel subunit. In contrast; the relatively slow
C-type inactivation involves some conformational changes near the outer
end of the ion conduction pathway (Hoshi et al., 1991; Yellen et al.,
1994). TEA, a common blocker of K+
channels that can act on the extracellular or intracellular side, is
often used to differentiate between these two types of inactivation. Blockade by extracellular TEA leads to a slowing of C-type
inactivation, presumably by interfering with conformational
rearrangements in the outer mouth of the pore, without affecting N-type
inactivation (Yellen et al., 1994). Intracellular TEA competes with the
N-terminal blocking particle slowing N-type inactivation, but it has no
effect on C-type inactivation (Choi et al., 1991). To determine whether the ultrafast inactivation process of EXP-2 might be of N or C type, we
investigated the effect of external and internal TEA on the current
amplitude and inactivation kinetics. Extracellular TEA blocked EXP-2
channels in a concentration-dependent manner with an
IC50 (at 70 mV) of 30 ± 13 mM
(n = 3) without affecting the kinetics of inactivation
(Fig. 6A). To study the
action of TEA from the inside, we injected TEA in EXP-2-expressing
oocytes to an intracellular concentration of ~50
mM. At this concentration, the inward potassium
current (at 70 mV) was reduced to 51 ± 6% of control
(n = 3). In contrast to extracellular TEA, which did not affect inactivation, intracellular TEA slowed inactivation approximately twofold (Fig. 6B). It appears therefore
that the ultrafast inactivation process in EXP-2 is similarly slowed by internal TEA like N-type inactivation in Shaker.
View larger version (27K):
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|
Figure 6.
Effect of external and internal TEA on
inactivation. Fully activated and inactivated EXP-2 channels were
allowed to recover from inactivation for 20 msec at 80 or 120 mV,
followed by a test pulse to 70 mV to determine the time course of
inactivation. A, External application of 50 mM TEA reduces the current amplitude after recovery from
inactivation to 31% of control without changing the kinetics of
current decay. The dotted line represents the current
trace in the presence of TEA scaled to match the control current at the
end of recovery from inactivation. Inset, Nearly
threefold reduction of current (to 41 ± 10% of control,
mean ± SEM; n = 3) without changing the
inactivation time constant. B, Internal application of
~50 mM TEA reduces the current to 53% of control and
slows inactivation. The dotted line represents the
scaled current trace in presence of TEA for direct comparison with the
control trace. Inset, Twofold current reduction and a
twofold increase in the inactivation time constant (mean ± SEM;
n = 4).
|
|
EXP-2 is a large conductance channel
We used cell-attached patches of EXP-expressing oocytes to measure
the conductance of single EXP-2 channels. From a holding potential at
80 mV, EXP-2 channels were activated (and inactivated) by 1 sec
prepulses to 20 mV, followed by test pulses ranging from 60 to 150
mV during which EXP-2 channels recover from inactivation (Fig.
7A). With 100 mM KCl in the recording pipette, EXP-2 channels showed unitary currents that increased with more negative test potentials. During subsequent steps to 40 mV, we were unable to detect
outward current through single channels, presumably because inactivation proceeds too rapidly (inact = 0.7 msec at 40 mV). The I-V relationship was linear from 150
to 0 mV with a chord conductance of 67 ± 2 pS and an extrapolated
reversal potential of 0.35 mV (n = 5). The ensemble
average for recovery from inactivation derived from 200 single-channel
traces was similar to that of traces obtained from macroscopic
recordings using the two-electrode voltage clamp (Fig.
7B).
View larger version (26K):
[in this window]
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|
Figure 7.
Single-channel currents from EXP-2 channels.
Single-channel activity was recorded during recovery from inactivation
with 100 mM KCl in the recording pipette. Channel
activation and inactivation were induced by a 1 sec prepulse to 20 mV.
A, The voltage for recovery was varied from 60 to
150 mV. Representative traces at all test voltages are
shown in the left panel. The right panel
shows the I-V relationship for single EXP-2 channels (5 different patches). Regression analysis yields a single-channel
conductance of 67 ± 2 pS. B, Ensemble average
currents for EXP-2 channels. Top trace (no prepulse)
shows the average of 20 traces when the activation-inactivation
prepulse to 20 mV was reduced to 4 msec. The middle
trace shows the average of 200 traces and the superimposed
single-exponential fit with a time constant of 3.1 msec. The
bottom trace depicts the voltage protocol during
sampling. Capacitive and leakage components were obtained from a
multiexponential fit to the average of 10 traces with no openings. The
idealized trace was then subtracted from each individual trace before
averaging. The remaining frequency transients were blanked.
|
|
| |
DISCUSSION |
EXP-2 is a novel K+ channel of the
Kv family
We describe the functional characterization of the novel,
voltage-gated K+ channel EXP-2 which is
expressed in pharyngeal muscle cells of the nematode C. elegans (Davis, 1999; Davis et al., 1999). Loss-of-function mutations in the exp-2 gene cause dramatic prolongations of
the muscle action potential; in contrast, a gain-of-function mutation leads to brief action potentials, indicating that exp-2 may
encode a voltage-controlled K+ channel
involved in rapid repolarization after the long plateau phase of the
action potential (Davis, 1999; Davis et al., 1999). When we had cloned
exp-2 DNA, it became apparent that the encoded EXP-2 protein
(528 amino acids) was very similar to voltage-gated K+ channels of the Kv family (35-43%
sequence identity to different Kv subfamilies) (Table 1). Although the
exp-2 gene had previously been placed in the Shab
subfamily (Wei et al., 1996), our analysis shows only a marginally
higher percentage of sequence identity to Shab or Kv2.1 (41 and 43%, respectively) compared with members of other Kv subfamilies
(35-40%). In contrast, different members of the same subfamily are
~61-84% identical (Table 1). It is possible, therefore, that EXP-2
represents the prototype of a novel K+
channel subfamily (to be designated Kv10) that has been found neither
in Drosophila nor in vertebrates. The degree of sequence identity that delineates subfamilies is somewhat arbitrary. It has been
shown that mainly channel subunits from the same subfamily form
heterotetrameric K+ channels (Christie et
al., 1990; Isacoff et al., 1990; Ruppersberg et al., 1990; Covarrubias
et al., 1991). More recently, it has become clear that members of the
subfamilies Kv5, Kv6, Kv8, and Kv9, which do not form functional
homotetramers, apparently associate with and modify the function of
subunits from the subfamilies Kv2 and Kv3 (Hugnot et al., 1996; Salinas
et al., 1997). It will be interesting to see whether EXP-2 subunits are
able to co-assemble with subunits from other Kv subfamilies.
The EXP-2 channel contains three consensus sites for phosphorylation by
protein kinase C (T131, T309, and S407) and two consensus sites for
glycosylation in the extracellular linker region connecting S1 and S2.
It is currently not known whether the activity of EXP-2 is modulated by
phosphorylation or whether EXP-2 channels are glycosylated. It has been
shown that two glycosylation sites at similar positions in the Shaker
K+ channel are indeed glycosylated
(Santacruz-Toloza et al., 1994).
EXP-2 is a Kv-type channel acting as an inward rectifier
The kinetic behavior of EXP-2 is highly unusual for a channel that
clearly belongs in the Kv family of voltage-gated
K+ channels (Table
2). Kv-type channels show relatively fast
voltage-dependent activation (with the exception of Shaw),
slow C-type inactivation, and voltage-dependent deactivation. In
addition, some Kv channels, e.g., Shaker, Kv1.4, and Kv3.4,
also show fast N-type inactivation. Activation of EXP-2 channels is
voltage-dependent but occurs relatively slowly even at positive
potentials (act = ~54 msec at 20 mV; Fig.
2). Deactivation is even slower and shows little or no voltage dependence between 80 and 120 mV (deact = ~80 msec). The greatest differences from Kv-type
K+ channels can be seen for inactivation
and recovery from inactivation. Both processes are voltage-dependent
and occur at very fast rates compared with most Kv channels (Fig. 5).
Inactivation begins to take place in the negative voltage range
(inact = ~8 msec at 60 mV) and occurs very
rapidly at positive voltages (inact = ~0.8
msec at 20 mV). Recovery from inactivation also occurs very rapidly
(rec = ~5 msec at 80 mV and ~1 msec at
120 mV).
The putative physiological function of EXP-2 in pharyngeal muscle cells
and the kinetics of oocyte-expressed channels are very reminiscent of
the role and kinetic properties of the human cardiac
K+ channel HERG (Trudeau et al., 1995;
Smith et al., 1996; Spector et al., 1996; Wang et al., 1996). Like
HERG, EXP-2 shows voltage-dependent slow activation, fast inactivation,
and fast recovery from inactivation. Interestingly, although EXP-2 and
HERG show very similar kinetics of activation and inactivation, the two
channels are not homologous and show no sequence similarity outside the
pore region. It is remarkable that the inward rectification mechanism
by rapid inactivation has evolved independently in two classes of
structurally unrelated voltage-gated K+ channels.
To our knowledge, EXP-2 is the only Kv-type
K+ channel that is known to function as an
inward rectifier in a normal physiological environment. The outwardly
rectifying Shaker channel was converted to an inward
rectifier by S4 mutations leading to channel activation at
approximately 200 mV (Miller and Aldrich, 1996). In contrast to
EXP-2, which is activated at approximately 20 mV and derives its
inward rectification from the ultrafast inactivation mechanism, the
Shaker S4 mutants are already activated and have undergone steady-state inactivation at the resting membrane potential. EXP-2 channels require depolarization before they can open; in contrast, the
Shaker S4 mutants can be activated by hyperpolarization.
Internal TEA blocks EXP-2 and slows inactivation
TEA blockade can be used to distinguish N-type from C-type
inactivation in voltage-gated K+ channels
(Choi et al., 1991). Although TEA blocks EXP-2 from either side, only
blockade from the inside slows inactivation (Fig. 6). The twofold
change, both in current reduction and in the slowing of inactivation,
is expected if internal TEA occluded ion flux by competing with the
N-terminal inactivation particle for binding to the inner vestibule.
The TEA effect on inactivation is clearly different between EXP-2 and
HERG, although the two channels behave similarly in many other aspects
(Table 2). In contrast to EXP-2, external TEA slows inactivation of
HERG, and this has been interpreted as a form of fast C-type
inactivation in HERG (Schönherr et al., 1996; Smith et al.,
1996). The finding that external TEA blocks EXP-2 without slowing
inactivation does not rule out the presence of C-type inactivation,
which might be slow and masked by faster N-type-like inactivation.
Also, it has been shown that some quaternary ammonium ions block from
the inside and affect conformational changes of the outer mouth that are involved in C-type inactivation (Baukrowitz and Yellen, 1996). Future experiments involving the removal of the N terminus will be
needed to determine whether a Shaker-type
"ball-and-chain" mechanism underlies the voltage-dependent,
ultrafast inactivation in EXP-2.
Besides the apparent difference in the underlying molecular mechanism
of fast inactivation, another difference from HERG (and other Kv
channels) is the large unit conductance of EXP-2. Single EXP-2 channels
show a unit conductance of 67 ± 2 pS. This is 5-10 times larger
than the conductances of many Kv channels (5-10 pS) with the exception
of Kv3.1 (~25 pS). It is also substantially larger then the 12 pS
conductance of HERG (Zou et al., 1997). We currently do not know the
sequence differences in EXP-2 that cause the relatively large conductance.
Are the properties of EXP-2 suitable for the rapid repolarization
after long-duration action potentials?
Intracellular recordings of pharyngeal muscle cells show action
potentials with long plateaus (150-200 msec) followed by a rapid phase
of repolarization. Several loss-of-function mutations in
exp-2 lead to dramatically prolonged action potentials, and a gain-of-function mutation results in brief action potentials (Davis
et al., 1995). These findings suggest strongly that EXP-2 channels are
directly involved in rapid repolarization, a role similar to HERG
channels in the human heart. The question remains, however, whether
homotetrameric EXP-2 channels are really suited for fast repolarization
after a long plateau. Although EXP-2 channels activate slowly
(act = ~54 msec at 20 mV), >90% of
channels become activated and inactivated
(inact = ~1 msec at 20 mV) during the ~150
msec plateau of the pharyngeal action potential. Hence, when the
membrane potential approaches ~0 mV toward the end of the plateau
phase, EXP-2 channels are "primed" for the rapid recovery from
inactivation. The rate of recovery from inactivation is, however, still
much slower than the inactivation rate (Fig. 5). This scenario is
reminiscent of voltage-sensitive Na+ channels in the
situation when the membrane potential approaches the threshold for
action potential generation. Under these threshold conditions, the rate
of Na+ channel activation is far slower than the
inactivation rate; however, local depolarization followed by transient
Na+ channel openings eventually lead to regenerative
channel openings resulting in the upstroke of the action potential. A
current with the properties of EXP-2 was originally described in
Ascaris by Byerly and Masuda (1979). These authors coined
the term negative spike channel because of its similar but
opposite action to voltage-sensitive Na+ channels.
Of course, we cannot rule out that the function of EXP-2 channels in
the pharyngeal muscle cells might be modified by other subunits. It is
currently not known whether EXP-2 subunits in C. elegans
pharyngeal muscle form homotetrameric channels, as assumed to be the
case in Xenopus oocytes, or whether they co-assemble with
other subunits in a heterotetrameric K+
channel complex that is responsible for the fast repolarization of
pharyngeal muscle cells.
| |
FOOTNOTES |
Received Aug. 23, 1999; revised Oct. 20, 1999; accepted Oct. 21, 1999.
This work was supported in part by National Institutes of Health Grants
NS28407 (R.H.J.), HL46154 (L.A.), and GM53196 (A.N.). We thank Dr. Gail
Robertson (University of Wisconsin Medical School) for the HERG cDNA
clone and Dr. Donald Hilgemann for insightful comments and critical
reading of this manuscript.
Correspondence should be addressed to Dr. Rolf H. Joho, Center for
Basic Neuroscience, The University of Texas Southwestern Medical
Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9111. E-mail:
joho{at}utsw.swmed.edu.
| |
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F. Espinosa, R. Fleischhauer, A. McMahon, and R. H. Joho
Dynamic Interaction of S5 and S6 during Voltage-Controlled Gating in a Potassium Channel
J. Gen. Physiol.,
August 1, 2001;
118(2):
157 - 170.
[Abstract]
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E. Loots and E. Y. Isacoff
Molecular Coupling of S4 to a K+ Channel's Slow Inactivation Gate
J. Gen. Physiol.,
November 1, 2000;
116(5):
623 - 636.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION
