(Received for publication, May 10, 1997, and in revised form, May 29, 1997)
From the Department of Pharmacology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan
The "ball and chain" model has been shown to be suitable for explaining the rapid inactivation of voltage-dependent K+ channels. For the Drosophila Shaker K+ channel (ShB), the first 20 residues of the amino terminus have been identified as the inactivation ball that binds to the open channel pore and blocks ion flow (Hoshi, T., Zagotta, W. N., and Aldrich, R. W. (1990) Science 250, 533-538; Zagotta, W. N., Hoshi, T., and Aldrich, R. W. (1990) Science 250, 568-571). We studied the structural elements responsible for rapid inactivation of a mammalian transient type K+ channel (rat Kv1.4) by constructing various mutants in the amino terminus and expressing them in Xenopus oocytes. Although it has been reported that the initial 37 residues might form the inactivation ball for rat Kv1.4 (Tseng-Crank, J., Yao, J.-A., Berman M. F., and Tseng, G.-N. (1993) J. Gen. Physiol. 102, 1057-1083), we found that not only the initial 37 residues, but also the following region, residues 40-68, could function independently as an inactivation gate. Like the Shaker inactivation ball, both potential inactivation domains have a hydrophobic amino-terminal region and a hydrophilic carboxyl-terminal region having net positive charge, which is essential for the domains to function as an inactivation gate.
Aldrich and co-workers have shown that a "ball and chain" model, originally proposed for Na+ channel inactivation (4), can also explain the rapid inactivation of a Drosophila Shaker K+ channel (1, 2). The amino-terminal domain (ball) tethered by the adjacent region (chain) to the channel protein binds to the channel pore after channel activation and blocks ion flow. In the Shaker K+ channel (ShB), the initial 20 amino acids have been identified as the inactivation ball. The following region preceding the assembly domain (5) has been identified as the chain tethering the ball to the channel (1). The 20-amino acid inactivation ball is composed of the 11 amino-terminal hydrophobic residues and the following 9 hydrophilic residues containing net positive charge. Both the hydrophobic stretch and the charged region are thought to be involved in the binding of the ball to its receptor via hydrophobic and electrostatic interactions. In contrast, in mammalian transient type K+ channels, the ball and chain structure had not been well defined, although it had been shown that deletion of various lengths from the amino-terminal region of Kv1.4 disrupted rapid inactivation suggesting the presence of a "ball" structure (6, 7). Tseng and co-workers (3) have studied this issue in more detail by deleting different domains in the amino-terminal region of rat Kv1.4. They have not identified "chain" structure but have shown that deletion of the amino-terminal hydrophobic domain, residues 3-25, resulted in loss of rapid inactivation. Deletion of the following hydrophilic region containing five positive and two negative charges, residues 26-37, greatly attenuated inactivation. Based on these and other findings, they suggested that the amino terminus of rat Kv1.4 might be similar to that of ShB in having one inactivation ball, which is composed of the initial 37 residues. In the present study, we investigated the structural elements responsible for rapid inactivation of rat Kv1.4 and have identified another domain that can produce rapid inactivation independently of the proposed inactivation ball.
Fig. 1 shows the amino-terminal
sequences of Kv1.4 and the mutants investigated in this study. Eleven
deletion mutants and one addition mutant were made in the
amino-terminal region of Kv1.4. In addition, one mutant in which amino
acid residues 40-68 of Kv1.4 were inverted in 2-39 &
69-162
was constructed. Fragments for all the mutants except the one with
inverted residues were generated by polymerase chain reaction
(PCR).1 The 20-22-base pair
sense primers used for generating
2-25,
2-26,
2-28,
2-30,
2-32,
2-39, and
2-61 corresponded to the appropriate region in Kv1.4 and contained an ApaI site,
unique within the multiple cloning site of the vector pBluescript II, and an ATG at the 5
-end. The antisense primer (AS1) complementary to
nucleotides (nt) 532-551 of Kv1.4 was used for the above seven mutants. The sense primer used for generating
29-162 corresponded to nucleotides 487-506 of Kv1.4 and contained a XhoI site
(which is unique in Kv1.4 at nt 80) at the 5
-end; the antisense primer was AS1. For constructing
2-39 &
69-162, the sense primer (S1), with an ApaI site at the 5
-terminus and corresponding to
nucleotides
35 to
16 of Kv1.4, was used with an antisense primer
complementary to nucleotides 180-199 with a XhoI site at
its 5
-end. To generate
38-162 and
2-39 &
61-162, two
fragments, amplified by PCR, were ligated into the mutants. The
upstream fragment for each mutant was designated fragment I; the
downstream fragment was fragment II. Fragment II for both mutants was
the same and corresponded to amino acid residues 163-185 of Kv1.4. The
sense primer for fragment I of
38-162 was S1, and that of
2-39
&
61-162 was the same one used for
2-39. The antisense primer
for the fragment I contained a StuI site at the 5
-end and
corresponded to nucleotides 96-114 for
38-162 and to nucleotides
161-181 for
2-39 &
61-162. The sense primer for fragment II
corresponded to nucleotides 490-508 and contained a SmaI
site at the 5
-end; the antisense primer was AS1. Amino acid residues
26-39 of Kv1.4 were added to the amino terminus of Kv1.4 in the
addition mutant. To make the addition mutant, two fragments (fragment I
was generated by PCR and fragment II was cut out from Kv1.4) were
ligated into the mutant. The sense primer for fragment I of the
addition mutant corresponded to nucleotides 76-99 and contained an
ApaI site and ATG at the 5
-end; the antisense primer was
complementary to nucleotides 97-116 and contained a NcoI
site at the 5
-end. To make amino acid residues 40-68
(AALAVAAATAAVEGTGGSGGGPHHHHQTR) invert in
2-39 &
69-162, the
sense oligonucleotide that codes for MRTQHHHHPGGGSGGTGEVAATAAAVALAA and
the antisense oligonucleotide complementary to it were used. They were
designed to produce an ApaI site at the 5
-end and a
MluI site at the 3
-end when annealed. The annealed fragment
was ligated to Kv1.4, which was digested with ApaI and
MluI. In a 100-µl PCR reaction, 100 pmol of each primer,
0.2 µg of template cDNA (Kv1.4 for all the mutants except
2-39 &
69-162;
2-39 for
2-39 &
69-162), and 5.0 units of Taq DNA polymerase (Perkin-Elmer) were used.
Reaction temperatures were varied using a thermal cycler
(Perkin-Elmer): 94 °C, 1 min; 55 °C, 2 min; and 72 °C, 3 min
for 25 cycles. The amplified fragment for
2-25,
2-26,
2-28,
2-30,
2-32,
2-39, and
2-61 was digested with
ApaI and MluI and ligated to Kv1.4 between the
ApaI and MluI sites. The amplified fragment for
29-162 was digested with XhoI and MluI and
ligated to Kv1.4 between the XhoI and MluI sites. The fragment for
2-39 &
69-162 was amplified using
2-39 as template and digested with ApaI and XhoI. The
digested fragment was ligated to
29-162, which was digested with
ApaI and XhoI. The amplified fragment I for
2-39 &
61-162 and
38-162 was digested with ApaI
and StuI, and the other fragment (fragment II) was digested with SmaI and MluI. These two fragments were
ligated to Kv1.4, which was digested with ApaI and
MluI. The amplified fragment I for the addition mutant,
which was digested with ApaI and NcoI, fragment
II, which was cut out from Kv1.4 with NcoI (at nt
2), and
PmaCI (at nt 200) were ligated to Kv1.4 digested with
ApaI and PmaCI. Sequences of all the fragments
generated by PCR in the mutants were verified on both strands by the
dideoxy chain termination method using an A.L.F. DNA Sequencer II
(Pharmacia Biotech Inc.).
Expression and Current Recording
Electrophysiological measurements were carried out essentially as reported previously (8, 9). The pBluescript II vectors containing the constructs were linearized with EcoRI, and cRNAs were prepared from these templates with T7 RNA polymerase (Stratagene). Transcribed RNAs were dissolved in water at a final concentration of 0.2 µg/µl for oocyte injection. The integrity of the cRNA products was checked by running the samples on formaldehyde containing agarose gels (10). Defolliculated Xenopus oocytes (stage V-VI) were injected with 40-50 nl (8-10 ng) of cRNA. The injected oocytes were incubated in Barth's medium supplemented with penicillin G (71.5 units/ml) and streptomycin (35.9 µg/ml) at 18 °C for 2-4 days before doing electrophysiological measurements. The K+ currents were recorded by a conventional two-microelectrode voltage clamp method with 3 M KCl-filled electrodes as described (8, 9). The basic bath recording solution consisted of ND 96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.5). For the bath solution containing high K+ (20K), Na+ was replaced with K+. All electrophysiological measurements were carried out at room temperature (21 ± 1 °C). Current records were low pass-filtered at 3 kHz.
All data are expressed as the mean ± S.E. The statistical significance was evaluated using Student's paired or unpaired t test. A p value smaller than 0.05 was considered to be significant.
Oocytes expressing Kv1.4 and all the mutant channels showed
voltage-dependent outward currents upon depolarization
(data not shown). They were held at 80 mV and depolarized to test
potentials.
Fig.
2A (upper panel) shows normalized currents of
2-28 and Kv1.4 recorded using a depolarizing pulse to +20 mV for
400 ms. The traces are superimposed to illustrate the differences in
their wave forms. The peak current of Kv1.4 and
2-28 at +20 mV was 2.05 ± 0.35 µA (n = 7) and 4.96 ± 0.54 µA (n = 5), respectively. The
2-28 current showed
little decline during the 400-ms test pulse, while the Kv1.4 current
inactivated almost completely.
inact of
2-28 current
was measured using a prolonged depolarization pulse (5000 ms). The
inact was 2090.9 ± 647.9 ms (n = 5), which was about 90 times larger than that of Kv1.4. The
inactivation of
2-28 seems to be qualitatively different from that
of Kv1.4. Since it has been shown for Shaker K+
channel and RHK1 (rat Kv1.4) that elevating [K+]o
can accelerate the recovery rate (11, 12), we investigated the effects
of changing [K+]o on the recovery rate. Recovery
from inactivation of
2-28 at 2 mM
[K+]o and 20 mM
[K+]o is shown in Fig. 2A (lower
panel). Elevating [K+]o had no effect on the
recovery time course (
rec: 3.43 ± 0.31 s at 2 mM [K+]o and 3.06 ± 0.42 s
at 20 mM [K+]o (n = 5)). Surprisingly, with further deletion (
2-39), rapid inactivation
was resumed. Currents of
2-39 and Kv1.4 recorded at +20 mV were
normalized and superimposed in Fig. 2B (upper
panel). The peak current of
2-39 at +20 mV was 0.71 ± 0.18 µA (n = 4). The
2-39 current showed rapid
inactivation. The
inact of
2-39 current was
30.33 ± 2.37 ms (n = 4), which is slightly larger than that of Kv1.4 (Fig. 1). Elevating
[K+]o accelerated the recovery from inactivation
of
2-39 (Fig. 2B,
lower panel). The
rec of
2-39 was
72.96 ± 8.25 s at 2 mM
[K+]o and 37.15 ± 2.48 s at 20 mM [K+]o (n = 4).
With further deletion (
2-61), fast inactivation disappeared again
(Fig. 2C, upper panel). The
2-61 current
showed little decline during a 400-ms test pulse. The peak current of
2-61 at +20 mV was 2.01 ± 0.51 µA (n = 6).
Inactivation was observed during a prolonged depolarization pulse (5000 ms). The recovery time course of the
2-61 current was not affected
by elevating [K+]o (Fig. 2C,
lower panel). The
rec was 3.17 ± 0.20 s at 2 mM [K+]o and
3.31 ± 0.82 s at 20 mM
[K+]o (n = 6). These results
suggested that Kv1.4 might have two potential inactivation balls.
Based on these findings, we constructed a deletion mutant that had only
the first potential inactivation ball, 38-162, and a mutant,
2-39 &
69-162, which had only the second potential inactivation
ball. Fig. 3 (A and
B, upper panel), shows the normalized currents
from
38-162 and
2-39 &
69-162 superimposed on the Kv1.4
current. The
38-162 current showed rapid inactivation. The
inact of
38-162 at +20 mV was 38.66 ± 1.46 ms
(n = 6), which was slightly larger than that of Kv1.4
(Fig. 1). Elevating [K+]o accelerated the
recovery of
38-162 (Fig. 3A, lower panel).
The
rec was 3.94 ± 0.32 s at 2 mM
[K+]o and 1.53 ± 0.08 s at 20 mM [K+]o (n = 6). The
peak current of
38-162 at +20 mV was 1.67 ± 0.25 µA
(n = 6).
2-39 &
69-162, with only the second potential inactivation ball, showed more rapid inactivation than Kv1.4
(Fig. 3B, upper panel). The
inact
of
2-39 &
69-162 at +20 mV was 12.05 ± 1.20 ms
(n = 6), which was significantly smaller than that of
Kv1.4 (p < 0.05)(Fig. 1). Elevating
[K+]o accelerated the recovery of
2-39 &
69-162 (Fig. 3B, lower panel). The
rec was 20.83 ± 2.46 s at 2 mM
[K+]o and 15.13 ± 1.07 s at 20 mM [K+]o (n = 6). The
peak current of
2-39 &
69-162 at +20 mV was 1.19 ± 0.37 µA (n = 6).
Effects of Deleting Positive Charges
Since both potential
inactivation balls contain net positive charge in the carboxyl termini,
we constructed mutants in which some positive charges were removed to
examine the contribution of charge to rapid inactivation. 29-162
was constructed to delete one net positive charge (3 arginine and 2 glutamic acid residues) from the first potential inactivation ball.
2-39 &
61-162 was constructed to delete one positive charge
(arginine) from the second potential inactivation ball. Fig.
4A (upper panel)
shows the normalized
29-162 current superimposed on the
38-162
current recorded at +20 mV. The peak current of
29-162 at +20 mV
was 1.80 ± 0.54 µA (n = 4). Inactivation of the
29-162 current was much slower than that of
38-162. The
inact of
29-162 was 361.09 ± 37.69 ms
(n = 4), which is about 9 times larger than that of
38-162 (Fig. 1). The
rec of
29-162 (1.55 ± 0.16 s at 2 mM [K+]o and
0.63 ± 0.10 s at 20 mM
[K+]o) was significantly smaller than that of
38-162 (3.94 ± 0.32 s at 2 mM
[K+]o and 1.53 ± 0.08 s at 20 mM [K+]o) (p < 0.01). Fig. 4A (lower panel) shows the effects of
elevating [K+]o on the recovery time course of
29-162. Currents of
2-39 &
61-162 and
2-39 &
69-162
recorded at +20 mV are normalized and superimposed in Fig.
4B (upper panel). The peak current of
2-39 &
61-162 at +20 mV was 1.41 ± 0.20 µA (n = 7). The
inact of
2-39 &
61-162 was 59.22 ± 1.77 ms (n = 7), which is 5 times larger than that of
2-39 &
69-162 (Fig. 1). Recovery from inactivation was much
faster in
2-39 &
61-162 than in
2-39 &
69-162 (Fig. 1).
The
rec of
2-39 &
61-162 and
2-39 &
69-162 at 2 mM [K+]o were
2.85 ± 0.31 s and 20.83 ± 2.46 s, respectively, and those at 20 mM [K+]o were
1.74 ± 0.12 s and 15.13 ± 1.07 s, respectively. Fig. 4B (lower panel) shows the effects of
elevating [K+]o on the
rec of
2-39 &
61-162.
Effects of Adding Positive Charges
When the structures of
2-28 and
2-39 are compared, they differ by a single net
positive charge (3 arginine and 2 glutamic acid residues) at the amino
terminus of the second potential inactivation ball. However, the
currents of the two channels were completely different (Fig.
5A, upper panel).
Therefore, we constructed four other mutants that varied in charge at
the amino terminus of the second potential ball,
2-32,
2-30,
2-26, and
2-25. The numbers of extra net positive charges are
one for
2-32 and
2-30, two for
2-26, and three for
2-25. The currents of all these mutants showed little inactivation
on the same time scale as Kv1.4 (400 ms) (Fig. 5A,
lower panel) and no significant differences in
inact measured during a test pulse of 5000 ms (data not
shown).
We also constructed a mutant ((+)Kv1.4) in which residues 26-39 of
Kv1.4 containing three net positive charges were added to the
amino-terminal end of Kv1.4. Adding positive charges greatly reduced
the rate of inactivation (Fig. 5B, upper panel).
The inact of (+)Kv1.4 was 155.9 ± 15.83 ms
(n = 6), which was significantly larger than that of
Kv1.4 (23.55 ± 3.48 ms; n = 7; p < 0.01) (Fig. 1). The
rec of (+)Kv1.4 at 2 mM [K+]o (7.10 ± 1.57 s)
was significantly larger than that at 20 mM
[K+]o (3.41 ± 0.45 s;
p < 0.05; n = 6) (Fig. 5B,
lower panel).
We constructed a mutant in which the second
potential inactivation ball was inverted (Inv(40-68)). The inverted
ball has positive charge at the amino terminus and a hydrophobic region
at the carboxyl terminus. This mutant showed little inactivation during
a 400-ms test pulse to +20 mV, whereas the parent mutant showed rapid
inactivation (Fig. 5C, upper panel). The
inact of Inv(40-68) recorded during a 5000-ms pulse was
2616.3 ± 252.8 ms (n = 5). Inverting the second potential ball resulted in the loss of rapid inactivation. There were
no differences in
rec of the mutant between 2 mM [K+]o (4.74 ± 0.16 s)
and 20 mM [K+]o (4.88 ± 0.48 s; n = 5) (Fig. 5C, lower
panel).
We found that there are two potential inactivation balls in the
amino-terminal region of rat Kv1.4. Deletion of amino acids 2-28
resulted in loss of rapid inactivation. This is consistent with the
finding of Tseng and co-workers (3), who found that deletion of
residues 3-25 disrupted rapid inactivation. Surprisingly, deletion of
11 more residues resulted in reappearance of rapid inactivation even
though the 2-39 mutant did not have the core hydrophobic region of
the inactivation ball. With further deletion of residues 40-61, rapid
inactivation disappeared again. It seems probable that besides the
inactivation ball proposed by Tseng and co-workers (the initial 37 residues), there exists a second potential inactivation ball having
residues 40-61 as an essential domain. To confirm the presence of two
potential balls, we made deletion mutants that had only one potential
ball and lacked most of the amino-terminal region preceding the
assembly domain (
38-162 and
2-39 &
69-162). As expected,
the currents of both the mutants showed rapid inactivation, which
indicated that the two potential ball, residues 2-37 and residues
40-68, respectively, could produce rapid inactivation independently.
Comparison of
38-162 and
2-39 &
69-162, both of which lack
most of the possible chain region, gave some information about the
characteristic differences between the first and the second ball. In
the case of the second ball (in
2-39 &
69-162), inactivation
was more rapid and the recovery from inactivation was slower than in
the case of the first ball (in
38-162) (Figs. 1 and 3). This
suggests that the second ball may have a higher affinity for the
receptor than the first ball. Compared with Kv1.4, binding between the
ball and the receptor seems to be much stronger for the second ball
than for the ball in wild type Kv1.4, as
2-39 currents recover
significantly more slowly than Kv1.4 currents (Fig. 1). Among the
mutants investigated, the ones that have the second ball recovered from
inactivation most slowly (
2-39 and
2-39 &
69-162). The
recovery rates of their currents were significantly slower than those
of the other mutants. The
2-39 &
69-162 currents recovered
faster than
2-39 currents, probably reflecting the influence of the
chain region on binding of the ball to the receptor. The presence of
residues 69-162 caused slowing of the recovery from inactivation of
the
2-39 current.
Similar to the structure of ShB inactivation ball, the two potential
balls in Kv1.4 have an amino-terminal hydrophobic region and a
carboxyl-terminal hydrophilic region containing net positive charge,
which is thought to be involved in the binding of the inactivation
particle to its receptor via electrostatic interactions (2, 13, 14).
Therefore we investigated the contribution of positive charge. Deletion
of positive charge from either ball greatly attenuated the inactivation
rates and accelerated the recovery rates, which probably reflects the
higher affinity of the ball to the receptor site with the
carboxyl-terminal positive charge. This result clearly indicates that
the positive charge at carboxyl-terminal region of the ball plays an
important role. The structural requirements for the inactivation ball
were further studied by deleting or adding positive charges in the
amino-terminal region of Kv1.4 and by inverting the amino acid sequence
of the potential inactivation ball. Since the structure of
non-inactivating 2-28 was just like having net positive charge (3 arginine and 2 glutamic acid residues) at the amino terminus of the
second potential ball of rapidly inactivating
2-39, the mutants
with different numbers of charges were constructed (
2-32,
2-30,
2-26 and
2-25). The currents through these mutants hardly
inactivated (Fig. 5A, lower panel). These results
suggest that one extra positive charge at the amino terminus of the
second inactivation ball is enough to disrupt its function. Therefore,
the influences of net positive charges at the amino terminus of the
inactivation ball of Kv1.4 were studied by adding residues 26-39
(three net positive charges) at the amino-terminal end of Kv1.4
((+)Kv1.4). The currents through (+)Kv1.4 inactivated but the rate of
inactivation was significantly slower than for wild type Kv1.4 (Fig.
5B). These results indicate that net positive charge at the
amino-terminal end of the inactivation ball of wild type Kv1.4 has a
profound effect on function. Together the results indicate the
structural requirements of the inactivation ball(s) are an
amino-terminal hydrophobic region and a carboxyl-terminal hydrophilic
region containing net positive charge.
In agreement with the results of Tseng and co-workers, changing [K+]o had no effects on the recovery rate for our mutants, which did not show rapid inactivation. Elevating [K+]o accelerated the recovery rate in mutants with rapid inactivation, which might reflect repulsion of the inactivation ball by K+ ions (11).
The most striking finding in the present study is that there exist two potential inactivation balls in the amino terminus of rat Kv1.4. It is not known how two inactivation balls could work in wild type Kv1.4. One of the two potential domains might function as the inactivation gate, or one inactivation gate might be composed of both domains. Alternatively, the redundancy of inactivation balls might be a safety device to ensure inactivation. The synthetic ShB inactivation ball peptide has been reported to block several types of K+ channels and also cyclic nucleotide gated channels (15-19). It will be of interest to synthesize the peptides corresponding to the first domain, the second domain and both the domains of Kv1.4, and to compare their effects on currents of the non-inactivating mutant of Kv1.4 and the other channels. Synthetic peptides could give useful information about how the two domains contribute to form the inactivation ball in wild type Kv1.4. Recently, NMR structures of the inactivation peptides of Kv3.4 (the initial 30 residues) and Kv1.4 (the initial 37 residues) have been reported. The inactivation peptides have a similar characteristic surface charge pattern with a positively charged, a hydrophobic, and a negatively charged region (20). The inactivation peptide of Kv1.4 whose NMR structure was determined corresponds to our first inactivation ball. It will be of interest to determine the NMR structure of the second domain and both the domains in the amino-terminal region of Kv1.4.
We thank Dr. Kazuo Nunoki for review of the manuscript.