A Mammalian Transient Type K+ Channel, Rat Kv1.4, Has Two Potential Domains That Could Produce Rapid Inactivation*

(Received for publication, May 10, 1997, and in revised form, May 29, 1997)

Shun-ichi Kondoh , Kuniaki Ishii Dagger , Yasuhiro Nakamura and Norio Taira

From the Department of Pharmacology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

In Vitro Mutagenesis

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 Delta 2-39 & Delta 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 Delta 2-25, Delta 2-26, Delta 2-28, Delta 2-30, Delta 2-32, Delta 2-39, and Delta 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 Delta 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 Delta 2-39 & Delta 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 Delta 38-162 and Delta 2-39 & Delta 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 Delta 38-162 was S1, and that of Delta 2-39 & Delta 61-162 was the same one used for Delta 2-39. The antisense primer for the fragment I contained a StuI site at the 5'-end and corresponded to nucleotides 96-114 for Delta 38-162 and to nucleotides 161-181 for Delta 2-39 & Delta 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 Delta 2-39 & Delta 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 Delta 2-39 & Delta 69-162; Delta 2-39 for Delta 2-39 & Delta 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 Delta 2-25, Delta 2-26, Delta 2-28, Delta 2-30, Delta 2-32, Delta 2-39, and Delta 2-61 was digested with ApaI and MluI and ligated to Kv1.4 between the ApaI and MluI sites. The amplified fragment for Delta 29-162 was digested with XhoI and MluI and ligated to Kv1.4 between the XhoI and MluI sites. The fragment for Delta 2-39 & Delta 69-162 was amplified using Delta 2-39 as template and digested with ApaI and XhoI. The digested fragment was ligated to Delta 29-162, which was digested with ApaI and XhoI. The amplified fragment I for Delta 2-39 & Delta 61-162 and Delta 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.).


Fig. 1. Diagram of the amino-terminal sequences of the mutants and the time constants for inactivation and recovery from inactivation of their currents. The first 190 amino acid residues of wild type Kv1.4 are shown at the top. Open circle shows positively charged amino acid residues, and closed circle shows negatively charged amino acid residues. Deleted regions in the mutants are shown by solid bars. Time constants for inactivation (tau inact) and recovery from inactivation (tau rec) at 2 mM [K+]o of the currents are shown on the right. The mutants whose currents did not rapidly inactivate during a 400-ms pulse are shown by (-) in the column of tau inact and tau rec. The values are expressed as the mean ± S.E. Differences between values were analyzed using Student's unpaired t test.
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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.


RESULTS

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.

Presence of Two Potential Inactivation Balls

Fig. 2A (upper panel) shows normalized currents of Delta 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 Delta 2-28 at +20 mV was 2.05 ± 0.35 µA (n = 7) and 4.96 ± 0.54 µA (n = 5), respectively. The Delta 2-28 current showed little decline during the 400-ms test pulse, while the Kv1.4 current inactivated almost completely. tau inact of Delta 2-28 current was measured using a prolonged depolarization pulse (5000 ms). The tau inact was 2090.9 ± 647.9 ms (n = 5), which was about 90 times larger than that of Kv1.4. The inactivation of Delta 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 Delta 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 (tau 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 (Delta 2-39), rapid inactivation was resumed. Currents of Delta 2-39 and Kv1.4 recorded at +20 mV were normalized and superimposed in Fig. 2B (upper panel). The peak current of Delta 2-39 at +20 mV was 0.71 ± 0.18 µA (n = 4). The Delta 2-39 current showed rapid inactivation. The tau inact of Delta 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 Delta 2-39 (Fig. 2B, lower panel). The tau rec of Delta 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 (Delta 2-61), fast inactivation disappeared again (Fig. 2C, upper panel). The Delta 2-61 current showed little decline during a 400-ms test pulse. The peak current of Delta 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 Delta 2-61 current was not affected by elevating [K+]o (Fig. 2C, lower panel). The tau 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.


Fig. 2. Rapid inactivation of Kv1.4 current disappeared with deletion of amino acid residues 2-28, but was resumed with further deletion (Delta 2-39). Upper panel, currents of Delta 2-28 (A), Delta 2-39 (B), and Delta 2-61 (C) are normalized and superimposed on Kv1.4 currents. Their currents were elicited by a depolarizing step to +20 mV from -80 mV. Lower panel, influences of changing [K+]o (open circle , 2 mM [K+]o; bullet , 20 mM [K+]o) on the recovery from inactivation are shown. A, Delta 2-28; B, Delta 2-39; C, Delta 2-61. A control pulse from -80 mV to +20 mV was followed by the identical second pulse after varying interpulse interval. Fraction recovery is plotted as a function of the interpulse interval.
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Based on these findings, we constructed a deletion mutant that had only the first potential inactivation ball, Delta 38-162, and a mutant, Delta 2-39 & Delta 69-162, which had only the second potential inactivation ball. Fig. 3 (A and B, upper panel), shows the normalized currents from Delta 38-162 and Delta 2-39 & Delta 69-162 superimposed on the Kv1.4 current. The Delta 38-162 current showed rapid inactivation. The tau inact of Delta 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 Delta 38-162 (Fig. 3A, lower panel). The tau 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 Delta 38-162 at +20 mV was 1.67 ± 0.25 µA (n = 6). Delta 2-39 & Delta 69-162, with only the second potential inactivation ball, showed more rapid inactivation than Kv1.4 (Fig. 3B, upper panel). The tau inact of Delta 2-39 & Delta 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 Delta 2-39 & Delta 69-162 (Fig. 3B, lower panel). The tau 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 Delta 2-39 & Delta 69-162 at +20 mV was 1.19 ± 0.37 µA (n = 6).


Fig. 3. Not only the initial 37 amino acid residues but also residues 40-68 have potential to work as an inactivation gate. Upper panel, normalized currents of Delta 38-162 (A) and Delta 2-39 & Delta 69-162 (B) are superimposed on Kv1.4 currents. Lower panel, influences of changing [K+]o (open circle , 2 mM [K+]o; bullet , 20 mM [K+]o) on the recovery from inactivation are shown. A, Delta 38-162; B, Delta 2-39 & Delta 69-162. Pulse protocols are same as described in the legend of Fig. 2.
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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. Delta 29-162 was constructed to delete one net positive charge (3 arginine and 2 glutamic acid residues) from the first potential inactivation ball. Delta 2-39 & Delta 61-162 was constructed to delete one positive charge (arginine) from the second potential inactivation ball. Fig. 4A (upper panel) shows the normalized Delta 29-162 current superimposed on the Delta 38-162 current recorded at +20 mV. The peak current of Delta 29-162 at +20 mV was 1.80 ± 0.54 µA (n = 4). Inactivation of the Delta 29-162 current was much slower than that of Delta 38-162. The tau inact of Delta 29-162 was 361.09 ± 37.69 ms (n = 4), which is about 9 times larger than that of Delta 38-162 (Fig. 1). The tau rec of Delta 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 Delta 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 Delta 29-162. Currents of Delta 2-39 & Delta 61-162 and Delta 2-39 & Delta 69-162 recorded at +20 mV are normalized and superimposed in Fig. 4B (upper panel). The peak current of Delta 2-39 & Delta 61-162 at +20 mV was 1.41 ± 0.20 µA (n = 7). The tau inact of Delta 2-39 & Delta 61-162 was 59.22 ± 1.77 ms (n = 7), which is 5 times larger than that of Delta 2-39 & Delta 69-162 (Fig. 1). Recovery from inactivation was much faster in Delta 2-39 & Delta 61-162 than in Delta 2-39 & Delta 69-162 (Fig. 1). The tau rec of Delta 2-39 & Delta 61-162 and Delta 2-39 & Delta 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 tau rec of Delta 2-39 & Delta 61-162.


Fig. 4. Influences of deleting net positive charge from the carboxyl-terminal region of the potential ball domains. Upper panel, currents of Delta 29-162 and its parent mutant (Delta 38-162) (A) and Delta 2-39 Delta 61-162 and its parent mutant (Delta 2-39 & Delta 69-162) (B) are normalized and superimposed. Lower panel, influences of changing [K+]o (open circle , 2 mM [K+]o; bullet , 20 mM [K+]o) on the recovery from inactivation are shown. A, Delta 29-162; B, Delta 2-39 & Delta 61-162. Pulse protocols are same as described in the legend of Fig. 2.
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Effects of Adding Positive Charges

When the structures of Delta 2-28 and Delta 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, Delta 2-32, Delta 2-30, Delta 2-26, and Delta 2-25. The numbers of extra net positive charges are one for Delta 2-32 and Delta 2-30, two for Delta 2-26, and three for Delta 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 tau inact measured during a test pulse of 5000 ms (data not shown).


Fig. 5. Influences of net positive charge at the amino-terminal region of the potential inactivation ball domains and by inverting the second potential ball domain. Upper panel, currents of Delta 2-28 and Delta 2-39 (A), (+)Kv1.4 and Kv1.4 (B), and Inv(40-68) and its parent mutant Delta 2-39 & Delta 69-162 (C) are normalized and superimposed. Lower panel, currents of Delta 2-25, Delta 2-26, Delta 2-30, and Delta 2-32 are normalized and superimposed (A). Influences of changing [K+]o ((open circle ) 2 mM [K+]o, (bullet ) 20 mM [K+]o) on the recovery from inactivation are shown. B, (+)Kv1.4; C, Inv(40-68). Pulse protocols are same as described in the legend of Fig. 2.
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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 tau 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 tau 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).

Effects of Inverting the Amino Acid Sequence of the Second Potential Ball

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 tau 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 tau 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).


DISCUSSION

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 Delta 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 (Delta 38-162 and Delta 2-39 & Delta 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 Delta 38-162 and Delta 2-39 Delta 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 Delta 2-39 & Delta 69-162), inactivation was more rapid and the recovery from inactivation was slower than in the case of the first ball (in Delta 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 Delta 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 (Delta 2-39 and Delta 2-39 & Delta 69-162). The recovery rates of their currents were significantly slower than those of the other mutants. The Delta 2-39 & Delta 69-162 currents recovered faster than Delta 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 Delta 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 Delta 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 Delta 2-39, the mutants with different numbers of charges were constructed (Delta 2-32, Delta 2-30, Delta 2-26 and Delta 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.


FOOTNOTES

*   This study was supported by Grants-in-aid 07557170 for Scientific Research and 08268202 for Scientific Research on Priority Areas of "Channel-Transporter Correlation" from the Ministry of Education, Science and Culture, Japan, and by the Sapporo Bioscience Foundation, Nishinomiya Basic Research Fund, Kanae Foundation of Research for New Medicine, and a Japan Heart Foundation Research Grant for 1995.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Present address: Dept. of Pharmacology, Yamagata University School of Medicine, 2-2-2 Iida-nishi, Yamagata 990-23, Japan. Tel.: 81-236-28-5234; Fax: 81-236-28-5235.
1   The abbreviations used are: PCR, polymerase chain reaction; nt, nucleotide(s).

ACKNOWLEDGEMENT

We thank Dr. Kazuo Nunoki for review of the manuscript.


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