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• W2019959686 abstract "The “ball and chain” model has been shown to be suitable for explaining the rapid inactivation of voltage-dependent K+ channels. For theDrosophila 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. The “ball and chain” model has been shown to be suitable for explaining the rapid inactivation of voltage-dependent K+ channels. For theDrosophila 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 (4Armstrong C.M. Bezanilla F. J. Gen. Physiol. 1977; 70: 567-590Crossref PubMed Scopus (764) Google Scholar), can also explain the rapid inactivation of a Drosophila Shaker K+ channel (1Hoshi T. Zagotta W.N. Aldrich R.W. Science. 1990; 250: 533-538Crossref PubMed Scopus (1277) Google Scholar, 2Zagotta W.N. Hoshi T. Aldrich R.W. Science. 1990; 250: 568-571Crossref PubMed Scopus (609) Google Scholar). 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 (5Li M. Jan Y.N. Jan L.Y. Science. 1992; 257: 1225-1230Crossref PubMed Scopus (395) Google Scholar) has been identified as the chain tethering the ball to the channel (1Hoshi T. Zagotta W.N. Aldrich R.W. Science. 1990; 250: 533-538Crossref PubMed Scopus (1277) Google Scholar). 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 (6Rettig J. Heinemann S.H. Wunder F. Lorra C. Parcej D.N. Dolly J.O. Pongs O. Nature. 1994; 369: 289-294Crossref PubMed Scopus (746) Google Scholar, 7Comer M.B. Campbell D.L. Rasmusson R.L. Lamson D.R. Morales M.J. Zhang Y. Strauss H.C. Am. J. Physiol. 1994; 267: H1383-H1395PubMed Google Scholar). Tseng and co-workers (3Tseng-Crank J. Yao J.-A. Berman M.F. Tseng G.-N. J. Gen. Physiol. 1993; 102: 1057-1083Crossref PubMed Scopus (54) Google Scholar) 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 Δ2–39 & Δ69–162 was constructed. Fragments for all the mutants except the one with inverted residues were generated by polymerase chain reaction (PCR). 1The abbreviations used are: PCR, polymerase chain reaction; nt, nucleotide(s). The 20–22-base pair sense primers used for generating Δ2–25, Δ2–26, Δ2–28, Δ2–30, Δ2–32, Δ2–39, and Δ2–61 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 Δ29–162 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 Δ2–39 & Δ69–162, 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 Δ38–162 and Δ2–39 & Δ61–162, 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 Δ38–162 was S1, and that of Δ2–39 & Δ61–162 was the same one used for Δ2–39. The antisense primer for the fragment I contained a StuI site at the 5′-end and corresponded to nucleotides 96–114 for Δ38–162 and to nucleotides 161–181 for Δ2–39 & Δ61–162. 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 anApaI 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 Δ2–39 & Δ69–162, 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 aMluI site at the 3′-end when annealed. The annealed fragment was ligated to Kv1.4, which was digested with ApaI andMluI. In a 100-μl PCR reaction, 100 pmol of each primer, 0.2 μg of template cDNA (Kv1.4 for all the mutants except Δ2–39 & Δ69–162; Δ2–39 for Δ2–39 & Δ69–162), 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 Δ2–25, Δ2–26, Δ2–28, Δ2–30, Δ2–32, Δ2–39, and Δ2–61 was digested withApaI and MluI and ligated to Kv1.4 between theApaI and MluI sites. The amplified fragment for Δ29–162 was digested with XhoI and MluI and ligated to Kv1.4 between the XhoI and MluI sites. The fragment for Δ2–39 & Δ69–162 was amplified using Δ2–39 as template and digested with ApaI and XhoI. The digested fragment was ligated to Δ29–162, which was digested withApaI and XhoI. The amplified fragment I for Δ2–39 & Δ61–162 and Δ38–162 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 andMluI. 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), andPmaCI (at nt 200) were ligated to Kv1.4 digested withApaI 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.). Electrophysiological measurements were carried out essentially as reported previously (8Ishii K. Nunoki K. Murakoshi H. Taira N. Biochem. Biophys. Res. Commun. 1992; 184: 1484-1489Crossref PubMed Scopus (19) Google Scholar,9Okada H. Ishii K. Nunoki K. Abe T. Taira N. Biochem. Biophys. Res. Commun. 1992; 189: 430-436Crossref PubMed Scopus (15) Google Scholar). 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 (10Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 7.43-7.45Google Scholar). 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 (8Ishii K. Nunoki K. Murakoshi H. Taira N. Biochem. Biophys. Res. Commun. 1992; 184: 1484-1489Crossref PubMed Scopus (19) Google Scholar, 9Okada H. Ishii K. Nunoki K. Abe T. Taira N. Biochem. Biophys. Res. Commun. 1992; 189: 430-436Crossref PubMed Scopus (15) Google Scholar). 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 unpairedt 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.2 A (upper panel) shows normalized currents of Δ2–28 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 Δ2–28 at +20 mV was 2.05 ± 0.35 μA (n = 7) and 4.96 ± 0.54 μA (n = 5), respectively. The Δ2–28 current showed little decline during the 400-ms test pulse, while the Kv1.4 current inactivated almost completely. τinact of Δ2–28 current was measured using a prolonged depolarization pulse (5000 ms). The τinact was 2090.9 ± 647.9 ms (n = 5), which was about 90 times larger than that of Kv1.4. The inactivation of Δ2–28 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 (11Demo S.D. Yellen G. Neuron. 1991; 7: 743-753Abstract Full Text PDF PubMed Scopus (248) Google Scholar, 12Tseng G.-N. Tseng-Crank J. Circ. Res. 1992; 71: 657-672Crossref PubMed Scopus (23) Google Scholar), we investigated the effects of changing [K+] o on the recovery rate. Recovery from inactivation of Δ2–28 at 2 mm[K+] o and 20 mm[K+] o is shown in Fig. 2 A (lower panel). Elevating [K+] o had no effect on the recovery time course (τrec: 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 (Δ2–39), rapid inactivation was resumed. Currents of Δ2–39 and Kv1.4 recorded at +20 mV were normalized and superimposed in Fig. 2 B (upper panel). The peak current of Δ2–39 at +20 mV was 0.71 ± 0.18 μA (n = 4). The Δ2–39 current showed rapid inactivation. The τinact of Δ2–39 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 Δ2–39 (Fig. 2 B,lower panel). The τrec of Δ2–39 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 (Δ2–61), fast inactivation disappeared again (Fig. 2 C, upper panel). The Δ2–61 current showed little decline during a 400-ms test pulse. The peak current of Δ2–61 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 Δ2–61 current was not affected by elevating [K+] o (Fig. 2 C,lower panel). The τrec 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, Δ38–162, and a mutant, Δ2–39 & Δ69–162, which had only the second potential inactivation ball. Fig. 3 (A andB, upper panel), shows the normalized currents from Δ38–162 and Δ2–39 & Δ69–162 superimposed on the Kv1.4 current. The Δ38–162 current showed rapid inactivation. The τinact of Δ38–162 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 Δ38–162 (Fig. 3 A, lower panel). The τrec 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 Δ38–162 at +20 mV was 1.67 ± 0.25 μA (n = 6). Δ2–39 & Δ69–162, with only the second potential inactivation ball, showed more rapid inactivation than Kv1.4 (Fig. 3 B, upper panel). The τinactof Δ2–39 & Δ69–162 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 Δ2–39 & Δ69–162 (Fig. 3 B, lower panel). The τrec 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 Δ2–39 & Δ69–162 at +20 mV was 1.19 ± 0.37 μA (n = 6). 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. Δ29–162 was constructed to delete one net positive charge (3 arginine and 2 glutamic acid residues) from the first potential inactivation ball. Δ2–39 & Δ61–162 was constructed to delete one positive charge (arginine) from the second potential inactivation ball. Fig.4 A (upper panel) shows the normalized Δ29–162 current superimposed on the Δ38–162 current recorded at +20 mV. The peak current of Δ29–162 at +20 mV was 1.80 ± 0.54 μA (n = 4). Inactivation of the Δ29–162 current was much slower than that of Δ38–162. The τinact of Δ29–162 was 361.09 ± 37.69 ms (n = 4), which is about 9 times larger than that of Δ38–162 (Fig. 1). The τrec of Δ29–162 (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 Δ38–162 (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. 4 A (lower panel) shows the effects of elevating [K+] o on the recovery time course of Δ29–162. Currents of Δ2–39 & Δ61–162 and Δ2–39 & Δ69–162 recorded at +20 mV are normalized and superimposed in Fig.4 B (upper panel). The peak current of Δ2–39 & Δ61–162 at +20 mV was 1.41 ± 0.20 μA (n = 7). The τinact of Δ2–39 & Δ61–162 was 59.22 ± 1.77 ms (n = 7), which is 5 times larger than that of Δ2–39 & Δ69–162 (Fig. 1). Recovery from inactivation was much faster in Δ2–39 & Δ61–162 than in Δ2–39 & Δ69–162 (Fig. 1). The τrec of Δ2–39 & Δ61–162 and Δ2–39 & Δ69–162 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. 4 B (lower panel) shows the effects of elevating [K+] o on the τrec of Δ2–39 & Δ61–162. When the structures of Δ2–28 and Δ2–39 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.5 A, upper panel). Therefore, we constructed four other mutants that varied in charge at the amino terminus of the second potential ball, Δ2–32, Δ2–30, Δ2–26, and Δ2–25. The numbers of extra net positive charges are one for Δ2–32 and Δ2–30, two for Δ2–26, and three for Δ2–25. The currents of all these mutants showed little inactivation on the same time scale as Kv1.4 (400 ms) (Fig. 5 A,lower panel) and no significant differences in τinact 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. 5 B, upper panel). The τinact 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 τrec 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. 5 B,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. 5 C, upper panel). The τinact 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 τrec 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. 5 C, 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 (3Tseng-Crank J. Yao J.-A. Berman M.F. Tseng G.-N. J. Gen. Physiol. 1993; 102: 1057-1083Crossref PubMed Scopus (54) Google Scholar), 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 Δ2–39 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 (Δ38–162 and Δ2–39 & Δ69–162). 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 Δ38–162 and Δ2–39 & Δ69–162, 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 Δ2–39 & Δ69–162), inactivation was more rapid and the recovery from inactivation was slower than in the case of the first ball (in Δ38–162) (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 Δ2–39 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 (Δ2–39 and Δ2–39 & Δ69–162). The recovery rates of their currents were significantly slower than those of the other mutants. The Δ2–39 & Δ69–162 currents recovered faster than Δ2–39 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 Δ2–39 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 (2Zagotta W.N. Hoshi T. Aldrich R.W. Science. 1990; 250: 568-571Crossref PubMed Scopus (609) Google Scholar, 13Murrell-Lagnado R.D. Aldrich R.W. J. Gen. Physiol. 1993; 102: 949-975Crossref PubMed Scopus (128) Google Scholar, 14Toro L. Ottolia M. Stefani E. Latorre R. Biochemistry. 1994; 33: 7220-7228Crossref PubMed Scopus (22) Google Scholar). 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 Δ2–28 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 Δ2–39, the mutants with different numbers of charges were constructed (Δ2–32, Δ2–30, Δ2–26 and Δ2–25). The currents through these mutants hardly inactivated (Fig. 5 A, 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.5 B). 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 (11Demo S.D. Yellen G. Neuron. 1991; 7: 743-753Abstract Full Text PDF PubMed Scopus (248) Google Scholar). 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 (15Dubinsky W.P. Mayorga-Wark O. Shultz S.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1770-1774Crossref PubMed Scopus (19) Google Scholar, 16Foster C.D. Chung S. Zagotta W.N. Aldrich R.W. Levitan I.B. Neuron. 1992; 9: 229-236Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 17Solaro C.R. Lingle C.J. Science. 1992; 257: 1694-1698Crossref PubMed Scopus (96) Google Scholar, 18Toro L. Stefani E. Latorre R. Neuron. 1992; 9: 237-245Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 19Kramer R.H. Goulding E. Siegelbaum S.A. Neuron. 1994; 12: 655-662Abstract Full Text PDF PubMed Scopus (46) Google Scholar). 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 (20Antz C. Geyer M. Fakler B. Schott M.K. Guy H.R. Frank R. Ruppersberg J.P. Kalbitzer H.R. Nature. 1997; 385: 272-275Crossref PubMed Scopus (99) Google Scholar). 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." @default.
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• W2019959686 date "1997-08-01" @default.
• W2019959686 modified "2023-10-18" @default.
• W2019959686 title "A Mammalian Transient Type K+ Channel, Rat Kv1.4, Has Two Potential Domains That Could Produce Rapid Inactivation" @default.
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