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Molecular Dissection of the Contribution of Negatively and Positively Charged Residues in S2, S3, and S4 to the Final Membrane Topology of the Voltage Sensor in the K+ Channel, KAT1

2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês

10.1074/jbc.m300431200

1083-351X

Yoko Sato, Masao Sakaguchi, Shinobu Goshima, Tatsunosuke Nakamura, Nobuyuki Uozumi,

Neuroscience and Neuropharmacology Research

Voltage-dependent ion channels control changes in ion permeability in response to membrane potential changes. The voltage sensor in channel proteins consists of the highly positively charged segment, S4, and the negatively charged segments, S2 and S3. The process involved in the integration of the protein into the membrane remains to be elucidated. In this study, we used in vitro translation and translocation experiments to evaluate interactions between residues in the voltage sensor of a hyperpolarization-activated potassium channel, KAT1, and their effect on the final topology in the endoplasmic reticulum (ER) membrane. A D95V mutation in S2 showed less S3-S4 integration into the membrane, whereas a D105V mutation allowed S4 to be released into the ER lumen. These results indicate that Asp95 assists in the membrane insertion of S3-S4 and that Asp105 helps in preventing S4 from being releasing into the ER lumen. The charge reversal mutation, R171D, in S4 rescued the D105R mutation and prevented S4 release into the ER lumen. A series of constructs containing different C-terminal truncations of S4 showed that Arg174 was required for correct integration of S3 and S4 into the membrane. Interactions between Asp105 and Arg171 and between negative residues in S2 or S3 and Arg174 may be formed transiently during membrane integration. These data clarify the role of charged residues in S2, S3, and S4 and identify posttranslational electrostatic interactions between charged residues that are required to achieve the correct voltage sensor topology in the ER membrane. Voltage-dependent ion channels control changes in ion permeability in response to membrane potential changes. The voltage sensor in channel proteins consists of the highly positively charged segment, S4, and the negatively charged segments, S2 and S3. The process involved in the integration of the protein into the membrane remains to be elucidated. In this study, we used in vitro translation and translocation experiments to evaluate interactions between residues in the voltage sensor of a hyperpolarization-activated potassium channel, KAT1, and their effect on the final topology in the endoplasmic reticulum (ER) membrane. A D95V mutation in S2 showed less S3-S4 integration into the membrane, whereas a D105V mutation allowed S4 to be released into the ER lumen. These results indicate that Asp95 assists in the membrane insertion of S3-S4 and that Asp105 helps in preventing S4 from being releasing into the ER lumen. The charge reversal mutation, R171D, in S4 rescued the D105R mutation and prevented S4 release into the ER lumen. A series of constructs containing different C-terminal truncations of S4 showed that Arg174 was required for correct integration of S3 and S4 into the membrane. Interactions between Asp105 and Arg171 and between negative residues in S2 or S3 and Arg174 may be formed transiently during membrane integration. These data clarify the role of charged residues in S2, S3, and S4 and identify posttranslational electrostatic interactions between charged residues that are required to achieve the correct voltage sensor topology in the ER membrane. endoplasmic reticulum dipeptidyl peptidase H1 segment of the E. coli leader peptidase prolactin rough microsomal membrane type I signal-anchor sequence type II signal-anchor sequence Voltage-dependent (gated) K+ channels contain six transmembrane segments (S1–S6) and the pore (1Jan L.Y. Jan Y.N. Annu. Rev. Neurosci. 1997; 20: 91-123Crossref PubMed Scopus (462) Google Scholar). The fourth transmembrane segment, S4, which contains several positively charged residues and is only weakly hydrophobic, is part of the voltage sensor. To date, there have been several reports on the operation of the voltage sensor in Shaker-type channels (2Gandhi C.S. Isacoff E.Y. J. Gen. Physiol. 2002; 120: 455-463Crossref PubMed Scopus (104) Google Scholar, 3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 4Cha A. Snyder G.E. Selvin P.R. Bezanilla F. Nature. 1999; 402: 809-813Crossref PubMed Scopus (435) Google Scholar, 5Glauner K.S. Mannuzzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (245) Google Scholar, 6Papazian D.M. Bezanilla F. Adv. Neurol. 1999; 79: 481-491PubMed Google Scholar, 7Männikkö R. Elinder F. Larsson H.P. Nature. 2002; 419: 837-841Crossref PubMed Scopus (165) Google Scholar). To express channel function, the voltage-dependent channel must be correctly integrated in the membrane, allowing the correct positioning of a set of amino acids involved in voltage sensing. Knowledge of the mechanism involved in the topogenesis of the voltage sensor in ion channels would provide great advances in our understanding of the voltage-sensing configuration. Only a limited number of reports have dealt with the membrane topogenesis of the different regions. Mutational analysis combined with electrophysiological measurements has identified the charged residues involved in the folding and function of theDrosophila Shaker K+ channel (8Perozo E. Santacruz-Toloza L. Stefani E. Bezanilla F. Papazian D.M. Biophys. J. 1994; 66: 345-354Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 9Papazian D.M. Shao X.M. Seoh S.A. Mock A.F. Huang Y. Wainstock D.H. Neuron. 1995; 14: 1293-1301Abstract Full Text PDF PubMed Scopus (321) Google Scholar). In addition, a model involving electrostatic interactions between charged residues in S2, S3, and S4 has been proposed on the basis of the time course of Shaker channel maturation in Xenopus laevis oocytes (10Tiwari-Woodruff S.K. Schulteis C.T. Mock A.F. Papazian D.M. Biophys. J. 1997; 72: 1489-1500Abstract Full Text PDF PubMed Scopus (198) Google Scholar). To address the question of how the final membrane topology of the K+ channel is reached, more experimental data on transmembrane biogenesis are needed.KAT1 is a well characterized plant hyperpolarization-activated K+ channel (11Uozumi N. Am. J. Physiol. 2001; 281: C733-C739Crossref PubMed Google Scholar, 12Schachtman D.P. Biochim. Biophys. Acta. 2000; 1465: 127-139Crossref PubMed Scopus (90) Google Scholar). Plant K+ channels play an important role in signal transduction and regulation of ion homeostatics (13Rodrı́guez-Navarro A. Biochim. Biophys. Acta. 2000; 1469: 1-30Crossref PubMed Scopus (409) Google Scholar), and their membrane topology resembles that of animal Shaker channels (14Uozumi N. Nakamura T. Schroeder J.I. Muto S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9773-9778Crossref PubMed Scopus (81) Google Scholar). Studies of the topogenic function of each of the six transmembrane regions, S1–S6, and of the pore region of KAT1 have demonstrated that the four hydrophobic segments, S1, S2, S5, and S6, are integrated sequentially into the endoplasmic reticulum (ER)1 membrane (15Ota K. Sakaguchi M. Hamasaki N. Mihara K. J. Biol. Chem. 1998; 273: 28286-28291Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 16Sato Y. Sakaguchi M. Goshima S. Nakamura T. Uozumi N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 60-65Crossref PubMed Scopus (50) Google Scholar), whereas S3 and S4 are suggested to be synergistically and posttranslationally integrated into the membrane only after a specific interaction occurs between them (16Sato Y. Sakaguchi M. Goshima S. Nakamura T. Uozumi N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 60-65Crossref PubMed Scopus (50) Google Scholar). Since S4 does not possess a stop-transfer function and is not released into the ER lumen (16Sato Y. Sakaguchi M. Goshima S. Nakamura T. Uozumi N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 60-65Crossref PubMed Scopus (50) Google Scholar), we wished to identify the residues and intramolecular interactions involved in its retention in the membrane. The integration involving the pairing of transmembrane units has been recognized as a second type of biogenesis of polytopic membrane proteins (17Chin C.N. von Heijne G. de Gier J.W. Trends Biochem. Sci. 2002; 27: 231-234Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The biogenesis process seems to be essential for the integration of charged transmembrane segments, which have low hydrophilic property. Further detailed study of the membrane insertion of the voltage sensor in the KAT1 channel will provide information on the dynamic membrane insertion process of closely spaced transmembrane segments throughout the polypeptide. To understand the molecular basis of the topogenic mechanism for the integration of S3 and S4 into the membrane, we have used in vitro translation and translocation experiments to evaluate the role of individual residues in KAT1 S2, S3, and S4 in the integration of the protein into the ER membrane and have identified specific interactions between charged residues that contribute to KAT1 membrane topology.DISCUSSIONIn this study, we used molecular dissection to assess the contribution of negatively and positively charged residues in S2–S4 to directing the final topology of the KAT1 voltage sensor. Single mutation of the negatively charged residues, Asp95, Asp105, and Asp141, had a clear effect on KAT1 topology, whereas single mutation of the positively charged residues, Arg165, Arg171, Arg174, Arg176, and Arg177, did not. However, the use of double mutations (Fig. 4D) or truncated proteins (Fig. 5) allowed us to analyze the role of S4 arginine residues in membrane topogenesis. For membrane integration of the voltage-sensing segments, negative amino acids in S2 are essential (Fig. 3B), but the positive residues in S4 can be compensated by neighboring positive residues (Fig. 4, A and B). Amino acid sequence alignment also showed that the negatively charged residues in S2 and in S3 are well conserved between animal and plant channels (Fig.1B), whereas positive residues in S4 vary in number and spacing (Fig. 1B).We found that several forces are involved in achieving the correct membrane integration of S3 and S4 as summarized in Fig.7. Replacement of S1 and S2 with H1 did not allow S3 and S4 to form the transmembrane structure (Fig. 2,A and B). Studies of voltage sensor operation in animal channels have predicted that negative residues in KAT1 are essential for channel function (2Gandhi C.S. Isacoff E.Y. J. Gen. Physiol. 2002; 120: 455-463Crossref PubMed Scopus (104) Google Scholar, 3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 6Papazian D.M. Bezanilla F. Adv. Neurol. 1999; 79: 481-491PubMed Google Scholar, 7Männikkö R. Elinder F. Larsson H.P. Nature. 2002; 419: 837-841Crossref PubMed Scopus (165) Google Scholar). In this study, we found that Asp95 and Asp105 are also involved in generating the correct membrane topology of the voltage sensor, each playing different roles. In the absence of a negative charge at Asp95 (D95V), the amount of S3 and S4 in the membrane was reduced (Figs. 3 and 6), suggesting that Asp95 assists in pulling the S3-S4 complex into the membrane. Replacement of Asp105 with valine or arginine caused release of S4 into the ER lumen (Figs. 3 and 6), and in the case of the D105R mutant, this effect was overcome by additional mutation of Arg171 in S4 to aspartate (Fig. 4D), showing that pairing of Asp105 and Arg171 occurs during the step of membrane integration of S3 and S4. Previous studies have indicated that electrostatic neutralization by Asp141 is a critical event for the initiation of membrane integration of S3 and S4 (14Uozumi N. Nakamura T. Schroeder J.I. Muto S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9773-9778Crossref PubMed Scopus (81) Google Scholar, 16Sato Y. Sakaguchi M. Goshima S. Nakamura T. Uozumi N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 60-65Crossref PubMed Scopus (50) Google Scholar). Fig.7 illustrates the interactions possibly involved in the membrane integration of S3 and S4.Interaction probably begins at N-terminal residues of S4 during their membrane insertion (17Chin C.N. von Heijne G. de Gier J.W. Trends Biochem. Sci. 2002; 27: 231-234Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Membrane integration of voltage sensor segments is thought to involve a series of interactions between positively and negatively charged residues. Arg174, Arg176, Arg177, and/or Arg184helped stabilize S4 in the membrane at the end of the S4 insertion event (Fig. 5). Since these residues reside within the membrane and Asp105 is located on the cytosolic side of the membrane according to a map of charged residues in the animal Shaker channel (2Gandhi C.S. Isacoff E.Y. J. Gen. Physiol. 2002; 120: 455-463Crossref PubMed Scopus (104) Google Scholar, 3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 4Cha A. Snyder G.E. Selvin P.R. Bezanilla F. Nature. 1999; 402: 809-813Crossref PubMed Scopus (435) Google Scholar, 5Glauner K.S. Mannuzzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (245) Google Scholar, 6Papazian D.M. Bezanilla F. Adv. Neurol. 1999; 79: 481-491PubMed Google Scholar), Asp105 and Arg171 may not be in close proximity to one another in the final configuration of KAT1 in the membrane. Moreover, in the animal Shaker channel, electrostatic interaction between charged residues in S2, S3, and S4 is involved in operating voltage sensing (10Tiwari-Woodruff S.K. Schulteis C.T. Mock A.F. Papazian D.M. Biophys. J. 1997; 72: 1489-1500Abstract Full Text PDF PubMed Scopus (198) Google Scholar). In the Drosophila Shaker K+ channel, residues Glu283, Arg368, and Arg371 (corresponding to Asp95, Arg171, and Arg174 in KAT1; Fig. 1B) form one charge network, whereas residues Glu293, Asp316, and Lys374(corresponding to Asp105, Asp141, and Arg177 in KAT1) form the other (10Tiwari-Woodruff S.K. Schulteis C.T. Mock A.F. Papazian D.M. Biophys. J. 1997; 72: 1489-1500Abstract Full Text PDF PubMed Scopus (198) Google Scholar). The charged residues that participate in the network are important for channel integration and function (3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 9Papazian D.M. Shao X.M. Seoh S.A. Mock A.F. Huang Y. Wainstock D.H. Neuron. 1995; 14: 1293-1301Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 10Tiwari-Woodruff S.K. Schulteis C.T. Mock A.F. Papazian D.M. Biophys. J. 1997; 72: 1489-1500Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 26Seoh S.A. Sigg D. Papazian D.M. Bezanilla F. Neuron. 1996; 16: 1159-1167Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 27Aggarwal S.K. MacKinnon R. Neuron. 1996; 16: 1169-1177Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). If the same applies to KAT1, Asp105 and Arg171 would belong to different networks (pairing groups) in the final topology, and the Asp105-Arg171 interaction, which was shown to affect the retention of S4 in the membrane (Fig. 4D), may represent a transient initial step in the insertion process. Further information on the final structure of voltage sensor segments will help us to uncover the membrane integration process completely.The use of proteins containing truncated S4 (Fig. 5) provided evidence for another electrostatic interaction between voltage-sensing segments and showed that Arg174 was a determining factor in S4 retention in the membrane (Fig. 5). We tried to determine which negative residue in S2 or in S3 interacted with Arg174using the mutant containing R174D, but this did not give the di-glycosylated band (data not shown). Taking into consideration the loss of function of D95V-, D105V-, and D141V-containing KAT1 and the retention of activity of R174V-containing KAT1 (Fig. 4C), Arg174 is unlikely to contribute a gating charge movement in the final topology by its interaction with Asp95, Asp105, or Asp141. The interaction of Arg174 with negative residues may be a transient event during the acquisition of the final topology. We estimate that, during membrane integration, the interaction between Arg171 and Asp105 is stronger than that between Arg174 and as yet undefined negative residues since the former interaction could be detected by the double mutation approach (Fig. 4D).Unlike the classical insertion of hydrophobic transmembrane segments into the membrane, the insertion process of S3-S4 in KAT1 into the membrane involves the posttranslational peptide binding of transmembrane segments, a newly recognized second type of membrane protein integration process (17Chin C.N. von Heijne G. de Gier J.W. Trends Biochem. Sci. 2002; 27: 231-234Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The charged residues in S2, S3, and S4 have been reported to be crucial to the voltage-sensing operation (2Gandhi C.S. Isacoff E.Y. J. Gen. Physiol. 2002; 120: 455-463Crossref PubMed Scopus (104) Google Scholar, 3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 4Cha A. Snyder G.E. Selvin P.R. Bezanilla F. Nature. 1999; 402: 809-813Crossref PubMed Scopus (435) Google Scholar, 5Glauner K.S. Mannuzzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (245) Google Scholar, 6Papazian D.M. Bezanilla F. Adv. Neurol. 1999; 79: 481-491PubMed Google Scholar, 7Männikkö R. Elinder F. Larsson H.P. Nature. 2002; 419: 837-841Crossref PubMed Scopus (165) Google Scholar). In this study, we have identified interactions between charged residues in voltage sensor segments and demonstrated their contribution to the membrane integration of S3 and S4, which shows that the charged residues also play a crucial role in the biogenesis of the voltage dependent KAT1 channel. Voltage-dependent (gated) K+ channels contain six transmembrane segments (S1–S6) and the pore (1Jan L.Y. Jan Y.N. Annu. Rev. Neurosci. 1997; 20: 91-123Crossref PubMed Scopus (462) Google Scholar). The fourth transmembrane segment, S4, which contains several positively charged residues and is only weakly hydrophobic, is part of the voltage sensor. To date, there have been several reports on the operation of the voltage sensor in Shaker-type channels (2Gandhi C.S. Isacoff E.Y. J. Gen. Physiol. 2002; 120: 455-463Crossref PubMed Scopus (104) Google Scholar, 3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 4Cha A. Snyder G.E. Selvin P.R. Bezanilla F. Nature. 1999; 402: 809-813Crossref PubMed Scopus (435) Google Scholar, 5Glauner K.S. Mannuzzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (245) Google Scholar, 6Papazian D.M. Bezanilla F. Adv. Neurol. 1999; 79: 481-491PubMed Google Scholar, 7Männikkö R. Elinder F. Larsson H.P. Nature. 2002; 419: 837-841Crossref PubMed Scopus (165) Google Scholar). To express channel function, the voltage-dependent channel must be correctly integrated in the membrane, allowing the correct positioning of a set of amino acids involved in voltage sensing. Knowledge of the mechanism involved in the topogenesis of the voltage sensor in ion channels would provide great advances in our understanding of the voltage-sensing configuration. Only a limited number of reports have dealt with the membrane topogenesis of the different regions. Mutational analysis combined with electrophysiological measurements has identified the charged residues involved in the folding and function of theDrosophila Shaker K+ channel (8Perozo E. Santacruz-Toloza L. Stefani E. Bezanilla F. Papazian D.M. Biophys. J. 1994; 66: 345-354Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 9Papazian D.M. Shao X.M. Seoh S.A. Mock A.F. Huang Y. Wainstock D.H. Neuron. 1995; 14: 1293-1301Abstract Full Text PDF PubMed Scopus (321) Google Scholar). In addition, a model involving electrostatic interactions between charged residues in S2, S3, and S4 has been proposed on the basis of the time course of Shaker channel maturation in Xenopus laevis oocytes (10Tiwari-Woodruff S.K. Schulteis C.T. Mock A.F. Papazian D.M. Biophys. J. 1997; 72: 1489-1500Abstract Full Text PDF PubMed Scopus (198) Google Scholar). To address the question of how the final membrane topology of the K+ channel is reached, more experimental data on transmembrane biogenesis are needed. KAT1 is a well characterized plant hyperpolarization-activated K+ channel (11Uozumi N. Am. J. Physiol. 2001; 281: C733-C739Crossref PubMed Google Scholar, 12Schachtman D.P. Biochim. Biophys. Acta. 2000; 1465: 127-139Crossref PubMed Scopus (90) Google Scholar). Plant K+ channels play an important role in signal transduction and regulation of ion homeostatics (13Rodrı́guez-Navarro A. Biochim. Biophys. Acta. 2000; 1469: 1-30Crossref PubMed Scopus (409) Google Scholar), and their membrane topology resembles that of animal Shaker channels (14Uozumi N. Nakamura T. Schroeder J.I. Muto S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9773-9778Crossref PubMed Scopus (81) Google Scholar). Studies of the topogenic function of each of the six transmembrane regions, S1–S6, and of the pore region of KAT1 have demonstrated that the four hydrophobic segments, S1, S2, S5, and S6, are integrated sequentially into the endoplasmic reticulum (ER)1 membrane (15Ota K. Sakaguchi M. Hamasaki N. Mihara K. J. Biol. Chem. 1998; 273: 28286-28291Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 16Sato Y. Sakaguchi M. Goshima S. Nakamura T. Uozumi N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 60-65Crossref PubMed Scopus (50) Google Scholar), whereas S3 and S4 are suggested to be synergistically and posttranslationally integrated into the membrane only after a specific interaction occurs between them (16Sato Y. Sakaguchi M. Goshima S. Nakamura T. Uozumi N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 60-65Crossref PubMed Scopus (50) Google Scholar). Since S4 does not possess a stop-transfer function and is not released into the ER lumen (16Sato Y. Sakaguchi M. Goshima S. Nakamura T. Uozumi N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 60-65Crossref PubMed Scopus (50) Google Scholar), we wished to identify the residues and intramolecular interactions involved in its retention in the membrane. The integration involving the pairing of transmembrane units has been recognized as a second type of biogenesis of polytopic membrane proteins (17Chin C.N. von Heijne G. de Gier J.W. Trends Biochem. Sci. 2002; 27: 231-234Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The biogenesis process seems to be essential for the integration of charged transmembrane segments, which have low hydrophilic property. Further detailed study of the membrane insertion of the voltage sensor in the KAT1 channel will provide information on the dynamic membrane insertion process of closely spaced transmembrane segments throughout the polypeptide. To understand the molecular basis of the topogenic mechanism for the integration of S3 and S4 into the membrane, we have used in vitro translation and translocation experiments to evaluate the role of individual residues in KAT1 S2, S3, and S4 in the integration of the protein into the ER membrane and have identified specific interactions between charged residues that contribute to KAT1 membrane topology. DISCUSSIONIn this study, we used molecular dissection to assess the contribution of negatively and positively charged residues in S2–S4 to directing the final topology of the KAT1 voltage sensor. Single mutation of the negatively charged residues, Asp95, Asp105, and Asp141, had a clear effect on KAT1 topology, whereas single mutation of the positively charged residues, Arg165, Arg171, Arg174, Arg176, and Arg177, did not. However, the use of double mutations (Fig. 4D) or truncated proteins (Fig. 5) allowed us to analyze the role of S4 arginine residues in membrane topogenesis. For membrane integration of the voltage-sensing segments, negative amino acids in S2 are essential (Fig. 3B), but the positive residues in S4 can be compensated by neighboring positive residues (Fig. 4, A and B). Amino acid sequence alignment also showed that the negatively charged residues in S2 and in S3 are well conserved between animal and plant channels (Fig.1B), whereas positive residues in S4 vary in number and spacing (Fig. 1B).We found that several forces are involved in achieving the correct membrane integration of S3 and S4 as summarized in Fig.7. Replacement of S1 and S2 with H1 did not allow S3 and S4 to form the transmembrane structure (Fig. 2,A and B). Studies of voltage sensor operation in animal channels have predicted that negative residues in KAT1 are essential for channel function (2Gandhi C.S. Isacoff E.Y. J. Gen. Physiol. 2002; 120: 455-463Crossref PubMed Scopus (104) Google Scholar, 3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 6Papazian D.M. Bezanilla F. Adv. Neurol. 1999; 79: 481-491PubMed Google Scholar, 7Männikkö R. Elinder F. Larsson H.P. Nature. 2002; 419: 837-841Crossref PubMed Scopus (165) Google Scholar). In this study, we found that Asp95 and Asp105 are also involved in generating the correct membrane topology of the voltage sensor, each playing different roles. In the absence of a negative charge at Asp95 (D95V), the amount of S3 and S4 in the membrane was reduced (Figs. 3 and 6), suggesting that Asp95 assists in pulling the S3-S4 complex into the membrane. Replacement of Asp105 with valine or arginine caused release of S4 into the ER lumen (Figs. 3 and 6), and in the case of the D105R mutant, this effect was overcome by additional mutation of Arg171 in S4 to aspartate (Fig. 4D), showing that pairing of Asp105 and Arg171 occurs during the step of membrane integration of S3 and S4. Previous studies have indicated that electrostatic neutralization by Asp141 is a critical event for the initiation of membrane integration of S3 and S4 (14Uozumi N. Nakamura T. Schroeder J.I. Muto S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9773-9778Crossref PubMed Scopus (81) Google Scholar, 16Sato Y. Sakaguchi M. Goshima S. Nakamura T. Uozumi N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 60-65Crossref PubMed Scopus (50) Google Scholar). Fig.7 illustrates the interactions possibly involved in the membrane integration of S3 and S4.Interaction probably begins at N-terminal residues of S4 during their membrane insertion (17Chin C.N. von Heijne G. de Gier J.W. Trends Biochem. Sci. 2002; 27: 231-234Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Membrane integration of voltage sensor segments is thought to involve a series of interactions between positively and negatively charged residues. Arg174, Arg176, Arg177, and/or Arg184helped stabilize S4 in the membrane at the end of the S4 insertion event (Fig. 5). Since these residues reside within the membrane and Asp105 is located on the cytosolic side of the membrane according to a map of charged residues in the animal Shaker channel (2Gandhi C.S. Isacoff E.Y. J. Gen. Physiol. 2002; 120: 455-463Crossref PubMed Scopus (104) Google Scholar, 3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 4Cha A. Snyder G.E. Selvin P.R. Bezanilla F. Nature. 1999; 402: 809-813Crossref PubMed Scopus (435) Google Scholar, 5Glauner K.S. Mannuzzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (245) Google Scholar, 6Papazian D.M. Bezanilla F. Adv. Neurol. 1999; 79: 481-491PubMed Google Scholar), Asp105 and Arg171 may not be in close proximity to one another in the final configuration of KAT1 in the membrane. Moreover, in the animal Shaker channel, electrostatic interaction between charged residues in S2, S3, and S4 is involved in operating voltage sensing (10Tiwari-Woodruff S.K. Schulteis C.T. Mock A.F. Papazian D.M. Biophys. J. 1997; 72: 1489-1500Abstract Full Text PDF PubMed Scopus (198) Google Scholar). In the Drosophila Shaker K+ channel, residues Glu283, Arg368, and Arg371 (corresponding to Asp95, Arg171, and Arg174 in KAT1; Fig. 1B) form one charge network, whereas residues Glu293, Asp316, and Lys374(corresponding to Asp105, Asp141, and Arg177 in KAT1) form the other (10Tiwari-Woodruff S.K. Schulteis C.T. Mock A.F. Papazian D.M. Biophys. J. 1997; 72: 1489-1500Abstract Full Text PDF PubMed Scopus (198) Google Scholar). The charged residues that participate in the network are important for channel integration and function (3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 9Papazian D.M. Shao X.M. Seoh S.A. Mock A.F. Huang Y. Wainstock D.H. Neuron. 1995; 14: 1293-1301Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 10Tiwari-Woodruff S.K. Schulteis C.T. Mock A.F. Papazian D.M. Biophys. J. 1997; 72: 1489-1500Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 26Seoh S.A. Sigg D. Papazian D.M. Bezanilla F. Neuron. 1996; 16: 1159-1167Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 27Aggarwal S.K. MacKinnon R. Neuron. 1996; 16: 1169-1177Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). If the same applies to KAT1, Asp105 and Arg171 would belong to different networks (pairing groups) in the final topology, and the Asp105-Arg171 interaction, which was shown to affect the retention of S4 in the membrane (Fig. 4D), may represent a transient initial step in the insertion process. Further information on the final structure of voltage sensor segments will help us to uncover the membrane integration process completely.The use of proteins containing truncated S4 (Fig. 5) provided evidence for another electrostatic interaction between voltage-sensing segments and showed that Arg174 was a determining factor in S4 retention in the membrane (Fig. 5). We tried to determine which negative residue in S2 or in S3 interacted with Arg174using the mutant containing R174D, but this did not give the di-glycosylated band (data not shown). Taking into consideration the loss of function of D95V-, D105V-, and D141V-containing KAT1 and the retention of activity of R174V-containing KAT1 (Fig. 4C), Arg174 is unlikely to contribute a gating charge movement in the final topology by its interaction with Asp95, Asp105, or Asp141. The interaction of Arg174 with negative residues may be a transient event during the acquisition of the final topology. We estimate that, during membrane integration, the interaction between Arg171 and Asp105 is stronger than that between Arg174 and as yet undefined negative residues since the former interaction could be detected by the double mutation approach (Fig. 4D).Unlike the classical insertion of hydrophobic transmembrane segments into the membrane, the insertion process of S3-S4 in KAT1 into the membrane involves the posttranslational peptide binding of transmembrane segments, a newly recognized second type of membrane protein integration process (17Chin C.N. von Heijne G. de Gier J.W. Trends Biochem. Sci. 2002; 27: 231-234Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The charged residues in S2, S3, and S4 have been reported to be crucial to the voltage-sensing operation (2Gandhi C.S. Isacoff E.Y. J. Gen. Physiol. 2002; 120: 455-463Crossref PubMed Scopus (104) Google Scholar, 3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 4Cha A. Snyder G.E. Selvin P.R. Bezanilla F. Nature. 1999; 402: 809-813Crossref PubMed Scopus (435) Google Scholar, 5Glauner K.S. Mannuzzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (245) Google Scholar, 6Papazian D.M. Bezanilla F. Adv. Neurol. 1999; 79: 481-491PubMed Google Scholar, 7Männikkö R. Elinder F. Larsson H.P. Nature. 2002; 419: 837-841Crossref PubMed Scopus (165) Google Scholar). In this study, we have identified interactions between charged residues in voltage sensor segments and demonstrated their contribution to the membrane integration of S3 and S4, which shows that the charged residues also play a crucial role in the biogenesis of the voltage dependent KAT1 channel. In this study, we used molecular dissection to assess the contribution of negatively and positively charged residues in S2–S4 to directing the final topology of the KAT1 voltage sensor. Single mutation of the negatively charged residues, Asp95, Asp105, and Asp141, had a clear effect on KAT1 topology, whereas single mutation of the positively charged residues, Arg165, Arg171, Arg174, Arg176, and Arg177, did not. However, the use of double mutations (Fig. 4D) or truncated proteins (Fig. 5) allowed us to analyze the role of S4 arginine residues in membrane topogenesis. For membrane integration of the voltage-sensing segments, negative amino acids in S2 are essential (Fig. 3B), but the positive residues in S4 can be compensated by neighboring positive residues (Fig. 4, A and B). Amino acid sequence alignment also showed that the negatively charged residues in S2 and in S3 are well conserved between animal and plant channels (Fig.1B), whereas positive residues in S4 vary in number and spacing (Fig. 1B). We found that several forces are involved in achieving the correct membrane integration of S3 and S4 as summarized in Fig.7. Replacement of S1 and S2 with H1 did not allow S3 and S4 to form the transmembrane structure (Fig. 2,A and B). Studies of voltage sensor operation in animal channels have predicted that negative residues in KAT1 are essential for channel function (2Gandhi C.S. Isacoff E.Y. J. Gen. Physiol. 2002; 120: 455-463Crossref PubMed Scopus (104) Google Scholar, 3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 6Papazian D.M. Bezanilla F. Adv. Neurol. 1999; 79: 481-491PubMed Google Scholar, 7Männikkö R. Elinder F. Larsson H.P. Nature. 2002; 419: 837-841Crossref PubMed Scopus (165) Google Scholar). In this study, we found that Asp95 and Asp105 are also involved in generating the correct membrane topology of the voltage sensor, each playing different roles. In the absence of a negative charge at Asp95 (D95V), the amount of S3 and S4 in the membrane was reduced (Figs. 3 and 6), suggesting that Asp95 assists in pulling the S3-S4 complex into the membrane. Replacement of Asp105 with valine or arginine caused release of S4 into the ER lumen (Figs. 3 and 6), and in the case of the D105R mutant, this effect was overcome by additional mutation of Arg171 in S4 to aspartate (Fig. 4D), showing that pairing of Asp105 and Arg171 occurs during the step of membrane integration of S3 and S4. Previous studies have indicated that electrostatic neutralization by Asp141 is a critical event for the initiation of membrane integration of S3 and S4 (14Uozumi N. Nakamura T. Schroeder J.I. Muto S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9773-9778Crossref PubMed Scopus (81) Google Scholar, 16Sato Y. Sakaguchi M. Goshima S. Nakamura T. Uozumi N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 60-65Crossref PubMed Scopus (50) Google Scholar). Fig.7 illustrates the interactions possibly involved in the membrane integration of S3 and S4. Interaction probably begins at N-terminal residues of S4 during their membrane insertion (17Chin C.N. von Heijne G. de Gier J.W. Trends Biochem. Sci. 2002; 27: 231-234Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Membrane integration of voltage sensor segments is thought to involve a series of interactions between positively and negatively charged residues. Arg174, Arg176, Arg177, and/or Arg184helped stabilize S4 in the membrane at the end of the S4 insertion event (Fig. 5). Since these residues reside within the membrane and Asp105 is located on the cytosolic side of the membrane according to a map of charged residues in the animal Shaker channel (2Gandhi C.S. Isacoff E.Y. J. Gen. Physiol. 2002; 120: 455-463Crossref PubMed Scopus (104) Google Scholar, 3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 4Cha A. Snyder G.E. Selvin P.R. Bezanilla F. Nature. 1999; 402: 809-813Crossref PubMed Scopus (435) Google Scholar, 5Glauner K.S. Mannuzzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (245) Google Scholar, 6Papazian D.M. Bezanilla F. Adv. Neurol. 1999; 79: 481-491PubMed Google Scholar), Asp105 and Arg171 may not be in close proximity to one another in the final configuration of KAT1 in the membrane. Moreover, in the animal Shaker channel, electrostatic interaction between charged residues in S2, S3, and S4 is involved in operating voltage sensing (10Tiwari-Woodruff S.K. Schulteis C.T. Mock A.F. Papazian D.M. Biophys. J. 1997; 72: 1489-1500Abstract Full Text PDF PubMed Scopus (198) Google Scholar). In the Drosophila Shaker K+ channel, residues Glu283, Arg368, and Arg371 (corresponding to Asp95, Arg171, and Arg174 in KAT1; Fig. 1B) form one charge network, whereas residues Glu293, Asp316, and Lys374(corresponding to Asp105, Asp141, and Arg177 in KAT1) form the other (10Tiwari-Woodruff S.K. Schulteis C.T. Mock A.F. Papazian D.M. Biophys. J. 1997; 72: 1489-1500Abstract Full Text PDF PubMed Scopus (198) Google Scholar). The charged residues that participate in the network are important for channel integration and function (3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 9Papazian D.M. Shao X.M. Seoh S.A. Mock A.F. Huang Y. Wainstock D.H. Neuron. 1995; 14: 1293-1301Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 10Tiwari-Woodruff S.K. Schulteis C.T. Mock A.F. Papazian D.M. Biophys. J. 1997; 72: 1489-1500Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 26Seoh S.A. Sigg D. Papazian D.M. Bezanilla F. Neuron. 1996; 16: 1159-1167Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 27Aggarwal S.K. MacKinnon R. Neuron. 1996; 16: 1169-1177Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar). If the same applies to KAT1, Asp105 and Arg171 would belong to different networks (pairing groups) in the final topology, and the Asp105-Arg171 interaction, which was shown to affect the retention of S4 in the membrane (Fig. 4D), may represent a transient initial step in the insertion process. Further information on the final structure of voltage sensor segments will help us to uncover the membrane integration process completely. The use of proteins containing truncated S4 (Fig. 5) provided evidence for another electrostatic interaction between voltage-sensing segments and showed that Arg174 was a determining factor in S4 retention in the membrane (Fig. 5). We tried to determine which negative residue in S2 or in S3 interacted with Arg174using the mutant containing R174D, but this did not give the di-glycosylated band (data not shown). Taking into consideration the loss of function of D95V-, D105V-, and D141V-containing KAT1 and the retention of activity of R174V-containing KAT1 (Fig. 4C), Arg174 is unlikely to contribute a gating charge movement in the final topology by its interaction with Asp95, Asp105, or Asp141. The interaction of Arg174 with negative residues may be a transient event during the acquisition of the final topology. We estimate that, during membrane integration, the interaction between Arg171 and Asp105 is stronger than that between Arg174 and as yet undefined negative residues since the former interaction could be detected by the double mutation approach (Fig. 4D). Unlike the classical insertion of hydrophobic transmembrane segments into the membrane, the insertion process of S3-S4 in KAT1 into the membrane involves the posttranslational peptide binding of transmembrane segments, a newly recognized second type of membrane protein integration process (17Chin C.N. von Heijne G. de Gier J.W. Trends Biochem. Sci. 2002; 27: 231-234Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The charged residues in S2, S3, and S4 have been reported to be crucial to the voltage-sensing operation (2Gandhi C.S. Isacoff E.Y. J. Gen. Physiol. 2002; 120: 455-463Crossref PubMed Scopus (104) Google Scholar, 3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (713) Google Scholar, 4Cha A. Snyder G.E. Selvin P.R. Bezanilla F. Nature. 1999; 402: 809-813Crossref PubMed Scopus (435) Google Scholar, 5Glauner K.S. Mannuzzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (245) Google Scholar, 6Papazian D.M. Bezanilla F. Adv. Neurol. 1999; 79: 481-491PubMed Google Scholar, 7Männikkö R. Elinder F. Larsson H.P. Nature. 2002; 419: 837-841Crossref PubMed Scopus (165) Google Scholar). In this study, we have identified interactions between charged residues in voltage sensor segments and demonstrated their contribution to the membrane integration of S3 and S4, which shows that the charged residues also play a crucial role in the biogenesis of the voltage dependent KAT1 channel.