A Central Role for the T1 Domain in Voltage-gated Potassium Channel Formation and Function
2001; Elsevier BV; Volume: 276; Issue: 30 Linguagem: Inglês
10.1074/jbc.m010540200
ISSN1083-351X
AutoresCandace Strang, Susan J. Cushman, David DeRubeis, David S. Peterson, Paul J. Pfaffinger,
Tópico(s)Chemical Synthesis and Analysis
ResumoTo interpret the recent atomic structures of the Kv (voltage-dependent potassium) channel T1 domain in a functional context, we must understand both how the T1 domain is integrated into the full-length functional channel protein and what functional roles the T1 domain governs. The T1 domain clearly plays a role in restricting Kv channel subunit heteromultimerization. However, the importance of T1 tetramerization for the assembly and retention of quarternary structure within full-length channels has remained controversial. Here we describe a set of mutations that disrupt both T1 assembly and the formation of functional channels and show that these mutations produce elevated levels of the subunit monomer that becomes subject to degradation within the cell. In addition, our experiments reveal that the T1 domain lends stability to the full-length channel structure, because channels lacking the T1 containing N terminus are more easily denatured to monomers. The integration of the T1 domain ultrastructure into the full-length channel was probed by proteolytic mapping with immobilized trypsin. Trypsin cleavage yields an N-terminal fragment that is further digested to a tetrameric domain, which remains reactive with antisera to T1, and that is similar in size to the T1 domain used for crystallographic studies. The trypsin-sensitive linkages retaining the T1 domain are cleaved somewhat slowly over hours. Therefore, they seem to be intermediate in trypsin resistance between the rapidly cleaved extracellular linker between the first and second transmembrane domains, and the highly resistant T1 core, and are likely to be partially structured or contain dynamic structure. Our experiments suggest that tetrameric atomic models obtained for the T1 domain do reflect a structure that the T1 domain sequence forms early in channel assembly to drive subunit protein tetramerization and that this structure is retained as an integrated stabilizing structural element within the full-length functional channel. To interpret the recent atomic structures of the Kv (voltage-dependent potassium) channel T1 domain in a functional context, we must understand both how the T1 domain is integrated into the full-length functional channel protein and what functional roles the T1 domain governs. The T1 domain clearly plays a role in restricting Kv channel subunit heteromultimerization. However, the importance of T1 tetramerization for the assembly and retention of quarternary structure within full-length channels has remained controversial. Here we describe a set of mutations that disrupt both T1 assembly and the formation of functional channels and show that these mutations produce elevated levels of the subunit monomer that becomes subject to degradation within the cell. In addition, our experiments reveal that the T1 domain lends stability to the full-length channel structure, because channels lacking the T1 containing N terminus are more easily denatured to monomers. The integration of the T1 domain ultrastructure into the full-length channel was probed by proteolytic mapping with immobilized trypsin. Trypsin cleavage yields an N-terminal fragment that is further digested to a tetrameric domain, which remains reactive with antisera to T1, and that is similar in size to the T1 domain used for crystallographic studies. The trypsin-sensitive linkages retaining the T1 domain are cleaved somewhat slowly over hours. Therefore, they seem to be intermediate in trypsin resistance between the rapidly cleaved extracellular linker between the first and second transmembrane domains, and the highly resistant T1 core, and are likely to be partially structured or contain dynamic structure. Our experiments suggest that tetrameric atomic models obtained for the T1 domain do reflect a structure that the T1 domain sequence forms early in channel assembly to drive subunit protein tetramerization and that this structure is retained as an integrated stabilizing structural element within the full-length functional channel. voltage-dependent potassium broad complex, tramtrack, bric-a-brac first transmembrane segment, S2, second transmembrane segment size exclusion chromatography green fluorescent protein enhanced GFP Chinese hamster ovary 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid fast protein liquid chromatography phosphate-buffered saline polyacrylamide gel electrophoresis activation domain DNA-binding domain pox virus and zinc finger The structural elements of potassium channels have begun to be characterized in atomic detail, allowing much increased sophistication in our understanding of their mechanism of action and biological function. For voltage-dependent potassium (Kv)1 channels, the structure of the highly conserved cytoplasmic N-terminal T1 domain has been determined as a rotationally symmetric tetramer from three different Kv channels and in complex with an auxiliary β-subunit protein (1Kreusch A. Pfaffinger P.J. Stevens C.F. Choe S. Nature. 1998; 392: 945-948Crossref PubMed Scopus (269) Google Scholar, 2Bixby K.A. Nanao M.H. Shen N.V. Kreusch A. Bellamy H. Pfaffinger P.J. Choe S. Nat. Struct. Biol. 1999; 6: 38-43Crossref PubMed Scopus (144) Google Scholar, 3Minor D.L. Lin Y.F. Mobley B.C. Avelar A. Jan Y.N. Jan L.Y. Berger J.M. Cell. 2000; 102: 657-670Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 4Gulbis J. Zhou M. Mann S. MacKinnon R. Science. 2000; 289: 123-127Crossref PubMed Scopus (285) Google Scholar). Because the T1 domain structures are determined from isolated soluble protein domains, questions arise as to the relevance of the determined structures to the ultrastructure of the full-length Kv channel and in how the tetrameric domain is integrated into the remainder of the channel. Recent published studies have suggested that the T1 structure within the channel is likely to be very similar to the tetrameric structure of the isolated domain (3Minor D.L. Lin Y.F. Mobley B.C. Avelar A. Jan Y.N. Jan L.Y. Berger J.M. Cell. 2000; 102: 657-670Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 5Shen N.V. Pfaffinger P.J. Neuron. 1995; 14: 625-633Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 6Cushman S. Manao M.H. Jahng A.W. DeRubeis D. Choe S. Pfaffinger P.J. Nat. Struct. Biol. 2000; 7: 403-407Crossref PubMed Scopus (92) Google Scholar, 7Kobertz W.R. Williams C. Miller C. Biochemistry. 2000; 39: 10347-10352Crossref PubMed Scopus (99) Google Scholar). However, crystallography also has shown that the T1 domain can adopt several different conformations within the channel that may be involved in regulating channel gating properties (3Minor D.L. Lin Y.F. Mobley B.C. Avelar A. Jan Y.N. Jan L.Y. Berger J.M. Cell. 2000; 102: 657-670Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 4Gulbis J. Zhou M. Mann S. MacKinnon R. Science. 2000; 289: 123-127Crossref PubMed Scopus (285) Google Scholar, 6Cushman S. Manao M.H. Jahng A.W. DeRubeis D. Choe S. Pfaffinger P.J. Nat. Struct. Biol. 2000; 7: 403-407Crossref PubMed Scopus (92) Google Scholar). Specifically, there is conformational variability in the C-terminal region that is anticipated to be against the membrane (4Gulbis J. Zhou M. Mann S. MacKinnon R. Science. 2000; 289: 123-127Crossref PubMed Scopus (285) Google Scholar, 6Cushman S. Manao M.H. Jahng A.W. DeRubeis D. Choe S. Pfaffinger P.J. Nat. Struct. Biol. 2000; 7: 403-407Crossref PubMed Scopus (92) Google Scholar). Furthermore, the T1 domain has been found to adopt a three-dimensional fold, called the BTB/POZ fold, that is observed in other protein complexes (8Choe S. Kreutsch A. Pfaffinger P.J. Trends Biochem. Sci. 1999; 24 (abstr.): 345-349Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 9Ahmad K.F. Engel C.K. Prive G.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95 (abstr.): 12123-12128Crossref PubMed Scopus (244) Google Scholar, 10Stebbins C.E. Kaelin W.G. Pavletich N.P. Science. 1999; 284 (abstr.): 455-461Crossref PubMed Scopus (690) Google Scholar, 11Schulman B.A. Carrano A.C. Jeffrey P.D. Bowen Z. Kinnucan E.R. Finnin M.S. Elledge S.J. Harper J.W. Pagano M. Pavletich N.P. Nature. 2000; 408: 381-386Crossref PubMed Scopus (490) Google Scholar, 12Aravind L. Koonin E.V. J. Mol. Biol. 1999; 285 (abstr.): 1353-1361Crossref PubMed Scopus (131) Google Scholar). Interestingly, other BTB/POZ subunits often exist in oligomeric association as monomeric or dimeric subunits, and there are multiple binding faces for other subunit proteins found within this fold as well. This particular domain then affords many possible binding interactions to itself, to other domains of the full-length channel, or to other proteins. Clearly, then, it becomes important to assess the ultrastructure of the T1 domain within the intact channel to formulate models for channel mechanism and biological activity. The T1 domain was identified originally as an important site for regulating intersubunit interactions during channel assembly, such that only a restricted number of heterotetramers are made from the many possible Kv channel subunit proteins that are synthesized within a single cell (5Shen N.V. Pfaffinger P.J. Neuron. 1995; 14: 625-633Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 13Lee T.E. Phillipson L.H. Kuznetzov A. Nelson D. Biophys. J. 1994; 66: 667-673Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 14Tu L. Wang J. Helm A. Skach W.R. Deutsch C. Biochemistry. 2000; 398: 24-36Google Scholar, 15Kobertz W.R. Miller C. Nat. Struct. Biol. 1999; 6: 1122-1125Crossref PubMed Scopus (66) Google Scholar, 16Jan L.Y. Jan Y.N. Trends Neurosci. 1990; 13: 415-420Abstract Full Text PDF PubMed Scopus (158) Google Scholar, 17Li M. Jan Y.N. Jan L.Y. Science. 1992; 257: 1225-1230Crossref PubMed Scopus (395) Google Scholar, 18Deal K.K. Lovinger D.M. Tamkun M.M. J. Neurosci. 1994; 14: 1666-1676Crossref PubMed Google Scholar, 19Xu J., Yu, W. Jan Y.N. Jan L.Y. Li M. J. Biol. Chem. 1995; 270: 24761-24768Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 20Hopkins W.F. Demas V. Tempel B.L. J. Neurosci. 1994; 14: 1385-1393Crossref PubMed Google Scholar, 21Pfaffinger P.J. DeRubeis D. J. Biol. Chem. 1995; 270: 28595-28600Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 22Schulteis C.T. Nagaya N. Papazian D.M. J. Biol. Chem. 1998; 273: 26210-26217Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Nevertheless, several groups have reported the functional expression of Kv channels after the deletion of the T1 domain in channel overexpression systems (13Lee T.E. Phillipson L.H. Kuznetzov A. Nelson D. Biophys. J. 1994; 66: 667-673Abstract Full Text PDF PubMed Scopus (81) Google Scholar, 15Kobertz W.R. Miller C. Nat. Struct. Biol. 1999; 6: 1122-1125Crossref PubMed Scopus (66) Google Scholar, 23Tu L. Sanarelli V. Sheng Z. Skach W. Pain D. Deutsch C. J. Biol. Chem. 1996; 271: 18904-18911Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), in contrast to other reports that T1 deletion precludes channel assembly (5Shen N.V. Pfaffinger P.J. Neuron. 1995; 14: 625-633Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 20Hopkins W.F. Demas V. Tempel B.L. J. Neurosci. 1994; 14: 1385-1393Crossref PubMed Google Scholar, 22Schulteis C.T. Nagaya N. Papazian D.M. J. Biol. Chem. 1998; 273: 26210-26217Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar,24Shen N.V. Chen X. Boyer M.M. Pfaffinger P.J. Neuron. 1993; 11: 67-76Abstract Full Text PDF PubMed Scopus (200) Google Scholar). The fact that channels can form without a T1 domain might be expected, because the membrane itself can serve to nucleate secondary structure into incorporated peptides via the hydrophobic effect (25White S.H. Wimley W.C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365Crossref PubMed Scopus (1483) Google Scholar), and the transmembrane core of the Kv channel is shared with a large number of homologous and tetrameric channel proteins that lack a T1 domain (26Mackinnon R. Nature. 1991; 350: 232-235Crossref PubMed Scopus (768) Google Scholar, 27Hille B. Ionic Channels of Excitable Membranes. 2nd Ed. Sinauer Associates, Inc., Sunderland, MA1992: 115-135Google Scholar). However, it does call into question what T1-T1 interactions might add to Kv channel formation and the resultant ultrastructure and whether these conformations might be a dynamic aspect of channel gating. We know from electrophysiological data (3Minor D.L. Lin Y.F. Mobley B.C. Avelar A. Jan Y.N. Jan L.Y. Berger J.M. Cell. 2000; 102: 657-670Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 4Gulbis J. Zhou M. Mann S. MacKinnon R. Science. 2000; 289: 123-127Crossref PubMed Scopus (285) Google Scholar) that there is a relationship between T1 conformation and gating, even if we do not know the exact details of how the T1 domain integrates into the intact channel ultrastructure, such as the number and nature of its contacts with other channel elements. Point mutations within the T1 domain of two different Shaker channels alter channel electrophysiology, suggesting that there is a very tight energetic and therefore conformational link between T1 and the channel gating elements. Based on the above electrophysiology and the rotational symmetry of the tetrameric T1, we suggest that the T1 domain lies in alignment with the pore and gating elements as has been proposed (3Minor D.L. Lin Y.F. Mobley B.C. Avelar A. Jan Y.N. Jan L.Y. Berger J.M. Cell. 2000; 102: 657-670Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 4Gulbis J. Zhou M. Mann S. MacKinnon R. Science. 2000; 289: 123-127Crossref PubMed Scopus (285) Google Scholar, 6Cushman S. Manao M.H. Jahng A.W. DeRubeis D. Choe S. Pfaffinger P.J. Nat. Struct. Biol. 2000; 7: 403-407Crossref PubMed Scopus (92) Google Scholar). This suggests that the T1 domain C terminus abuts S4 and possibly interacts with S6 and the channel C terminus, but this has not yet been demonstrated. Recent data from the Miller laboratory (7Kobertz W.R. Williams C. Miller C. Biochemistry. 2000; 39: 10347-10352Crossref PubMed Scopus (99) Google Scholar) have shown that the atomic structure of the isolated tetramer can be used to correctly predict cross-linking pairs of amino acids in the full-length channel, indicating that the crystal structure of the T1 domain does approximate the ultrastructure in this portion of the full-length channel. Additionally, they suggest that the T1 domain is a "hanging gondola" tethered to the membranous channel portions by "cables" of unknown structural characteristics. Unfortunately, relatively little is known about how the T1 domain is integrated into the remainder of the channel. It is expected that the T1-S1 linkage lies near the outer edge of the channel, because S1 appears to be a lipid-facing transmembrane segment (28Monks S.A. Needleman D.J. Miller C. J. Gen. Physiol. 1999; 113: 415-423Crossref PubMed Scopus (122) Google Scholar, 29Hong K.H. Miller C. J. Gen. Physiol. 2000; 115: 51-58Crossref PubMed Scopus (114) Google Scholar, 30Shi G. Trimmer J.S. J. Membr. Biol. 1999; 168: 265-273Crossref PubMed Scopus (71) Google Scholar), but structural details of the linkage between the T1 domain and the first transmembrane segment, S1, are lacking because of solubility problems encountered with crystallization attempts on this construct. In a recent article from the Jan laboratory (31Zerangue N. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. 2000; 97: 3591-3595Crossref PubMed Scopus (88) Google Scholar), a completely distinct tetrameric motif of roughly the same dimensions as the T1 domain was found to provide a scaffolding platform for the efficient and high yield formation of a surface channel even though the permuted channel itself had altered kinetic and gating properties. Based on similarities in the electron micrographs of Kv channels and the nicotinic receptor (32Li M. Unwin N. Stauffer K.A. Jan Y.N. Jan L.Y. Curr. Biol. 1994; 4: 110-115Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), it has been proposed that an aqueous gap exists between the T1 domain and the transmembrane domains in the open channel (33Biggin P.C. Roosild T. Choe S. Curr. Opin. Struct. Biol. 2000; 10: 456-461Crossref PubMed Scopus (46) Google Scholar, 34Miyazawa A. Fujiyoshi Y. Stowell M. Unwin N. J. Mol. Biol. 1999; 288: 765-786Crossref PubMed Scopus (429) Google Scholar) and that the linkage here corresponds to the cables of the hanging gondola model (7Kobertz W.R. Williams C. Miller C. Biochemistry. 2000; 39: 10347-10352Crossref PubMed Scopus (99) Google Scholar). If it is the primary link from T1 to the membrane integrated channel and itself contains little structure or it folds independently of the T1 domain, this linker may explain why the Jan laboratory successfully substituted a completely separate tetrameric motif for T1. We have taken a mutational approach toward the study of T1 subunit interactions and their role in the structure and stability of the intact channel. We have generated a set of point mutations that affect subunit interactions in T1 and subsequently disrupt tetramer formation. These mutations were generated from a random screen, yet the mutated residues are the very same amino acids that are conserved among either all Kv channels or the Shaker subfamily. We next examined the ability of full length Kv subunit proteins to form channels when the T1 region contained these same mutations and found disruptions in tetrameric channel assembly, as would be expected if T1 tetramer formation drives the assembly and cell surface expression of the intact channel. Within the crystal structure of T1, these mutated amino acids were found either at subunit interfaces or at critical locations for subunit folding, and thus, the perturbations in channel assembly provide independent confirmation of the absolute requirement for correct T1 folding and assembly to occur in the intact channel. We then present further biochemical analysis of these mutants and the native intact channel to examine the role of T1 in the dynamic regulation of channel assembly and in the ultrastructure of the intact channel. Yeast two-hybrid analysis was conducted according to the method of Fields and Song (35Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4860) Google Scholar), using a Gal4 two-hybrid phagemid vector kit (Stratagene), and the manufacturer's instructions for the quantitative determination of β-galactosidase expression, witho-nitrophenyl-β-d-galactopyranoside as a colorimetric substrate. Region-specific random mutagenesis was performed using randomly mutagenizing oligonucleotides of ∼90 bases (Ref. 36Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 15.96Google Scholar). (Oligonucleotides are synthesized to a specific region with doped bases, in which each base is doped at the 0.15% level with the other three bases. This doping creates a low probability of misincorporation at every position along the oligonucleotide. If each base has an equal probability of incorporation, then the calculated mutation rate is low enough to produce most full-length oligonucleotides with 0–2 random point mutations per oligonucleotide.) Mutagenizing oligonucleotides were used in primer extension reactions, made double-stranded, and cassette-cloned into Gal4-AD-T1. Libraries of mutant T1 domains were made by transforming the ligations into bacteria and preparing the DNA from >98% colonies in the plate of transformants, taking care to avoid disparity in colony growth. These cDNA libraries were then transformed into Gal4-BD-T1 yeast and plated at a low density to allow clear growth of single colonies. T1 assembly mutants were identified by the inability of a yeast colony containing both vectors to express β-galactosidase. The mutated T1 cassettes, which resulted in white colonies as Gal4-AD-T1 mutants, were swapped into the Gal4-BD-T1 vector and retested against wild-type Gal4-AD-T1. For most mutations, the results were very similar when the mutation was placed in either the AD or BD fusion construct. Mutations were identified by DNA sequencing of the entire cassette. Further characterization by in vitro translation of mutant T1 domains and identification of monomer and tetrameric fractions by size exclusion chromatography (SEC) was performed as described previously (21Pfaffinger P.J. DeRubeis D. J. Biol. Chem. 1995; 270: 28595-28600Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). For making the full-length constructs, mutant T1 domains were cassette-cloned into the Aplysia Shaker Kv1.1 coding sequence either with or without an EGFP tag made by in-frame cloning of EGFP after residue 477 of the aKv1.1 C terminus. To enhance characterization in physiology studies, the first four N-terminal amino acids of aKv1.1 were removed to reduce N-type inactivation. For the analysis of monomer and tetramer fractions of T1 mutant full-length channels proteins were transfected into CHO cells, and membranes were isolated as described below and then solubilized in 1% CHAPS either with or without added SDS at 1%. SEC-FPLC separation was performed with an Amersham Pharmacia Biotech FPLC over a Superose-6 column with added CHAPS to 0.5%. Full-length constructs were expressed inXenopus oocytes by either microinjection of cytomegalovirus promoter cDNA expression constructs into the nucleus ofXenopus oocytes or injection of cRNA made in vitro with T7 RNA polymerase. For each batch of oocytes, a control wild-type construct expression was used to determine the expected amount of channel expression for a given length of time. Recordings were performed from 1 to 3 days after injection as described previously (6Cushman S. Manao M.H. Jahng A.W. DeRubeis D. Choe S. Pfaffinger P.J. Nat. Struct. Biol. 2000; 7: 403-407Crossref PubMed Scopus (92) Google Scholar). Expression levels were compared by measuring peak current levels in response to a voltage pulse to +60 mV for 100–200 ms. All tissue culture supplies were purchased from Life Technologies, Inc. except for serum that was purchased from Hyclone, Inc. The microscopic analysis of cells transfected with Kv1.1-EGFP and its mutants was performed on a Zeiss LSM-510 confocal microscope, using the recommended settings for EGFP detection, with parallel capturing of the optical image. Serial sections were taken in 0.8-micron steps starting at the cell–coverslip interface using an ×63 oil-immersion objective lens. Representative sections are shown. COS7 cells plated on glass coverslips were transfected with Fugene™ (Roche Molecular Biochemicals) as recommended by the manufacturer using the same cDNA constructions that were tested in oocyte expression studies. At 24–30 h post-transfection, the cells were washed with ice-cold PBS, fixed in 3% formaldehyde (EMS, Ft. Washington, PA) in PBS/0.15 mm Ca2+ for 30 min, washed again with ice-cold PBS, and mounted with VectaShield™ (Vector Laboratories, Burlingame, CA). Colocalization experiments to demonstrate that the monomeric subunits are retained in the endoplasmic reticulum were conducted using fluorescence immunocytochemistry with antisera to an endoplasmic reticulum protein standard, calnexin, and a secondary antibody tagged with rhodamine (both antisera from Santa Cruz Biotechnology, Inc.). Briefly, fixed cells were permeabilized with a CHAPS wash of 0.5% CHAPS in PBS for 30 min, blocked with normal donkey serum (Cappel Laboratories) at a dilution of 1:10 in PBS, reacted with the primary antiserum at a dilution of 1:100 for 30 min, washed with PBS (three changes over 30 min), and finally reacted with the secondary antiserum at a dilution of 1:100 prior to microscopy. Membrane preparations of aKv1.1 (37Pfaffinger P.J. Furukawa Y. Zhoe B. Dugan D. Kandel E.R. J. Neurosci. 1991; 11: 918-927Crossref PubMed Google Scholar) from a stable CHO-derived cell line expressing a C-terminal EGFP-tagged aKv1.1 channel were made by low ionic strength lysis of the cells (1 ml of 50 mm Tris-HCl and 1 mm EDTA, pH 7.5 in a 100-mm dish of cells at >85% confluency with the following protease inhibitors added: 2 µg/ml leupeptin, 1 mmphenylmethylsulfonyl fluoride, and a mixture of EDTA-free protease inhibitors from Roche Molecular Biochemicals) at 0 °C, followed by brief sonication, a low speed spin of 1500 rpm for 2 min to remove organelles, and a high speed spin of 60,000 rpm for 20 min to pellet the membranes. The pellet was resuspended in PBS, sonicated briefly to obtain a uniform opalescent suspension, and subjected to trypsin digestion. Trypsin immobilized onto agarose beads in a 1:1 suspension with H2O added at a ratio of 1:20 (ml of membrane preparation:beads), and the mixture was rotated for 16 h at room temperature. The digested Kv1.1 preparation was passed through a 0.2-micron filter to remove trypsin and membrane-associated proteins before separation on a Superose-12 SEC-FPLC column (Amersham Pharmacia Biotech) run in 50 mm Tris-HCl, 1 mm EDTA, pH 7.5, and 150 mm NaCl. Fractions were acetone-precipitated and run on 15% SDS-PAGE gels, and the locations of proteins were determined by Western blotting (21Pfaffinger P.J. DeRubeis D. J. Biol. Chem. 1995; 270: 28595-28600Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). For chemical cross-linking, the released trypsin-resistant tetramer was isolated as described above and compared with the T1 domain purified from bacterial expression. All cross-linking reagents were from Pierce. Tested cross-linkers includedN-α-[maleimidoacetoxy]succinimide ester, bismaleimidoethane, disuccinimidyl glutarate, N-succinimidyl iodoacetate. Cross-linking was carried out for 30 min at room temperature in the FPLC buffer, using 1 mm cross-linking reagent, and then stopped using l-cysteine at 2.5 mm. Proteins were separated on 15% SDS-PAGE gels (38Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207180) Google Scholar) to determine the extent of cross-linking. We first sought to examine how the formation of the functional channel is affected by T1 domain point mutations that demonstrate altered intersubunit T1 domain interactions. To address this question, we first identified point mutations within the T1 domain that disrupt the ability of the domain to tetramerize. We then determined whether these point mutations disrupt intact Kv channel assembly and the formation of functional channels. To rapidly characterize a large number of such point mutations, we performed a genetic screen for T1 assembly mutations using the yeast two-hybrid system. We first confirmed that the natural specificities for homomeric and heteromeric T1 assembly were reproduced in the standard yeast two-hybrid screen, as previously described (16Jan L.Y. Jan Y.N. Trends Neurosci. 1990; 13: 415-420Abstract Full Text PDF PubMed Scopus (158) Google Scholar, 17Li M. Jan Y.N. Jan L.Y. Science. 1992; 257: 1225-1230Crossref PubMed Scopus (395) Google Scholar, 18Deal K.K. Lovinger D.M. Tamkun M.M. J. Neurosci. 1994; 14: 1666-1676Crossref PubMed Google Scholar, 19Xu J., Yu, W. Jan Y.N. Jan L.Y. Li M. J. Biol. Chem. 1995; 270: 24761-24768Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). To identify point mutations of the Shaker T1 domain that disrupt assembly, we created a library of T1 domain mutations, using a randomly doped oligonucleotide-based approach (36Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 15.96Google Scholar), fused to Gal4-AD. We then cotransfected this library with a wild-type Gal4-BD-T1 fusion construct and tested it for activation of the LacZ reporter gene using X-gal. White colonies, which are indicative of a failure of the T1 domains to interact in these yeast, were characterized to determine the molecular basis for the deficit. The mutant Gal4-AD-T1 domain clones were isolated and sequenced, and their phenotype was confirmed by retransformation, transfer of the deficiency to the Gal4-BD-T1 construct by subcloning of the mutant T1 domain sequences, and failure of the mutant T1 domains to tetramerize after in vitrotranslation (5Shen N.V. Pfaffinger P.J. Neuron. 1995; 14: 625-633Abstract Full Text PDF PubMed Scopus (159) Google Scholar). In addition to frameshift mutations and stop codons, 12 different missense point mutations that satisfied these criteria were identified (see Fig. 1A). These mutations are distributed throughout the T1 domain from residues 75–168 and are found to encode a variety of changes to conserved amino acids (Fig. 1 B). Based on the T1 structure, these mutations likely produce either steric blocks to subunit tetramerization or alter the packing between layers of secondary structure within the monomer (Table I).Table IResidues important for tetramer formation in Kv1.1 T1 domain generated from a yeast two-hybrid screen used to randomly generate inhibitory mutationsResidueLocation, T1 structureInterpretation for loss of tetramer formation1L75PLayer 1, N terminus, β-strand 2Interrupts β1→β2 loop to β2 strand interaction at subunit interface2F87SLayer 1, interhelical loop α1–α2 conserved among all KvDestabilizes intradomain packing between layers 1 and 33P88LLayer 1, interhelical loop α1–α2 conserved among all Kv1.xChange polypeptide chain angle, destabilizes intradomain packing between layers 1 and 34F110SLayer 1, C terminus, β-strand 4 conserved among all KvDestabilizes intradomain packing within layer 15D119YLayer 2, mid-helix, α3 conserved among all Kv1.x and adjacent to F118, which is conserved among all KvInterrupts polar subunit interface between Asp-119, Arg-115′, Gln-126, and Ser-73′6I121TLayer 2, mid-helix, α3 conserved among all KvInterrupts polar subunit interface betw
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