Tim17p Regulates the Twin Pore Structure and Voltage Gating of the Mitochondrial Protein Import Complex TIM23
2006; Elsevier BV; Volume: 282; Issue: 6 Linguagem: Inglês
10.1074/jbc.m607551200
ISSN1083-351X
AutoresSonia Martinez‐Caballero, Sergey M. Grigoriev, Johannes M. Herrmann, María Luisa Campo, Kathleen W. Kinnally,
Tópico(s)ATP Synthase and ATPases Research
ResumoThe TIM23 complex mediates import of preproteins into mitochondria, but little is known of the mechanistic properties of this translocase. Here patch clamping reconstituted inner membranes allowed for first time insights into the structure and function of the preprotein translocase. Our findings indicate that the TIM23 channel has "twin pores" (two equal sized pores that cooperatively gate) thereby strikingly resembling TOM, the translocase of the outer membrane. Tim17p and Tim23p are homologues, but their functions differ. Tim23p acts as receptor for preproteins and may largely constitute the preprotein-conducting passageway. Conversely depletion of Tim17p induces a collapse of the twin pores into a single pore, whereas N terminus deletion or C terminus truncation results in variable sized pores that cooperatively gate. Further analysis of Tim17p mutants indicates that the N terminus is vital for both voltage sensing and protein sorting. These results suggest that although Tim23p is the main structural unit of the pore Tim17p is required for twin pore structure and provides the voltage gate for the TIM23 channel. The TIM23 complex mediates import of preproteins into mitochondria, but little is known of the mechanistic properties of this translocase. Here patch clamping reconstituted inner membranes allowed for first time insights into the structure and function of the preprotein translocase. Our findings indicate that the TIM23 channel has "twin pores" (two equal sized pores that cooperatively gate) thereby strikingly resembling TOM, the translocase of the outer membrane. Tim17p and Tim23p are homologues, but their functions differ. Tim23p acts as receptor for preproteins and may largely constitute the preprotein-conducting passageway. Conversely depletion of Tim17p induces a collapse of the twin pores into a single pore, whereas N terminus deletion or C terminus truncation results in variable sized pores that cooperatively gate. Further analysis of Tim17p mutants indicates that the N terminus is vital for both voltage sensing and protein sorting. These results suggest that although Tim23p is the main structural unit of the pore Tim17p is required for twin pore structure and provides the voltage gate for the TIM23 channel. Because more than 95% of the ∼700 yeast mitochondrial proteins are encoded in the nucleus, newly synthesized proteins need to be translocated to their final destinations in the outer and inner membranes, the matrix, or the intermembrane space (1Reichert A.S. Neupert W. Trends Genet. 2004; 20: 555-562Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Three multisubunit complexes or translocases mediate this routing of preproteins. All precursor proteins cross the outer membrane through the translocase of the outer membrane (TOM). 3The abbreviations used are: TOM, translocase of the outer membrane; DHFR, dihydrofolate reductase; PAM, presequence translocase-associated motor; TIM, translocase of the inner membrane; pS, picosiemens; PEG, polyethylene glycol. The TIM22 and TIM23 complexes are two translocases in the inner membrane (for reviews, see Refs. 2Chacinska A. Lind M. Frazier A.E. Dudek J. Meisinger C. Geissler A. Sickmann A. Meyer H.E. Truscott K.N. Guiard B. Cell. 2005; 120: 817-829Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 3Grigoriev S.M. Muro C. Dejean L.M. Campo M.L. Martinez-Caballero S. Kinnally K.W. Int. Rev. Cytol. 2004; 238: 227-274Crossref PubMed Scopus (26) Google Scholar, 4Peixoto P.M.V. Martínez-Caballero S. Grigoriev S.M. Kinnally K.W. Campo M.L. Rec. Res. Dev. Biophys. 2004; 3: 413-474Google Scholar, 5Rehling P. Brandner K. Pfanner N. Nat. Rev. Mol. Cell. 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Meyer H.E. Meisinger C. Pfanner N. Rehling P. Cell. 2002; 111: 507-518Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 9Yamamoto H. Esaki M. Kanamori T. Tamura Y. Nishikawa S. Endo T. Cell. 2002; 111: 519-528Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 10Mokranjac D. Paschen S.A. Kozany C. Prokisch H. Hoppins S.C. Nargang F.E. Neupert W. Hell K. EMBO J. 2003; 22: 816-825Crossref PubMed Scopus (152) Google Scholar). Tim23p is embedded in the inner membrane and putatively forms the translocation pore of the complex. Tim23p also contains a hydrophilic domain of about 100 amino acids exposed to the intermembrane space that has receptor-like properties for the recognition of preproteins (11Emtage J.L. Jensen R.E. J. Cell Biol. 1993; 122: 1003-1012Crossref PubMed Scopus (131) Google Scholar, 12Bauer M.F. Sirrenberg C. Neupert W. Brunner M. Cell. 1996; 87: 33-41Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 13Lohret T.A. 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Biol. 2004; 11: 234-241Crossref PubMed Scopus (134) Google Scholar, 18Li Y. Dudek J. Guiard B. Pfanner N. Rehling P. Voos W. J. Biol. Chem. 2004; 279: 38047-38054Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 19Mokranjac D. Sichting M. Neupert W. Hell K. EMBO J. 2003; 22: 4945-4956Crossref PubMed Scopus (170) Google Scholar, 20Truscott K.N. Voos W. Frazier A.E. Lind M. Li Y. Geissler A. Dudek J. Muller H. Sickmann A. Meyer H.E. Meisinger C. Guiard B. Rehling P. Pfanner N. J. Cell Biol. 2003; 163: 707-713Crossref PubMed Scopus (163) Google Scholar). Until recently, little was known of the function of the integral membrane protein Tim17p. The high degree of homology of Tim17p with Tim23p led to speculation that these two proteins form the protein-translocating channel (21Kubrich M. Keil P. Rassow J. Dekker P.J. Blom J. Meijer M. Pfanner N. FEBS Lett. 1994; 349: 222-228Crossref PubMed Scopus (69) Google Scholar, 22Moro F. Sirrenberg C. Schneider H.C. Neupert W. Brunner M. EMBO J. 1999; 18: 3667-3675Crossref PubMed Scopus (83) Google Scholar, 23Ryan K.R. Leung R.S. Jensen R.E. Mol. Cell. Biol. 1998; 18: 178-187Crossref PubMed Scopus (49) Google Scholar). It has also been hypothesized that Tim17p might form a Tim23p-independent channel that mediates incorporation of proteins into the inner membrane by a stop-transfer mechanism (24Pfanner N. Chacinska A. Biochim. Biophys. Acta. 2002; 1592: 15-24Crossref PubMed Scopus (46) Google Scholar). Recently Tim17p was shown to be essential for both sorting of proteins into the inner membrane and translocation of precursor proteins into the matrix where Tim17p may provide a link between the TIM23 and PAM complexes (2Chacinska A. Lind M. Frazier A.E. Dudek J. Meisinger C. Geissler A. Sickmann A. Meyer H.E. Truscott K.N. Guiard B. Cell. 2005; 120: 817-829Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Moreover Tim17p specifically interacts with the purified N-terminal domain of Pam18/Tim14p, which is exposed to the intermembrane space (2Chacinska A. Lind M. Frazier A.E. Dudek J. Meisinger C. Geissler A. Sickmann A. Meyer H.E. Truscott K.N. Guiard B. Cell. 2005; 120: 817-829Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). At the core of each of the translocases is a channel, or pore, that provides the aqueous pathway for the transit of unfolded proteins. The TOM and TIM22 complexes were found to have twin pore structures by single particle analysis (25Kunkele K.-P. Heins S. Dembowski M. Nargang F.E. Benz R. Thieffry M. Walz J. Lill R. Nussberger S. Neupert W. Cell. 1998; 93: 1009-1019Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 26Rehling P. Model K. Brandner K. Kovermann P. Sickmann A. Meyer H.E. Kuhlbrandt W. Wagner R. Truscott K.N. Pfanner N. Science. 2003; 299: 1747-1751Crossref PubMed Scopus (234) Google Scholar); this approach has not yet been successfully applied to the TIM23 complex. In previous electrophysiological studies, the channel activities associated with the TOM and TIM23 complexes were found to be remarkably similar (13Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (93) Google Scholar, 27Muro C. Grigoriev S.M. Pietkiewicz D. Kinnally K.W. Campo M.L. Biophys. J. 2003; 84: 2981-2989Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In this study, we provide evidence that the TIM23 channel has a twin pore structure, like the TOM and TIM22 channels, and that Tim17p is vital to maintaining this structure. Analysis of several mutants revealed that the N terminus of Tim17p acts as the voltage sensor for the TIM23 complex. Isolation of Mitochondria and Preparation of Proteoliposomes—A mutant strain of Saccaromyces cerevisiae, Tim17(Gal10), in which the expression of TIM17 gene is controlled by a Gal10 promoter was used (28Milisav I. Moro F. Neupert W. Brunner M. J. Biol. Chem. 2001; 276: 25856-25861Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Cells were cultivated at 30 °C on standard defined lactate medium with 2% glucose in the presence or absence of 1% galactose for 24 h as described by Milisav et al. (28Milisav I. Moro F. Neupert W. Brunner M. J. Biol. Chem. 2001; 276: 25856-25861Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Three additional Tim17 mutants were analyzed and include versions lacking the C-terminal 24 (Tim17ΔC) or N-terminal 11 (Tim17ΔN) amino acids, double point mutant D4R/D8K (Tim17DD→ RK), and a four-point mutant with an additional replacement at amino acids 80 and 83 (Tim17DD→ RK/KR→ DD). Cells were grown at 30 °C on semisynthetic lactate medium as described by Meier et al. (29Meier S. Neupert W. Herrmann J.M. J. Biol. Chem. 2005; 280: 7777-7785Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Mitochondria were isolated from logarithmically growing cells as described previously (13Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (93) Google Scholar). Homogenization buffer was 0.6 m sorbitol, 10 mm Tris, 1 mm EDTA, 0.2% bovine serum albumin, 1 mm phenylmethylsulfonyl fluoride (pH 7.4) containing protease inhibitor mixture (Sigma catalog number P 8215). Mitoplasts were prepared from isolated mitochondria by the French press method (30Decker G.L. Greenawalt J.W. J. Ultrastruct. Res. 1977; 59: 44-56Crossref PubMed Scopus (61) Google Scholar), and the inner membranes were further purified according to Mannella (31Mannella C. J. Cell Biol. 1982; 94: 680-687Crossref PubMed Scopus (129) Google Scholar) as described previously (32Pavlov E.V. Priault M. Pietkiewicz D. Cheng E.H.-Y. Antonsson B. Manon S. Korsmeyer S.J. Mannella C.A. Kinnally K.W. J. Cell Biol. 2001; 155: 725-732Crossref PubMed Scopus (238) Google Scholar). Membrane purity was routinely assessed, and cross-contamination was typically less than 5%. Inner membranes were reconstituted into giant proteoliposomes by dehydration-rehydration as described previously (13Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (93) Google Scholar, 33Lohret T.A. Kinnally K.W. Biophys. J. 1995; 68: 2299-2309Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 34Lohret T.A. Murphy R.C. Drgon T. Kinnally K.W. J. Biol. Chem. 1996; 271: 4846-4849Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) using soybean l-α-phosphatidylcholine (Sigma Type IV-S). Patch Clamping Techniques—Patch clamp experiments were carried out on reconstituted TIM23 channels of proteoliposomes containing purified mitochondrial inner membranes (13Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (93) Google Scholar, 32Pavlov E.V. Priault M. Pietkiewicz D. Cheng E.H.-Y. Antonsson B. Manon S. Korsmeyer S.J. Mannella C.A. Kinnally K.W. J. Cell Biol. 2001; 155: 725-732Crossref PubMed Scopus (238) Google Scholar). Briefly membrane patches were excised from giant proteoliposomes after formation of a gigaseal using microelectrodes with ∼0.4-μm-diameter tips and resistances of 10–30 megaohms. Unless otherwise indicated, the solution in the microelectrode and bath was 150 mm KCl, 5 mm HEPES, pH 7.4, at ∼23 °C. Voltage clamp was established with the inside-out excised configuration (35Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15174) Google Scholar) using a Dagan 3900 patch clamp amplifier in the inside-out mode. Voltages across excised patches were reported as bath potentials. The open probability, Po, was calculated as the fraction of the total time the channel spent in the open state from total amplitude histograms generated with WinEDR Software (courtesy of J. Dempster, University of Strathclyde, Glasgow, UK) from 20–40 s of current traces. V0 is the voltage at which the channel spends half of the time open (Po is 0.5). Mean open time was measured by analyzing >1000 transition events per patch. Filtration was 2 kHz with 5-kHz sampling for all analysis and currents traces shown unless otherwise stated. Simulations were generated by WinEDR Software as described previously (27Muro C. Grigoriev S.M. Pietkiewicz D. Kinnally K.W. Campo M.L. Biophys. J. 2003; 84: 2981-2989Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) by providing single channel parameters including transition amplitude, mean open and closed times, and designating five openings/burst for each data set. The distribution of time spent in each of the three states (O, two open (PO1O2 = Po2); S, one open and one closed (PO1C2 or C1O2 = 2Po(1 – Po)); and C, two closed (PC1C2 = (1 – Po)2)) was fit to the open state of two independent channels. Permeability ratios were calculated from the reversal potential in the presence of a 150:30 mm KCl gradient as described previously (13Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (93) Google Scholar). Peptides were introduced by perfusion of the 0.5-ml bath with 3–5 ml of medium. Flicker rates were determined from 20–40 s of current traces as the number of transition events/s from the open to lower conductance states with a 50% threshold of the predominant event (∼250 pS). The pore size was estimated using the polymer exclusion method (36Bezrukov S.M. Kasianowicz J.J. Eur. Biophys. J. 1997; 26: 471-476Crossref PubMed Scopus (77) Google Scholar, 37Krasilnikov O.V. Sabirov R.Z. Ternovsky V.I. Merzliak P.G. Muratkhodjaev J.N. FEMS Microbiol. Immunol. 1992; 5: 93-100Crossref PubMed Google Scholar). The transition size and peak conductance in the presence of a series of polyethylene glycols (PEGs; molecular mass, 200–8000 Da) was determined. PEG solutions were 15% (w/v) in 150 mm KCl, 5 mm HEPES, pH 7.4, and were added to the bath by perfusion. The radius of the pore of the TIM23 channel was also estimated from the peak conductance assuming a pore length of 7 nm (38Szabo I. Bernardi P. Zoratti M. J. Biol. Chem. 1992; 267: 2940-2946Abstract Full Text PDF PubMed Google Scholar). That is, Rpore = (l + (π a)/2) (ρ/π a2) where R and ρ are the resistivity of the pore and solution, l is pore length, and a is pore radius (39Hille B. Ionic Channels of Excitable Membranes. 4th Ed. Sinauer Associates, Sunderland, MA2001: 351-352Google Scholar). Immunoblotting—Mitochondrial proteins were separated by SDS-PAGE (40Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and electrotransferred (32Pavlov E.V. Priault M. Pietkiewicz D. Cheng E.H.-Y. Antonsson B. Manon S. Korsmeyer S.J. Mannella C.A. Kinnally K.W. J. Cell Biol. 2001; 155: 725-732Crossref PubMed Scopus (238) Google Scholar) onto polyvinylidene difluoride membranes. Indirect immunodetection used chemiluminescence (ECL by Amersham Biosciences) using horseradish peroxidase-coupled secondary antibodies. Membrane proteins (0.5–12 μg/line) were decorated with antibodies against Tim23p, Tim17p, and Tim44p (gift of M. Brunner and I. Milisav). Scion imaging and densitometry were used to semiquantify the amount of Tim17p and Tim23p from the signal intensities of bands on Western blots that were normalized relative to 1 μg of total protein of cells grown in the presence of galactose. Peptides—Peptides were prepared by the New York State Department of Health Wadsworth Center Peptide Synthesis Core Facility (Albany, NY) using an Applied Biosystems 431A automated peptide synthesizer as described previously (13Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (93) Google Scholar). The presequence peptides used were based on amino acids 1–13 and 1–22 from the N terminus of cytochrome oxidase subunit IV of S. cerevisiae (yCoxIV-(1–13) and yCoxIV-(1–22)) and a synthetic mitochondrial presequence, SynB2 (46Allison D.S. Schatz G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9011-9015Crossref PubMed Scopus (125) Google Scholar). Peptides were subjected to mass spectroscopy to determine impurities and proper composition and were typically >90% pure. Considerable evidence links TIM23 channel activity to protein import and the TIM23 complex. Antibodies against Tim23p specifically block TIM23 channel activity in patch clamp experiments and protein import in mitoplasts (13Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (93) Google Scholar). A tim23.1 strain that is import-deficient displays altered TIM23 channel activity (13Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (93) Google Scholar). TIM23 channel activity is reversibly regulated by synthetic presequence peptides (13Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (93) Google Scholar, 33Lohret T.A. Kinnally K.W. Biophys. J. 1995; 68: 2299-2309Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 41Kushnareva Y.E. Campo M.L. Kinnally K.W. Sokolove P.M. Arch. Biochem. Biophys. 1999; 366: 107-115Crossref PubMed Scopus (35) Google Scholar, 42Kushnareva Y.E. Polster B.M. Sokolove P.M. Kinnally K.W. Fiskum G. Arch. Biochem. Biophys. 2001; 386: 251-260Crossref PubMed Scopus (36) Google Scholar). The frequency of detecting TIM23 channels is directly coupled to the amount of Tim17p (supplemental Fig. S1) or Tim23p (47Martinez-Caballero S. Peixoto P.M.V. Kinnally K.W. Campo M.L. Anal. Biochem. 2007; (in press)PubMed Google Scholar) present in the membrane. Moreover the single channel properties of TIM23 are surprisingly similar to those of TOM channel, the import channel of the outer membrane (27Muro C. Grigoriev S.M. Pietkiewicz D. Kinnally K.W. Campo M.L. Biophys. J. 2003; 84: 2981-2989Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). To determine whether TIM23 channels have a twin pore structure like that of TOM channels, the architecture of the TIM23 channel was investigated by characterizing normal and Tim17 mutant strains of yeast with patch clamp techniques. Mitochondrial inner membranes were purified and fused with small liposomes to form giant proteoliposomes. Membrane patches were excised from proteoliposomes with a micropipette, and the conductance (which is proportional to the resistance to the current flow of ions and the pore size) was measured at various voltages in the presence and absence of molecules that can size the channel pore. Single channel properties were routinely measured to verify TIM23 channel identity. Like TOM, normal TIM23 channels have an open state conductance of 1000 pS and a half-open substate of 500 pS; most transitions in the current traces are 500 pS (Fig. 1, A and B, and Table 1) (13Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (93) Google Scholar, 27Muro C. Grigoriev S.M. Pietkiewicz D. Kinnally K.W. Campo M.L. Biophys. J. 2003; 84: 2981-2989Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar).TABLE 1Comparison of the electrophysiological properties of TIM23 channelsWild-type mitoplastsaData from Lohret et al. (13, 33, 34, 45); data not leak-subtracted.Wild typebData from Muro et al. (27).Tim17(Gal10)+GalTim17(Gal10)-GalPeak conductance (pS)12001160 ± 140 (n = 10)985 ± 74 (n = 8)679 ± 114 (n = 15)Transition size (pS)500490 ± 43 (n = 10)494 ± 39 (n = 10)0 (n = 15)Mean open time (ms) (+20 mV)710.6 ± 4.3 (n = 13)11.14 ± 4.9 (n = 11)∞ (n = 15)Voltage-dependentYesYesYesNoGating charge-4.7 ± 1.1-4.2 ± 0.7 (n = 20)-3.9 ± 0.7 (n = 7)0V0 (mV)cVoltage at which the channel spends half of the time open (open probability, Po, is 0.5).5-3550 ± 10 (n = 20)38.5 ± 7.7 (n = 7)NAPermeability, K+/Cl-6.05.0 ± 0.3 (n = 20)5.8 ± 1.6 (n = 6)5.4 ± 1.9 (n = 10)Cooperatively gateYesYesYesNAPeptide sensitivity↑ Flicker↑ Flicker↑ FlickerClosurePore structureNDTwinTwinSinglea Data from Lohret et al. (13Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (93) Google Scholar, 33Lohret T.A. Kinnally K.W. Biophys. J. 1995; 68: 2299-2309Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 34Lohret T.A. Murphy R.C. Drgon T. Kinnally K.W. J. Biol. Chem. 1996; 271: 4846-4849Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 45Lohret T.A. Kinnally K.W. J. Biol. Chem. 1995; 270: 15950-15953Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar); data not leak-subtracted.b Data from Muro et al. (27Muro C. Grigoriev S.M. Pietkiewicz D. Kinnally K.W. Campo M.L. Biophys. J. 2003; 84: 2981-2989Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar).c Voltage at which the channel spends half of the time open (open probability, Po, is 0.5). Open table in a new tab The Putative Twin Pore Structure of the TIM23 Channel—The single channel behavior can shed light on the pore structure of the TIM23 channel. TIM23 behavior could be described by any of the three models in Fig. 1, C–E. Changes in the radius of the "iris-like" pore of Fig. 1C could account for the open (1000 pS), half-open (500 pS), and closed (0 pS) states. This type of behavior is typified by voltage-dependent anion-selective channel where large conformational changes reduce the radius of the open pore to one that is half-open or closed (43Colombini M. Blachly-Dyson E. Forte M. Ion Channels. 1996; 4: 169-202Crossref PubMed Scopus (251) Google Scholar). Alternatively the TIM23 channel may have two pores of equal size (Fig. 1, D and E). In both cases, the two pores are open when the conductance is ∼1000 pS, one pore is open and one is closed when the conductance is 500 pS, and both are closed when the conductance is 0 pS. The transition size corresponding to opening or closing of one pore in current traces is typically 500 pS (Fig. 1, A and B). The difference between the models of Fig. 1, D and E, is that gating of the twin-sized pores of Fig. 1D is independent and that of Fig. 1E is cooperative. The model with a single pore that changes diameter (iris-like) can be distinguished experimentally from the twin pore models by differences in the pore size of the open state. The iris-like model predicts a significant change in pore radius as the channel transits between the open and half-open states. Both twin pore models predict that the open and half-open states have the same pore size. The conductance of TIM23 channels was used to estimate the size of large pores by the method of Hille (39Hille B. Ionic Channels of Excitable Membranes. 4th Ed. Sinauer Associates, Sunderland, MA2001: 351-352Google Scholar) (see "Experimental Procedures" for formula) from two different control strains. (The single channel behavior of TIM23 channels from both strains was identical (Fig. 1 and Table 1) (13Lohret T.A. Jensen R.E. Kinnally K.W. J. Cell Biol. 1997; 137: 377-386Crossref PubMed Scopus (93) Google Scholar, 27Muro C. Grigoriev S.M. Pietkiewicz D. Kinnally K.W. Campo M.L. Biophys. J. 2003; 84: 2981-2989Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar).) Assuming a pore length of 7 nm (38Szabo I. Bernardi P. Zoratti M. J. Biol. Chem. 1992; 267: 2940-2946Abstract Full Text PDF PubMed Google Scholar), this approach indicates that the iris-like single pore would have an open state radius of 1.43 ± 0.08 nm and half-open radius of 0.93 ± 0.03 nm (Table 2). Both twin pores are predicted to have a radius of 0.93 ± 0.03 nm. The polymer exclusion method was then used to measure the pore size to determine whether the radius actually changed during transitions between the open and half-open states.TABLE 2Sizing the radius of the TIM23 channelMethodWild typeTim17(Gal10)+GalTim17(Gal10)-GalPolymer exclusionaCalculated using the method of Bezrukov and Kasianowicz (36).Transition size (nm)0.81-0.91 (n = 5)0.88-0.94 (n = 5)NAPeak conductance (nm)ND0.90-0.94 (n = 5)0.95-1.14 (n = 7)Pore resistancebCalculated from pore resistance using the method of Hille (39) from the transition size or peak conductance assuming a pore length of 7 nm (38).Transition size (nm)0.93 ± 0.03 (n = 10)0.94 ± 0.04 (n = 10)NAPeak conductance (nm)1.43 ± 0.08 (n = 10)1.33 ± 0.05 (n = 8)1.10 ± 0.09 (n = 15)a Calculated using the method of Bezrukov and Kasianowicz (36Bezrukov S.M. Kasianowicz J.J. Eur. Biophys. J. 1997; 26: 471-476Crossref PubMed Scopus (77) Google Scholar).b Calculated from pore resistance using the method of Hille (39Hille B. Ionic Channels of Excitable Membranes. 4th Ed. Sinauer Associates, Sunderland, MA2001: 351-352Google Scholar) from the transition size or peak conductance assuming a pore length of 7 nm (38Szabo I. Bernardi P. Zoratti M. J. Biol. Chem. 1992; 267: 2940-2946Abstract Full Text PDF PubMed Google Scholar). Open table in a new tab The polymer exclusion method is a common means of measuring pore sizes and is based on an observed decrease in conductance when non-electrolytes, e.g. PEG of various molecular weights and known radii, enter the pore of the channel (3Grigoriev S.M. Muro C. Dejean L.M. Campo M.L. Martinez-Caballero S. Kinnally K.W. Int. Rev. Cytol. 2004; 238: 227-274Crossref PubMed Scopus (26) Google Scholar, 36Bezrukov S.M. Kasianowicz J.J. Eur. Biophys. J. 1997; 26: 471-476Crossref PubMed Scopus (77) Google Scholar, 37Krasilnikov O.V. Sabirov R.Z. Ternovsky V.I. Merzliak P.G. Muratkhodjaev J.N. FEMS Microbiol. Immunol. 1992; 5: 93-100Crossref PubMed Google Scholar, 44Sabirov R.Z. Krasilnikov O.V. Ternovsky V.I. Merzliak P.G. Gen. Physiol. Biophys. 1993; 12: 95-111PubMed Google Scholar). The presence of non-electrolytes in the pore reduces the "room" available for the current-carrying electrolytes K+ and Cl–, reducing the conductance. Impermeable non-electrolytes have no effect on the conductance once corrected for differences in the conductivity of media. Both the transition size and the peak conductance of TIM23 channels decreased in the presence of 200–600-Da PEGs (0.5–0.8 nm) indicating that these PEG were permeable (Fig. 1, F–H). Neither were affected by the 1000–8000 molecular weight PEG (0.94–3.05 nm) indicating that these PEGs were not permeable in either the half-open or open state. A pore radius of 0.81–0.94 nm was calculated from the transition size (Fig. 1, I and J), and 0.90–0.94 nm was calculated from the peak conductance data (Fig. 1K and Table 2). Both values are similar to the 0.93 ± 0.03-nm radius estimated from the transition size by the method of Hille (39Hille B. Ionic Channels of Excitable Membranes. 4th Ed. Sinauer Associates, Sunderland, MA2001: 351-352Google Scholar). However, these values are significantly different from 1.43 ± 0.08 nm predicted from the peak conductance (Table 2). Hence the TIM23 channel is not an iris-like single pore (Fig. 1C) because the fully open and half-open states have the same permeability for various sized PEGs, i.e. their radii are the same. Further investigations were needed to distinguish between the two twin pore models. Do the twin pores open and close independently (Fig. 1D) or cooperatively (Fig. 1E)? If multiple, independent channels are in a membrane patch, the total amplitude histograms should fit a binomial distributi
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