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Depolarization Induces Intersubunit Cross-linking in a S4 Cysteine Mutant of the Shaker Potassium Channel

2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês

10.1074/jbc.m207258200

ISSN

1083-351X

Autores

Qadeer Aziz, Christopher J. Partridge, Tim S. Munsey, Asipu Sivaprasadarao,

Tópico(s)

Neuroscience and Neural Engineering

Resumo

Voltage-gated potassium (Kv) channels are integral membrane proteins, composed of four subunits, each comprising six (S1–S6) transmembrane segments. S1–S4 comprise the voltage-sensing domain, and S5–S6 with the linker P-loop forms the ion conducting pore domain. During activation, S4 undergoes structural rearrangements that lead to the opening of the channel pore and ion conduction. To obtain details of these structural changes we have used the engineered disulfide bridge approach. For this we have introduced the L361C mutation at the extracellular end of S4 of the Shaker K channel and expressed the mutant channel inXenopus oocytes. When exposed to mild oxidizing conditions (ambient oxygen or copper phenanthroline), Cys-361 formed an intersubunit disulfide bridge as revealed by the appearance of a dimeric band on Western blotting. As a consequence, the mutant channel suffered a significant loss in conductance (measured by two-electrode voltage clamp). Removal of native cysteines failed to prevent the disulfide formation, indicating that Cys-361 forms a disulfide with its counterpart in the neighboring subunit. The effect was voltage-dependent and occurred during channel activation after Cys-361 has been exposed to the extracellular phase. Although the disulfide bridge reduced the maximal conductance, it caused a hyperpolarizing shift in the conductance-voltage relationship and reduced the deactivation kinetics of the channel. The latter two effects suggest stabilization of the open state of the channel. In conclusion, we report that during activation the intersubunit distance between the N-terminal ends of the S4 segments of the L361C mutant Shaker K channel is reduced. Voltage-gated potassium (Kv) channels are integral membrane proteins, composed of four subunits, each comprising six (S1–S6) transmembrane segments. S1–S4 comprise the voltage-sensing domain, and S5–S6 with the linker P-loop forms the ion conducting pore domain. During activation, S4 undergoes structural rearrangements that lead to the opening of the channel pore and ion conduction. To obtain details of these structural changes we have used the engineered disulfide bridge approach. For this we have introduced the L361C mutation at the extracellular end of S4 of the Shaker K channel and expressed the mutant channel inXenopus oocytes. When exposed to mild oxidizing conditions (ambient oxygen or copper phenanthroline), Cys-361 formed an intersubunit disulfide bridge as revealed by the appearance of a dimeric band on Western blotting. As a consequence, the mutant channel suffered a significant loss in conductance (measured by two-electrode voltage clamp). Removal of native cysteines failed to prevent the disulfide formation, indicating that Cys-361 forms a disulfide with its counterpart in the neighboring subunit. The effect was voltage-dependent and occurred during channel activation after Cys-361 has been exposed to the extracellular phase. Although the disulfide bridge reduced the maximal conductance, it caused a hyperpolarizing shift in the conductance-voltage relationship and reduced the deactivation kinetics of the channel. The latter two effects suggest stabilization of the open state of the channel. In conclusion, we report that during activation the intersubunit distance between the N-terminal ends of the S4 segments of the L361C mutant Shaker K channel is reduced. dithiothreitol cysteine-less Tris(2-carboxyethyl)phosphine hydrochloride para-chloromercuribenzenesulphonate current voltage copper (II) phenanthroline Voltage-gated potassium (Kv) channels are transmembrane proteins made up of two domains, a central pore domain and a surrounding voltage-sensing domain. The pore domain forms the water-filled, potassium ion-selective pore across the plasma membrane of the cell, whereas the voltage-sensing domain regulates the opening and closing of activation gates situated at the cytoplasmic end of the pore domain (1Sigworth F.J. Q. Rev. Biophys. 1994; 27: 1-40Crossref PubMed Scopus (347) Google Scholar, 2Yellen G. Q. Rev. Biophys. 1998; 31: 239-295Crossref PubMed Scopus (401) Google Scholar, 3Bezanilla F. Physiol. Rev. 2000; 80: 555-592Crossref PubMed Scopus (720) Google Scholar, 4Hille B. Ion Channels of Excitable Membranes. 3rd. Sinauer Associates, Inc., Sunderland, Massachusetts2001Google Scholar). The gates are closed at negative (resting) membrane potentials but open upon membrane depolarization to allow K+ ions to enter the pore. Due to the lack of three-dimensional structural data for any of the Kv channel proteins, it is not clear how the voltage sensor detects changes in membrane potential and transmits the signal to the activation gates. Kv channels are made up of four subunits, each of which contains six transmembrane segments, named S1–S6. The S5–S6 and the “P-loop” connecting these segments form the central pore domain in Kv channels (2Yellen G. Q. Rev. Biophys. 1998; 31: 239-295Crossref PubMed Scopus (401) Google Scholar). The structure of the pore domain is thought to be similar to that of the bacterial potassium channel, KcsA, whose structure has been determined by x-ray diffraction (5Doyle D.A. Morais C.J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5769) Google Scholar, 6MacKinnon R. Cohen S.L. Kuo A. Lee A. Chait B.T. Science. 1998; 280: 106-109Crossref PubMed Scopus (371) Google Scholar, 7Zhou Y. Morais-Cabral J.H. Kaufman A. MacKinnon R. Nature. 2001; 414: 43-48Crossref PubMed Scopus (1737) Google Scholar). The remaining transmembrane segments, in particular the S2 to S4 segments, are thought to comprise the voltage-sensing domain of the channel (2Yellen G. Q. Rev. Biophys. 1998; 31: 239-295Crossref PubMed Scopus (401) Google Scholar,9Tiwari-Woodruff S.K. Schulteis C.T. Mock A.F. Papazian D.M. Biophys. J. 1997; 72: 1489-1500Abstract Full Text PDF PubMed Scopus (199) Google Scholar, 10Durell S.R. Hao Y. Guy H.R. J. Struct. Biol. 1998; 121: 263-284Crossref PubMed Scopus (94) Google Scholar). Of the four segments, S4 plays a pivotal role. When the membrane is depolarized, it moves out of the membrane, thereby carrying its charged residues (arginine and lysine), known as gating charges, across the membrane electric field (11Larsson H.P. Baker O.S. Dhillon D.S. Isacoff E.Y. Neuron. 1996; 16: 387-397Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 12Yusaf S.P. Wray D. Sivaprasadarao A. Pflugers Arch. 1996; 433: 91-97Crossref PubMed Scopus (119) Google Scholar, 13Yang N. Horn R. Neuron. 1995; 15: 213-218Abstract Full Text PDF PubMed Scopus (351) Google Scholar, 14Baker O.S. Larsson H.P. Mannuzzu L.M. Isacoff E.Y. Neuron. 1998; 20: 1283-1294Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 15Wang M.H. Yusaf S.P. Elliott D.J. Wray D. Sivaprasadarao A. J. Physiol. (Lond). 1999; 521: 315-326Crossref Scopus (30) Google Scholar, 16Cha A. Snyder G.E. Selvin P.R. Bezanilla F. Nature. 1999; 402: 809-813Crossref PubMed Scopus (435) Google Scholar, 17Glauner K.S. Mannuzzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (246) Google Scholar). It is this movement that appears to trigger the opening of the activation gates. Molecular modeling (10Durell S.R. Hao Y. Guy H.R. J. Struct. Biol. 1998; 121: 263-284Crossref PubMed Scopus (94) Google Scholar) and mutagenic (18Monks S.A. Needleman D.J. Miller C. J. Gen. Physiol. 1999; 113: 415-423Crossref PubMed Scopus (122) Google Scholar, 19Li-Smerin Y. Hackos D.H. Swartz K.J. J. Gen. Physiol. 2000; 115: 33-50Crossref PubMed Scopus (155) Google Scholar, 20Li-Smerin Y. Hackos D.H. Swartz K.J. Neuron. 2000; 25: 411-423Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) studies suggest that one face of the S4 segment is in direct contact with the pore domain, whereas the rest is surrounded by S1–S3, which seem to protect the charged S4 segment from the energetically unfavorable lipid environment by providing counter charges. Because of these extensive interactions that S4 appears to be engaged in, when S4 moves one would expect major changes in residue-residue contacts with the neighboring helices. Indirect evidence indicates that the S4 movement is accompanied by changes in the electrostatic interactions between its positive charges and the negatively charged residues present in the S2 and S3 segments (21Tiwari-Woodruff S.K. Lin M.A. Schulteis C.T. Papazian D.M. J. Gen. Physiol. 2000; 115: 123-138Crossref PubMed Scopus (120) Google Scholar). Defining the residue-residue contacts between S4 and the segments with which it is in contact and how they change during channel activation is critical for an appreciation of the molecular mechanism by which S4 is able to sense changes in membrane potential and transmit the signal to the pore domain. Toward this end, we set out to use the engineered disulfide approach. This approach allows determination of the residue-residue contacts within the three-dimensional context of a protein (22Chervitz S.A. Falke J.J. J. Biol. Chem. 1995; 270: 24043-24053Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and also allows structural changes underlying the activation of a protein to be elucidated (23Liu Y. Jurman M.E. Yellen G. Neuron. 1996; 16: 859-867Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). The approach involves the introduction of pairs of cysteine residues at positions that are thought to lie in close proximity and then investigating which cysteine pairs can be induced to form a disulfide bridge. The formation of disulfide bridges will often impair or alter the course of further motions, thereby producing a change in the functional properties of the protein (23Liu Y. Jurman M.E. Yellen G. Neuron. 1996; 16: 859-867Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). In the absence of measurable functional changes, however, disulfides can be detected biochemically (22Chervitz S.A. Falke J.J. J. Biol. Chem. 1995; 270: 24043-24053Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Formation of a disulfide bridge is interpreted in terms of the residues being in close proximity. Any changes in the pattern of disulfide formation between the resting and activated states of the protein will reflect structural motions underlying the activation. The power of this approach has been illustrated with a number of membrane proteins, including potassium channels (23Liu Y. Jurman M.E. Yellen G. Neuron. 1996; 16: 859-867Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 24Krovetz H.S. van Dongen H.M. van Dongen A.M. Biophys. J. 1997; 72: 117-126Abstract Full Text PDF PubMed Scopus (45) Google Scholar). In the present study, we set out to identify which residues (from other segments) are in close proximity to Lys-361 of the Shaker potassium channel. We focused our attention on this residue because it occupies a critical position in the channel. It is located within the bilayer yet close to the extracellular boundary (11Larsson H.P. Baker O.S. Dhillon D.S. Isacoff E.Y. Neuron. 1996; 16: 387-397Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 12Yusaf S.P. Wray D. Sivaprasadarao A. Pflugers Arch. 1996; 433: 91-97Crossref PubMed Scopus (119) Google Scholar, 14Baker O.S. Larsson H.P. Mannuzzu L.M. Isacoff E.Y. Neuron. 1998; 20: 1283-1294Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 15Wang M.H. Yusaf S.P. Elliott D.J. Wray D. Sivaprasadarao A. J. Physiol. (Lond). 1999; 521: 315-326Crossref Scopus (30) Google Scholar). This means that when S4 moves out this residue is expected to sever all interactions with the neighboring membrane embedded segments. More importantly, its substitution with cysteine does not alter the net charge of S4, and hence would not be expected to disrupt electrostatic interactions that may be critical for the helical packing and normal functioning of the channel. Our results show that the mutant channels are susceptible to oxidation and that this oxidation occurs during depolarization of the membrane. Western blotting showed a dimeric Shaker protein band, indicating that oxidation leads to a disulfide bond between neighboring subunits. Removal of all the native cysteines failed to prevent the oxidation, which led us to suggest that the cysteine at position 361 forms an intersubunit disulfide with its counterpart from a neighboring subunit. Finally, we show that oxidation occurs at potentials where C-type inactivation is absent, indicating that the disulfide formation occurs during the activation of the mutant channel. Taken together, data presented here suggests that the N-terminal ends of the S4 segments move toward each other during the activation of the channel. Amino acid substitutions were introduced into the inactivation ball (residues 6–46)-removed Shaker potassium channel (25Hoshi T. Zagotta W.N. Aldrich R.W. Science. 1990; 250: 533-538Crossref PubMed Scopus (1280) Google Scholar), or into its cysteine-less version (C-less Shaker), by site directed mutagenesis. cRNA transcripts were made fromHindIII-linearized plasmid (pKS-Bluescript) constructs containing the wild-type and mutant cDNA sequences using the MEGAScript kit (Ambion). All methods are as previously described (12Yusaf S.P. Wray D. Sivaprasadarao A. Pflugers Arch. 1996; 433: 91-97Crossref PubMed Scopus (119) Google Scholar). Oocytes were isolated fromXenopus laevis and anesthetized by immersion in 0.2% 3-aminobenzoic acid ethyl ester (Sigma). The animals were then killed by cervical dislocation. Dumont stage V or VI oocytes were selected, defolliculated, and injected with 5–20 ng of cRNA. The oocytes were incubated at 19 °C in modified Barth's solution containing 88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 0.82 mmMgSO4, 0.33 mmCa(NO3)2, 0.41 mmCaCl2, 0.1 mm dithiothreitol (DTT),1 and 5 mmHEPES, pH 7.4, supplemented with penicillin (10 units ml−1) and streptomycin (0.1 mg ml−1). Whole cell currents were recorded between 2 and 3 days after injection using the standard two-electrode voltage clamp configuration (12Yusaf S.P. Wray D. Sivaprasadarao A. Pflugers Arch. 1996; 433: 91-97Crossref PubMed Scopus (119) Google Scholar) in Ringer's solution containing 82 mm NaCl, 2 mmKCl, 5 mm Tris-Cl, 1 mm MgCl2, pH 7.2. Microelectrodes were made from borosilicate glass and filled with 3 m KCl and had resistance that varied between 0.5 and 2.0mΩ. Steady-state currents were measured from the injected oocytes during brief (50–500 ms) depolarising steps to +40 mV, given at 10 s intervals, from a holding potential of −80 mV. Currents were filtered at 2 kHz and sampled at 4 kHz. To examine the effect of modification reagents, after measuring currents in Ringer's solution (control recordings), cells were superfused with 1–100 μm copper (II) phenanthroline (Cu (II) Phe, 1:3 ratio) or 100 μm para-chloromercuribenzenesulphonate (pCMBS) solution made up in Ringer's solution. Current (I)-voltage (V) relationships were measured from oocytes by stepping to positive potentials in 10 mV increments from a holding potential of −80 mV. The steps were applied at 10 s intervals and lasted for 50–500 ms. A series of 20 hyperpolarizing steps of 10 mV were also applied to measure leak currents. The leak currents were averaged and subtracted from the current records. The leak subtracted I-V data were fitted to the Boltzmann function, I/Imax = 1/(1+exp(V0.5−Vtest)/k), where V0.5 is the midpoint of half-maximal current and k is the slope factor (= RT/zF, where R is the gas constant, T is the absolute temperature, z is the valence, and F is the Faraday's constant). Activation time constants were calculated by fitting the upper 50% of the raising phase of the current data to an exponential function. Deactivation kinetic parameters were obtained by fitting the tail current traces (measured in 100K (100 mmK+) Ringer's solution) to a bi-exponential decay equation. Oocytes expressing wild-type or L361C Shaker RNA were superfused with 100 μmCu (II) phe while the cells were repeatedly pulsed to +40 mV (from −80 mV), until the currents at +40 mV were maximally inhibited. Following this, oocytes were perfused with Ringer's solution supplemented with 2 mm EDTA (to chelate any metals that could potentially induce disulfides in the subsequent steps). The oocytes were then gently homogenized in a buffer containing 10 mm HEPES, 83 mm NaCl, 1 mm MgCl2, pH 7.9, and a mixture of protease inhibitors (Sigma, P-2714). Yolk and other debris were removed from the homogenate by repeated 10-min centrifugation at 13,000 × g (all operations were performed at 4 °C). The supernatant containing crude cell membranes was subjected to Western blotting. Oocyte membranes were solubilized in a buffer containing 62 mm Tris, pH 6.8, 10% glycerol, 2% SDS, and 0.2% bromphenol blue. Equal amounts of membranes were treated with water (non-reduced) or 1 mm DTT (reduced) and incubated at 37 °C for 30 min. Proteins were separated by SDS-polyacrylamide gel (7%) electrophoresis and transferred on to a polyvinylidene fluoride membrane (Immobilon-P, Millipore). Following transfer the membrane was soaked in 100% methanol for 15 s and allowed to dry for 30 min at 37 °C. The membrane was then incubated for 1 h in blocking solution (10% nonfat dried milk in 20 mm sodium phosphate, 150 mm NaCl, pH 7.4, phosphate-buffered saline) containing a 1:500 dilution of affinity purified anti-Shaker antibodies, raised in the rabbit against the synthetic peptide, CKKSSLSESSSDIMDLDDGID (residues 517–536). The membranes were then washed three times for 2 min each in phosphate-buffered saline, followed by incubation in blocking buffer containing a 1:2500 dilution of the secondary antibody (horseradish peroxidase-conjugate of goat-anti rabbit antiserum, Bio-Rad). Protein was detected using the ECL plus chemiluminiscence kit (AmershamBiosciences). Oxidation of closely placed cysteine thiols to a disulfide bridge can be enhanced by using Cu (II) Phe as a catalyst (22Chervitz S.A. Falke J.J. J. Biol. Chem. 1995; 270: 24043-24053Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Because the Shaker potassium channel contains 28 cysteines (7 cysteines per subunit) that could potentially form disulfides, we have first examined the effect of Cu (II) Phe on the wild-type channel. For this, the channel was expressed in Xenopus oocytes, and the effect of perfusion of Cu (II) Phe on the properties of the channel was examined by two-electrode voltage clamp. Fig.1 shows that the reagent has no effect on the current-voltage (I-V) relationship (Fig. 1 C) or the activation (Fig. 1, B and D) and deactivation (Fig. 1 E) kinetics of the channel. This suggests that under these experimental conditions, none of the native cysteines are close enough to undergo disulfide oxidation. An alternative explanation would be that any disulfides formed have no effect on the functional properties of the channel. Fig. 2 shows that the application of Cu (II) Phe to oocytes expressing the L361C mutant channel caused rapid inhibition (time constant, τ = 1.46 ± 0.16 min; n = 4) of currents. The inhibition could not be reversed by Cu (II) Phe removal alone (Ringer's wash), but could be fully reversed with DTT (1 mm) (Fig. 2 A), a reagent capable of reducing disulfide bridges to cysteines. The inhibition was incomplete, with about 30% of the currents remaining, when the inhibition reached a steady state. The residual currents displayed slowed activation kinetics (τa at +40 mV, before = 1.73 ± 0.1 ms; after Cu (II) Phe = 18.6 ± 1.9 ms) (Fig. 2, D and E) and a negative shift (13 mV) in the current-voltage relationship (Fig. 2 C). There was also a significant decrease in the effective gating valence (z values were 1.96 ± 0.25 before and 1.08 ± 0.07 after oxidation). In addition, the deactivation kinetics were dramatically reduced (Fig. 2 F), with nearly 50% of the current remaining even after a 500 ms deactivating pulse at −110 mV. There was also some reduction in the rate of C-type inactivation (Fig. 2 E) (inactivation time constants measured at +40 mV before and after Cu (II) Phe treatment are 4.38 ± 0.3 s and 6.6 ± 0.7 s, respectively). These changes in the properties argue that the residual currents are not due to channels that escaped oxidation but due to channels modified by Cu (II) Phe. The reagent used in the above experiment has 100 μmCu2+ and 300 μm phenanthroline (commonly used concentrations) (22Chervitz S.A. Falke J.J. J. Biol. Chem. 1995; 270: 24043-24053Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). At this metal ion to chelating agent ratio (1:3), a significant amount of free, uncomplexed Cu2+ would be expected to be present in the reagent. To eliminate the possibility that the inhibition could be due to the binding of free copper (II) to cysteines, we have reduced the concentration of Cu (II) to 5 μm and increased the concentration of phenanthroline to 200 μm. The resulting reagent (ratio of Cu (II) to Phe 1:40), which would contain a negligible amount of free copper, showed no significant effect on the rate (1.46 ± 0.3 min with 1:3 Cu (II) Phe and 1.28 ± 0.18 min with 1:40 Cu (II) Phe) or the extent of inhibition (66.7 ± 4.0% with 1:3 Cu (II) Phe and 63.1 ± 4.4% with 1:40 Cu (II) Phe) of the mutant channel current (Fig.2 G), suggesting that the effect is not due to the binding of free Cu2+ to the cysteine at position 361. Free Cu2+ also inhibits, but the effect, unlike that produced by Cu (II) Phe, is fully reversed by Ringer's wash alone (data not shown). Taken together, these data suggest that the observed inhibition is likely to be due to the oxidation of cysteine thiols to disulfides. Data in Fig. 2 showed that Cu (II) Phe produces very rapid oxidation of the mutant channel. We wondered if the ambient oxygen itself is adequate to induce oxidation. To test this, we have incubated the injected oocytes in ND-96 medium without DTT (we routinely included 50 μm DTT in our medium). These oocytes expressed low currents and displayed slow activation kinetics with a time constant (20.8 ± 1.2 ms) (Fig. 3,C and D) that is similar to the Cu (II) Phe oxidized mutant channel (18.6 ± 1.9 ms) (Fig. 2, D andE). Application of DTT caused a rapid increase in steady-state currents (Fig. 3 A), which was accompanied by an increase in the rate of activation (τa = 5.9 ± 2.8 ms) (Fig. 3, C and D). We found that when the oocytes were incubated in ND-96 lacking DTT currents at the end of DTT application were routinely higher than the currents seen at the beginning of the recording, although there were differences from oocyte to oocyte and batch to batch with respect to the extent of increase. These observations argue against the possibility that the reduced currents are due to oxidation of cysteine thiols to sulfinic and sulfonic acid derivatives, which generally require harsh oxidative conditions. The data thus support the idea that the oxidation of cysteines at position 361 leads to disulfides. As mentioned above, there are seven native cysteines in each of the subunits of the Shaker channel. Of these, three are in the transmembrane portion of the channel (at positions 245 in S2, 286 in S2, and 462 in S6), which are potentially available for disulfide bonding with the cysteine at position 361. The disulfide could be with a cysteine in the same subunit or from a neighboring subunit. Although detection of intrasubunit disulfides is hard by biochemical means, an intersubunit disulfide can be readily detected, as the size of the cross-linked species will increase 2-, 3-, or 4-times depending upon the manner in which the disulfide bridges occur. To investigate this, we treated the oocytes expressing the L361C mutant channels with Cu (II) Phe while pulsing to +40 mV and subjected the membranes isolated from them to Western blotting using an anti-Shaker antibody. Fig.4 A shows a dimeric band of ∼220 kDa (lane 3), which could be reduced to the monomeric form (∼110 kDa) with DTT (lane 4). No dimeric band was detectable with wild-type Shaker (lane 1) or L361C channels not treated with Cu (II) Phe (lane 5), indicating that S4 cysteines may form an intersubunit disulfide. Because we found that L361C channels undergo spontaneous oxidation when the oocytes are incubated in a medium lacking DTT (Fig. 3), we have also subjected the membranes isolated from these oocytes to Western blotting. Fig.4 B shows that dimers are indeed formed through spontaneous oxidation by the ambient oxygen. A significant amount of monomeric protein was also seen; this is consistent with the expectation that oxidation by ambient oxygen would be slow and incomplete. These data indicate that Cys-361 forms an intersubunit disulfide bridge with a cysteine in a neighboring subunit. To identify the cysteine with which the cysteine at position 361 forms the intersubunit disulfide bridge, we have introduced a single cysteine at position 361 of a Shaker mutant channel from which all native cysteines have been removed (referred to as C-less Shaker) (26Boland L.M. Jurman M.E. Yellen G. Biophys. J. 1994; 66: 694-699Abstract Full Text PDF PubMed Scopus (33) Google Scholar). The idea was to subsequently introduce cysteines at the native positions and examine the effect of Cu (II) Phe. However, when we tested for the effect, Cu (II) Phe caused inhibition of currents through this mutant channel (Fig.5, A and B). Moreover, oocytes (expressing the Cys-361 C-less Shaker channels) incubated in DTT minus medium elicited currents that increased with the application of DTT (Fig. 5 D), indicating that ambient oxygen can also inhibit the mutant channel currents. The ability of DTT to reverse the effects of ambient oxygen and Cu (II) Phe suggests that, as in the wild-type channel, cysteines (at 361) in the C-less background also undergo oxidation to disulfides. The low levels of expression of C-less Shaker channels in oocytes prevented us from confirming this by Western blotting; thus, we were unable to completely rule out the possibility that oxidation might lead to products other than disulfides. As an alternative, we have tested the ability of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to reverse the effect of Cu (II) Phe. The mechanism of action of TCEP is different from that of DTT, and TCEP, unlike DTT, is highly specific for reducing disulfide bridges (27Ruegg U.T. Rudinger J. Methods Enzymol. 1977; 47: 111-116Crossref PubMed Scopus (261) Google Scholar). Fig. 5 (E andF) shows that TCEP fully reversed the effect of Cu (II) Phe, confirming the fact that disulfide formation is the cause of inhibition by Cu (II) Phe. Because this mutant channel contains no cysteines other than those at position 361, we conclude that Cys-361 of one subunit forms a disulfide bridge with its counterpart in the neighboring subunit. We next investigated the voltage-dependence of current inhibition by Cu (II) Phe for the L361C mutant channel (Fig. 6). In these experiments, oocytes were held at various potentials (−120 mV to −20 mV) for 200 s while superfusing the oocyte with the reagent. This was followed by repeated pulsing to +40 mV (from a holding potential of −80 mV) to record the currents. Fig. 6 (A and B) shows that inhibition of currents through L361C was highly voltage-dependent and occurred with a V0.5(voltage at which 50% of the channels were inhibited) value of −77 mV and a slope factor of 3.08 ± 0.16 mV. The data suggest that it is during depolarization that S4 segments move close enough toward each other to result in a disulfide between the cysteines at position 361. It may be noted that the voltage-dependence is not due to the effect of the electric field on the reagent, because the reagent used here is a catalyst (neutral or slightly positive), which generates free radicals from the ambient oxygen. During depolarization the Δ(6–46) Shaker channel undergoes fast activation followed by slow C-type inactivation (2Yellen G. Q. Rev. Biophys. 1998; 31: 239-295Crossref PubMed Scopus (401) Google Scholar,28Olcese R. Latorre R. Toro L. Bezanilla F. Stefani E. J. Gen. Physiol. 1997; 110: 579-589Crossref PubMed Scopus (156) Google Scholar, 29Loots E. Isacoff E.Y. J. Gen. Physiol. 1998; 112: 377-389Crossref PubMed Scopus (155) Google Scholar, 30Loots E. Isacoff E.Y. J. Gen. Physiol. 2000; 116: 623-636Crossref PubMed Scopus (94) Google Scholar, 31Larsson H.P. Elinder F. Neuron. 2000; 27: 573-583Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 32Elinder F. Arhem P. Larsson H.P. Biophys. J. 2001; 80: 1802-1809Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 33Elinder F. Mannikko R. Larsson H.P. J. Gen. Physiol. 2001; 118: 1-10Crossref PubMed Scopus (67) Google Scholar). There is evidence that S4 undergoes distinct conformational changes during both these steps (29Loots E. Isacoff E.Y. J. Gen. Physiol. 1998; 112: 377-389Crossref PubMed Scopus (155) Google Scholar, 30Loots E. Isacoff E.Y. J. Gen. Physiol. 2000; 116: 623-636Crossref PubMed Scopus (94) Google Scholar, 34Gandhi C.S. Loots E. Isacoff E.Y. Neuron. 2000; 27: 585-595Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Thus the observed disulfide bridge formation can occur during either of these steps. To distinguish between these two possibilities, we have followed C-type inactivation of L361C as a function of membrane voltage. As can be seen from Fig. 6 B, C-type inactivation occurs at much more (∼90 mV) positive potentials (V0.5 = 13.6 ± 1.4 mV) than disulfide cross-linking (V0.5 = −77 ± 0.17 mV), suggesting that oxidation to disulfides must have occurred during activation. To provide further evidence, we have allowed the channels to inactivate maximally (∼80% current loss) by using a long pulse to +40 mV and then applied Cu (II) Phe for 2 min while holding the cells at 0 mV to prevent recovery from inactivation (Fig.7). Washing with Ringer's solution reversed over 40% of the lost current. Because the Cu (II) Phe effect is not reversible by Ringer's solution, this reversal must represent recovery from inactivation. It also suggests that the inactivated channels were unaffected by Cu (II) Phe. Subsequent application of DTT produced further recovery from inhibition (∼20%), which might represent channels that have not undergone inactivation during the long inactivation pulse, and hence are susceptible to the Cu (II) Phe effect. Following maximal reversal of currents, when Cu (II) Phe was re-applied it produced the normal rapid inhibition (reversible by DTT), the magnitude (∼55%) of which is larger than that (20%) produced by the reagent when applied to channels during the long inactivating pulse. These data argue that cross-linking of adjacent cysteines at position 361 occurs during activation rather than during C-type inactivation. We also investigated whether the intersubunit disulfide formation occurs before or after S4 begins to move out of the membrane electric field. For this, we studied the movement of cysteine at position 361 out of the membrane bilayer, as a function of voltage, using pCMBS. pCMBS, a membrane-impermeable cysteine reagent, like the water-soluble methanethiosulphonate reagents (11Larsson H.P. Baker O.S. Dhillon D.S. Isacoff E.Y. Neuron. 1996; 16: 387-397Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 13Yang N. Horn R. Neuron. 1995; 15: 213-218Abstract Full Text PDF PubMed Scopus (351) Google Scholar), reacts with S4 cysteines only when they move out of the membrane bilayer, thereby reporting the outward movement of an S4 residue (12Yusaf S.P. Wray D. Sivaprasadarao A. Pflugers Arch. 1996; 433: 91-97Crossref PubMed Scopus (119) Google Scholar). The data (Fig. 6 B) show that the outward movement of the cysteine at position 361 begins at more negative potentials (by ∼30 mV) compared with the disulfide cross-linking, suggesting that the exposure of S4 to the extracellular phase may precede cross-linking. Previous studies (11Larsson H.P. Baker O.S. Dhillon D.S. Isacoff E.Y. Neuron. 1996; 16: 387-397Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 12Yusaf S.P. Wray D. Sivaprasadarao A. Pflugers Arch. 1996; 433: 91-97Crossref PubMed Scopus (119) Google Scholar, 14Baker O.S. Larsson H.P. Mannuzzu L.M. Isacoff E.Y. Neuron. 1998; 20: 1283-1294Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 15Wang M.H. Yusaf S.P. Elliott D.J. Wray D. Sivaprasadarao A. J. Physiol. (Lond). 1999; 521: 315-326Crossref Scopus (30) Google Scholar) have shown that in response to membrane depolarization the S4 segment of the Shaker potassium channel moves out of the transmembrane field by exposing over 7 residues to the extracellular phase. It is believed that this movement is accompanied by changes in its interaction with the other segments of the channel that ultimately lead to the activation of the channel. To investigate this, we have used the engineered disulfide method (22Chervitz S.A. Falke J.J. J. Biol. Chem. 1995; 270: 24043-24053Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 23Liu Y. Jurman M.E. Yellen G. Neuron. 1996; 16: 859-867Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar), an approach that has been successfully used to reveal changes in residue-residue interactions during the activation of channels (23Liu Y. Jurman M.E. Yellen G. Neuron. 1996; 16: 859-867Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar) and receptors (35Farrens D.L. Altenbach C. Yang K. Hubbell W.L. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1116) Google Scholar). Our data reveal that during activation the intersubunit distance between the N-terminal ends of S4 decreases such that cysteines engineered at position 361 form an intersubunit disulfide. When the Shaker channel containing a cysteine at position 361 was expressed in Xenopus oocytes and exposed to ambient oxygen or to the mild oxidising agent Cu (II) Phe, there was a reduction in the current flowing through the channel (Fig. 2). This was due to the formation of a disulfide bridge as the effect was reversed by DTT (Figs. 2 and 3) and TCEP (Fig. 5), and a dimeric band (Fig. 4) was detected when the oxidized L361C channel protein was subjected to Western blotting. The latter finding also indicates that the disulfide was formed between cysteines from neighboring subunits, rather than from within a subunit, of the channel. In an attempt to identify the cysteine with which Cys-361 forms the disulfide bridge, we have first examined the effect of Cu (II) Phe on the Shaker channel containing cysteines at position 361 but depleted of all native cysteines (Fig.5). Rather unexpectedly, this mutant was also inhibited by Cu (II) Phe. The most plausible interpretation of this finding is that the cysteine at position 361 forms an intersubunit disulfide with its counterpart in a neighboring subunit. We found that the intersubunit disulfide formation between the cysteines at position 361 does not occur at −90 mV, where the channels are in their closed state. Upon depolarization, however, they undergo rapid oxidation to disulfides (Fig. 6). These data suggest that in the closed state of the channel the 361 cysteines were not close to one another, but during depolarization, when the channel begins to open (and may also begin to inactivate), they move close enough to undergo disulfide oxidation. Previous studies (11Larsson H.P. Baker O.S. Dhillon D.S. Isacoff E.Y. Neuron. 1996; 16: 387-397Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 12Yusaf S.P. Wray D. Sivaprasadarao A. Pflugers Arch. 1996; 433: 91-97Crossref PubMed Scopus (119) Google Scholar, 14Baker O.S. Larsson H.P. Mannuzzu L.M. Isacoff E.Y. Neuron. 1998; 20: 1283-1294Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 15Wang M.H. Yusaf S.P. Elliott D.J. Wray D. Sivaprasadarao A. J. Physiol. (Lond). 1999; 521: 315-326Crossref Scopus (30) Google Scholar) have shown that the cysteine at position 361 can be fully exposed to the extracellular phase at −90 mV (also see Fig. 6 B). This means that the cysteines from the neighboring subunits move toward each other's proximity after the residues have been exposed to the extracellular phase, i.e.after S4s have, at least partially, moved out of the membrane electric field. Consistent with this suggestion, we found that cross-linking reduces the effective gating charge (z) from 1.96 ± 0.25 to 1.08 ± 0.07. Depolarization of the membrane has two effects on the Shaker channel (N-type inactivation-removed), a fast activation followed by a slow C-type inactivation (2Yellen G. Q. Rev. Biophys. 1998; 31: 239-295Crossref PubMed Scopus (401) Google Scholar). Previous studies (2Yellen G. Q. Rev. Biophys. 1998; 31: 239-295Crossref PubMed Scopus (401) Google Scholar,28Olcese R. Latorre R. Toro L. Bezanilla F. Stefani E. J. Gen. Physiol. 1997; 110: 579-589Crossref PubMed Scopus (156) Google Scholar, 29Loots E. Isacoff E.Y. J. Gen. Physiol. 1998; 112: 377-389Crossref PubMed Scopus (155) Google Scholar, 30Loots E. Isacoff E.Y. J. Gen. Physiol. 2000; 116: 623-636Crossref PubMed Scopus (94) Google Scholar, 31Larsson H.P. Elinder F. Neuron. 2000; 27: 573-583Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 32Elinder F. Arhem P. Larsson H.P. Biophys. J. 2001; 80: 1802-1809Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 33Elinder F. Mannikko R. Larsson H.P. J. Gen. Physiol. 2001; 118: 1-10Crossref PubMed Scopus (67) Google Scholar) have shown that the structural changes associated with the activation motion of S4 are distinct from those occurring during the inactivation process. The motions observed in this study are more likely to occur during activation rather than during inactivation. The reasons are as follows: (i) C-type inactivation for L361C begins to occur at more positive potentials (∼ 90 mV) than disulfide cross-linking (Fig. 6 B); (ii) cross-linking reduces rather than stabilizing C-type inactivation in the L361C mutant channel (Fig.2 E); (iii) and finally, and more importantly, application of Cu (II) Phe to L361C channels that had already undergone maximal C-type inactivation does not cause irreversible inhibition of channel currents (Fig. 7). Thus we conclude that the movement of cysteine residues at position 361 into each other's proximity occurs during the activation of the channel rather than during C-type inactivation. Our data suggesting that during activation the intersubunit distance between positions 361 is reduced are obtained from the L361C mutant channel. It therefore raises the critical question: does this occur in the native channel? Could the effect be due to structural change imparted by the mutation? In the absence of direct structural data, this is a very difficult question to address. However, the facts that residues at position 361 are not conserved among Kvchannels and that substitution of cysteine at this position does not affect the net charge of S4 and, more importantly, the functional properties of the channel (11Larsson H.P. Baker O.S. Dhillon D.S. Isacoff E.Y. Neuron. 1996; 16: 387-397Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 12Yusaf S.P. Wray D. Sivaprasadarao A. Pflugers Arch. 1996; 433: 91-97Crossref PubMed Scopus (119) Google Scholar), suggest that any structural change induced by the mutation is likely to be subtle rather than substantial. Thus, we are tempted to suggest that the depolarization-induced motions may reduce the intersubunit distance between positions 361 of S4 in the native channel.

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