Methanethiosulfonate Ethylsulfonate Research Articles

Identification of transmembrane domain 6 & 7 residues that contribute to the binding pocket of the urotensin II receptor

Urotensin II (U-II), a cyclic undecapeptide, is the natural ligand of the urotensin II (UT) receptor, a G protein-coupled receptor. In the present study, we used the substituted-cysteine accessibility method to identify specific residues in transmembrane domains (TMDs) six and seven of the rat urotensin II receptor (rUT) that contribute to the formation of the binding pocket of the receptor. Each residue in the R256 (6.32)-Q283 (6.59) fragment of TMD6 and the A295 (7.31)-T321 (7.57) fragment of TMD7 was mutated, individually, to a cysteine. The resulting mutants were expressed in COS-7 cells, which were subsequently treated with the positively charged methanethiosulfonate-ethylammonium (MTSEA) or the negatively charged methanethiosulfonate-ethylsulfonate (MTSES) sulfhydryl-specific alkylating agents. MTSEA treatment resulted in a significant reduction in the binding of TMD6 mutants F268C (6.44) and W278C (6.54) and TMD7 mutants L298C (7.34), T302C (7.38), and T303C (7.39) to 125I-U-II. MTSES treatment resulted in a significant reduction in the binding of two additional mutants, namely L282C (6.58) in TMD6 and Y300C (7.36) in TMD7. These results suggest that specific residues orient themselves within the water-accessible binding pocket of the rUT receptor. This approach, which allowed us to identify key determinants in TMD6 and TMD7 that contribute to the UT receptor binding pocket, enabled us to further refine our homology-based model of how U-II interacts with its cognate receptor.

Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins

This protocol describes a detailed method to study the static and dynamic features of membrane proteins, as well as solvent accessibility, by utilizing the lactose permease of Escherichia coli (LacY) as a model. The method relies on the use of functional single-Cys mutants, an affinity tag and a PhosphoImager. The membrane-permeant, radioactive thiol reagent N-[ethyl-1-14C]ethylmaleimide ([14C]NEM) is used to detect site-directed alkylation of engineered single-Cys mutants in situ. The solvent accessibility of the Cys residues is also determined by blockage of [14C]NEM labeling with membrane-impermeant thiol reagents such as methanethiosulfonate ethylsulfonate (MTSES). The labeled proteins are purified by mini-scale affinity chromatography and analyzed by gel electrophoresis. Gels are dried and exposed to a PhosphoImager screen for 1-5 d, and incorporation of radioactivity is visualized. Initial results can be obtained in 24 h.

APETx1 from Sea Anemone Anthopleura elegantissima Is a Gating Modifier Peptide Toxin of the Human Ether-a-go-go- Related Potassium Channel

We studied the mechanism of action and the binding site of APETx1, a peptide toxin purified from sea anemone, on the human ether-a-go-go-related gene (hERG) channel. Similar to the effects of gating modifier toxins (hanatoxin and SGTx) on the voltage-gated potassium (Kv) 2.1 channel, APETx1 shifts the voltage-dependence of hERG activation in the positive direction and suppresses its current amplitudes elicited by strong depolarizing pulses that maximally activate the channels. The APETx1 binding site is distinctly different from that of a pore-blocking peptide toxin, BeKm-1. Mutations in the S3b region of hERG have dramatic impact on the responsiveness to APETx1: G514C potentiates whereas E518C abolishes the APETx1 effect. Restoring the negative charge at position 518 (methanethiosulfonate ethylsulfonate modification of 518C) partially restores APETx1 responsiveness, supporting an electrostatic interaction between E518 and APETx1. Among the three hERG isoforms, hERG1 and hERG3 are equally responsive to APETx1, whereas hERG2 is insensitive. The key feature seems to be an arginine residue uniquely present at the 514-equivalent position in hERG2, where the other two isoforms possess a glycine. Our data show that APETx1 is a gating modifier toxin of the hERG channel, and its binding site shares characteristics with those of gating modifier toxin binding sites on other Kv channels.

Cysteine Accessibility in the Hydrophilic Cleft of Human Organic Cation Transporter 2

Organic cation transporters (OCTs) are involved in the renal elimination of many cationic drugs and toxins. A hypothetical three-dimensional structure of OCT2 based on a homology model that used the Escherichia coli glycerol 3-phosphate transporter as a template has been described (Zhang, X., Shirahatti, N. V., Mahadevan, D., and Wright, S. H. (2005) J. Biol. Chem. 280, 34813-34822). To further define OCT structure, the accessibility to hydrophilic thiol-reactive reagents of the 13 cysteine residues contained in the human ortholog of OCT2 was examined. Maleimide-PEO2-biotin precipitated (surface biotinylation followed by Western blotting) and reduced tetraethylammonium transport by OCT2 expressed in Chinese hamster ovary cells, effects that were largely reversed by co-exposure to substrates and transport inhibitors, suggesting interaction with cysteines that are near to or part of a substrate-binding surface. Cysteines at amino acid position 437, 451, 470, and 474 were identified from the model as being located in transmembrane helices that participate in forming the hydrophilic cleft, the proposed region of substrate-protein interaction. To determine which residues are exposed to the solvent, a mutant with all four of these cysteines converted to alanine, along with four variants of this mutant each with an individual cysteine restored, were created. Maleimide-PEO2-biotin was only effective at precipitating and reducing transport by wild-type OCT2 and the mutant with cysteine 474 restored. Additionally, the smaller thiol-reactive reagent, methanethiosulfonate ethylsulfonate, reduced transport by wild-type OCT2 and the mutant with cysteine 474 restored. These data demonstrate that cysteine 474 of OCT2 is exposed to the aqueous milieu of the cleft and contributes to forming a pathway for organic cation transport.

Transmembrane Domain VII of the Human Apical Sodium-Dependent Bile Acid Transporter ASBT (SLC10A2) Lines the Substrate Translocation Pathway

Recent evidence implicating transmembrane (TM) segment 7 of the apical sodium-dependent bile acid transporter (ASBT) in substrate interaction warranted examination of its aqueous accessibility. Therefore, cysteine substitution of 22 consecutive amino acids was performed against a methanethiosulfonate (MTS)-resistant background (C270A). Activity and susceptibility to polar MTS derivatives [(2-aminoethyl)-methanethiosulfonate (MTSEA), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and methanethiosulfonate ethylsulfonate (MTSES)] of mutants were evaluated in COS-1 cells. Thr289, Tyr293, Gln297, Ala301, Phe307, and Tyr308 represented loss-of-function mutants; furthermore, the measurable residual activities for T289C, Y293C, and A301C (<or=20% control) proved insensitive to MTS treatment. MTSES and MTSET inhibition was confined to residues lining the extracellular half of TM7; amino acids situated deeper within the membrane were unaffected. In contrast, the entire length of TM7 was susceptible to the relatively smaller MTSEA; moreover, MTSEA sensitivity was significantly amended by coapplication with substrates. This selective pattern of modification suggests that the highly conserved lower half of TM7 lies within a water-filled cavity easily accessible from the extracellular milieu, whereas residues approaching the cytosolic/membrane interface reside in pores for which accessibility is modulated by molecular volume. Functionally inactive and MTS-inaccessible residues (T289C, Y293C, Q297C, and A301C) within TM7 may play a structural role critical to transporter function; conversely, MTS-sensitive residues are spatially distinct and may demarcate a face of the TM involved in substrate translocation. In addition, computational analysis of solvent-accessible domains identified five key solvent pockets that predominantly line the hydrophilic face of TM7. Combined, our data suggest that TM7 plays a dominant role in the hASBT translocation process.

Site-Directed Alkylation of Cysteine Replacements in the Lactose Permease of Escherichia coli: Helices I, III, VI, and XI

To complete a study on site-directed alkylation of Cys replacements in the lactose permease of Escherichia coli (LacY), the reactivity of single-Cys mutants in helices I, III, VI, and XI, as well as some of the adjoining loops, with N-[14C]ethylmaleimide (NEM) or methanethiosulfonate ethylsulfonate (MTSES) was studied in right-side-out membrane vesicles. With the exception of several positions in the middle of helix I, which either face the bilayer or are in close proximity to other helices, the remaining Cys replacements react with the membrane-permeant alkylating agent NEM. In helices III and XI, most Cys replacements are also alkylated by NEM except for positions that face the bilayer. The reactivity of Cys replacements in helix VI is noticeably lower and only 45% of the replacements label. Binding of sugar leads to significant increases in the reactivity of Cys residues that are located primarily at the same level as the sugar-binding site or in the periplasmic half of each helix. Remarkably, studies with small, impermeant MTSES show that single-Cys replacements in the cytoplasmic portions of helices I and XI, which line the inward-facing cavity, are accessible to solvent from the periplasmic surface of the membrane. Moreover, addition of ligand results in increased accessibility of Cys residues to the aqueous milieu in the periplasmic region of the helices, which may reflect structural rearrangements leading to opening of an outward-facing cavity. The findings are consistent with the X-ray structure of LacY and with the alternating access model [Abramson, J., Smirnova, I., et al. (2003) Science 301, 610-615].

Outer Pore Architecture of a Ca2+-selective TRP Channel

The TRP superfamily forms a functionally important class of cation channels related to the product of the Drosophila trp gene. TRP channels display an unusual diversity in activation mechanisms and permeation properties, but the basis of this diversity is unknown, as the structure of these channels has not been studied in detail. To obtain insight in the pore architecture of TRPV6, a Ca(2+)-selective member of the TRPV subfamily, we probed the dimensions of its pore and determined pore-lining segments using cysteine-scanning mutagenesis. Based on the permeability of the channel to organic cations, we estimated a pore diameter of 5.4 A. Mutating Asp(541), a residue involved in high affinity Ca(2+) binding, altered the apparent pore diameter, indicating that this residue lines the narrowest part of the pore. Cysteines introduced in a region preceding Asp(541) displayed a cyclic pattern of reactivity to Ag(+) and cationic methylthio-sulfanate reagents, indicative of a pore helix. The anionic methanethiosulfonate ethylsulfonate showed only limited reactivity in this region, consistent with the presence of a cation-selective filter at the outer part of the pore helix. Based on these data and on homology with the bacterial KcsA channel, we present the first structural model of a TRP channel pore. We conclude that main structural features of the outer pore, namely a selectivity filter preceded by a pore helix, are conserved between K(+) channels and TRPV6. However, the selectivity filter of TRPV6 is wider than that of K(+) channels and lined by amino acid side chains rather than main chain carbonyls.

Site-directed sulfhydryl labeling of helix IX in the lactose permease of Escherichia coli.

Site-directed sulfhydryl modification of transmembrane helix IX in the lactose permease of Escherichia coli was studied in right-side-out membrane vesicles with the thiol-specific reagents N-[(14)C]ethylmaleimide (NEM) and methanethiosulfonate ethylsulfonate (MTSES) which are permeant and impermeant, respectively. Out of approximately 20 mutants with a single Cys residue at each position in the helix, only five mutants label with NEM. (i) Cys residues at positions 291, 308, and 310 label at 25 degrees C, and binding of substrate has no effect. (ii) Cys residues at positions 295 and 298 label only in the presence of substrate. NEM labeling at 0 degrees C indicates that alkylation of Cys residues at positions 295 and 308 is dependent on the thermal motion of the protein. In contrast, temperature has little effect on labeling of Cys residues at positions 291, 298, and 310. Interestingly, pretreatment with MTSES blocks NEM labeling of all the mutants. The findings demonstrate that the face of helix IX on which Arg302 is located is involved in ligand-induced conformational changes and accessible to water from the periplasmic surface of the membrane. Since Arg302 facilitates deprotonation of Glu325 (helix X) during turnover [Sahin-Tóth, M., and Kaback, H. R. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 6068-6073], the findings are consistent with the idea that this face of helix IX may comprise part of the H(+) translocation pathway.

An External Determinant in the S5-P Linker of the Pacemaker (HCN) Channel Identified by Sulfhydryl Modification

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels underlie spontaneous rhythmic activities in the heart and brain. Sulfhydryl modification of ion channels is a proven approach for studying their structure-function relationships; here we examined the effects of the hydrophilic sulfhydryl-modifying agents methanethiosulfonate ethylammonium (MTSEA(+)) and methanethiosulfonate ethylsulfonate (MTSES(-)) on wild-type (WT) and engineered HCN1 channels. External application of MTSEA(+) to WT channels irreversibly reduced whole-cell currents (I(MTSEA)/I(Control) = 42 +/- 2%), slowed activation and deactivation kinetics ( approximately 7- and approximately 3-fold at -140 and -20 mV, respectively), and produced hyperpolarizing shifts of steady-state activation (V(12)((MTSEA)) = -125.8 +/- 9.0 mV versus V(12)((Control)) = -76.4 +/- 1.6 mV). Sequence inspection revealed the presence of five endogenous cysteines in the transmembrane domains of HCN1: three are putatively close to the extracellular milieu (Cys(303), Cys(318), and Cys(347) in the S5, S5-P, and P segments, respectively), whereas the remaining two are likely to be cytoplasmic or buried. To identify the molecular constituent(s) responsible for the effects of MTSEA(+), we mutated the three "external" cysteines individually to serine. C303S did not yield measurable currents. Whereas C347S channels remained sensitive to MTSEA(+), C318S was not modified (I(MTSEA)/I(Control) = 101 +/- 2%, V(12)((MTSEA)) = -78.4 +/- 1.1 mV, and V(12)((Control)) = -79.8 +/- 2.3 mV). Likewise, WT (but not C318S) channels were sensitive to MTSES(-). Despite their opposite charges, MTSES(-) produced changes directionally similar to those effected by MTSEA(+) (I(MTSES)/I(Control) = 22 +/- 1.6% and V(12)((MTSES)) = -145.9 +/- 4.9 mV). We conclude that S5-P Cys(318) of HCN1 is externally accessible and that the external pore vestibule and activation gating of HCN channels are allosterically coupled.

Transport function of the renal type IIa Na+/P(i) cotransporter is codetermined by residues in two opposing linker regions.

Two highly similar regions in the predicted first intracellular (ICL-1) and third extracellular loop (ECL-3) of the type IIa Na+/Pi cotransporter (NaPi-IIa) have been shown previously to contain functionally important sites by applying the substituted cysteine accessibility method (SCAM). Incubation in methanethiosulfonate (MTS) reagents of mutants that contain novel cysteines in both loops led to full inhibition of cotransport activity. To elucidate further the role these regions play in defining the transport mechanism, a double mutant (A203C-S460C) was constructed with novel cysteines in each region. The effect of cysteine modification by different MTS reagents on two electrogenic transport modes (leak and cotransport) was investigated. MTSEA (2-aminoethyl MTS hydrobromide) and MTSES (MTS ethylsulfonate) led to full inhibition of cotransport and increased the leak, whereas incubation in MTSET (2-[trimethylammonium]ethyl MTS bromide) inhibited only cotransport. The behavior of other double mutants with a cysteine retained at one site and hydrophobic or hydrophilic residues substituted at the other site, indicated that most likely only Cys-460 was modifiable, but the residue at Ala-203 was critical for conferring the leak and cotransport mode behavior. Substrate interaction with the double mutant was unaffected by MTS exposure as the apparent Pi and Na+ affinities for Pi-induced currents and respective activation functions were unchanged after cysteine modification. This suggested that the modified site did not interfere with substrate recognition/binding, but prevents translocation of the fully loaded carrier. The time-dependency of cotransport loss and leak growth during modification of the double cysteine mutant was reciprocal, which suggested that the modified site is a kinetic codeterminant of both transport modes. The behavior is consistent with a kinetic model for NaPi-IIa that predicts mutual exclusiveness of both transport modes. Together, these findings suggest that parts of the opposing linker regions are associated with the NaPi-IIa transport pathway.

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helices IV and V that contain the major determinants for substrate binding.

Helices IV and V in the lactose permease of Escherichia coli contain the major determinants for substrate binding [Glu126 (helix IV), Arg144 (helix V), and Cys148 (helix V)]. Structural and dynamic features of this region were studied by using site-directed sulfhydryl modification of 48 single-Cys replacement mutants with N-[(14)C]ethylmaleimide (NEM) in the absence or presence of ligand. In right-side-out membrane vesicles, Cys residues in the cytoplasmic halves of both helices react with NEM in the absence of ligand, while Cys residues in the periplasmic halves do not. Five Cys replacement mutants at the periplasmic end of helix V and one at the cytoplasmic end of helix V label only in the presence of ligand. Interestingly, in addition to native Cys148, a known binding-site residue, labeling of mutant Ala122 --> Cys, which is located in helix IV across from Cys148, is markedly attenuated by ligand. Furthermore, alkylation of the Ala122 --> Cys mutant blocks transport, and protection is afforded by substrate, indicating that Ala122 is also a component of the sugar binding site. Methanethiosulfonate ethylsulfonate, an impermeant thiol reagent shown clearly in this paper to be impermeant in E. coli spheroplasts, was used to identify substituted Cys side chains exposed to water and accessible from the periplasmic side. Most of the Cys mutants in the cytoplasmic halves of helices IV and V, as well as two residues in the intervening loop, are accessible to the aqueous phase from the periplasmic face of the membrane. The findings indicate that the cytoplasmic halves of helices IV and V are more reactive/accessible to thiol reagents and more exposed to solvent than the periplasmic half. Furthermore, positions that exhibit ligand-induced changes are located for the most part in the vicinity of the residues directly involved in substrate binding, as well as the cytoplasmic loop between helices IV and V.

The Surface Accessibility of the Glycine Receptor M2–M3 Loop Is Increased in the Channel Open State

Mutations in the extracellular M2-M3 loop of the glycine receptor (GlyR) alpha1 subunit have been shown previously to affect channel gating. In this study, the substituted cysteine accessibility method was used to investigate whether a structural rearrangement of the M2-M3 loop accompanies GlyR activation. All residues from R271C to V277C were covalently modified by both positively charged methanethiosulfonate ethyltrimethylammonium (MTSET) and negatively charged methanethiosulfonate ethylsulfonate (MTSES), implying that these residues form an irregular surface loop. The MTSET modification rate of all residues from R271C to K276C was faster in the glycine-bound state than in the unliganded state. MTSES modification of A272C, L274C, and V277C was also faster in the glycine-bound state. These results demonstrate that the surface accessibility of the M2-M3 loop is increased as the channel transitions from the closed to the open state, implying that either the loop itself or an overlying domain moves during channel activation.

Structural and gating changes of the sodium channel induced by mutation of a residue in the upper third of IVS6, creating an external access path for local anesthetics.

Membrane-impermeant quaternary amine local anesthetics QX314 and QX222 can access their binding site on the cytoplasmic side of the selectivity filter from the outside in native cardiac Na(+) channels. Mutation of domain IV S6 Ile-1760 of rat brain IIA Na(+) channel or the equivalent (Ile-1575) in the adult rat skeletal muscle isoform (mu 1) creates an artificial access path for QX. We examined the characteristics of mutation of mu 1-I1575 and the resulting QX path. In addition to allowing external QX222 access, I1575A accelerated decay of Na(+) current and shifted steady-state availability by -27 mV. I1575A had negligible effects on inorganic or organic cation selectivity and block by tetrodotoxin (TTX), saxitoxin (STX), or mu-conotoxin (mu-CTX). It exposed a site within the protein that binds membrane-permeant methanethiosulfonate ethylammonium (MTSEA), but not membrane-impermeant methanethiosulfonate ethyltrimethylammonium (MTSET) and methanethiosulfonate ethylsulfonate (MTSES). MTSEA binding abolished the QX path created by this mutation, without effects on toxin binding. The mu-CTX derivative R13N, which partially occluded the pore, had no effect on QX access. I1575A exposed two Cys residues because a disulfide bond was formed under oxidative conditions, but the exposed Cys residues are not those in domain IV S6, adjacent to Ile-1575. The Cys mutant I1575C was insensitive to external Cd(2+) and MTS compounds (MTSEA, MTSET, MTSES), and substitution of Ile with a negatively charged residue (I1575E) did not affect toxin binding. Ile-1575 seems to be buried in the protein, and its mutation disrupts the protein structure to create the QX path without disturbing the outer vestibule and its selectivity function.

Novel Structural Determinants of μ-Conotoxin (GIIIB) Block in Rat Skeletal Muscle (μ1) Na+ Channels

mu-Conotoxin (mu-CTX) specifically occludes the pore of voltage-dependent Na(+) channels. In the rat skeletal muscle Na(+) channel (mu1), we examined the contribution of charged residues between the P loops and S6 in all four domains to mu-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC(50) for mu-CTX block by approximately 100-fold (wild-type = 22.3 +/- 7.0 nm; D762C = 2558 +/- 250 nm; E765C = 2020 +/- 379 nm). Restoration or reversal of charge by external modification of the cysteine-substituted channels with methanethiosulfonate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect mu-CTX block (D762C: IC(50, MTSEA+) = 2165.1 +/- 250 nm; IC(50, MTSES-) = 2753.5 +/- 456.9 nm; E765C: IC(50, MTSEA+) = 2200.1 +/- 550.3 nm; IC(50, MTSES-) = 3248.1 +/- 2011.9 nm) compared with their unmodified counterparts. In contrast, the charge-conserving mutations D762E (IC(50) = 21.9 +/- 4.3 nm) and E765D (IC(50) = 22.0 +/- 7.0 nm) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC(50) = 4139.9 +/- 687.9 nm) and E765K (IC(50) = 4202.7 +/- 1088.0 nm) destabilized mu-CTX block even further, suggesting a prominent electrostatic component of the interactions between these DII residues and mu-CTX. Kinetic analysis of mu-CTX block reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k(off)) rates with little changes in association (k(on)) rates. We conclude that the acidic residues at positions 762 and 765 are key determinants of mu-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MTSEA side chain to modify mu-CTX sensitivity places steric constraints on the sites of toxin interaction.

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: N-ethylmaleimide-sensitive face of helix II.

Cys-scanning mutagenesis of helix II in the lactose permease of Escherichia coli [Frillingos, S., Sun, J. et al. (1997) Biochemistry 36, 269-273] indicates that one face contains positions where Cys replacement or Cys replacement followed by treatment with N-ethylmaleimide (NEM) significantly inactivates the protein. In this study, site-directed sulfhydryl modification is utilized in situ to study this face of helix II. [(14)C]NEM labeling of 13 single-Cys mutants, including the nine NEM-sensitive Cys replacements, in right-side-out membrane vesicles is examined. Permease mutants with a single-Cys residue in place of Gly46, Phe49, Gln60, Ser67, or Leu70 are alkylated by NEM at 25 degrees C in 10 min, and mutants with Cys in place of Thr45 and Ser53 are labeled only in the presence of ligand, while mutants with Cys in place of Ile52, Ser56, Leu57, Leu62, Phe63, or Leu65 do not react. Binding of substrate leads to a marked increase in labeling of Cys residues at positions 45, 49, or 53 in the periplasmic half of helix II and a slight decrease in labeling of Cys residues at positions 60 or 67 in the cytoplasmic half. Labeling studies with methanethiosulfonate ethylsulfonate (MTSES) show that positions 45 and 53 are accessible to solvent in the presence of ligand only, while positions 46, 49, 67, and 70 are accessible to solvent in the absence or presence of ligand. Position 60 is also exposed to solvent, and substrate binding causes a decrease in solvent accessibility. The findings demonstrate that the NEM-sensitive face of helix II participates in ligand-induced conformational changes. Remarkably, this membrane-spanning face is accessible to the aqueous phase from the periplasmic side of the membrane. In the following paper in this issue [Venkatesan, P., Hu, Y., and Kaback, H. R. (2000) Biochemistry 39, 10656-10661], the approach is applied to helix X.

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helix VII.

Site-directed sulfhydryl modification in situ is employed to investigate structural and dynamic features of transmembrane helix VII and the beginning of the periplasmic loop between helices VII and VIII (loop VII/VIII). Essentially all of the Cys-replacement mutants in the periplasmic half of the helix and the portion of loop VII/VIII tested are labeled by N-[(14)C]ethylmaleimide (NEM). In contrast, with the exception of two mutants at the cytoplasmic end of helix VII, none of the mutants in the cytoplasmic half react with the alkylating agent. Labeling of most of the mutants is unaltered by ligand at 25 degrees C. However, at 4 degrees C, conformational changes induced by substrate binding become apparent. In the presence of ligand, permease mutants with a Cys residue at position 241, 242, 244, 245, 246, or 248 undergo a marked increase in labeling, while the reactivity of a Cys at position 238 is slightly decreased. Labeling of the remaining Cys-replacement mutants is unaffected by ligand. Studies with methanethiosulfonate ethylsulfonate (MTSES), a hydrophilic impermeant thiol reagent, show that most of the positions that react with NEM are accessible to MTSES; however, the two NEM-reactive mutants at the cytoplasmic end of helix VII and position 236 in the middle of the membrane-spanning domain are not. The findings demonstrate that positions in helix VII that reflect ligand-induced conformational changes are located in the periplasmic half and accessible to the aqueous phase from the periplasmic face of the membrane. In the following papers in this issue (Venkatesan, P., Lui, Z., Hu, Y., and Kaback H. R.; Venkatesan, P., Hu, Y., and Kaback H. R.), the approach is applied to helices II and X.

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helix X.

Helix X in the lactose permease of Escherichia coli contains two residues that are irreplaceable with respect to active transport, His322 and Glu325, as well as Lys319, which is charge-paired with Asp240 in helix VII. Structural and dynamic features of transmembrane helix X are investigated here by site-directed thiol modification of 14 single-Cys replacement mutants with N-[(14)C]ethylmaleimide (NEM) in right-side-out membrane vesicles. Permease mutants with a Cys residue at position 326, 327, 329, 330, or 331 in the cytoplasmic half of the transmembrane domain are alkylated by NEM at 25 degrees C, a mutant with Cys at position 315 at the periplasmic surface is labeled in the presence of substrate exclusively, and mutants with Cys at positions 317, 318, 320, 321, 324, 328, 332, or 333 do not react with NEM under the conditions tested. Binding of substrate causes increased labeling of a Cys residue at position 315 and decreased labeling of Cys residues at positions 326, 327, and 329. Studies with methanethiosulfonate ethylsulfonate indicate that Cys residues at positions 326, 329, 330, and 331 in the cytoplasmic half are accessible to the aqueous phase from the periplasmic face of the membrane. Ligand binding results in clear attenuation of solvent accessibility of Cys at position 326 and a marginal increase in accessibility of Cys at position 327 to solvent. The findings indicate that the cytoplasmic half of helix X is more reactive/accessible to thiol reagents and more exposed to solvent than the periplasmic half. Furthermore, positions that reflect ligand-induced conformational changes are located on the same face of helix X as Lys319, His322, and Glu325.

Role of domain 4 in sodium channel slow inactivation.

Depolarization of sodium channels initiates at least three gating pathways: activation, fast inactivation, and slow inactivation. Little is known about the voltage sensors for slow inactivation, a process believed to be separate from fast inactivation. Covalent modification of a cysteine substituted for the third arginine (R1454) in the S4 segment of the fourth domain (R3C) with negatively charged methanethiosulfonate-ethylsulfonate (MTSES) or with positively charged methanethiosulfonate-ethyltrimethylammonium (MTSET) produces a marked slowing of the rate of fast inactivation. However, only MTSES modification produces substantial effects on the kinetics of slow inactivation. Rapid trains of depolarizations (2-20 Hz) cause a reduction of the peak current of mutant channels modified by MTSES, an effect not observed for wild-type or unmodified R3C channels, or for mutant channels modified by MTSET. The data suggest that MTSES modification of R3C enhances entry into a slow-inactivated state, and also that the effects on slow inactivation are independent of alterations of either activation or fast inactivation. This effect of MTSES is observed only for cysteine mutants within the middle of this S4 segment, and the data support a helical secondary structure of S4 in this region. Mutation of R1454 to the negatively charged residues aspartate or glutamate cannot reproduce the effects of MTSES modification, indicating that charge alone cannot account for these results. A long-chained derivative of MTSES has similar effects as MTSES, and can produce these effects on a residue that does not show use-dependent current reduction after modification by MTSES, suggesting that the sulfonate moiety can reach a critical site affecting slow inactivation. The effects of MTSES on R3C are partially counteracted by a point mutation (W408A) that inhibits slow inactivation. Our data suggest that a region near the midpoint of the S4 segment of domain 4 plays an important role in slow inactivation.

Effect of sulfhydryl reagents on the regulatory system of the L-type Ca channel in frog ventricular myocytes.

The effects of sulfhydryl (SH) reagents on the L-type Ca current (ICa) were studied in frog ventricular myocytes using the whole-cell patch-clamp method. Methanethiosulfonate ethylammonium (MTSEA+) was found to enter the cell through the membrane and cause a remarkable increase in Ica from the intracellular side. Methanethiosulfonate ethyltrimethylammonium (MTSET+) and methanethiosulfonate ethylsulfonate (MTSES-) could not penetrate the membrane and were effective only when directly applied to the intracellular side. In addition, suppressive effects on ICa of these MTS reagents were indicated by the following observation. A progressive decay in the peak amplitude of ICa after establishing maximal ICa, stimulated by intracellular MTSET+, was prevented by adding extracellular dithiothreitol (DTT). The SH-oxidizing agents N-ethylmaleimide (NEM), chloramine-T (CL-T), 2,2'-dithiodipyridine (DTDP) and 2,2'-dithio-bis-5-nitropyridine (DTBNP) also exerted a stimulatory effect on Ica. The effect of SH reagents persisted even when cAMP production was inhibited with Rp-cAMP-S, or when G-protein was inhibited with 1 mM GDPbetaS, indicating that the effect is not due to cAMP production or G-protein stimulation. It is concluded that there are sites on the Ca channels that are subject to direct modification by SH reagents.

Identification of acetylcholine receptor channel-lining residues in the M1 segment of the beta-subunit.

The substituted cysteine accessibility method (SCAM) was applied to the first membrane-spanning segment (M1) of the mouse-muscle acetylcholine (ACh) receptor beta subunit. One at a time, each residue from betaR219 to betaP247, except betaC233, was mutated to Cys, and the mutant beta subunits were expressed together with wild-type alpha, gamma, and delta in Xenopus oocytes. All 28 mutants yielded functional receptors. The accessibility of the substituted Cys to the methanethiosulfonate (MTS) derivatives, MTS ethylammonium (MTSEA), MTS ethyltrimethylammonium (MTSET), and MTS ethylsulfonate (MTSES), added extracellularly in the absence or the presence of ACh, was inferred from their irreversible effects on ACh-induced current. Three consecutive residues close to the extracellular end of M1, betaF224C, betaY225C, and betaL226C, reacted both in the absence and presence of ACh, and one deeper residue, betaV229C reacted only in the presence of ACh. betaV229C also reacted with 2-aminoethyl-2-aminoethanethiosulfonate (AEAETS) and with 2-hydroxyethyl MTS (MTSEH). The rate constants for the reactions of betaV229C with MTSEA, which permeates the open channel, and with MTSEH, which is uncharged, were independent of membrane potential. The rate constant for the reaction of the doubly positively charged AEAETS, however, was dependent on membrane potential, consistent with the exposure of betaV229C in the open channel. The N-terminal third of betaM1, like that of alphaM1, contributes to the lining of the channel and undergoes structural changes during gating.