The conserved cysteine 7.38 residue is differentially accessible in the binding-site crevices of the mu, delta, and kappa opioid receptors

Binding pockets of the opioid receptors are presumably formed among the transmembrane domains (TMDs) and are accessible from the extracellular medium. In this study, we determined the sensitivity of binding of [(3)H]diprenorphine, an antagonist, to mu, delta, and kappa opioid receptors to charged methanethiosulfonate (MTS) derivatives and identified the cysteine residues within the TMDs that conferred the sensitivity. Incubation of the mu opioid receptor expressed in HEK293 cells with MTS ethylammonium (MTSEA), MTS ethyltrimethylammonium (MTSET), or MTS ethylsulfonate (MTSES) inhibited [(3)H]diprenorphine binding with the potency order of MTSEA > MTSET > MTSES. Pretreatment of mu, delta, and kappa opioid receptors with MTSEA dose-dependently inhibited [(3)H]diprenorphine binding with MTSEA sensitivity in the order of kappa > mu > delta. The effects of MTSEA occurred rapidly, reaching the maximal inhibition in 10 min. (-)-Naloxone, but not (+)-naloxone, prevented the MTSEA effect, demonstrating that the reaction occurs within or in the vicinity of the binding pockets. Each cysteine residue in the TMDs of the three receptors was mutated singly, and the effects of MTSEA treatment were examined. The mutants had similar affinities for [(3)H]diprenorphine, and C7. 38(321)S, C7.38(303)S, and C7.38(315)S mutations rendered mu, delta, and kappa opioid receptors less sensitive to the effect of MTSEA, respectively. These results indicate that the conserved Cys7.38 is differentially accessible in the binding-site crevice of these receptors. The second extracellular loop of the kappa receptor, which contains several acidic residues, appears to play a role, albeit small, in its higher sensitivity to MTSEA, whereas the negative charge of Glu6.58(297) did not. To the best of our knowledge, this is the first report to show that a conserved residue among highly homologous G protein-coupled receptors is differentially accessible in the binding-site crevice. In addition, this represents the first successful generation of MTSEA-insensitive mutants of mu, delta, and kappa opioid receptors, which will allow determination of residues accessible in the binding-site crevices of these receptors by the substituted cysteine accessibility method.     

Constitutive activation of the mu opioid receptor by mutation of D3.49(164), but not D3.32(147): D3.49(164) is critical for stabilization of the inactive form of the receptor and for its expression

The roles of conserved aspartates in the third transmembrane domain of the rat mu opioid receptor (RMOR) were explored with mutations of D3.32(147) and D3.49(164). D3.49(164) in the highly conserved DRY motif was mutated to 13 amino acids. Except for the D3.49(164)E mutant, each mutant displayed little or no detectable [(3)H]diprenorphine binding, and pretreatment with naloxone greatly enhanced binding. D3.49(164)H, -Q, -Y, -M, and -E mutants were further studied. D3.32(147) was substituted with A or N. All seven mutants exhibited similar binding affinities for the antagonist [(3)H]diprenorphine as the wild-type. The D3.49(164)H, -Q, -Y, and -M mutants, but not the D3.49(164)E and D3.32(147) mutants, exhibited enhanced basal [(35)S]GTPgammaS binding which was comparable to the maximally activated level of the wild-type and was related to expression levels. Naloxone, naltrexone, and naloxone methiodide significantly inhibited the basal [(35)S]GTPgammaS binding of the D3.49(164) mutants, indicating inverse agonist activities. Treatment of the D3.49(164)Y mutant with pertussis toxin greatly reduced the basal [(35)S]GTPgammaS binding, demonstrating constitutive activation of Galpha(i)/Galpha(o). The D3.49(164)H, -Y, -M, and -Q mutants had higher affinities for DAMGO than the wild-type, which were not significantly lowered by GTPgammaS. Thus, mutation of D3.49(164) to H, Y, M, or Q in RMOR resulted in receptor assuming activated conformations. In contrast, the D3.49(164)E mutant displayed significantly lower basal [(35)S]GTPgammaS binding and reduced affinity for DAMGO. Upon incubation of membranes at 37 degrees C, the constitutively active D3.49(164)Y mutant was structurally less stable, whereas the inactivated D3.49(164)E mutant was more stable, than the wild-type. Computational simulations showed that the E3.49 side chain interacted strongly with the conserved R3.50 in the DRY motif and stabilized the inactive form of the receptor. Taken together, these results indicate that D3.49 plays an important role in constraining the receptor in inactive conformations.     

Comparison of the amino acid residues in the sixth transmembrane domains accessible in the binding-site crevices of mu, delta, and kappa opioid receptors.

We have mapped the residues in the sixth transmembrane domains (TMs 6) of the mu, delta, and kappa opioid receptors that are accessible in the binding-site crevices by the substituted cysteine accessibility method (SCAM). We previously showed that ligand binding to the C7.38S mutants of the mu and kappa receptors and the wild-type delta receptor was relatively insensitive to methanethiosulfonate ethylammonium (MTSEA), a positively charged sulfhydryl-specific reagent. These MTSEA-insensitive constructs were used as the templates, and 22 consecutive residues in TM6 (excluding C6.47) of each receptor were mutated to cysteine, 1 at a time. Most mutants retained binding affinities for [3H]diprenorphine, a nonselective opioid antagonist, similar to that of the template receptors. Treatment with MTSEA significantly inhibited [3H]diprenorphine binding to 11 of 22 mutants of the rat mu receptor and 9 of 22 mutants of the human delta receptor and 10 of 22 mutants of the human kappa receptor. Naloxone or diprenorphine protected all sensitive mutants, except the A6.42(287)C mu mutant. Thus, V6.40, F6.44, W6.48, I6.51, Y6.54, V6.55, I6.56, I6.57, K6.58, and A6.59 of the mu receptor; F6.44, I6.51, F6.54, V6.55, I6.56, V6.57, W6.58, T6.59, and L6.60 of the delta receptor; and F6.44, W6.48, I6.51, F6.54, I6.55, L6.56, V6.57, E6.58, A6.59, and L6.60 of the kappa receptor are on the water-accessible surface of the binding-site crevices. The accessibility patterns of residues in the TMs 6 of the mu, delta, and kappa opioid receptors are consistent with the notion that the TMs 6 are in alpha-helical conformations with a narrow strip of accessibility on the intracellular side of 6.54 and a wider area of accessibility on the extracellular side of 6.54, likely due to a proline kink at 6.50 that bends the helix in toward the binding pocket and enables considerable motion in this region. The wider exposure of residues 6.55-6.60 to the binding-site crevice, combined with the divergent amino acid sequences, is consistent with the inferred role of residues in this region in determining ligand binding selectivity. The conservation of the accessibility pattern on the cytoplasmic side of 6.54 suggests that this region may be important for receptor activation. This accessibility pattern is similar to that of the D2 dopamine receptor, the only other GPCR in which TM6 has been mapped by SCAM. That opioid receptors and the remotely related D2 dopamine receptor have similar accessibility patterns in TM6 suggest that these segments of GPCRs in the rhodopsin-like subfamily not only share secondary structure but also are packed similarly into the transmembrane bundle and thus have similar tertiary structure.     

The seventh transmembrane domains of the delta and kappa opioid receptors have different accessibility patterns and interhelical interactions

We applied the substituted cysteine accessibility method (SCAM) to map the residues of the transmembrane helices (TMs) 7 of delta and kappa opioid receptors (deltaOR and kappaOR) that are on the water-accessible surface of the binding-site crevices. A total of 25 consecutive residues (except C7.38) in the TMs 7 were mutated to Cys, one at a time, and each mutant was expressed in HEK 293 cells. Most mutants displayed similar binding affinity for [(3)H]diprenorphine, an antagonist, as the wild types. Pretreatment with (2-aminoethyl)methanethiosulfonate (MTSEA) inhibited [(3)H]diprenorphine binding to eight deltaOR and eight kappaOR mutants. All mutants except deltaOR L7.52(317)C were protected by naloxone from the MTSEA effect, indicating that the side chains of V7.31(296), A7.34(299), I7.39(304), L7.41(306), G7.42(307), P7.50(315), and Y7.53(318) of deltaOR and S7.34(311), F7.37(314), I7.39(316), A7.40(317), L7.41(318), G7.42(319), Y7.43(320), and N7.49(326) of kappaOR are on the water-accessible surface of the binding pockets. Combining the SCAM data with rhodopsin-based molecular models of the receptors led to the following conclusions. (i) The residues of the extracellular portion of TM7 predicted to face TM1 are sensitive to MTSEA in kappaOR but are not in deltaOR. Thus, TM1 may be closer to TM7 in deltaOR than in kappaOR. (ii) MTSEA-sensitive mutants start at position 7.31(296) in deltaOR and at 7.34(311) in kappaOR, suggesting that TM7 in deltaOR may have an additional helical turn (from 7.30 to 7.33). (iii) There is a conserved hydrogen-bond network linking D2.50 of the NLxxxD motif in TM2 with W6.48 of the CWxP motif in TM6. (iv) The NPxxY motif in TM7 interacts with TM2, TM6, and helix 8 to maintain receptors in inactive states. To the best of our knowledge, this represents the first such comparison of the structures of two highly homologous GPCRs.     

The local environment at the cytoplasmic end of TM6 of the mu opioid receptor differs from those of rhodopsin and monoamine receptors: introduction of an ionic lock between the cytoplasmic ends of helices 3 and 6 by a L6.30(275)E mutation inactivates the mu opioid receptor and reduces the constitutive activity of its T6.34(279)K mutant

Activation of rhodopsin and monoamine G protein-coupled receptors (GPCRs) has been proposed to involve in part the disruption of a conserved E6.30-R3.50 ionic interaction between transmembrane segments (TMs) 3 and 6. However, this interaction does not occur in the opioid receptors, which have L275 at 6.30. On the basis of our findings that mutations of T6.34(279) to K and D produced, respectively, a constitutively active and an inactive form of the mu opioid receptor, we previously suggested that the functional role of the 6.30(275) residue could be assumed by T6.34(279), but the interplay between residues at positions 6.30 and 6.34 remained unresolved. In this study, we examined the effects of introducing an E in position 6.30(275) of the wild type (WT) and of the T6.34(279) mutants of the mu opioid receptor to compare the participation of the 6.30 locus in molecular events during activation in this receptor with its role in other GPCRs. The L6.30(275)E and the L6.30(275)E/T6.34(279)D mutants displayed no constitutive activity and could not be activated by the agonist DAMGO or morphine. The L6.30(275)E/T6.34(279)K mutant had some constitutive activity, but much less than the T6.34(279)K mutant, and could be activated by both agonists. The rank order of affinity for the agonist DAMGO is as follows: T6.34(279)K > WT congruent with L6.30(275)E/T6.34(279)K > L6.30(275)E congruent with T6.34(279)D > L6.30(275)E/T6.34(279)D; however, all constructs have a similar affinity for the antagonist [(3)H]diprenorphine. These data are interpreted in the context of interactions with the conserved R3.50(165) in TM3. When L6.30(275) is mutated to E, the favorable E6.30(275)-R3.50(165) interaction stabilizes an inactive state, as in rhodopsin, and hence reduces the activities of T6.34(279) mutants. Thus, the mu opioid receptor is shown to be different from rhodopsin and monoamine GPCRs, of which the WTs with native E6.30 can be activated, and the 6.34D or 6.34K mutants display enhanced constitutive activities. Our molecular modeling results suggest that some specific differences in local geometry at the cytoplasmic ends of TM5 and TM6 may account in part for the observed differences in the molecular mechanisms of receptor activation.     

Functional role of a conserved motif in TM6 of the rat mu opioid receptor: constitutively active and inactive receptors result from substitutions of Thr6.34(279) with Lys and Asp.

Mutations within the "X1BBX2X3B" motif or its variants in the junction of the third intracellular (i3) loop and the sixth transmembrane domain (TM6) have been shown to lead to constitutive activation of several G protein-coupled receptors (GPCRs). In this study, T6.34(279) at the X3 locus of the rat mu opioid receptor was mutated to Lys and Asp, and the mutants were examined for binding and signaling properties. The T6.34(279)K mutant was poorly expressed, and pretreatment with naloxone greatly enhanced its expression. This construct exhibited properties identified previously with constitutive activation: (1) compared with the wild type, it produced much higher agonist-independent [35S]GTPgammaS binding, which was abolished by pertussis toxin treatment; (2) it displayed an enhanced affinity for the agonist DAMGO similar to that of the high-affinity state of the wild type, which was not altered by GTPgammaS, while having unchanged affinity for the antagonist diprenorphine. The T6.34(279)K mutant displayed a higher intracellular receptor pool than the wild type. Naloxone inhibited the basal [35S]GTPgammaS binding of the T6.34(279)K mutant, demonstrating inverse agonist activity at this mutant receptor. In contrast, the T6.34(279)D substitution did not increase basal [35S]GTPgammaS binding, greatly reduced agonist-promoted [35S]GTPgammaS binding, and markedly decreased affinity for DAMGO. Thus, the T6.34(279)D mutant adopts conformations corresponding to inactive states of the receptor. The results were interpreted in the structural context of a model for the mu opioid receptor that incorporates the information from the crystal structure of rhodopsin. The interaction of T6.34(279) with R3.50(165) in the mu opioid receptor is considered to stabilize the inactive conformations. The T6.34(279)K substitution would then disrupt this interaction and support agonist-free activation, while T6.34(279)D mutation should strengthen this interaction which keeps the receptor in inactive states. T6.34(279) may, in addition, interact with the neighboring R6.35(280) to help constrain the receptor in inactive states, and T6.34(279)K and T6.34(279)D mutations would affect this interaction by disrupting or strengthening it, respectively. To the best of our knowledge, the results presented here represent the first structurally rationalized demonstration that mutations of this locus can lead to dramatically different properties of a GPCR.     

Arginine-349 and aspartate-373 of the Na(+)/dicarboxylate cotransporter are conformationally sensitive residues.

The conserved residues, Arg-349 and Asp-373, of the renal Na(+)/dicarboxylate cotransporter (NaDC-1) have been shown in our previous studies to affect substrate affinity and cation binding. In this study, amino acids surrounding Arg-349 and Asp-373 were individually mutated to cysteines and their sensitivity to methanethiosulfonate reagents (MTS) was tested. Only three of the 21 mutants were sensitive to MTS reagents: R349C, S372C, and D373C. The R349C mutant had reduced activity which was restored by chemical modification with MTSEA. The effect of MTSEA was only observed in the presence of sodium, indicating that Arg-349 is conformationally accessible. The succinate transport activity of the S372C mutant was stimulated by both MTSEA and MTSET. The D373C mutant was very sensitive to inhibition by MTSET (K(i) = 0.5 microM) in sodium buffer. The inhibition of D373C by MTSET was prevented by substrate, suggesting that the substrate-induced conformational change occludes the residue. We conclude that the accessibility of Arg-349 and Asp-373 is likely to change with the conformational states of the transport cycle.     

The transport activity of the Na+-Ca2+ exchanger NCX1 expressed in HEK 293 cells is sensitive to covalent modification of intracellular cysteine residues by sulfhydryl reagents

Membrane permeable N-ethylmaleimide (NEM) and (2-aminoethyl)methanethiosulfonatehydrobromide (MTSEA) inhibited the rat brain Na(+)-Ca(2+) exchanger RBE-2 (NCX1.5) expressed in HEK 293 cells in a dose dependent manner. 50% inhibition was obtained at 1 mm MTSEA and 1.65 mm NEM. External application of membrane impermeable [2-(trimethylammonium) ethyl]methanethiosulfonatebromide (MTSET) and sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES) did not inhibit the transport activity in whole cells. Following reconstitution, however, of RBE-2 transfected cell proteins into proteoliposomes, external application of MTSET and MTSES led to a decrease in transport activity to 42.7 (S.D. = 9.1) and 51% (S.D. = 10.14), respectively. Similar results were obtained also when the rat heart isoform RHE-1 (NCX1.1) or the rat brain isoform RBE-1 (NCX1.4) was expressed. NEM and MTSEA inhibited Na(+) gradient-dependent Ca(2+) uptake also in HEK 293 cells expressing RBE-2/C14A/C20S/ C122S/C780S (numbering corresponds to RBE-2), a mutant in which all putative extracellular cysteines were exchanged. To study the accessibility of different cysteines to covalent modification, surface biotinylation of cells expressing the wild type exchanger and its mutants was carried out using 3-(N-maleimidylpropionyl)biocytin. Surface biotinylation revealed immunoreactive protein derived from the wild type Na(+)-Ca(2+) exchanger only if the transfected cells were exposed to the reducing agent Tris(2-carboxyethyl)phosphine. No reduction was needed when the single cysteine mutants of RBE-2, C14A, C20S, and C780S, were expressed. Treatment of the cells expressing these mutants with MTSET before biotinylation, led to a decrease in the amount of exchanger protein that was revealed. No immunoreactive protein was detected when the quadruple mutant RBE-2, C14A/C20S/C122S/C780S, was biotinylated, suggesting that no additional cysteines are accessible directly from the extracellular face of the membrane. Permeabilizing the cells expressing RBE-2/C14A/C20S/ C122S/C780S with streptolysin O resulted in biotinylation of the exchanger protein. Its amount decreased if exposure to NEM preceded streptolysin O treatment. Our results suggest that Na(+)-Ca(2+) exchange activity is inhibited by covalent modification with sulfhydryl reagents of cysteine residues that are accessible from the cytoplasmic face of the membrane.     

Slow-inactivation induced conformational change in domain 2-segment 6 of cardiac Na+ channel

To examine conformational changes during slow inactivation involving domain 2-segment 6 (D2-S6) of human cardiac Na(+) channel (hNav1.5), we applied the substituted-cysteine accessibility method (SCAM) using methanethiosulfonate ethylammonium (MTSEA). We substituted cysteine (C) for native valine (V) at position 930 of D2-S6 in the MTSEA-resistant hNav1.5 mutant C373Y to produce the double mutant C373Y-V930C. Whole-cell Na(+) currents were recorded using patch-clamp techniques in transiently transfected HEK cells. In C373Y-V930C, we find that MTSEA (1.5 mM) applied in the closed state (-160 mV) has no significant effect on whole-cell Na(+) current, while MTSEA applied in the slow-inactivated state (prolonged depolarization at 0 mV) decreases current. We propose that D2-S6 in hNav1.5 undergoes molecular rearrangement during slow inactivation exposing the side chain of residue 930 such that it becomes accessible to modification by MTSEA.     

Substituted cysteine accessibility of the third transmembrane domain of the creatine transporter: defining a transport pathway

Twenty-two amino acid residues from transmembrane domain 3 of the creatine transporter were replaced, one at a time, with cysteine. The background for mutagenesis was a C144S mutant retaining approximately 75% of wild-type transport activity but resistant to methanethiosulfonate (MTS) reagents. Each substitution mutant was tested for creatine transport activity and sensitivity to the following MTS reagents: 2-aminoethyl methanethiosulfonate (MTSEA), 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and 2-sulfonatoethyl methanethiosulfonate (MTSES). Two mutants (G134C and Y148C) were inactive, but most mutants showed significant levels of creatine transport. Treatment with MTSEA inhibited the activity of the W154C, Y147C, and I140C mutants. Creatine partially protected I140C from inactivation, and this residue, like Cys-144 in the wild-type CreaT, is predicted to be close to a creatine binding site. MTSEA inactivation of Y147C was dependent on Na+ and Cl- suggesting that solvent accessibility was ion-dependent. Helical wheel and helical net projections indicate that the three MTSEA-sensitive mutants (W154C, Y147C, and I140C) and two inactive mutants (V151C and Y148C) are aligned on a face of an alpha-helix, suggesting that they form part of a substrate pathway. The W154C mutant, located near the external face of the membrane, was accessible to the larger MTS reagents, whereas those implicated in creatine binding were only accessible to the smaller MTSEA. Consideration of our data, together with a study on the serotonin transporter (Chen, J. G., Sachpatzidis, A., and Rudnick, G. (1997) J. Biol. Chem. 272, 28321-28327), suggests that involvement of residues from transmembrane domain 3 is a common feature of the substrate pathway of Na+- and Cl- -dependent neurotransmitter transporters.     

Functional role of the spatial proximity of Asp114(2.50) in TMH 2 and Asn332(7.49) in TMH 7 of the mu opioid receptor

We examined whether a proposed spatial proximity between Asp114(2.50) and Asn332(7.49) affected the functional properties of the mu opioid receptor. The D114(2.50)N mutant had reduced binding affinities for morphine, DAMGO and CTAP, but not for naloxone and [3H]diprenorphine; this mutation also abolished agonist-induced increase in [35S]GTPgammaS binding. The N332(7.49)D mutation eliminated detectable binding of either [3H]diprenorphine or [3H]DAMGO. The combined D114(2.50)N-N332(7.49)D mutation restored high affinity binding for [3H]diprenorphine, CTAP and naloxone, and restored partially the binding affinities, potencies and efficacies of morphine and DAMGO. Thus, reciprocal mutations of Asp114(2.50) and Asn332(7.49) compensate for the detrimental effects of the single mutations, indicating that the residues are adjacent in space and that their chemical functionalities are important for ligand binding and receptor activation.     

Chloroethylclonidine and 2-aminoethyl methanethiosulfonate recognize two different conformations of the human alpha(2A)-adrenergic receptor.

The substituted cysteine-accessibility method and two sulfhydryl-specific reagents, the methane-thiosulfonate derivative 2-aminoethyl methanethiosulfonate (MTSEA) and the alpha(2)-adrenergic receptor (alpha(2)-AR) agonist chloroethylclonidine (CEC), were used to determine the relative accessibility of engineered cysteines in the fifth transmembrane domain of the human alpha(2A)-AR (Halpha2A). The second-order rate constants for the reaction of the receptor with MTSEA and CEC were determined with the wild type Halpha2A (cysteine at position 201) and receptor mutants containing accessible cysteines at other positions within the binding-site crevice (positions 197, 200, and 204). The rate of reaction of CEC was similar to that of MTSEA at residues Cys-197, Cys-201, and Cys-204. The rate of reaction of CEC with Cys-200, however, was more than 5 times that of MTSEA, suggesting that these compounds may interact with two different receptor conformations. MTSEA, having no recognition specificity for the receptor, likely reacts with the predominant inactive receptor conformation (R), whereas the agonist CEC may stabilize and react preferentially with the active receptor conformation (R*). This hypothesis was consistent with three-dimensional receptor-ligand models, which further suggest that alpha(2A)-AR activation may involve the clockwise rotation of transmembrane domain 5.     

A role for the middle C terminus of G-protein-activated inward rectifier potassium channels in regulating gating

We have used sulfhydryl-modifying reagents to investigate the regulation of G-protein-activated inward rectifier potassium (GIRK) channels via their cytoplasmic domains. Modification of either the conserved N-terminal cysteines (GIRK1C53 and GIRK2C65) or the middle C-terminal cysteines (GIRK1C310 and GIRK2C321) independently inhibited GIRK1/GIRK2 heteromeric channels. With the exception of GIRK2C65, these cysteines were relatively inaccessible to large modifying reagents. The accessibility was further reduced by a mutation at the end of the second transmembrane domain that stabilized the open state of the channel. Thus it is unlikely that these cysteines line the permeation pathway of the open pore. Cysteines introduced 3 and 6 amino acids upstream of GIRK2C321 (G318C and E315C) were considerably more accessible. The effect of modification was dependent on the charge of the reagent. Modification of E315C in GIRK2 and E304C in GIRK1 by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increased the current by approximately 17-fold, whereas modification by 2-aminoethyl methanethiosulfonate hydrochloride (MTSEA(+)), abolished the current. There was no effect on single-channel conductance. Thus a switch in charge at this middle C-terminal position was sufficient to gate the channel open and closed. This glutamate is conserved in all members of the Kir family. The E303K mutation in Kir2.1 inhibits channel function and causes Andersen's syndrome in humans (Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., Clark, J., Deymeer, F., George, A. L., Jr., Fish, F. A., Hahn, A., Nitu, A., Ozdemir, C., Serdaroglu, P., Subramony, S. H., Wolfe, G., Fu, Y. H., and Ptacek, L. J. (2001) Cell 105, 511-519 and Preisig-Muller, R., Schlichthorl, G., Goerge, T., Heinen, S., Bruggemann, A., Rajan, S., Derst, C., Veh, R. W., and Daut, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7774-7779). Our results suggest that this residue regulates channel gating through an electrostatic mechanism.     

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.     

A conformationally sensitive residue on the cytoplasmic surface of serotonin transporter

Serotonin transporter (SERT) contains a single reactive external cysteine residue at position 109 (Chen, J. G., Liu-Chen, S., and Rudnick, G. (1997) Biochemistry 36, 1479-1486) and seven predicted cytoplasmic cysteines. A mutant of rat SERT (X8C) in which those eight cysteine residues were replaced by other amino acids retained approximately 32% of wild type transport activity and approximately 56% of wild type binding activity. In contrast to wild-type SERT or the C109A mutant, X8C was resistant to inhibition of high affinity cocaine analog binding by the cysteine reagent 2-(aminoethyl)methanethiosulfonate hydrobromide (MTSEA) in membrane preparations from transfected cells. Each predicted cytoplasmic cysteine residue was reintroduced, one at a time, into the X8C template. Reintroduction of Cys-357, located in the third intracellular loop, restored MTSEA sensitivity similar to that of C109A. Replacement of only Cys-109 and Cys-357 was sufficient to prevent MTSEA sensitivity. Thus, Cys-357 was the sole cytoplasmic determinant of MTSEA sensitivity in SERT. Both serotonin and cocaine protected SERT from inactivation by MTSEA at Cys-357. This protection was apparently mediated through a conformational change following ligand binding. Although both ligands bind in the absence of Na(+) and at 4 degrees C, their ability to protect Cys-357 required Na(+) and was prevented at 4 degrees C. The accessibility of Cys-357 to MTSEA inactivation was increased by monovalent cations. The K(+) ion, which is believed to serve as a countertransport substrate for SERT, was the most effective ion for increasing Cys-357 reactivity.     

Structural determinants of the closed KCa3.1 channel pore in relation to channel gating: results from a substituted cysteine accessibility analysis

In this work we address the question of the KCa3.1 channel pore structure in the closed configuration in relation to the contribution of the C-terminal end of the S6 segments to the Ca(2+)-dependent gating process. Our results based on SCAM (substituted cysteine accessibility method) experiments first demonstrate that the S6 transmembrane segment of the open KCa3.1 channel contains two distinct functional domains delimited by V282 with MTSEA and MTSET binding leading to a total channel inhibition at positions V275, T278, and V282 and to a steep channel activation at positions A283 and A286. The rates of modification by MTSEA (diameter 4.6 A) of the 275C (central cavity) and 286C residues (S6 C-terminal end) for the closed channel configuration were found to differ by less than sevenfold, whereas experiments performed with the larger MTSET reagent (diameter 5.8 A) resulted in modification rates 10(3)-10(4) faster for cysteines at 286 compared with 275. Consistent with these results, the modification rates of the cavity lining 275C residue by MTSEA, Et-Hg(+), and Ag(+) appeared poorly state dependent, whereas modification rates by MTSET were 10(3) faster for the open than the closed configuration. A SCAM analysis of the channel inner vestibule in the closed state revealed in addition that cysteine residues at 286 were accessible to MTS reagents as large as MTS-PtrEA, a result supported by the observation that binding of MTSET to cysteines at positions 283 or 286 could neither sterically nor electrostatically block the access of MTSEA to the closed channel cavity (275C). It follows that the closed KCa3.1 structure can hardly be accountable by an inverted teepee-like structure as described for KcsA, but is better represented by a narrow passage centered at V282 (equivalent to V474 in Shaker) connecting the channel central cavity to the cytosolic medium. This passage would not be however restrictive to the diffusion of small reagents such as MTSEA, Et-Hg(+), and Ag(+), arguing against the C-terminal end of S6 forming an obstructive barrier to the diffusion of K(+) ions for the closed channel configuration.     

Involvement of surface cysteines in activity and multimer formation of thimet oligopeptidase

Thimet oligopeptidase is a metalloenzyme involved in regulating neuropeptide processing. Three cysteine residues (246, 248, 253) are known to be involved in thiol activation of the enzyme. In contrast to the wild-type enzyme, the triple mutant (C246S/C248S/C253S) displays increased activity in the absence of dithiothreitol. Dimers, purportedly formed through cysteines 246, 248 and 253, have been thought to be inactive. However, analysis of the triple mutant by native gel electrophoresis reveals the existence of dimers and multimers, implying that oligomer formation is mediated by other cysteines, probably on the surface, and that some of these forms are enzymatically active. Isolation and characterization of iodoacetate-modified monomers and dimers of the triple mutant revealed that, indeed, certain dimeric forms of the enzyme are still fully active, whereas others show reduced activity. Cysteine residues potentially involved in dimerization were identified by modeling of thimet oliogopeptidase to its homolog, neurolysin. Five mutants were constructed; all contained the triple mutation C246S/C248S/C253S and additional substitutions. Substitutions at C46 or C682 and C687 prevented multimer formation and inhibited dimer formation. The C46S mutant had enzymatic activity comparable to the parent triple mutant, whereas that of C682S/C687S was reduced. Thus, the location of intermolecular disulfide bonds, rather than their existence per se, is relevant to activity. Dimerization close to the N-terminus is detrimental to activity, whereas dimerization near the C-terminus has little effect. Altering disulfide bond formation is a potential regulatory factor in the cell owing to the varying oxidation states in subcellular compartments and the different compartmental locations and functions of the enzyme.     

Influence of agonists and antagonists on the segmental motion of residues near the agonist binding pocket of the acetylcholine-binding protein

Using the Lymnaea acetylcholine-binding protein as a surrogate of the extracellular domain of the nicotinic receptor, we combined site-directed labeling with fluorescence spectroscopy to assess possible linkages between ligand binding and conformational dynamics. Specifically, 2-[(5-fluoresceinyl)aminocarbonyl]ethyl methanethiosulfonate was conjugated to a free cysteine on loop C and to five substituted cysteines at strategic locations in the subunit sequence, and the backbone flexibility around each site of conjugation was measured with time-resolved fluorescence anisotropy. The sites examined were in loop C (Cys-188 using a C187S mutant), in the beta9 strand (T177C), in the beta10 strand (D194C), in the beta8-beta9 loop (N158C and Y164C), and in the beta7 strand (K139C). Conjugated fluorophores at these locations show distinctive anisotropy decay patterns indicating different degrees of segmental fluctuations near the agonist binding pocket. Ligand occupation and decay of anisotropy were assessed for one agonist (epibatidine) and two antagonists (alpha-bungarotoxin and d-tubocurarine). The Y164C and Cys-188 conjugates were also investigated with additional agonists (nicotine and carbamylcholine), partial agonists (lobeline and 4-hydroxy,2-methoxy-benzylidene anabaseine), and an antagonist (methyllycaconitine). With the exception of the T177C conjugate, both agonists and antagonists perturbed the backbone flexibility of each site; however, agonist-selective changes were only observed at Y164C in loop F where the agonists and partial agonists increased the range and/or rate of the fast anisotropy decay processes. The results reveal that agonists and antagonists produced distinctive changes in the flexibility of a portion of loop F.     

Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore

Different transmembrane (TM) α helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl(-) channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84-T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.