Structure,Function, and Modification of the Voltage Sensor in Voltage-Gated Ion Channels

Voltage-gated ion channels are crucial for both neuronal and cardiac excitability. Decades of research have begun to unravel the intriguing machinery behind voltage sensitivity. Although the details regarding the arrangement and movement in the voltage-sensor domain are still debated, consensus is slowly emerging. There are three competing conceptual models: the helical-screw, the transporter, and the paddle model. In this review we explore the structure of the activated voltage-sensor domain based on the recent X-ray structure of a chimera between Kv1.2 and Kv2.1. We also present a model for the closed state. From this we conclude that upon depolarization the voltage sensor S4 moves approximately 13 A outwards and rotates approximately 180 degrees, thus consistent with the helical-screw model. S4 also moves relative to S3b which is not consistent with the paddle model. One interesting feature of the voltage sensor is that it partially faces the lipid bilayer and therefore can interact both with the membrane itself and with physiological and pharmacological molecules reaching the channel from the membrane. This type of channel modulation is discussed together with other mechanisms for how voltage-sensitivity is modified. Small effects on voltage-sensitivity can have profound effects on excitability. Therefore, medical drugs designed to alter the voltage dependence offer an interesting way to regulate excitability.     

Membrane-perturbing properties of two Arg-rich paddle domains from voltage-gated sensors in the KvAP and HsapBK K(+) channels

Voltage-gated K(+) channels are gated by displacement of basic residues located in the S4 helix that together with a part of the S3 helix, S3b, forms a "paddle" domain, whose position is altered by changes in the membrane potential modulating the open probability of the channel. Here, interactions between two paddle domains, KvAPp from the K(v) channel from Aeropyrum pernix and HsapBKp from the BK channel from Homo sapiens, and membrane models have been studied by spectroscopy. We show that both paddle domains induce calcein leakage in large unilamellar vesicles, and we suggest that this leakage represents a general thinning of the bilayer, making movement of the whole paddle domain plausible. The fact that HsapBKp induces more leakage than KvAPp may be explained by the presence of a Trp residue in HsapBKp. Trp residues generally promote localization to the hydrophilic-hydrophobic interface and disturb tight packing. In magnetically aligned bicelles, KvAPp increases the level of order along the whole acyl chain, while HsapBKp affects the morphology, also indicating that KvAPp adapts more to the lipid environment. Nuclear magnetic resonance (NMR) relaxation measurements for HsapBKp show that overall the sequence has anisotropic motions. The S4 helix is well-structured with restricted local motion, while the turn between S4 and S3b is more flexible and undergoes slow local motion. Our results indicate that the calcein leakage is related to the flexibility in this turn region. A possibility by which HsapBKp can undergo structural transitions is also shown by relaxation NMR, which may be important for the gating mechanism.     

Double gaps along Shaker S4 demonstrate omega currents at three different closed states

The aim of the present study was to investigate in detail how the voltage sensor in the Shaker potassium channel moves during the gating process. After the publication of the open channel structure from the crystallized KvAP channel in 2003, an alternative so-called "paddle" model was put forward in contrast to the existing helical screw model. The voltage sensor S4 contains 4 arginine residues relevant for gating, R1(362), R2(365), R3(368) and R4(371), each separated by 2 neutral residues. These charged residues coil as one of three threads on the S4-α-helix. Based on a previous finding that the mutation R1S leads to the so-called omega leak current through a "gating-pore" in the closed state, we introduced gaps systematically along the arginine thread substituting long arginines by short serines. Mutations R2S or R3S did neither create transient nor steady leaks. The fact that the native residue A359, which is located three amino acids in front of R1, is a short one, motivated us to check its role. Mutation of A359 to arginine blocked the omega current in R1S mutant indicating that the omega pore is occupied by A359 and R1. Introducing further double gaps (RR to SS) at sequential positions (0+1, 1+2, 2+3), produced clear leak currents which were remarkably stable over a wide voltage range. These leaks contradict that S4 would swing together with S3 in lipid according to the paddle hypothesis. Rather, our results show that during gating the S4 segment moves in 3 helical steps through a fixed pore formed by the channel protein.     

Changes in local S4 environment provide a voltage-sensing mechanism for mammalian hyperpolarization-activated HCN channels

The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitutions in the COOH-terminal half of S4 selectively reacted with internal MTSET when the channel was open. In the open state, the boundary between externally and internally accessible residues was remarkably narrow (approximately 3 residues). This suggests that S4 lies in a water-filled gating canal with a very narrow barrier between the external and internal solutions, similar to depolarization-gated channels. However, the pattern of reactivity is incompatible with either classical gating models, which postulate a large translational or rotational movement of S4 within a gating canal, or with a recent model in which S4 forms a peripheral voltage-sensing paddle (with S3b) that moves within the lipid bilayer (the KvAP model). Rather, we suggest that voltage sensing is due to a rearrangement in transmembrane segments surrounding S4, leading to a collapse of an internal gating canal upon channel closure that alters the shape of the membrane field around a relatively static S4 segment.     

Functional Identification of Close Proximity Amino Acid Side Chains within the Transmembrane-Spanning Helixes of the P2X2 Receptor

The transition from the closed to open state greatly alters the intra- and inter-subunit interactions of the P2X receptor (P2XR). The interactions that occur in the transmembrane domain of the P2X2R remain unclear. We used substituted cysteine mutagenesis disulfide mapping to identify pairs of residues that are in close proximity within the transmembrane domain of rP2X2R and compared our results to the predicted positions of these amino acids obtained from a rat P2X2R homology model of the available open and closed zebrafish P2X4R structures. Alternations in channel function were measured as a change in the ATP-gated current before and after exposure to dithiothreitol. Thirty-six pairs of double mutants of rP2X2R expressed in HEK293 cells produced normal functioning channels. Thirty-five pairs of these mutants did not exhibit a functionally detectable disulfide bond. The double mutant H33C/S345C formed redox-dependent cross-links in the absence of ATP. Dithiothreitol ruptured the disulfide bond of H33C/S345C and induced a 2 to 3-fold increase in current. The EC₅₀ for H33C/S345C before dithiothreitol treatment was ∼2-fold higher than that after dithiothreitol treatment. Dithiothreitol reduced the EC₅₀ to wild-type levels. Furthermore, expression of trimeric concatamer receptors with Cys mutations at some but not all six positions showed that the more disulfide bond formation sites within the concatamer, the greater current potentiation after dithiothreitol incubation. Immunoblot analysis of H33C/S345C revealed one monomer band under nonreducing conditions strongly suggesting that disulfide bonds are formed within single subunits (intra-subunit) and not between two subunits (inter-subunit). Taken together, these data indicate that His33 and Ser345 are proximal to each other across an intra-subunit interface. The relative movement between the first transmembrane and the second transmembrane in the intra-subunit is likely important for transmitting the action of ATP binding to the opening of the channel.     

Position and role of the BK channel alpha subunit S0 helix inferred from disulfide crosslinking

The position and role of the unique N-terminal transmembrane (TM) helix, S0, in large-conductance, voltage- and calcium-activated potassium (BK) channels are undetermined. From the extents of intra-subunit, endogenous disulfide bond formation between cysteines substituted for the residues just outside the membrane domain, we infer that the extracellular flank of S0 is surrounded on three sides by the extracellular flanks of TM helices S1 and S2 and the four-residue extracellular loop between S3 and S4. Eight different double cysteine-substituted alphas, each with one cysteine in the S0 flank and one in the S3-S4 loop, were at least 90% disulfide cross-linked. Two of these alphas formed channels in which 90% cross-linking had no effect on the V(50) or on the activation and deactivation rate constants. This implies that the extracellular ends of S0, S3, and S4 are close in the resting state and move in concert during voltage sensor activation. The association of S0 with the gating charge bearing S3 and S4 could contribute to the considerably larger electrostatic energy required to activate the BK channel compared with typical voltage-gated potassium channels with six TM helices.     

Molecular dynamics simulation of Kv channel voltage sensor helix in a lipid membrane with applied electric field

In this article, we present the results of the molecular dynamics simulations of amphiphilic helix peptides of 13 amino-acid residues, placed at the lipid-water interface of dipalmitoylphosphatidylcholine bilayers. The peptides are identical with, or are derivatives of, the N-terminal segment of the S4 helix of voltage-dependent K channel KvAP, containing four voltage-sensing arginine residues (R1-R4). Upon changing the direction of the externally applied electric field, the tilt angle of the wild-type peptide changes relative to the lipid-water interface, with the N-terminus heading up with an outward electric field. These movements were not observed using an octane membrane in place of the dipalmitoylphosphatidylcholine membrane, and were markedly suppressed by 1), substituting Phe located one residue before the first arginine (R1) with a hydrophilic residue (Ser, Thr); or 2), changing the periodicity rule of Rs from at-every-third to at-every-fourth position; or 3), replacing R1 with a lysine residue (K). These and other findings suggest that the voltage-dependent movement requires deep positioning of Rs when the resting (inward) electric field is present. Later, we performed simulations of the voltage sensor domain (S1-S4) of Kv1.2 channel. In simulations with a strong electric field (0.1 V/nm or above) and positional restraints on the S1 and S2 helices, S4 movement was observed consisting of displacement along the S4 helix axis and a screwlike axial rotation. Gating-charge-carrying Rs were observed to make serial interactions with E183 in S1 and E226 in S2, in the outer water crevice. A 30-ns-backward simulation started from the open-state model gave rise to a structure similar to the recent resting-state model, with S4 moving vertically ∼6.7 Å. The energy landscape around the movement of S4 appears very ragged due to salt bridges formed between gating-charge-carrying residues and negatively charged residues of S1, S2, and S3 helices. Overall, features of S3 and S4 movements are consistent with the recent helical-screw model. Both forward and backward simulations show the presence of at least two stable intermediate structures in which R2 and R3 form salt bridges with E183 or E226, respectively. These structures are the candidates for the states postulated in previous gating kinetic models, such as the Zagotta-Hoshi-Aldrich model, to account for more than one transition step per subunit for activation.     

Demonstration of physical proximity between the N terminus and the S4-S5 linker of the human ether-a-go-go-related gene (hERG) potassium channel

Potassium channels encoded by the human ether-à-go-go-related gene (hERG) contribute to cardiac repolarization as a result of their characteristic gating properties. The hERG channel N terminus acts as a crucial determinant in gating. It is also known that the S4-S5 linker couples the voltage-sensing machinery to the channel gate. Moreover, this linker has been repeatedly proposed as an interaction site for the distal portion of the N terminus controlling channel gating, but direct evidence for such an interaction is still lacking. In this study, we used disulfide bond formation between pairs of engineered cysteines to demonstrate the close proximity between the beginning of the N terminus and the S4-S5 linker. Currents from channels with introduced cysteines were rapidly and strongly attenuated by an oxidizing agent, this effect being maximal for cysteine pairs located around amino acids 3 and 542 of the hERG sequence. The state-dependent modification of the double-mutant channels, but not the single-cysteine mutants, and the ability to readily reverse modification with the reducing agent dithiothreitol indicate that a disulfide bond is formed under oxidizing conditions, locking the channels in a non-conducting state. We conclude that physical interactions between the N-terminal-most segment of the N terminus and the S4-S5 linker constitute an essential component of the hERG gating machinery, thus providing a molecular basis for previous data and indicating an important contribution of these cytoplasmic domains in controlling its unusual gating and hence determining its physiological role in setting the electrical behavior of cardiac and other cell types.     

Crystal structure of soybean proglycinin A1aB1b homotrimer

Soybean glycinin is a member of the 11 S globulin family. The crystal structure of proglycinin was determined by X-ray crystallography at 2.8 A resolution with an R-factor of 0.199 and a free R-factor of 0.250. A trimer molecule was found in an asymmetric unit of crystals. The trimer model contains three A1aB1b subunits and comprises 1128 amino acid residues and 34 water molecules. The constituent protomers of the homo-trimeric protein are arranged around a 3-fold symmetry axis with dimensions of 95 Ax95 Ax40 A. The protomer model is composed of five fragments which correspond roughly to conserved regions based on the sequence alignment of various 11 S globulins. The core of the protomer consists of two jelly-roll beta-barrels and two extended helix domains. This structure of proglycinin is similar to those of canavalin and phaseolin belonging to the 7 S globulin family, strongly supporting the hypothesis that both 7 S and 11 S globulins are derived from a common ancestor. The inter and intra-chain disulfide bonds conserved in the 11 S globulin family are clearly observed. It is found that the face with the inter-chain disulfide bond (IE face) contains more hydrophobic residues than that with the intra-chain disulfide bond. This suggests that a mature hexamer is formed by the interaction between the IE faces after processing.     

Resin-acid derivatives bind to multiple sites on the voltage-sensor domain of the Shaker potassium channel

Voltage-gated potassium (K_V) channels can be opened by negatively charged resin acids and their derivatives. These resin acids have been proposed to attract the positively charged voltage-sensor helix (S4) toward the extracellular side of the membrane by binding to a pocket located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. By contrast to this proposed mechanism, neutralization of the top gating charge of the Shaker K_V channel increased resin-acid–induced opening, suggesting other mechanisms and sites of action. Here, we explore the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated/open state of the Shaker K_V channel by a combination of in silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resin-acid–binding sites around S4: (1) the S3/S4 site previously suggested, in which positively charged residues introduced at the top of S4 are critical to keep the compound bound, (2) a site in the cleft between S4 and the pore domain (S4/pore site), in which a tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound, and (3) a site located on the extracellular side of the voltage-sensor domain, in a cleft formed by S1–S4 (the top-VSD site). The multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation make the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding dynamics and the types of interactions it can form with the protein in a state-specific manner.     

Interaction between extracellular Hanatoxin and the resting conformation of the voltage-sensor paddle in Kv channels

In voltage-activated potassium (Kv) channels, basic residues in S4 enable the voltage-sensing domain to move in response to membrane depolarization and thereby trigger the activation gate to open. In the X-ray structure of the KvAP channel, the S4 helix is located near the intracellular boundary of the membrane where it forms a "voltage-sensor paddle" motif with the S3b helix. It has been proposed that the paddle is lipid-exposed and that it translocates through the membrane as it activates. We studied the interaction of externally applied Hanatoxin with the voltage-sensor paddle in Kv channels and show that the toxin binds tightly even at negative voltages where the paddle is resting and the channel is closed. Moreover, measurements of gating charge movement suggest that Hanatoxin interacts with and stabilizes the resting paddle. These findings point to an extracellular location for the resting conformation of the voltage-sensor paddle and constrain its transmembrane movements during activation.     

Relative Movements of Transmembrane Regions at the Outer Mouth of the Cystic Fibrosis Transmembrane Conductance Regulator Channel Pore during Channel Gating

Multiple transmembrane (TM) segments line the pore of the cystic fibrosis transmembrane conductance regulator Cl(-) channel; however, the relative alignment of these TMs and their relative movements during channel gating are unknown. To gain three-dimensional structural information on the outer pore, we have used patch clamp recording to study the proximity of pairs of cysteine side chains introduced into TMs 6 and 11, using both disulfide cross-linking and Cd(2+) coordination. Following channel activation, disulfide bonds could apparently be formed between three cysteine pairs (of 15 studied): R334C/T1122C, R334C/G1127C, and T338C/S1118C. To examine the state dependence of cross-linking, we combined these cysteine mutations with a nucleotide-binding domain mutation (E1371Q) that stabilizes the channel open state. Investigation of the effects of the E1371Q mutation on disulfide bond formation and Cd(2+) coordination suggests that although R334C/T1122C and T338C/S1118C are closer together in the channel open state, R334C/G1127C are close together and can form disulfide bonds only when the channel is closed. These results provide important new information on the three-dimensional structure of the outer mouth of the cystic fibrosis transmembrane conductance regulator channel pore: TMs 6 and 11 are close enough together to form disulfide bonds in both open and closed channels. Moreover, the altered relative locations of residues in open and in closed channels that we infer allow us to propose that channel opening and closing may be associated with a relative translational movement of TMs 6 and 11, with TM6 moving "down" (toward the cytoplasm) during channel opening.     

Depolarization induces intersubunit cross-linking in a S4 cysteine mutant of the Shaker potassium channel

Voltage-gated potassium (K(v)) 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 in Xenopus 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.     

The orientation and molecular movement of a k(+) channel voltage-sensing domain

Voltage-gated channels operate through the action of a voltage-sensing domain (membrane segments S1-S4) that controls the conformation of gates located in the pore domain (membrane segments S5-S6). Recent structural studies on the bacterial K(v)AP potassium channel have led to a new model of voltage sensing in which S4 lies in the lipid at the channel periphery and moves through the membrane as a unit with a portion of S3. Here we describe accessibility probing and disulfide scanning experiments aimed at determining how well the K(v)AP model describes the Drosophila Shaker potassium channel. We find that the S1-S3 helices have one end that is externally exposed, S3 does not undergo a transmembrane motion, and S4 lies in close apposition to the pore domain in the resting and activated state.     

Assembly of functional rhodopsin requires a disulfide bond between cysteine residues 110 and 187

Cysteine residues 110 and 187 are essential for the formation of the correct bovine rhodopsin structure (Karnik, S. S., Sakmar, T. P., Chen, H.-B., and Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8459-8463). We now show that the sulfhydryl groups of these 2 cysteine residues interact to form a disulfide bond. Rhodopsin mutants containing cysteine----serine substitutions were prepared as follows. In one mutant, CysVII, all the 10 cysteine residues of rhodopsin were replaced by serines. A second mutant, CysVIII, contained only C110 and C185; a third mutant, CysIX, contained only C185 and C187 while the fourth mutant, CysX, contained only C110 and C187. Only mutant CysX formed functional rhodopsin. Mutants CysVIII and CysIX reacted with [3H]iodoacetic acid showing the presence of free sulfhydryl groups while mutant CysX was inert to this reagent. CysX reacted with cyanide ion to form a thiocyanate derivative showing the presence of a disulfide bond. The C110-C187 disulfide bond is buried in rhodopsin because reactions with disulfide reducing agents and cyanide ion require prior treatment with denaturants.     

Computer simulation of the KvAP voltage-gated potassium channel: steered molecular dynamics of the voltage sensor

The recent crystal structures of the voltage-gated potassium channel KvAP and its isolated voltage-sensing 'paddle' (composed of segments S1-S4) challenge existing models of voltage gating and raise a number of questions about the structure of the physiologically relevant state. We investigate a possible gating mechanism based on the crystal structures in a 10 ns steered molecular dynamics simulation of KvAP in a membrane-mimetic octane layer. The structure of the full KvAP protein has been modified by restraining the S2-S4 domain to the conformation of the isolated high-resolution paddle structure. After an initial relaxation, the paddle tips are pulled through the membrane from the intracellular to the extracellular side, corresponding to a putative change from closed to open. We describe the effect of this large-scale motion on the central pore domain, which remains largely unchanged, on the protein hydrogen-bonding network and on solvent. We analyze the motion of the S3b-S4 portion of the protein and propose a possible coupling mechanism between the paddle motion and the opening of the channel. Interactions between the arginine residues in S4, solvent and chloride ions are likely to play a role in the gating charge.     

Coupling between Residues on S4 and S1 Defines the Voltage-Sensor Resting Conformation in NaChBac

The voltage sensor is a four-transmembrane helix bundle (S1-S4) that couples changes in membrane potential to conformational alterations in voltage-gated ion channels leading to pore opening and ion conductance. Although the structure of the voltage sensor in activated potassium channels is available, the conformation of the voltage sensor at rest is still obscure, limiting our understanding of the voltage-sensing mechanism. By employing a heterologously expressed Bacillus halodurans sodium channel (NaChBac), we defined constraints that affect the positioning and depolarization-induced outward motion of the S4 segment. We compared macroscopic currents mediated by NaChBac and mutants in which E43 on the S1 segment and the two outermost arginines (R1 and R2) on S4 were substituted. Neutralization of the negatively charged E43 (E43C) had a significant effect on channel gating. A double-mutant cycle analysis of E43 and R1 or R2 suggested changes in pairing during channel activation, implying that the interaction of E43 with R1 stabilizes the voltage sensor in its closed/available state, whereas interaction of E43 with R2 stabilizes the channel open/unavailable state. These constraints on S4 dynamics that define its stepwise movement upon channel activation and positioning at rest are novel, to the best of our knowledge, and compatible with the helical-screw and electrostatic models of S4 motion.     

Conformational changes in the parathyroid hormone receptor associated with activation by agonist

Binding of hormones to their cognate G protein-coupled receptors (GPCRs) induces conformational shifts within the receptor based on evidence from a few hormone-receptor systems. Employing an engineered disulfide bond formation strategy and guided by a previously established model of the PTH-PTH receptor (PTHR)1 bimolecular complex, we set out to document and characterize the nature of agonist-induced changes in this family B GPCR. A mutant PTHR1 was generated which incorporates a Factor Xa cleavage site in the third intracellular loop. Treatment with Factor Xa fragments the receptor. However, if a new disulfide bond was formed before exposure to the enzyme, the fragments remain held together. A set of double cysteine-containing mutants were designed to probe the internal relative movements of transmembrane (TM) helices 2 and TM7. PTH enhanced formation of disulfide bonds in the K240C/F447C and A242C/F447C mutants. For the F238C/F447C mutant, a disulfide bond is formed in the basal state, but is disrupted by interaction with PTH. For the D241C/F447C PTHR1 construct, no disulfide bond formation was observed in either the basal or hormone-bound state. These findings demonstrate that the conformation of PTHR1 is altered from the basal state when PTH is bound. Novel information regarding spatial proximities between TM2 and TM7 of PTHR1 and the nature of relative movements between the two transmembrane regions was revealed. The data confirm and extend the experimentally derived model of the PTH-PTHR1 complex and provide insights at a new level of detail into the early events in PTHR1 activation by PTH.     

Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6

The large-conductance, voltage- and Ca²⁺-gated K⁺ (BK) channel consists of four α subunits, which form a voltage- and Ca²⁺-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance–voltage (G-V) curve to the left at [Ca²⁺] > 2 µM. In addition to the six transmembrane (TM) helices, S1–S6, conserved in all voltage-dependent K⁺ channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2–S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2–S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2–S3) has no effect on V₅₀ for activation, implying that the cytoplasmic end of S0 and the S2–S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V₅₀.     

A molecular mechanism for the cleavage of a disulfide bond as the primary function of agonist binding to G-protein-coupled receptors based on theoretical calculations supported by experiments.

A model of the binding site of delta-opioids in the extracellular region of the G-protein-coupled opioid receptor based on modelling studies is presented. The distance between Asp288 and the disulfide bridge (Cys121-Cys198) formed between the first and second extracellular loops was found to be short. This model is consistent with site-directed mutagenesis studies. The arrangement of the ligands found in the receptor led to the development of a reaction mechanism for the cleavage of the disulfide bond catalysed by the ligands. Semi-empirical quantum chemical PM3 and AM1 calculations as well as ab initio studies showed that the interaction between the carboxylic acid side chain of aspartic acid and the disulfide bond leads to the polarization of, and withdrawal of a proton from, the protonated nitrogen of the ligand to one of the sulfur atoms. A mixed sulfenic acid and carboxylic acid anhydrate is formed as an intermediate as well as a thiol. The accompanying cleavage of the disulfide bond may produce a conformational change in the extracellular loops such that the pore formed by the seven-helix bundle opens allowing entrance of the ligand, water and ions into the cell. Cleavage of the disulfide bond after opioid administration was demonstrated experimentally by flow-cytometric measurements employing CMTMR and monobromobimane-based analyses of membrane-located thiols. The suggested mechanism may explain, in a consistent way, the action of agonists and antagonists and is assumed to be common for many G-protein coupled receptors.