Mutant Cycle Analysis Research Articles

Gating Transitions in the Palm Domain of ASIC1a*

Acid-sensing ion channels (ASICs) are trimeric cation-selective proton-gated ion channels expressed in the central and peripheral nervous systems. The pore-forming transmembrane helices in these channels are linked by short loops to the palm domain in the extracellular region. Here, we explore the contribution to proton gating and desensitization of Glu-79 and Glu-416 in the palm domain of ASIC1a. Engineered Cys, Lys, and Gln substitutions at these positions shifted apparent proton affinity toward more acidic values. Double mutant cycle analysis indicated that Glu-79 and Glu-416 cooperatively facilitated pore opening in response to extracellular acidification. Channels bearing Cys at position 79 or 416 were irreversibly modified by thiol-reactive reagents in a state-dependent manner. Glu-79 and Glu-416 are located in β-strands 1 and 12, respectively. The covalent modification by (2-(trimethylammonium)ethyl) methanethiosulfonate bromide of Cys at position 79 impacted conformational changes associated with pore closing during desensitization, whereas the modification of Cys at position 416 affected conformational changes associated with proton gating. These results suggest that β-strands 1 and 12 contribute antagonistically to activation and desensitization of ASIC1a. Site-directed mutagenesis experiments indicated that the lower palm domain contracts in response to extracellular acidification. Taken together, our studies suggest that the lower palm domain mediates conformational movements that drive pore opening and closing events.

Nicotinic Receptor Transduction Zone: Invariant Arginine Couples to Multiple Electron-Rich Residues

Gating of the muscle-type acetylcholine receptor (AChR) channel depends on communication between the ACh-binding site and the remote ion channel. A key region for this communication is located within the structural transition zone between the ligand-binding and pore domains. Here, stemming from β-strand 10 of the binding domain, the invariant αArg209 lodges within the hydrophobic interior of the subunit and is essential for rapid and efficient channel gating. Previous charge-reversal experiments showed that the contribution of αArg209 to channel gating depends strongly on αGlu45, also within this region. Here we determine whether the contribution of αArg209 to channel gating depends on additional anionic or electron-rich residues in this region. Also, to reconcile diverging findings in the literature, we compare the dependence of αArg209 on αGlu45 in AChRs from different species, and compare the full agonist ACh with the weak agonist choline. Our findings reveal that the contribution of αArg209 to channel gating depends on additional nearby electron-rich residues, consistent with both electrostatic and steric contributions. Furthermore, αArg209 and αGlu45 show a strong interdependence in both human and mouse AChRs, whereas the functional consequences of the mutation αE45R depend on the agonist. The emerging picture shows a multifaceted network of interdependent residues that are required for communication between the ligand-binding and pore domains.

Proline Residues at the Nicotinic Acetylcholine Transmitter Binding Sites

The two neuromuscular acetylcholine receptor (AChR) transmitter binding sites are located in the extracellular domain of the protein at the α-e and α-δ subunit interfaces. At each site there are two vicinal prolines in the complimentary e/δ subunit (ProD1 and ProD2). We estimated for dozens of mutations of e/δ ProD1 and ProD2 (single-channels, HEK cells, +100mV, 23 oC) the gating energies with ACh molecules bound (G2 and G1) and without any agonist (G0). From these we calculated the energy for gating arising from the affinity change for ACh (GB1+GB2=G2-G0). Mutation of ProD2 had larger effects than of ProD1. The eProD2-L mutation, which causes a congenital myasthenic syndrome, increases αGB (+3.4 kcal/mol, throughout) but does not change G0. For the same mutation in the δ subunit the change in GB was smaller (+1.9). All of the side chain substitutions at e/δProD2 increased both GB and G0 compared to the wild-type (which has the values −5.1 and +8.4). The largest increase in αGB was for eProD2-R (+4.7) and in G0 for δProD2-Q (+2.5). We used mutant cycle analysis to estimate αGB¬ coupling between side chains. The two ProD2 residues interact weakly with each other (−0.6), but there is a strong interaction between eProD2-αGlyB1 (+2.7) but not between δProD2- αGlyB2. It is possible that a concerted, Pro-Gly ‘switch' at α-e occurs near to the onset of channel-opening. We engineered AChRs having only one functional binding site and quantified GB(ACh) for either the α-e (by using the δProD2-R knockout or α-δ (by using the eProD2-R knockout) site. The sites provide +5.2 and −4.7 kcal/mol with ACh. One-site AChRs will be useful for studying the energy sources for gating by agonists at the two different binding sites.

Defining the Interactive Surfaces of Tarantula Toxins for Voltage Sensors in Kv Channels

Voltage-gated ion channels play crucial roles in the generation and propagation of electrical signals within the nervous system. Peptide toxins from tarantula venom interact with voltage-sensing domains in Kv channels and have provided useful tools for exploring their structures and gating mechanisms. However, relatively little is known about the specific molecular interactions between these toxins and voltage sensors. In the present study we set out to define the surface of GxTx-1E that interacts with the voltage sensors in the Kv2.1 channel. GxTx-1E is a particularly attractive candidate for further study because the structure of this amphipathic toxin has been solved, and it binds with high affinity to the voltage-sensor paddle motif in the Kv2.1 channel to inhibit opening, providing the opportunity to undertake mutant cycle analysis between toxin and channel. Towards this goal we mutated 28 of the 36 non-Cys residues in GxTx-1E to Ala using solid-phase chemical synthesis, folded the mutants in vitro and studied their interactions with the Kv2.1 channel using voltage-clamp electrophysiological recording techniques. Of all the mutations studied thus far, none of the polar residue mutants produced changes in toxin affinity greater than 5-fold, whereas five hydrophobic residue mutants produced > 10-fold changes in toxin affinity. In the structure of the toxin, all of these hydrophobic residues are positioned to form a contiguous surface. We are currently undertaking mutant cycle analysis between these mutants and those within the paddle motif to define the protein-protein interface between toxin and channel.

Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

Gating Interactions between the Cytoplasmic T1 Domain and Pore of a Kv Channel

Whereas the gating roles of the voltage-sensing and pore domains of Kv channels have been extensively investigated, much less is known about the mechanisms governing the regulation of gating by the cytoplasmic T1 domain. Previous work from this lab strongly suggests that the Kv4.1 T1 domain undergoes conformational changes directly linked to gating (J. Gen. Physiol., 127:391-400, 2006); however, the structural basis of the communication between the activation gate and the T1 domain has remained elusive. To tackle this problem, we applied cysteine scanning mutagenesis, voltage-clamp electrophysiology, double mutant cycle analysis (DMCA) and oxidizing reagents to probe specific interactions between the cytoplasmic T1 domain L4 helix, the S4-S5 linker and S6 tail. The energetic impact of the mutations on activation gating is apparently small. By comparison, the energetic impact of the mutations on inactivation gating was significantly greater. DMCA of K141C(L4)+A422C(S6) revealed a significant interaction energy (4 kcal/mol), which suggests an interaction between K141 in the L4 helix and A422 in S6. Supporting this possibility, the currents induced by K141C(L4)+A422C(S6) are inhibited (∼65% of control) by an oxidizing agent (tBuHO2), whereas the single mutants are not; and the effect is reversed by the reducing agent DTT. These results indicate the formation of a disulfide bond between the interacting side chains, which is consistent with physical interaction between K141(L4) and A422(S6). Overall, we conclude that regulation of inactivation gating by the T1 domain involves a physical interaction between the T1 L4 helix and the distal segment of S6. Supported by NIH grant R01 NS032337 (MC).

Salt Bridge Switching from Arg290/Glu167 to Arg290/ATP Promotes the Closed-to-Open Transition of the P2X2 Receptor

P2X receptors are trimeric adenosine-5'-triphosphate (ATP)-gated cation channels involved in fast signal transduction in many cell types. In this study, we used homology modeling of the rat P2X2 receptor with the zebrafish P2X4 X-ray template to determine that the side chains of the Glu167 and Arg290 residues are in close spatial vicinity within the ATP-binding pocket when the rat P2X2 channel is closed. Through charge reversal mutation analysis and mutant cycle analysis, we obtained evidence that Glu167 and Arg290 form an electrostatic interaction. In addition, disulfide trapping indicated the close proximity of Glu167 and Arg290 when the channel is in the closed state, but not in the ATP-bound open state. Consistent with a gating-induced movement that disrupts the Glu167/Arg290 salt bridge, a comparison of the closed and open rat P2X2 receptor models revealed a significant rearrangement of the protein backbone and the side chains of the Glu167 and Arg290 residues during the closed-to-open transition. The associated release of the Glu167/Arg290 salt bridge during channel opening allows a strong ionic interaction between Arg290 and a γ-phosphate oxygen of ATP. We conclude from these results that the state-dependent salt bridge switching from Arg290/Glu167 to Arg290/ATP fulfills a dual role: to destabilize the closed state of the receptor and to promote the ionic coordination of ATP in the ATP-binding pocket.

KCNQ1 Channels Do Not Undergo Concerted but Sequential Gating Transitions in Both the Absence and the Presence of KCNE1 Protein

The co-assembly of KCNQ1 with KCNE1 produces I(KS), a K(+) current, crucial for the repolarization of the cardiac action potential. Mutations in these channel subunits lead to life-threatening cardiac arrhythmias. However, very little is known about the gating mechanisms underlying KCNQ1 channel activation. Shaker channels have provided a powerful tool to establish the basic gating mechanisms of voltage-dependent K(+) channels, implying prior independent movement of all four voltage sensor domains (VSDs) followed by channel opening via a last concerted cooperative transition. To determine the nature of KCNQ1 channel gating, we performed a thermodynamic mutant cycle analysis by constructing a concatenated tetrameric KCNQ1 channel and by introducing separately a gain and a loss of function mutation, R231W and R243W, respectively, into the S4 helix of the VSD of one, two, three, and four subunits. The R231W mutation destabilizes channel closure and produces constitutively open channels, whereas the R243W mutation disrupts channel opening solely in the presence of KCNE1 by right-shifting the voltage dependence of activation. The linearity of the relationship between the shift in the voltage dependence of activation and the number of mutated subunits points to an independence of VSD movements, with each subunit incrementally contributing to channel gating. Contrary to Shaker channels, our work indicates that KCNQ1 channels do not experience a late cooperative concerted opening transition. Our data suggest that KCNQ1 channels in both the absence and the presence of KCNE1 undergo sequential gating transitions leading to channel opening even before all VSDs have moved.

Functionally important aromatic-aromatic and sulfur-π interactions in the D2 dopamine receptor.

The recently published crystal structure of the D3 dopamine receptor shows a tightly packed region of aromatic residues on helices 5 and 6 in the space bridging the binding site and what is thought to be the origin of intracellular helical motion. This highly conserved region also makes contacts with residues on helix 3, and here we use double mutant cycle analysis and unnatural amino acid mutagenesis to probe the functional role of several residues in this region of the closely related D2 dopamine receptor. Of the eight mutant pairs examined, all show significant functional coupling (Ω > 2), with the largest coupling coefficients observed between residues on different helices, C3.36/W6.48, T3.37/S5.46, and F5.47/F6.52. Additionally, three aromatic residues examined, F5.47, Y5.48, and F5.51, show consistent trends upon progressive fluorination of the aromatic side chain. These trends are indicative of a functionally important electrostatic interaction with the face of the aromatic residue examined, which is likely attributed to aromatic-aromatic interactions between residues in this microdomain. We also propose that the previously determined fluorination trend at W6.48 is likely due to a sulfur-π interaction with the side chain of C3.36. We conclude that these residues form a tightly packed structural microdomain that connects helices 3, 5, and 6, thus forming a barrier that prevents dopamine from binding further toward the intracellular surface. Upon activation, these residues likely do not change their relative conformation, but rather act to translate agonist binding at the extracellular surface into the large intracellular movements that characterize receptor activation.

A Coupled Array of Noncovalent Interactions Impacts the Function of the 5-HT3A Serotonin Receptor in an Agonist-Specific Way

The serotonin type 3A (5-HT(3)A) receptor is a Cys-loop (pentameric) neurotransmitter-gated ion channel found in the central and peripheral nervous systems and implicated in numerous diseases. In previous studies with the endogenous agonist serotonin, we identified two interactions critical for receptor function: a cation-π interaction with W183 in loop B (TrpB) and a hydrogen bond to E129 in loop A. Here we employ mutant cycle analyses utilizing conventional and unnatural amino acid mutagenesis to demonstrate that a third residue, D124 of loop A, forms two functionally important hydrogen bonds to the backbone of loop B. We also show that these three interactions, the cation-π interaction, the backbone hydrogen bonds, and the E129 hydrogen bond, are tightly coupled to each other, suggesting they function as a single unit. We also identify key functional differences between serotonin and the competitive partial agonist m-chlorophenyl biguanide (mCPBG) at these residues. mCPBG displays no cation-π at TrpB and extreme sensitivity to the positioning of E129, on which it is reliant for initiation of channel gating.

Myasthenic syndrome AChRα C-loop mutant disrupts initiation of channel gating

Congenital myasthenic syndromes (CMSs) are neuromuscular disorders that can be caused by defects in ace-tylcholine receptor (AChR) function. Disease-associated point mutants can reveal the unsuspected functional significance of mutated residues. We identified two pathogenic mutations in the extracellular domain of the AChR α subunit (AChRα) in a patient with myasthenic symptoms since birth: a V188M mutation in the C-loop and a heteroallelic G74C mutation in the main immunogenic region. The G74C mutation markedly reduced surface AChR expression in cultured cells, whereas the V188M mutant was expressed robustly but had severely impaired kinetics. Single-channel patch-clamp analysis indicated that V188M markedly decreased the apparent AChR channel opening rate and gating efficiency. Mutant cycle analysis of energetic coupling among conserved residues within or dispersed around the AChRα C-loop revealed that V188 is functionally linked to Y190 in the C-loop and to D200 in β-strand 10, which connects to the M1 transmembrane domain. Furthermore, V188M weakens inter-residue coupling of K145 in β-strand 7 with Y190 and with D200. Cumulatively, these results indicate that V188 of AChRα is part of an interdependent tetrad that contributes to rearrangement of the C-loop during the initial coupling of agonist binding to channel gating.

Binding interface of cardiac potassium channel proteins identified by hydrogen deuterium exchange of synthetic peptides

Three synthetic peptides, derived from the human potassium channel proteins Ether-a-go-go-related gene (HERG), KCNQ1, and KCNE1, were investigated by hydrogen deuterium exchange coupled with electron-transfer dissociation mass spectrometry at single residue resolution. Each amino acid residue in the first half of the HERG peptide incorporated deuterons with a higher rate than those in the second half of the peptide, consistent with the nuclear magnetic resonance structure of this peptide, with amino acids 1-10 being a flexible coil, whereas amino acids 11-24 are a stable amphipathic helix. The binding interface of KCNQ1 and KCNE1 was determined by comparing the difference of sequential fragment ions before and after binding. The residues determined to be involved in binding were consistent with a cysteine cross-linking study and confirmed by double mutant cycle analysis.

A single amino acid determines the toxicity of Ginkgo biloba extracts

Ginkgo biloba extracts are currently used for a wide range of health-related conditions. Some of the medical benefits of these extracts are controversial, but their lack of toxicity in humans is not in doubt. These extracts are, however, highly toxic to insects. Their active components (ginkgolides and bilobalide) have structures similar to the convulsant picrotoxin, a GABAA receptor antagonist, so their lack of toxicity in mammals is puzzling. Here, we show that the different compositions of insect and vertebrate GABA receptor pores are responsible for the differing toxicities. Insect GABA receptors contain Ala at their 2′ position in the pore. Substitution with Val, which is the equivalent residue in vertebrate GABAA receptor α-subunits, decreases ginkgolide potency by up to 10,000-fold. The reverse mutation in vertebrate GABAA α1 subunits increased the sensitivity of α1β2 and α1β2γ2 receptors to ginkgolides. Mutant cycle analysis demonstrates a strong interaction between the ginkgolides and the 2′ residue, a result supported by in silico docking of compounds into a model of the pore. We conclude that the insecticidal activity of G. biloba extracts can be attributed to their effects at insect GABA receptors, and the presence of a Val at the 2′ position in vertebrate GABAA receptors explains why these compounds are not similarly toxic to humans.—Thompson, A. J., McGonigle, I., Duke, R., Johnston, G. A. R., Lummis, S. C. R. A single amino acid determines the toxicity of Ginkgo biloba extracts.

A single GluN2 subunit residue controls NMDA receptor channel properties via intersubunit interaction

NMDA receptors (NMDARs) are glutamate-gated ion channels present at most excitatory mammalian synapses. The four GluN2 subunits (GluN2A–D) contribute to four diheteromeric NMDAR subtypes that play divergent physiological and pathological roles. Channel properties fundamental to NMDAR function vary among subtypes. We investigated the amino acid residues responsible for variations in channel properties by creating and examining NMDARs containing mutant GluN2 subunits. Unexpectedly, we found that the NMDAR subtype specificity of three crucial channel properties, Mg2+ block, selective permeability to Ca2+, and single-channel conductance, all are controlled primarily by the residue at a single GluN2 site in the M3 transmembrane region. Mutant cycle analysis guided by molecular modeling revealed that a GluN2-GluN1 subunit interaction mediates the site’s effects. We conclude that a single GluN2 subunit residue couples with the pore-forming loop of the GluN1 subunit to create naturally-occurring variations in NMDAR properties that are critical to synaptic plasticity and learning.

Two dyad-free Shaker-type K+ channel blockers from scorpion venom

Most of scorpion toxins affecting voltage-gated K+ channels (KTxs) contain a functional dyad composed of a lysine and an aromatic amino acid separated by a suitable distance. By means of two-electrode voltage clamp technique, we describe functional characterization of two Mesobuthus martensii KTxs (BmP02 and BmP03) without the dyad. These two toxins differ by only one single residue at site 16 (K16N) but they display differential affinities on insect and mammalian Shaker-type K+ channels expressed in Xenopus oocytes. At 1 μM concentration, BmP02 and BmP03 inhibited currents of rKv1.1, rKv1.2, rKv1.3, and Shaker IR, but lacked detectable activity on rKv1.4. The half-inhibitory concentrations (IC50) of BmP02 for rKv1.1, rKv1.2, rKv1.3 and Shaker IR channels are 1.95 μM, 4.40 μM, 7 nM and 20.44 μM, respectively. For BmP03, the corresponding IC50 values for these channels are 5.48 μM, 530 nM, 85.4 nM, and 4.64 μM, respectively. Affinity variation (more than 10-fold) between BmP02 and BmP03 on rKv1.3 indicates functional importance of a cationic side chain at site 16. A pH-dependent experiment and a double mutant cycle analysis suggest that the residue K16 resides on the channel-facing surface of the toxin and within 5 Å of rKv1.3 position 401. These two toxins block rKv1.3 in a weak voltage-dependent manner and both slightly shift the current activation curve to positive potentials. Our work is thus crucial to further understanding structure–function relationship of KTxs without a functional dyad.

Differential Functional Requirements at the Binding Site Between Serotonin and the Partial Agonist M-Chlorophenylbiguanide in Serotonin 3A Receptors

The Serotonin 3a Receptor (5HT3aR) is a pentameric ligand gated ion channel found in the central and peripheral nervous systems and implicated in numerous diseases. In previous studies with the endogenous agonist serotonin, we have identified two interactions critical for receptor function: a cation-π interaction at W183 in loop B and a hydrogen bond at E129 in loop A. Here we employ mutant cycle analyses utilizing conventional and unnatural amino acid mutagenesis to investigate how a third residue, D124 of loop A, orients the aforementioned residues for proper receptor function. We also identify differences in receptor binding and the initiation of channel gating between serotonin and the competitive partial agonist m-chlorophenylbiguanide (mCPBG) at these and adjacent residues.

Tailoring μ-Conotoxin-KIIIA to Selectively Inhibit NaV1.7

Voltage-gated sodium(NaV) channels are critical in initiating and propagating nerve impulses in a variety of tissues including, skeletal(NaV1.4) and cardiac(NaV1.5) muscle, brain(NaV1.2), and peripheral nerve(NaV1.7). Of special interest as a potential drug target is NaV1.7, which mediates propagation of pain signals from receptors to the CNS. Selective blockers of this channel could offer an important analgesic treatment. μ-Conotoxin KIIIA blocks NaV1.2, 1.4 and 1.7, expressed in mammalian cells(Kds: 5, 37, and 97nM respectively). We found two positions in KIIIA, where substitutions alter its selectivity among these channels. KIIIA-H12A shows increased potency for NaV1.7 compared with NaV1.2 and 1.4, but remains more potent against NaV1.2 than NaV1.7(Kds, μM: NaV1.2,10.8; NaV1.4,110 and NaV1.7,19. The second derivative, KIIIA-R14A, inhibits NaV1.7 more strongly than both NaV1.2 and NaV1.4 with Kds (μM): 0.5, 1.1 and 5.7 for NaVs 1.7, 1.2, and 1.4, respectively. These changes in toxin selectivity are associated with differences in the outer ring charges of these channels. The “outer ring” pore-vestibule residues in all NaV channels, except hNaV1.7, are EEDD. In hNaV1.7, the third aspartate is replaced by isoleucine. We have tested reciprocal mutations at this position(NaV1.4D/I and NaV1.7I/D), and these mutations account for the differences in Kd for the wild-type toxin. These channel mutants show no difference from the native channels for block by KIIIA-R14A, suggesting the interaction between the toxin/channel pair has been removed (double mutant cycle analysis yields ΔΔG=∼1.7kT). We further examined these two positions by synthesizing the double mutant, KIIIA-H12Q/R14A, to examine its potency against NaV1.7. Preliminary data indicate that the double mutant(KIIIA-H12Q/R14A) inhibits NaV1.7 more strongly than either NaV1.2 or NaV1.4(Kds:23, 286, and 70 μM, respectively). Thus, interactions with this channel residue could be important in designing blockers that differentially interact with the outer ring to increase their selectively for NaV1.7.

The Interaction of Voltage-Sensor and Gate in BK Channels

To study the molecular basis and mechanism of voltage sensor/gate interaction in BK channels we performed an alanine-scan of the S4-S5 linker and the C-terminal end of S6 in mSlo1. Potassium conductance was measured over a wide range of voltage and calcium and fit to the Horrigan-Aldrich (HA) gating model. Open probability was measured at extreme negative voltages to determine if mutations altered the stability of the gate when voltage sensors are not activated (L in the HA model). Mutations in both linker and S6 were identified that alter L and therefore could be involved in voltage-sensor/gate interaction. The largest inhibitory effects were observed at the ends of S4-S5 linker (Q222A, I233A and K234A) and P319A in S6, each of which decreased L by 10- to 100-fold and shifted V0.5 to more positive voltages. I233A, unlike Q222A or K234A, also produced a decrease in voltage-dependence that is consistent with a decrease in voltage-sensor/gate coupling (D in the HA model). A structural model, based on Kv1.2, suggests inter- or intra-subunit interactions could potentially exist between these sites. Similarly, mutations near the end of S6 (R329A, K330A) that produce an approximate 3 fold increase in L without changing V0.5, may decrease voltage-sensor/gate coupling and could potentially interact with the N-terminal end of the linker where mutations F223A, L227A also produced a moderate increase in L. On the other hand, the largest increases in L were observed with mutations in S6 (P320A, E321A, E324A) that have no obvious interaction partner in S4-S5 and potentially alter the stability of the gate directly. In summary, we have identified several candidates that may be involved in voltage-sensor/gate interaction and will provide a basis for future experiments involving double mutant cycle analysis.

Structural modelling and mutant cycle analysis predict pharmacoresponsiveness of a Nav1.7 mutant channel

Sodium channel Na(V)1.7 is critical for human pain signalling. Gain-of-function mutations produce pain syndromes including inherited erythromelalgia, which is usually resistant to pharmacotherapy, but carbamazepine normalizes activation of Na(V)1.7-V400M mutant channels from a family with carbamazepine-responsive inherited erythromelalgia. Here we show that structural modelling and thermodynamic analysis predict pharmacoresponsiveness of another mutant channel (S241T) that is located 159 amino acids distant from V400M. Structural modelling reveals that Na(v)1.7-S241T is ~2.4 Å apart from V400M in the folded channel, and thermodynamic analysis demonstrates energetic coupling of V400M and S241T during activation. Atomic proximity and energetic coupling are paralleled by pharmacological coupling, as carbamazepine (30 μM) depolarizes S214T activation, as previously reported for V400M. Pharmacoresponsiveness of S241T to carbamazepine was further evident at a cellular level, where carbamazepine normalized the hyperexcitability of dorsal root ganglion neurons expressing S241T. We suggest that this approach might identify variants that confer enhanced pharmacoresponsiveness on a variety of channels.

Evidence for an Extended Hydrogen Bond Network in the Binding Site of the Nicotinic Receptor: ROLE OF THE VICINAL DISULFIDE OF THE α1 SUBUNIT

The defining feature of the α subunits of the family of nicotinic acetylcholine receptors is a vicinal disulfide between Cys-192 and Cys-193. Although this structure has played a pivotal role in a number of pioneering studies of nicotinic receptors, its functional role in native receptors remains uncertain. Using mutant cycle analysis and unnatural residue mutagenesis, including backbone mutagenesis of the peptide bond of the vicinal disulfide, we have established the presence of a network of hydrogen bonds that extends from that peptide NH, across a β turn to another backbone hydrogen bond, and then across the subunit interface to the side chain of a functionally important Asp residue in the non-α subunit. We propose that the role of the vicinal disulfide is to distort the β turn and thereby properly position a backbone NH for intersubunit hydrogen bonding to the key Asp.