Residues In Transmembrane Segments Research Articles

Two rare variants that affect the same amino acid in CFTR have distinct responses to ivacaftor.

Some residues in the cystic fibrosis transmembrane conductance regulator (CFTR) channel are the site of more than one CFTR variant that cause cystic fibrosis. Here, we investigated the function of S1159F and S1159P, two variants associated with different clinical phenotypes, which affect the same pore-lining residue in transmembrane segment 12 that are both strongly potentiated by ivacaftor when expressed in CFBE41o- bronchial epithelial cells. To study the single-channel behaviour of CFTR, we applied the patch-clamp technique to Chinese hamster ovary cells heterologously expressing CFTR variants incubated at 27°C to enhance channel residence at the plasma membrane. S1159F- and S1159P-CFTR formed Cl- channels activated by cAMP-dependent phosphorylation and gated by ATP that exhibited thermostability at 37°C. Both variants modestly reduced the single-channel conductance of CFTR. By severely attenuating channel gating, S1159F- and S1159P-CFTR reduced the open probability (Po ) of wild-type CFTR by ≥75% at ATP (1mM); S1159F-CFTR caused the greater decrease in Po consistent with its more severe clinical phenotype. Ivacaftor (10-100nM) doubled the Po of both CFTR variants without restoring Po values to wild-type levels, but concomitantly, ivacaftor decreased current flow through open channels. For S1159F-CFTR, the reduction of current flow was marked at high (supersaturated) ivacaftor concentrations (0.5-1μM) and voltage-independent, identifying an additional detrimental action of elevated ivacaftor concentrations. In conclusion, S1159F and S1159P are gating variants, which also affect CFTR processing and conduction, but not stability, necessitating the use of combinations of CFTR modulators to optimally restore their channel activity. KEY POINTS: Dysfunction of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes the genetic disease cystic fibrosis (CF). This study investigated two rare pathogenic CFTR variants, S1159F and S1159P, which affect the same amino acid in CFTR, to understand the molecular basis of disease and response to the CFTR-targeted therapy ivacaftor. Both rare variants diminished CFTR function by modestly reducing current flow through the channel and severely inhibiting ATP-dependent channel gating with S1159F exerting the stronger adverse effect, which correlates with its association with more severe disease. Ivacaftor potentiated channel gating by both rare variants without restoring their activity to wild-type levels, but concurrently reduced current flow through open channels, particularly those of S1159F-CFTR. Our data demonstrate that S1159F and S1159P cause CFTR dysfunction by multiple mechanisms that require combinations of CFTR-targeted therapies to fully restore channel function.

Charge-voltage curves of Shaker potassium channel are not hysteretic at steady state.

Charge-voltage curves of many voltage-gated ion channels exhibit hysteresis but such curves are also a direct measure of free energy of channel gating and, hence, should be path-independent. Here, we identify conditions to measure steady-state charge-voltage curves and show that these are curves are not hysteretic. Charged residues in transmembrane segments of voltage-gated ion channels (VGICs) sense and respond to changes in the electric field. The movement of these gating charges underpins voltage-dependent activation and is also a direct metric of the net free-energy of channel activation. However, for most voltage-gated ion channels, the charge-voltage (Q-V) curves appear to be dependent on initial conditions. For instance, Q-V curves of Shaker potassium channel obtained by hyperpolarizing from 0 mV is left-shifted compared to those obtained by depolarizing from a holding potential of -80 mV. This hysteresis in Q-V curves is a common feature of channels in the VGIC superfamily and raises profound questions about channel energetics because the net free-energy of channel gating is a state function and should be path independent. Due to technical limitations, conventional gating current protocols are limited to test pulse durations of <500 ms, which raises the possibility that the dependence of Q-V on initial conditions reflects a lack of equilibration. Others have suggested that the hysteresis is fundamental thermodynamic property of voltage-gated ion channels and reflects energy dissipation due to measurements under non-equilibrium conditions inherent to rapid voltage jumps (Villalba-Galea. 2017. Channels. https://doi.org/10.1080/19336950.2016.1243190). Using an improved gating current and voltage-clamp fluorometry protocols, we show that the gating hysteresis arising from different initial conditions in Shaker potassium channel is eliminated with ultra-long (18-25 s) test pulses. Our study identifies a modified gating current recording protocol to obtain steady-state Q-V curves of a voltage-gated ion channel. Above all, these findings demonstrate that the gating hysteresis in Shaker channel is a kinetic phenomenon rather than a true thermodynamic property of the channel and the charge-voltage curve is a true measure of the net-free energy of channel gating.

Successful Application of Site-directed Mutagenesis Polymerase Chain Reaction to Mutate TMS 11 of the Staphylococcal Multidrug Efflux Protein QacA

Staphylococcus aureus is a major problem in both the clinical setting and within the community. S. aureus can quickly develop resistance to a wide range of antibiotics through a number of different mechanisms, of which, using transporters located in the cell membrane to pump antibiotics out of the cell is the most serious concern. In staphylococcal species, QacA, one such important transporter, is encoded by qacA. QacA is 55kD in size and has 14 transmembrane segments (TMS) (TMS 1-TMS 14). This research describes the mutation process of the amino acid residues in TMS 11 of QacA using site-direct PCR. In this research, 15 primers were successfully designed for site-directed mutagenesis PCR. The site-mutagenesis PCR was successfully conducted to create 15 qacA mutants. These mutants will be used in further functional research of QacA.

Residues in Transmembrane Segments of the P2X4 Receptor Contribute to Channel Function and Ethanol Sensitivity.

Mouse models of alcohol use disorder (AUD) revealed purinergic P2X4 receptors (P2X4Rs) as a promising target for AUD drug development. We have previously demonstrated that residues at the transmembrane (TM)–ectodomain interface and within the TM1 segment contribute to the formation of an ethanol action pocket in P2X4Rs. In the present study, we tested the hypothesis that there are more residues in TM1 and TM2 segments that are important for the ethanol sensitivity of P2X4Rs. Using site-directed mutagenesis and two electrode voltage-clamp electrophysiology in Xenopus oocytes, we found that arginine at position 33 (R33) in the TM1 segment plays a role in the ethanol sensitivity of P2X4Rs. Molecular models in both closed and open states provided evidence for interactions between R33 and aspartic acid at position 354 (D354) of the neighboring TM2 segment. The loss of ethanol sensitivity in mixtures of wild-type (WT) and reciprocal single mutants, R33D:WT and D354R:WT, versus the WT-like response in R33D-D354R:WT double mutant provided further support for this interaction. Additional findings indicated that valine at TM1 position 49 plays a role in P2X4R function by providing flexibility/stability during channel opening. Collectively, these findings identified new activity sites and suggest the importance of TM1-TM2 interaction for the function and ethanol sensitivity of P2X4Rs.

Functional analyses of the UDP-galactose transporter SLC35A2 using the binding of bacterial Shiga toxins as a novel activity assay

SLC35A2 transports UDP-galactose from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum for glycosylation. Mutations in SLC35A2 induce a congenital disorder of glycosylation. Despite the biomedical relevance, mechanisms of transport via SLC35A2 and the impact of disease-associated mutations on activity are unclear. To address these issues, we generated a predicted structure of SLC35A2 and assayed for the effects of a set of structural and disease-associated mutations. Activity assays were performed using a rescue approach in ΔSLC35A2 cells and took advantage of the fact that SLC35A2 is required for expression of the glycosphingolipid globotriaosylceramide (Gb3), the cell surface receptor for Shiga toxin 1 (STx1) and 2 (STx2). The N- and C-terminal cytoplasmic loops of SLC35A2 were dispensable for activity, but two critical glycine (Gly-202 and Gly-214) and lysine (Lys-78 and Lys-297) residues in transmembrane segments were required. Residues corresponding to Gly-202 and Gly-214 in the related transporter SLC35A1 form a substrate-translocating channel, suggesting that a similar mechanism may be involved in SLC35A2. Among the eight disease-associated mutations tested, SLC35A2 function was completely inhibited by two (S213F and G282R) and partially inhibited by three (R55L, G266V, and S304P), providing a straight-forward mechanism of disease. Interestingly, the remaining three (V331I, V258M, and Y267C) did not impact SLC35A2 function, suggesting that complexities beyond loss of transporter activity may underlie disease due to these mutations. Overall, our results provide new insights into the mechanisms of transport of SLC35A2 and improve understanding of the relationship between SLC35A2 mutations and disease.

Loss-of-function variants of SCN8A in intellectual disability without seizures

Objective:To determine the functional effect of SCN8A missense mutations in 2 children with intellectual disability and developmental delay but no seizures.Methods:Genomic DNA was analyzed by next-generation sequencing. SCN8A variants were introduced into the Nav1.6 complementary DNA by site-directed mutagenesis. Channel activity was measured electrophysiologically in transfected ND7/23 cells. The stability of the mutant channels was assessed by Western blot.Results:Both children were heterozygous for novel missense variants that altered conserved residues in transmembrane segments of Nav1.6, p.Gly964Arg in D2S6 and p.Glu1218Lys in D3S1. Both altered amino acids are evolutionarily conserved in vertebrate and invertebrate channels and are predicted to be deleterious. Neither was observed in the general population. Both variants completely prevented the generation of sodium currents in transfected cells. The abundance of Nav1.6 protein was reduced by the Glu1218Lys substitution.Conclusions:Haploinsufficiency of SCN8A is associated with cognitive impairment. These observations extend the phenotypic spectrum of SCN8A mutations beyond their established role in epileptic encephalopathy (OMIM#614558) and other seizure disorders. SCN8A should be considered as a candidate gene for intellectual disability, regardless of seizure status.

Familial gain-of-function Nav1.9 mutation in a painful channelopathy

ObjectiveGain-of-function mutations in Nav1.9 have been identified in three families with rare heritable pain disorders, and in patients with painful small-fibre neuropathy. Identification and functional assessment of new Nav1.9 mutations...

Analysis of conserved NCS2 motifs in the Escherichia coli xanthine permease XanQ.

The xanthine permease XanQ of Escherichia coli is a paradigm for transporters of the evolutionarily broad family nucleobase-cation symporter-2 (NCS2) that transport key metabolites or anti-metabolite analogs. Most functionally known members are xanthine/uric acid transporters related to XanQ and belong to a distinct phylogenetic cluster of the family. Here, we present a comprehensive mutagenesis of XanQ based on the identification and Cys-scanning analysis of conserved sequence motifs in this cluster. Results are interpreted in relation to homology modeling on the structurally known template of UraA and previous data on critical binding-site residues in transmembrane segments (TMs) 3, 8 and 10. The current analysis, of motifs distant to the binding site, revealed a set of functionally important residues in TMs 2, 5, 12 and 13, including seven irreplaceable ones, of which six are Gly residues in the gate domain (159, 369, 370, 383, 409) and in TM2 (Gly-71), and one is polar (Gln-75). Gln-75 (TM2) is probably crucial in a network of hydrogen-bonding interactions in the middle of the core domain involving another essential residue, Asp-304 (TM9). Although the two residues are irreplaceable individually, combinatorial replacement of Gln-75 with Asn and of Asp-304 with Glu rescues significant transport activity.

Cysteine Modification: Probing Channel Structure, Function and Conformational Change.

Cysteine substitution has been a powerful tool to investigate the structure and function of proteins. It has been particularly useful for studies of membrane proteins in their native environment, embedded in phospholipid membranes. Among the 20 amino acids, cysteine is uniquely reactive. This reactivity has motivated the synthesis of a wide array of sulfhydryl reactive chemicals. The commercially available array of sulfhydryl reactive reagents has allowed investigators to probe the local steric and electrostatic environment around engineered cysteines and to position fluorescent, paramagnetic and mass probes at specific sites within proteins and for distance measurements between pairs of sites. Probing the reactivity and accessibility of engineered cysteines has been extensively used in Substituted Cysteine Accessibility Method (SCAM) investigations of ion channels, membrane transporters and receptors. These studies have successfully identified the residues lining ion channels, agonist/antagonist and allosteric modulator binding sites, and regions whose conformation changes as proteins transition between different functional states. The thousands of cysteine-substitution mutants reported in the literature demonstrate that, in general, mutation to cysteine is well tolerated. This has allowed systematic studies of residues in transmembrane segments and in other parts of membrane proteins. Finally, by inserting pairs of cysteines and assaying their ability to form disulfide bonds, changes in proximity and mobility relationships between specific positions within a protein can be inferred. Thus, cysteine mutagenesis has provided a wealth of data on the structure of membrane proteins in their functional environment. This data can complement the structural insights obtained from the burgeoning number of crystal structures of detergent solubilized membrane proteins whose functional state is often uncertain. This article will review the use of cysteine mutagenesis to probe structure-function relationships in ion channels focusing mainly on Cys-loop receptors.

Claudin-3 and Claudin-5 Protein Folding and Assembly into the Tight Junction Are Controlled by Non-conserved Residues in the Transmembrane 3 (TM3) and Extracellular Loop 2 (ECL2) Segments

The mechanism of tight junction (TJ) assembly and the structure of claudins (Cldn) that form the TJ strands are unclear. This limits the molecular understanding of paracellular barriers and strategies for drug delivery across tissue barriers. Cldn3 and Cldn5 are both common in the blood-brain barrier but form TJ strands with different ultrastructures. To identify the molecular determinants of folding and assembly of these classic claudins, Cldn3/Cldn5 chimeric mutants were generated and analyzed by cellular reconstitution of TJ strands, live cell confocal imaging, and freeze-fracture electron microscopy. A comprehensive screening was performed on the basis of the rescue of mutants deficient for strand formation. Cldn3/Cldn5 residues in transmembrane segment 3, TM3 (Ala-127/Cys-128, Ser-136/Cys-137, Ser-138/Phe-139), and the transition of TM3 to extracellular loop 2, ECL2 (Thr-141/Ile-142) and ECL2 (Asn-148/Asp-149, Leu-150/Thr-151, Arg-157/Tyr-158), were identified to be involved in claudin folding and/or assembly. Blue native PAGE and FRET assays revealed 1% n-dodecyl β-d-maltoside-resistant cis-dimerization for Cldn5 but not for Cldn3. This homophilic interaction was found to be stabilized by residues in TM3. The resulting subtype-specific cis-dimer is suggested to be a subunit of polymeric TJ strands and contributes to the specific ultrastructure of the TJ detected by freeze-fracture electron microscopy. In particular, the Cldn5-like exoplasmic face-associated and particle-type strands were found to be related to cis-dimerization. These results provide new insight into the mechanisms of paracellular barrier formation by demonstrating that defined non-conserved residues in TM3 and ECL2 of classic claudins contribute to the formation of TJ strands with differing ultrastructures.

The Unwound Portion Dividing Helix IV of NhaA Undergoes a Conformational Change at Physiological pH and Lines the Cation Passage

pH and Na(+) homeostasis in all cells requires Na(+)/H(+) antiporters. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and their pH regulation. Functional studies of NhaA in the membrane have yielded valuable information regarding its functionality in situ at physiological pH. Here, we Cys-scanned the discontinuous transmembrane segment (TM) IV (helices IVp and IVc connected by an extended chain) of NhaA to explore its functionality at physiological pH. We then tested the accessibility of the Cys replacements to the positively charged SH reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) and the negatively charged 2-sulfonatoethyl methanethiosulfonate (MTSES) in intact cells at pH 8.5 and 6.5 and in parallel tested their accessibility to MTSET in high-pressure membranes at both pH values. We found that the outer membrane of E. coli TA16 acts as a partially permeable barrier to MTSET. Overcoming this technical problem, we revealed that (a) Cys replacement of the most conserved residues of TM IV strongly increases the apparent K(m) of NhaA to both Na(+) and Li(+), (b) the cationic passage of NhaA at physiological pH is lined by the most conserved and functionally important residues of TM IV, and (c) a pH shift from 6.5 to 8.5 induces conformational changes in helix IVp and in the extended chain at physiological pH.

Mutational Analysis and Modeling Reveal Functionally Critical Residues in Transmembrane Segments 1 and 3 of the UapA Transporter

Earlier, we identified mutations in the first transmembrane segment (TMS1) of UapA, a uric acid-xanthine transporter in Aspergillus nidulans, that affect its turnover and subcellular localization. Here, we use one of these mutations (H86D) and a novel mutation (I74D) as well as genetic suppressors of them, to show that TMS1 is a key domain for proper folding, trafficking and turnover. Kinetic analysis of mutants further revealed that partial misfolding and deficient trafficking of UapA does not affect its affinity for xanthine transport, but reduces that of uric acid and confers a degree of promiscuity towards the binding of other purines. This result strengthens the idea that subtle interactions among domains not directly involved in substrate binding refine the selectivity of UapA. Characterization of second-site suppressors of H86D revealed a genetic interaction of TMS1 with TMS3, the latter segment shown for the first time to be important for UapA function. Systematic mutational analysis of polar and conserved residues in TMS3 showed that Ser154 is crucial for UapA transport activity. Our results are in agreement with a topological model of UapA built on the recently published structure of UraA, a bacterial homolog of UapA.

Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A

NBCe1-A and AE1 both belong to the SLC4 HCO(3)(-) transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala(800)-Lys(967). Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met(858) accessible to both biotin maleimide and TAMRA and Thr(926)-Ala(929) only to TAMRA labeling. The intracellular surface contains a highly exposed (Met(813)-Gly(828)) region and a cryptic (Met(887)-Arg(904)) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp(960). Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro(868)-Leu(967) (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1.

Transmembrane Segment Packing of the Na+/Ca2+ Exchanger Investigated with Chemical Cross-Linkers

The Na(+)/Ca(2+) exchanger (NCX1) is a plasma membrane protein important in regulating Ca(2+) in cardiac myocytes. The topological model is comprised of nine transmembrane segments (TMSs). To gain insights into the TMS packing arrangement of NCX1, we performed cysteine cross-linking experiments. Pairs of amino acids in different TMSs were mutated to cysteine on the backbone of a cysteineless NCX1. The mutated exchangers were expressed in an insect cell line and treated with cysteine-specific chemical cross-linkers followed by SDS-PAGE to determine the proximity of the introduced cysteines. Previously, we showed that TMSs 2, 3, 7, and 8 are near one another and that residues in TMSs 1 and 2 are close to TMS 6. In this report, we use the same approach to provide evidence for the arrangement of the remaining three TMSs (4, 5, and 9). We present a computer-generated two-dimensional model of transmembrane packing that minimizes the lengths of all cross-links.

Importance of lipid-exposed residues in transmembrane segment four for family B calcitonin receptor homo-dimerization

Dimerization of the prototypic family B G protein-coupled secretin receptor is determined by the lipid-exposed face of transmembrane segment four (TM4), and has substantial functional importance, facilitating G protein coupling. Recently, we demonstrated that the human secretin receptor elicits an inter-receptor bioluminescence resonance energy transfer (BRET) signal with most other human family B peptide receptors, except for the calcitonin receptor. In this study we have explored the occurrence and importance of calcitonin receptor oligomerization. Static and saturation receptor BRET were utilized to demonstrate that, unlike the human calcitonin receptor that does not yield a significant homomeric BRET signal, the rabbit calcitonin receptor exhibits strong resonance energy transfer. Within the lipid-exposed face of TM4, rabbit and human calcitonin receptors differ by a single amino acid (Arg236 in human; His in rabbit), while Thr253 that occurs in human and rabbit calcitonin receptors is unique across family B receptors. Mutating Arg236 or Thr253 of the human calcitonin receptor to residues found in the rabbit calcitonin receptor or the human secretin receptor (R236H, R236Y and T253A) resulted in generation of significant BRET signals. Similarly, mutation of Val250 of the human calcitonin receptor to another key lipid-facing residue found in the secretin receptor (V250I) also increased the receptor BRET signal. These data support the consistent theme of lipid-exposed residues of TM4 being important for the dimerization of the calcitonin receptor. However, rabbit and human calcitonin receptor constructs bound calcitonin and stimulated cAMP similarly, suggesting that differences in BRET could reflect differences in orientation or in the stability of homo-dimeric receptor complexes, which were nevertheless similarly effective in eliciting the functions attributed to that complex. The likelihood of human calcitonin receptor dimerization, even in the absence of a significant BRET signal, was further supported by data demonstrating that the peptide representing TM4 of this receptor that disrupts the rabbit receptor BRET signal, produced a right shift in the cAMP concentration–response curves for both rabbit and human receptors.

Residues Contributing to the Na+-binding Pocket of the SLC24 Na+/Ca2+-K+ Exchanger NCKX2

Na(+)/Ca(2+)-K(+) exchangers (NCKX; gene family SLC24) are plasma membrane Ca(2+) transporters that mediate the extrusion of one Ca(2+) ion and one K(+) ion in exchange for four Na(+) ions. NCKX is modeled to have two sets of five transmembrane segments separated by a large cytosolic loop; within each set of transmembrane segments are regions of internal symmetry termed alpha(1) and alpha(2) repeats. The central residues that are important for Ca(2+) and K(+) liganding and transport have been identified in NCKX2, and they comprise three central acidic residues, Glu(188) in alpha(1) and Asp(548) and Asp(575) in alpha(2), as well as Ser/Thr residues one-helical turn away from these residues. In this study, we have scanned through more than 100 single-residue substitutions of NCKX2 for shifts in Na(+) affinity using a fluorescence assay to monitor changes in free Ca(2+) in HEK293 cells treated with gramicidin to control intracellular Na(+). We have identified 31 residues that, when substituted, result in shifts in Na(+) affinity, either toward higher or lower K(m) values when compared with wild type NCKX2 (K(m) for Na(+) 58 mm). These residues include the central acidic residues Glu(188), Asp(548), and Asp(575), and their neighboring residues in alpha(1) and alpha(2), in addition to a number of newly investigated residues in transmembrane segment 3. Our results relate the identification of residues important for Na(+) transport in this study to those previously identified as important in the counter-transport of Ca(2+) and K(+), lending support to the alternating access model of transmembrane transport.

Transmembrane Segment II of NhaA Na+/H+ Antiporter Lines the Cation Passage, and Asp65 Is Critical for pH Activation of the Antiporter

The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 5.5-8.5), many questions related to the active state of NhaA have remained elusive. Our experimental results at physiological pH and computational analyses reveal that amino acid residues in transmembrane segment II contribute to the cation pathway of NhaA and its pH regulation: 1) transmembrane segment II is a highly conserved helix and the conserved amino acid residues are located on one side of the helix facing either the cytoplasmic or periplasmic funnels of NhaA structure. 2) Cys replacements of the conserved residues and measuring their antiporter activity in everted membrane vesicles showed that D65C, L67C, E78C, and E82C increased the apparent K(m) to Na+ and Li+ and changed the pH response of the antiporter. 3) Introduced Cys replacements, L60C, N64C, F71C, F72C, and E78C, were significantly alkylated by [14C]N-ethylmaleimide implying the presence of water-filled cavities in NhaA. 4) Several Cys replacements were modified by MTSES and/or MTSET, membrane impermeant, negatively and positively charged reagents, respectively, that could reach Cys replacements from the periplasm only via water-filled funnel(s). Remarkably, the reactivity of D65C to MTSES increased with increasing pH and chemical modification by MTSES but not by MTSET, decreased the apparent K(m) of the antiporter at pH 7.5 (10-fold) but not at pH 8.5, implying the importance of Asp(65) negative charge for pH activation of the antiporter.

Identification of a cluster of residues in transmembrane segment 6 of domain III of the cockroach sodium channel essential for the action of pyrethroid insecticides.

A phenylalanine residue (Phe1519) in the sixth transmembrane segment of domain III (IIIS6) of the cockroach BgNa(v) sodium channel is required for the binding and action of pyrethroids. However, whether or not other residues in IIIS6 participate in the action of pyrethroids remains to be determined. In the present study, we conducted a systematic analysis of 20 residues in IIIS6 of the BgNa(v) channel using alanine-scanning mutagenesis. Our results show that alanine substitutions of four residues, Ile1514, Gly1516, Phe1518 and Asn1522, altered sodium channel sensitivity to pyrethroid insecticides. Whereas the G1516A, F1518A and N1522A substitutions diminished sodium channel sensitivity to all seven pyrethroids examined, including four type I (lacking the alpha-cyano group at the phenoxybenzyl alcohol) and three type II (containing the alpha-cyano group) pyrethroids, the I1514A substitution enhanced sodium channel sensitivity to four type I and type II pyrethroids that contain the phenoxybenzyl alcohol only. We also show that alanine/lysine substitutions of Leu1521 and Ser1517 affected the action of BTX (batrachotoxin), but not pyrethroids. In the Kv1.2-based homology model of the open sodium channel, side chains of Ile1514, Phe1518 and Asn1522 are exposed towards helix IIS5 and linker IIS4-IIS5, which contain previously identified pyrethroid-interacting residues, whereas Ser1517 and Leu1521 face the inner pore where the BTX receptor is located. Thus the present study provides further evidence for structural models in which pyrethroids bind to the lipid-exposed interface formed by helices IIIS6, IIS5 and linker helix IIS4-IIS5, whereas BTX binds to the pore-exposed side of the IIIS6 helix.

CFTR: Differential Reactivity, State-dependent Accessibility And Blocker Occlusion Of Cysteines Substituted For Adjacent Residues In TM6

We and others have identified residues in transmembrane segment six (TM6) of the CFTR chloride channel where substituted cysteines react with externally applied, polar, thiol-directed probes. A scan of TM6 showed that the profile of reactivity differed for channel-permeant reagents, such as [Au(CN)2]− , and channel-impermeant reagents, such as MTSET+ and MTSES−. A cysteine at 338 (T338C) reacted with both channel-impermeant and -permeant probes while a cysteine at 337 (F337C) was unreactive toward channel-impermeant probes but reacted with [Au(CN)2]−. The reaction rate of [Au(CN)2]− was 200 times faster with T338C than with F337C. Furthermore, the reactivity of F337C was highly dependent on the activation state of the channel, being greatly reduced prior to channel activation. T338C exhibited much less pronounced dependence on the activation state. We also compared the ability of a presumed open-channel blocker, GlyH-101, to occlude reactions at the two cysteines. F337C was effectively protected from reaction toward [Au(CN)2]−, whereas the reactivity of T338C was much less sensitive to the presence of the blocker. Finally we used a homology model of CFTR to estimate the relative positions of 337 and 338 in relation to the anion-selective pore. The model predicts that, whereas 338 is positioned such that it would be expected to directly line the pore, 337 could be partially occluded by other helices. Further, a molecular dynamics simulation suggests that movement of the transmembrane helices would significantly alter occlusion of F337C and have much less effect on T338C. These results demonstrate that by combining a quantitative analysis of the reactivity of substituted cysteines with an atomic-scale CFTR model we can begin to reveal the architecture of the CFTR anion-selective pore. Supported by NIH, CF Foundation and American Lung Association.

Prediction of helix–helix contacts and interacting helices in polytopic membrane proteins using neural networks

Despite rapidly increasing numbers of available 3D structures, membrane proteins still account for less than 1% of all structures in the Protein Data Bank. Recent high-resolution structures indicate a clearly broader structural diversity of membrane proteins than initially anticipated, motivating the development of reliable structure prediction methods specifically tailored for this class of molecules. One important prediction target capturing all major aspects of a protein's 3D structure is its contact map. Our analysis shows that computational methods trained to predict residue contacts in globular proteins perform poorly when applied to membrane proteins. We have recently published a method to identify interacting alpha-helices in membrane proteins based on the analysis of coevolving residues in predicted transmembrane regions. Here, we present a substantially improved algorithm for the same problem, which uses a newly developed neural network approach to predict helix-helix contacts. In addition to the input features commonly used for contact prediction of soluble proteins, such as windowed residue profiles and residue distance in the sequence, our network also incorporates features that apply to membrane proteins only, such as residue position within the transmembrane segment and its orientation toward the lipophilic environment. The obtained neural network can predict contacts between residues in transmembrane segments with nearly 26% accuracy. It is therefore the first published contact predictor developed specifically for membrane proteins performing with equal accuracy to state-of-the-art contact predictors available for soluble proteins. The predicted helix-helix contacts were employed in a second step to identify interacting helices. For our dataset consisting of 62 membrane proteins of solved structure, we gained an accuracy of 78.1%. Because the reliable prediction of helix interaction patterns is an important step in the classification and prediction of membrane protein folds, our method will be a helpful tool in compiling a structural census of membrane proteins.