marine drugs

Article The Role of Individual Disulfide Bonds of µ- GIIIA in the Inhibition of NaV1.4

Penggang Han 1,2, Kang Wang 2, Xiandong Dai 2, Ying Cao 2, Shangyi Liu 2, Hui Jiang 2, Chongxu Fan 2,*, Wenjian Wu 1 and Jisheng Chen 1,2 1 College of Science, National University of Defense Technology, Changsha 410073, Hunan, China; [email protected] (P.H.); [email protected] (W.W.); [email protected] (J.C.) 2 Beijing Institute of Pharmaceutical Chemistry, Beijing 102205, China; [email protected] (K.W.); [email protected] (X.D.); [email protected] (Y.C.); [email protected] (S.L.); [email protected] (H.J.) * Correspondence: [email protected]

Academic Editor: Peer B. Jacobson Received: 28 September 2016; Accepted: 11 November 2016; Published: 18 November 2016 Abstract: µ-Conotoxin GIIIA, a peptide isolated from Conus geographus, preferentially blocks the skeletal muscle NaV1.4. GIIIA folds compactly to a pyramidal structure stabilized by three disulfide bonds. To assess the contributions of individual disulfide bonds of GIIIA to the blockade of NaV1.4, seven disulfide-deficient analogues were prepared and characterized, each with one, two, or three pairs of disulfide-bonded Cys residues replaced with Ala. The inhibitory potency of the analogues against NaV1.4 was assayed by whole cell patch-clamp on rNaV1.4, heterologously expressed in HEK293 cells. The corresponding IC50 values were 0.069 ± 0.005 µM for GIIIA, 2.1 ± 0.3 µM for GIIIA-1, 3.3 ± 0.2 µM for GIIIA-2, and 15.8 ± 0.8 µM for GIIIA-3 (-1, -2 and -3 represent the removal of disulfide bridges Cys3–Cys15, Cys4–Cys20 and Cys10–Cys21, respectively). Other analogues were not active enough for IC50 measurement. Our results indicate that all three disulfide bonds of GIIIA are required to produce effective inhibition of NaV1.4, and the removal of any one significantly lowers its sodium channel binding affinity. Cys10–Cys21 is the most important for the NaV1.4 potency.

Keywords: µ-conotoxin; GIIIA; disulfide bond; NaV1.4; electrophysiology

1. Introduction are small, cysteine-rich bioactive peptides derived from the of tropical marine cone snails, genus Conus. Conotoxins have a wide range of pharmacological targets, including different isoforms of ion channels and membrane receptors [1]. Thus, conotoxins are useful research tools and promising drug leads for diseases related to these targets. , ω-conotoxin MVIIA, has been approved by the US Food and Drug administration as a local analgesic for severe chronic pain treatment [2], and several other pain relief drugs based on conotoxins are under preclinical or clinical testing [3,4]. µ-Conotoxins belong to the M superfamily of conopeptides and comprise 16 to 26 residues with six Cys residues arranged in a class III framework (–C1C2–C3–C4–C5C6–) [5]. µ-Conotoxins are the only venom peptides that selectively inhibit voltage-gated sodium channels by sterically and electrostatically blocking the ion flux via binding to the outer vestibule of the channel [6]. The µ-conotoxin GIIIA isolated from Conus geographus was among the first to be characterized. GIIIA consists of 22 amino acids (see Figure1) packed in a pyramidal structure, with three disulfide bonds and three hydroxyproline residues [7]. GIIIA preferentially blocks skeletal muscle sodium channel subtype NaV1.4 by a rather complex toxin–pore interaction, with several residues in the toxin contributing to the high-affinity binding [8].

Mar. Drugs 2016, 14, 213; doi:10.3390/md14110213 www.mdpi.com/journal/marinedrugs Mar. Drugs 2016, 14, 213 2 of 9

Mar.subtype Drugs 2016 Na, V141.4, 213 by a rather complex toxin–pore interaction, with several residues in the 2toxin of 9 contributing to the high‐affinity binding [8].

FigureFigure 1. 1.Amino Amino acid acid sequence sequence andand disulfidedisulfide bonds of of μ‐µ-conotoxinconotoxin GIIIA. GIIIA. *: *: amidated amidated CC‐terminus;-terminus; O: O:trans trans-4-hydroxyproline.‐4‐hydroxyproline.

The most challenging step in the chemical synthesis of disulfide‐rich peptides, such as μ‐ The most challenging step in the chemical synthesis of disulfide-rich peptides, such as µ-conotoxin, conotoxin, is oxidative folding, which is often time‐consuming, low in efficiency and poor in yield is oxidative folding, which is often time-consuming, low in efficiency and poor in yield [9]. [9]. The disulfide bonds in the peptide sequences give rise to a well‐defined and constrained The disulfide bonds in the peptide sequences give rise to a well-defined and constrained framework framework and generally play an important role in their biological action [10]. However, growing and generally play an important role in their biological action [10]. However, growing evidence evidence suggests that many disulfide‐rich peptides do not need all their native disulfide bonds to suggests that many disulfide-rich peptides do not need all their native disulfide bonds to retain retain biological activity [11–13]. For example, the removal of the Cys1–Cys16 bridge in ω‐conotoxin biological activity [11–13]. For example, the removal of the Cys1–Cys16 bridge in ω-conotoxin MVIIA MVIIA did not abolish its binding affinity [14]. The deletion of a disulfide bond can did not abolish its calcium channel binding affinity [14]. The deletion of a disulfide bond can no doubt no doubt accelerate the chemical synthesis of the small neuropeptide and benefit the structure‐ accelerate the chemical synthesis of the small neuropeptide and benefit the structure-function studies. function studies. As a valuable probe for sodium channel research, GIIIA has attracted considerable attention. As a valuable probe for sodium channel research, GIIIA has attracted considerable attention. Previous studies have mainly focused on the key residues in the toxin-channel interaction, but the role Previous studies have mainly focused on the key residues in the toxin‐channel interaction, but the of individual disulfide bonds in the inhibition of NaV1.4 remains ambiguous. In this study, we assessed role of individual disulfide bonds in the inhibition of NaV1.4 remains ambiguous. In this study, we the contributions of the three individual disulfide bonds to the inhibitory activity against Na 1.4. GIIIA assessed the contributions of the three individual disulfide bonds to the inhibitory activityV against and seven analogues, each with one, two, or three pairs of disulfide-bonded Cys residues replaced NaV1.4. GIIIA and seven analogues, each with one, two, or three pairs of disulfide‐bonded Cys with Ala, were prepared, and their blocking potency was assayed using the whole cell patch-clamp residues replaced with Ala, were prepared, and their blocking potency was assayed using the whole technique on HEK293 cells transiently expressing sodium channel subtype rNaV1.4. cell patch‐clamp technique on HEK293 cells transiently expressing sodium channel subtype rNaV1.4. 2. Materials and Methods 2. Materials and Methods 2.1. Materials and Apparatus 2.1. Materials and Apparatus Rink amide resins, Fmoc-protected amino acids, and other reagents for peptide synthesis were purchasedRink from amide GL resins, Biochem, Fmoc Shanghai,‐protected China. amino HPLC-grade acids, and other ACN reagents was obtained for peptide from Sigma-Aldrich, synthesis were St.purchased Louis, MO, from USA. GL LipofectamineBiochem, Shanghai, 2000 China. was purchased HPLC‐grade from ACN Life Technologies,was obtained from Chagrin Sigma Falls,‐Aldrich, OH, USA.St. Louis, All reagents MO, USA. used Lipofectamine were analytical 2000 grade. was Thepurchased HEK293 from cell strainLife Technologies, was offered byChagrin the Cell Falls, Center OH, ofUSA. Chinese All reagents Academy used of Sciences, were analytical Shanghai, grade. China. The Standard HEK293 µcell-conotoxin strain was GIIIA offered was by purchased the Cell Center from Bachem,of Chinese Bubendorf, Academy Switzerland. of Sciences, Shanghai, China. Standard μ‐conotoxin GIIIA was purchased from Bachem,Solid-phase Bubendorf, peptide Switzerland. synthesis was performed on a CEM Liberty peptide synthesizer (CEM, Matthews,Solid‐phase NC, USA). peptide MALDI-TOF-MS synthesis was performed was measured on a onCEM a BrukerLiberty ultraflexpeptide synthesizer TOF/TOF mass(CEM, spectrometerMatthews, NC, (Bruker USA). Daltonics, MALDI Billerica,‐TOF‐MS MA, was USA) measured with α on-cyano-4-hydroxycinnamic a Bruker ultraflex TOF/TOF acid as mass the matrix.spectrometer Reversed-phase (Bruker Daltonics, HPLC was Billerica, performed MA, on USA) an Agilent with α‐ 1100cyano system‐4‐hydroxycinnamic with a dual wavelength acid as UV the detectormatrix. (Agilent,Reversed Santa‐phase Clara, HPLC CA, was USA). performed C18 Vydac on an columns Agilent 1100 were system purchased with from a dual Grace, wavelength Deerfield, UV IL,detector USA. Whole (Agilent, cell Santa patch-clamp Clara, CA, recordings USA). C18 were Vydac conducted columns on were an Axon700Bpurchased amplifierfrom Grace, (Molecular Deerfield, Devices,IL, USA. Sunnyvale, Whole cell CA, patch USA).‐clamp The recordings MF-900 Microforge were conducted and Shutter on an P-97 Axon700B Micropipette amplifier puller (Molecular were the productsDevices, of Sunnyvale, Narishige Group,CA, USA). Tokyo, The Japan MF‐900 and Microforge Sutter Instrument, and Shutter Novato, P‐97 CA, Micropipette USA, respectively. puller were the products of Narishige Group, Tokyo, Japan and Sutter Instrument, Novato, CA, USA, respectively. 2.2. Chemical Synthesis of Peptides 2.2. Chemical Synthesis of Peptides Peptides were synthesized on Rink amide resin using Fmoc chemistry and standard side chain protectionPeptides except were on the synthesized cysteine residues. on Rink Toamide prepare resin GIIIA using analogues Fmoc chemistry containing and two standard disulfide side bonds, chain theprotection peptides except were synthesized on the cysteine using residues. a selective To Cys prepare protection GIIIA scheme. analogues One containing pair of connected two disulfide Cys residuesbonds, the was peptides protected were by tritylsynthesized groups, using and thea selective other pair Cys was protection protected scheme. by Acm One groups. pair of For connected GIIIA andCys other residues analogues, was protected all Cys residues by trityl were groups, trityl-protected. and the other pair was protected by Acm groups. For GIIIAFollowing and other chain analogues, assembly, all Cys the peptidesresidues were partiallytrityl‐protected. deprotected and cleaved from the resin with a mixture of TFA/TIS/H2O (95:2.5:2.5, by volume) for 2 h at room temperature. The cleaved Mar. Drugs 2016, 14, 213 3 of 9 peptides were filtered, precipitated with cold ethyl ether, and washed several times with cold ethyl ether. The released peptides were purified by reversed-phase HPLC on a semi-preparative C18 Vydac column (218TP510, 10 mm × 250 mm) eluted with a gradient of 0%–50% ACN in 0.1% TFA over 50 min. The flow rate was 3 mL/min, and absorbance was monitored at 214 nm. The masses of the linear peptides were validated by MALDI-TOF MS.

2.3. Oxidative Folding of Analogues Containing Two Disulfide Bonds The purified linear peptides contain two Acm-protected Cys and two free thiols. A two-step oxidation protocol was used to selectively fold the peptides. To promote disulfide bond formation between the free thiols, the peptides were diluted to a final concentration of 0.2 mg/mL with 0.2 M aqueous ammonium bicarbonate (pH 7.8) and stirred moderately at room temperature. Oxidation reactions were analyzed by HPLC and judged to be complete when the HPLC profiles reached a steady equilibrium state with one main peak. After the first oxidation step, the peptides were concentrated and purified by semi-preparative HPLC as described above. To remove the Acm groups from the other pair of cysteines and close the remaining disulfide bridge, the monocyclic peptides were treated with 2 mM iodine dissolved in 50% aqueous ACN containing 0.1% TFA. The progress of Acm-deprotection and subsequent disulfide bond formation was monitored by analytical HPLC at regular intervals until completion (approximately 30 min). The reaction was then quenched by addition of a 5 mg/mL ascorbic acid solution, resulting in decolorization of the solution. The fully oxidized peptides were then subjected to semi-preparative HPLC. The purities of the folded peptides were assessed by analytical HPLC using a C18 Vydac column (218TP54, 4.6 mm × 250 mm) with a gradient of 0%–30% ACN in 0.1% TFA in 30 min at 1 mL/min. GIIIA and other analogues were oxidized by one-step air oxidation and purified by HPLC as described above. The identities of all the final products were confirmed by MALDI-TOF MS.

2.4. Plasmid Constructions

SCN4A, encoding the α subunit of rNaV1.4, was cloned from rat skeletal muscle cDNA and inserted into the multiple cloning sites of the pcDNA3.1+ vector using a PCR-based strategy. + The pcDNA3.1 -rNaV1.4 plasmid was purified and confirmed by DNA sequencing analysis. The pcDNA3.1+-EGFP plasmid was constructed similarly.

2.5. Cell Culture and Transient Expression in HEK293 Cells HEK293 cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum and 1% penicillin–streptomycin. Cells were maintained at 37 ◦C in a humidified incubator with 95% air and 5% CO2. HEK293 cells were trypsinized, diluted with culture medium, and grown in 35-mm culture dishes. When grown to >60% confluence, cells were transfected with + + a 5:1 ratio of pcDNA3.1 -rNaV1.4 and pcDNA3.1 -EGFP using the Lipofectamine 2000 transfection kit. Approximately 24 h after transfection, cells displaying green fluorescence were selected for electrophysiological recordings.

2.6. Electrophysiology Whole cell patch-clamp recordings from HEK293 cells were performed using an Axon700B amplifier controlled by a pClamp10.2 data acquisition system [15,16]. For the recordings, the cells were perfused with extracellular solution containing 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM D-glucose, and 10 mM HEPES. The pH of the solution was adjusted to 7.4 using NaOH. Patch pipettes had resistances of 2–4 MΩ, and the series resistance was compensated for by approximately 60% to minimize voltage errors. The pipettes were filled with intracellular solution containing 50 mM CsCl, 10 mM NaCl, 60 mM CsF, 2 mM MgCl2, 20 mM EGTA, and 10 mM HEPES with the pH adjusted to 7.2 by the addition of CsOH. Stock solutions of GIIIA and the analogues were diluted to the final concentrations in extracellular solution and applied through the perfusion system. Mar. Drugs 2016, 14, 213 4 of 9 Mar.Mar. Drugs Drugs 20162016,, 14,,, 14213, 213 4 of4 9 of 9 Mar. Drugs 2016, 14, 213 4 of 9 diluteddiluted toto thethe finalfinal concentrationsconcentrations inin extracellularextracellular solutionsolution andand appliedapplied throughthrough thethe perfusionperfusion system.system. diluted to the final concentrations in extracellular solution and applied through the perfusion system. SodiumSodiumSodium currentscurrents currents werewere were evokedevoked evoked byby by aa depolarizationdepolarization a depolarization pulsepulse pulse fromfrom from aa holdingholding a holding potentialpotential potential ((−−80 (80− 80mV)mV) mV) toto a toa testtest a test Sodium currents were evoked by a depolarization pulse from a holding potential (−80 mV) to a test voltagevoltagevoltage ((−−1010 (− mV).mV).10 mV). TheThe The pulsepulse pulse durationduration duration waswas was 5050 ms 50ms ms withwith with anan interval aninterval interval ofof 55 of s.s. 5 OutputOutput s. Output signalssignals signals werewere werew w lolo low-pass‐‐passpass voltage (−10 mV). The pulse duration was 50 ms with an interval of 5 s. Output signals werew lo ‐pass filteredvoltage at(− 105 kHzmV). and The sampled pulse duration at 10 kHz. was All50 ms experiments with an interval were performedof 5 s. Output at constantsignals were temperature,w lo ‐pass filteredfiltered at at5 kHz 5 kHz and and sampled sampled at at10 10kHz. kHz. All All experiments experiments were were performed performed at constant at constant temperature, temperature, filtered at◦ 5 kHz and sampled at 10 kHz. All experiments were performed at constant temperature, 21–2421–2421–24 °C.°C. C. 21–24 °C. Individual IC5050 values were calculated by fitting dose–response data to the following Hillslope IndividualIndividual IC50 IC values50 values were werecalculated calculated by fitting by dose–response fitting dose–response data to the data following to the Hillslope following Individual IC50 values were calculated by fitting dose–response data to the following Hillslope equation:equation:Hillslope equation: equation: I/Imax = 1/(1 + ([toxin]/IC50) ˆ Hillslope) I/Imax = 1 (1 + ([toxin]/IC5050) ^ Hillslope) I/Imax = 1 (1 + ([toxin]/IC50) ^ Hillslope) I/Imax = 1 (1 + ([toxin]/IC50) ^ Hillslope) wherewhere Imax Imax and and I are I are the the peak peak sodium sodium currents currents in thethe absenceabsence and and presence presence of of toxin, toxin, [toxin] [toxin] is theis the drug where Imax and I are the peak sodium currents in the absence and presence of toxin, [toxin] is the whereconcentration, Imax and I and are Hillslope the peak issodium the fitting currents coefficients. in the absence and presence of toxin, [toxin] is the drugdrug concentration,concentration, andand HillslopeHillslope isis thethe fittingfitting coefficients.coefficients. drug concentration, and Hillslope is the fitting coefficients. drugElectrophysiological concentration,Electrophysiological and Hillslope data data were were is analyzed the analyzed fitting using coefficients. using the the Clampfit10.2 Clampfit10.2 (Molecular (Molecular Devices, Devices, Sunnyvale, Sunnyvale, Electrophysiological data were analyzed using the Clampfit10.2 (Molecular Devices, Sunnyvale, CA,Electrophysiological USA) and Sigmaplot10.0 data were (Systat analyzed Software, using Inc., the San Clampfit10.2 Jose, CA, USA) (Molecular software. Devices, Data were Sunnyvale, presented CA,CA, USA)USA) andand Sigmaplot10.0Sigmaplot10.0 (Systat(Systat Software,Software, Inc.,Inc., SanSan Jose,Jose, CA,CA, USA)USA) sofsoftware.tware. DataData werewere CA, USA) and Sigmaplot10.0 (Systat Software, Inc., San Jose, CA, USA) software.tware.tware.tware. Data were presentedCA,as theUSA) mean as and the± Sigmaplot10.0meanSEM with ± SEM n = with number (Systat n = of numberSoftware, independent of independentInc., measurements. San Jose, measurements. CA, Datasets USA) were sofDatasetstware.tware. tested wereData for statistical testedwere presented as the mean ± SEM with n = number of independent measurements. Datasets were tested presentedsignificance as the using mean the ± Student’sSEM witht ntest, = number with p

The linear forms of the analogues were chemically synthesized with an automated peptide TheThe linearlinear formsforms ofof thethe analoguesanalogues werewere chemicallychemically synthesizedsynthesized withwith anan automatedautomated peptidepeptide synthesizerThe linear using forms Fmoc-based of the analogues protocols, were as chemically described insynthesized Materials andwith Methods. an automated The Rink peptide amide synthesizersynthesizer usingusing FmocFmoc‐‐basedbased protocols,protocols, asas describeddescribed inin MaterialsMaterials andand Methods.Methods. TheThe RinkRink amideamide synthesizerresin was using used asFmoc a solid‐based support, protocols, on whichas described the first in amino Materials acid and was Methods. loaded. The The linear Rink amide peptides resinresin waswas usedused asas aa solidsolid support,support, onon whichwhich thethe firstfirst aminoamino acidacid waswas loaded.loaded. TheThe linearlinear peptidespeptides resinprepared was used by reversed-phase as a solid support, HPLC on were which characterized the first amino by mass acid spectrometry was loaded. to determineThe linear their peptides identity. preparedprepared byby reversedreversed‐‐phasephase HPLCHPLC werewere charcharacterizedacterizedacterized byby massmass spectrometryspectrometry toto determinedetermine theirtheir preparedFor analogues by reversed containing‐phase two HPLC disulfide were bonds,characterizedacterizedacterizedacterizedacterized a strategy by combiningmass spectrometry orthogonal to cysteinedetermine protection their identity.identity. ForFor analoguesanalogues containingcontaining twotwo disulfidedisulfide bonds,bonds, aa strategystrategy combiningcombining orthogonalorthogonal cysteinecysteine identity.with two-step For analogues oxidation containing was adopted two to disulfide ensure the bonds, correct a formationstrategy combining of the native orthogonal disulfide bridges cysteine [17 ], protectionprotection withwith twotwo‐‐stepstep oxidationoxidation waswas adoptedadopted toto ensureensure thethe correctcorrect formationformation ofof thethe nativenative protectionas illustrated with fortwo GIIIA-3‐step oxidation in Figure was2. Formation adopted ofto theensure first disulfidethe correct bridge formation was accomplished of the native by disulfidedisulfide bridgesbridges [17],[17], asas illustratedillustrated forfor GIIIAGIIIA‐‐33 inin FigureFigure 2.2. FormationFormation ofof thethe firstfirst disulfidedisulfide bridgebridge disulfideammonium bridges bicarbonate-mediated [17], as illustrated for air GIIIA oxidation,‐3 in whereasFigure 2. the Formation second bridge of the wasfirst closed disulfide using bridge iodine. waswas accomplishedaccomplished byby amammoniummonium bicarbonatebicarbonate‐‐mediatedmediated airair oxidation,oxidation, whereaswhereas thethe secondsecond bridgebridge was accomplished by ammonium bicarbonate‐mediated air oxidation, whereas the second bridge waswas closedclosed usingusing iodine.iodine. GIIIAGIIIA andand remainingremaining analoguesanalogues werewere foldedfolded directlydirectly byby airair oxidation.oxidation. TheThe was closed using iodine. GIIIA and remaining analogues were folded directly by air oxidation. The Mar. Drugs 2016, 14, 213 5 of 9

Mar. Drugs 2016, 14, 213 5 of 9 GIIIAMar. Drugs and 2016 remaining, 14, 213 analogues were folded directly by air oxidation. The final products were5 of 9 purifiedfinal products by HPLC were and purified confirmed by HPLC by MALDI-TOF-MS and confirmed with by MALDI GIIIA: ‐mTOF/z 2606.9‐MS with (calculated GIIIA: m =/z 2607.2), 2606.9 final products were purified by HPLC and confirmed by MALDI‐TOF‐MS with GIIIA: m/z 2606.9 GIIIA-1/GIIIA-2:(calculated = 2607.2),m/z GIIIA2545.1‐1/GIIIA (calculated‐2: m =/z 2545.2),2545.1 (calculated GIIIA-3: m =/ z2545.2),2545.2 (calculatedGIIIA‐3: m/z = 2545.2 2545.2), (calculated GIIIA-12: (calculated = 2607.2), GIIIA‐1/GIIIA‐2: m/z 2545.1 (calculated = 2545.2), GIIIA‐3: m/z 2545.2 (calculated m= /2545.2),z 2483.1 GIIIA (calculated‐12: m/z =2483.1 2483.3), (calculated GIIIA-13: = 2483.3),m/z 2483.3 GIIIA (calculated‐13: m/z 2483.3 = 2483.3), (calculated GIIIA-23: = 2483.3),m/z 2483.2GIIIA‐ = 2545.2), GIIIA‐12: m/z 2483.1 (calculated = 2483.3), GIIIA‐13: m/z 2483.3 (calculated = 2483.3), GIIIA‐ (calculated23: m/z 2483.2 = 2483.3), (calculated GIIIA-123: = 2483.3),m/ zGIIIA2422.3‐123: (calculated m/z 2422.3 = 2422.3).(calculated GIIIA = 2422.3). was identical GIIIA was to the identical standard to 23: m/z 2483.2 (calculated = 2483.3), GIIIA‐123: m/z 2422.3 (calculated = 2422.3). GIIIA was identical to GIIIAthe standard according GIIIA to HPLC according analysis. to FigureHPLC 3analysis. shows the Figure HPLC 3 elution shows profiles the HPLC and correspondingelution profiles mass and the standard GIIIA according to HPLC analysis. Figure 3 shows the HPLC elution profiles and spectracorresponding of GIIIA mass and GIIIA-3.spectra of GIIIA and GIIIA‐3. corresponding mass spectra of GIIIA and GIIIA‐3.

Figure 2. Synthesis and oxidation strategy for GIIIA‐3. GIIIA‐3 was chemically synthesized using two Figure 2. Synthesis and oxidation strategy for GIIIA-3.GIIIA‐3. GIIIA GIIIA-3‐3 was chemically synthesized using two sets of Cys thiol‐protecting groups. Cys3 and Cys15 were protected with trityl groups, while Cys4 setssets ofof CysCys thiol-protecting thiol‐protecting groups. groups. Cys3 Cys3 and and Cys15 Cys15 were were protected protected with with trityl trityl groups, groups, while while Cys4 Cys4 and and Cys20 were protected with Acm groups. A two‐step oxidation protocol was used to selectively Cys20and Cys20 were were protected protected with Acmwith groups.Acm groups. A two-step A two oxidation‐step oxidation protocol protocol was used was to used selectively to selectively fold the linearfold the peptide. linear peptide. Air oxidation Air oxidation was applied was to applied form the to disulfideform the bonddisulfide Cys3–Cys15, bond Cys3–Cys15, and iodine and oxidation iodine fold the linear peptide. Air oxidation was applied to form the disulfide bond Cys3–Cys15, and iodine wasoxidation used towas remove used to Acm remove protection Acm protection and close and the secondclose the disulfide second bonddisulfide Cys4–Cys20. bond Cys4–Cys20. * denotes * oxidation was used to remove Acm protection and close the second disulfide bond Cys4–Cys20. * Cdenotes-terminal C‐terminal amide. amide. denotes C‐terminal amide.

Figure 3. HPLC elution profiles and MALDI‐TOF mass spectra of GIIIA and GIIIA‐3. Final folded Figure 3. HPLC elution profiles and MALDI‐TOF mass spectra of GIIIA and GIIIA‐3. Final folded Figurepeptides 3. wereHPLC dissolved elution profilesin 0.1% TFA, and MALDI-TOFthen applied to mass a C18 spectra analytical of GIIIA column and and GIIIA-3. eluted Finalwith a folded linear peptides were dissolved in 0.1% TFA, then applied to a C18 analytical column and eluted with a linear peptidesgradient wereof 0%–30% dissolved ACN in in 0.1% 0.1% TFA, TFA thenat 1 mL/min applied over to a C1830 min. analytical Peaks were column collected and eluted for MALDI with a‐ gradient of 0%–30% ACN in 0.1% TFA at 1 mL/min over 30 min. Peaks were collected for MALDI‐ linearTOF‐MS gradient analysis. of 0%–30%The MS data ACN (MH in 0.1%+, 2607.9 TFA for at GIIIA, 1 mL/min 2546.2 over for 30GIIIA min.‐3) Peakswere consistent were collected with forthe TOF‐MS analysis. The MS data (MH+, 2607.9 +for GIIIA, 2546.2 for GIIIA‐3) were consistent with the MALDI-TOF-MScalculated mass, indicating analysis. The that MS the datapeptides (MH were, 2607.9 correctly for GIIIA, synthesized. 2546.2 for GIIIA-3) were consistent withcalculated the calculated mass, indicating mass, indicating that the peptides that the peptides were correctly were correctly synthesized. synthesized.

3.2. Effect of the Analogues on rNaV1.4 Expressed in HEK293 Cells 3.2. Effect of the Analogues on rNaV1.4 Expressed in HEK293 Cells 3.2. Effect of the Analogues on rNaV1.4 Expressed in HEK293 Cells It has been demonstrated that GIIIA is a selective blocker of the skeletal muscle subtype NaV1.4 It has been demonstrated that GIIIA is a selective blocker of the skeletal muscle subtype NaV1.4 [7]. Therefore,It has been we demonstrated assessed the that biological GIIIA is activity a selective of seven blocker disulfide of the skeletal‐deficient muscle analogues subtype by measuring NaV1.4 [7]. [7]. Therefore, we assessed the biological activity of seven disulfide‐deficient analogues by measuring Therefore,their potency we assessedfor blocking the biological rNaV1.4, expressed activity of sevenin HEK293 disulfide-deficient cells by whole analogues cell patch by‐clamp measuring protocols. their their potency for blocking rNaV1.4, expressed in HEK293 cells by whole cell patch‐clamp protocols. potencyControl forexperiments blocking rNa wereV1.4, performed expressed on in more HEK293 than cells 100 cells by whole not transfected cell patch-clamp with rNa protocols.V1.4 expressing Control Control experiments were performed on more than 100 cells not transfected with rNaV1.4 expressing experimentsplasmids, and were no performedNa+ currents on were more detected. than 100 cells not transfected with rNaV1.4 expressing plasmids, plasmids, and no Na+ currents were detected. and noRepresentative Na+ currents current were detected. traces in the absence and presence of 100 nM GIIIA, 5 μM GIIIA‐1, GIIIA‐ Representative current traces in the absence and presence of 100 nM GIIIA, 5 μM GIIIA‐1, GIIIA‐ 2, GIIIA‐3 are shown in Figure 4. The three analogues inhibit the Na+ currents in a similar way to 2, GIIIA‐3 are shown in Figure 4. The three analogues inhibit the Na+ currents in a similar way to GIIIA. The remaining currents do not show any obvious alterations in kinetics. GIIIA. The remaining currents do not show any obvious alterations in kinetics. Mar. Drugs 2016, 14, 213 6 of 9

Representative current traces in the absence and presence of 100 nM GIIIA, 5 µM GIIIA-1, GIIIA-2, GIIIA-3 are shown in Figure4. The three analogues inhibit the Na + currents in a similar way to GIIIA. TheMar. remainingDrugs 2016, 14 currents, 213 do not show any obvious alterations in kinetics. 6 of 9

Figure 4. Effect of GIIIA and its analogues on rNaV1.4 expressed in HEK293 cells. Sodium currents Figure 4. Effect of GIIIA and its analogues on rNaV1.4 expressed in HEK293 cells. Sodium currents were elicited by a depolarizing step from a holding potential of −80 mV to a test voltage of −10 mV were elicited by a depolarizing step from a holding potential of −80 mV to a test voltage of −10 mV from HEK293 cells expressing rNaV1.4. Representative current traces are shown in the absence from HEK293 cells expressing rNaV1.4. Representative current traces are shown in the absence (Control) (Control) and presence of (A) 100 nM GIIIA; (B) 5 μM GIIIA‐1, GIIIA‐2, GIIIA‐3. and presence of (A) 100 nM GIIIA; (B) 5 µM GIIIA-1, GIIIA-2, GIIIA-3.

The inhibitory potency of the analogues was measured at various concentrations, and the The inhibitory potency of the analogues was measured at various concentrations, and the electrophysiological data were fitted to the Hillslope equation, yielding corresponding IC50 values, as electrophysiological data were fitted to the Hillslope equation, yielding corresponding IC50 values, listed in Table 2. GIIIA inhibits the depolarization‐evoked NaV1.4 currents in a concentration‐ as listed in Table2. GIIIA inhibits the depolarization-evoked Na V1.4 currents in a concentration- dependent manner (Figure 5) with an IC50 of 0.069 ± 0.005 μM, similar to the values reported dependent manner (Figure5) with an IC of 0.069 ± 0.005 µM, similar to the values reported previously [18,19]. GIIIA‐12, GIIIA‐13, and50 GIIIA‐23, which lost two disulfide bonds, and the linear previously [18,19]. GIIIA-12, GIIIA-13, and GIIIA-23, which lost two disulfide bonds, and the linear peptide GIIIA‐123, did not exhibit any significant blockade at 100 μM, suggesting that they cannot peptide GIIIA-123, did not exhibit any significant blockade at 100 µM, suggesting that they cannot adopt an appropriate conformation to inhibit the sodium channel. The potency of the analogues adopt an appropriate conformation to inhibit the sodium channel. The potency of the analogues containing two of three disulfide bonds against NaV1.4 decreased in the following order: GIIIA‐1> containing two of three disulfide bonds against NaV1.4 decreased in the following order: GIIIA-1 > GIIIA‐2 > GIIIA‐3. The corresponding IC50 values were 2.1 ± 0.3 μM for GIIIA‐1, 3.3 ± 0.2 μM for GIIIA-2 > GIIIA-3. The corresponding IC values were 2.1 ± 0.3 µM for GIIIA-1, 3.3 ± 0.2 µM for GIIIA‐2, and 15.8 ± 0.8 μM for GIIIA‐3, indicating50 that among the three individual disulfide bridges, GIIIA-2, and 15.8 ± 0.8 µM for GIIIA-3, indicating that among the three individual disulfide bridges, Cys10–Cys21 is the most important for the blockade of NaV1.4, while Cys3–Cys15 is the least Cys10–Cys21 is the most important for the blockade of Na 1.4, while Cys3–Cys15 is the least important. important. However, the most potent analogue, GIIIA‐1,V missing the Cys3–Cys15 disulfide bridge, However, the most potent analogue, GIIIA-1, missing the Cys3–Cys15 disulfide bridge, still reduced still reduced the inhibition of NaV1.4 by more than 30‐fold, and the removal of Cys10–Cys21 reduced thethe inhibitionpotency approximately of NaV1.4 by more200‐fold. than This 30-fold, result and suggests the removal that all of three Cys10–Cys21 disulfide reduced bonds of the GIII potency A are approximately 200-fold. This result suggests that all three disulfide bonds of GIII A are needed to needed to produce effective inhibition of NaV1.4, and the specific conformation stabilized by the three producedisulfide effective bonds is inhibition essential for of Na theV 1.4,blockade. and the specific conformation stabilized by the three disulfide bonds is essential for the blockade.

Table 2. The inhibitory potency of GIIIA and its analogues against rNaV1.4.

Peptides IC50 (µM) * Inhibitory Potency GIIIA 0.069 ± 0.005 1 GIIIA-1 2.1 ± 0.3 0.033 GIIIA-2 3.3 ± 0.2 0.021 GIIIA-3 15.8 ± 0.8 0.005 GIIIA-12 >100 <0.0007 GIIIA-13 >100 <0.0007 GIIIA-23 >100 <0.0007 GIIIA-123 >100 <0.0007 * Data are mean ± SEM from at least three cells.

Figure 5. Concentration–response curves obtained for the inhibition of rNaV1.4‐mediated Na+ currents

by GIIIA (), GIIIA‐1(), GIIIA‐2 (★) and GIIIA‐3 (). Corresponding IC50 values were 0.069 ± 0.005 μM for GIIIA, 2.1 ± 0.3 μM for GIIIA‐1, 3.3 ± 0.2 μM for GIIIA‐2, and 15.8 ± 0.8 μM for GIIIA‐3. Values are mean ± SEM from three to five separate cells. Mar. Drugs 2016, 14, 213 6 of 9

Figure 4. Effect of GIIIA and its analogues on rNaV1.4 expressed in HEK293 cells. Sodium currents were elicited by a depolarizing step from a holding potential of −80 mV to a test voltage of −10 mV

from HEK293 cells expressing rNaV1.4. Representative current traces are shown in the absence (Control) and presence of (A) 100 nM GIIIA; (B) 5 μM GIIIA‐1, GIIIA‐2, GIIIA‐3.

The inhibitory potency of the analogues was measured at various concentrations, and the electrophysiological data were fitted to the Hillslope equation, yielding corresponding IC50 values, as listed in Table 2. GIIIA inhibits the depolarization‐evoked NaV1.4 currents in a concentration‐ dependent manner (Figure 5) with an IC50 of 0.069 ± 0.005 μM, similar to the values reported previously [18,19]. GIIIA‐12, GIIIA‐13, and GIIIA‐23, which lost two disulfide bonds, and the linear peptide GIIIA‐123, did not exhibit any significant blockade at 100 μM, suggesting that they cannot adopt an appropriate conformation to inhibit the sodium channel. The potency of the analogues containing two of three disulfide bonds against NaV1.4 decreased in the following order: GIIIA‐1> GIIIA‐2 > GIIIA‐3. The corresponding IC50 values were 2.1 ± 0.3 μM for GIIIA‐1, 3.3 ± 0.2 μM for GIIIA‐2, and 15.8 ± 0.8 μM for GIIIA‐3, indicating that among the three individual disulfide bridges, Cys10–Cys21 is the most important for the blockade of NaV1.4, while Cys3–Cys15 is the least important. However, the most potent analogue, GIIIA‐1, missing the Cys3–Cys15 disulfide bridge, still reduced the inhibition of NaV1.4 by more than 30‐fold, and the removal of Cys10–Cys21 reduced the potency approximately 200‐fold. This result suggests that all three disulfide bonds of GIII A are needed to produce effective inhibition of NaV1.4, and the specific conformation stabilized by the three Mar. Drugs 2016, 14, 213 7 of 9 disulfide bonds is essential for the blockade.

+ Figure 5. Concentration–response curves obtained for the inhibition of rNaV1.4‐mediated Na currents + Figure 5. Concentration–response curves obtained for the inhibition of rNaV1.4-mediated Na by GIIIA (), GIIIA‐1(), GIIIA‐2 (★) and GIIIA‐3 (). Corresponding IC50 values were 0.069 ± 0.005 currents by GIIIA (), GIIIA-1 ( ), GIIIA-2 (F) and GIIIA-3 (+). Corresponding IC50 values were μM for GIIIA, 2.1 ± 0.3 μM for GIIIA‐1, 3.3 ± 0.2 μM for GIIIA‐2, and 15.8 ± 0.8 μM for GIIIA‐3. Values 0.069 ± 0.005 µM for GIIIA, 2.1 ± 0.3 µM for GIIIA-1, 3.3 ± 0.2 µM for GIIIA-2, and 15.8 ± 0.8 µM for are mean ± SEM from three to five separate cells. GIIIA-3. Values are mean ± SEM from three to five separate cells.

4. Discussion This research investigated the role of individual disulfide bonds of GIIIA in the inhibition of NaV1.4. Our data demonstrate that all three disulfide bonds are important in forming the specific conformation of the molecule for exerting the biological activity, and depleting any one would significantly reduce its NaV1.4 potency. In particular, the removal of the Cys10–Cys21 bridge produced an approximately 200-fold loss in binding affinity for NaV1.4. Mutational studies on GIIIA have proven that Arg13 is a key residue for the blockade of channel function [20–22]. Although Arg13 plays a prominent role, it has been verified that other residues including Arg1, Lys11, Gln14, Lys16, Hyp17 and Arg19 also affect the bioactivity [23–25]. Recently, homology models of mammalian NaV1.4 based on the X-ray crystal structures of several bacteria voltage-gated sodium channels were constructed to study GIIIA binding to NaV1.4 [26,27]. The binding mode of the GIIIA-NaV1.4 complex indicates that the toxin blocks the pore through multiple interactions of Arg13, Lys16, and Arg19 with the acidic residues in the channel vestibule [26–28]. We deduce that the rigidity of GIIIA maintained by three disulfide bonds are important for NaV1.4 inhibition. The removal of any one disulfide bridge would cause a loss of rigidity, reducing the affinity of the peptide to the pore. NaV1.4 blockade by different disulfide isomers of µ-conotoxin PIIIA was investigated [29]. Tietze and coworkers [8] found that three disulfide-bridged peptide isomers, termed PIIIA-1, -2 and -3, were able to block NaV1.4 with considerable, yet distinguishable potency. PIIIA-1, with 1–5/2–4/3–6 connectivity, was twice as potent as PIIIA-2, which adopted the same 1–4/2–5/3–6 pattern as PIIIA [30]. PIIIA-3, with 1–2/3–4/5–6 connectivity, retained about 50% activity of PIIIA. NMR analysis and molecular dynamic simulations revealed that the key residue R14 of PIIIA-1 binds much deeper in the channel pore than PIIIA and PIIIA-3 [29]. This result offered straightforward proof that disulfide bridges affect the docking of amino acid residues to the pore. Moreover, consistently with our results, the reduced PIIIA was not active [29]. This finding also suggested that the rigid structure maintained by three disulfide bridges is necessary for the sodium channel block. In another study, the contributions of individual disulfide bridges in µ-conotoxin BuIIIB to NaV1.3 inhibition were assessed [31]. BuIIIB shares the 1–4/2–5/3–6 disulfide pattern with the following disulfide bridges: Cys5–Cys17 (1–4), Cys6–Cys23 (2–5), and Cys13–Cys24 (3–6) [31,32]. In contrast to GIIIA, the study suggested that the removal of the Cys5–Cys17 bridge reduced the blockade potency by 80-fold, whereas removal of either the Cys6–Cys23 or Cys13–Cys24 bridges caused only a slight impact on affinity. This result indicated that the Cys5–Cys17 bridge in BuIIIB is the most important disulfide Mar. Drugs 2016, 14, 213 8 of 9

bridge for NaV1.3 inhibition [31]. The systematic study of the binding modes of GIIIA and BuIIIB to the NaV1 channels reveals a common binding motif for the blockade [28]. The binding motif consists of three corresponding basic residues that form contacts with the channel pore [28]. Intriguingly, despite sharing a common motif and same disulfide pattern, the roles of the individual disulfides bridges of GIIIA and BuIIIB in the NaV1 inhibition are different. This finding may be attributed to the differences in their tertiary structure. In conclusion, our study clearly shows that all three disulfide bonds in GIIIA are needed to produce effective inhibition of NaV1.4 and validates the important contribution of individual disulfide bonds to toxin-NaV1.4 interactions. These results will provide valuable information, making it possible to elucidate the mechanisms of GIIIA binding to the sodium channel NaV1.4 and design analogues with better affinity and selectivity for therapeutic use.

Author Contributions: J.C. and C.F. conceived and designed the experiments; P.H. performed the experiments; K.W. and S.L. analyzed the data; X.D. and Y.C. assisted with sample preparation; H.J. contributed analysis tools; W.W. revised and edited the paper; C.F. and P.H. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

References

1. Lewis, R.J.; Dutertre, S.; Vetter, I.; Christie, M.J. Conus venom peptide pharmacology. Pharmacol. Rev. 2012, 64, 259–298. [CrossRef][PubMed] 2. Wermeling, D.P. Ziconotide, an intrathecally administered N-type calcium channel antagonist for the treatment of chronic pain. Pharmacotherapy 2005, 25, 1084–1094. [CrossRef][PubMed] 3. Callaghan, B.; Haythornthwaite, A.; Berecki, G.; Clark, R.J.; Craik, D.J.; Adams, D.J. Analgesic alpha-conotoxins Vc1.1 and Rg1A inhibit N-type calcium channels in rat sensory neurons via GABAB receptor activation. J. Neurosci. 2008, 28, 10943–10951. [CrossRef][PubMed] 4. Carstens, B.B.; Clark, R.J.; Daly, N.L.; Harvey, P.J.; Kaas, Q.; Craik, D.J. Engineering of conotoxins for the treatment of pain. Curr. Pharm. Des. 2011, 17, 4242–4253. [CrossRef][PubMed] 5. Knapp, O.; McArthur, J.R.; Adams, D.J. Conotoxins targeting neuronal voltage-gated sodium channel subtypes: Potential analgesics? (Basel) 2012, 4, 1236–1260. [CrossRef][PubMed] 6. Hui, K.; Lipkind, G.; Fozzard, H.A.; French, R.J. Electrostatic and steric contributions to block of the skeletal muscle sodium channel by mu-conotoxin. J. Gen. Physiol. 2002, 119, 45–54. [CrossRef][PubMed] 7. Hidaka, Y.; Sato, K.; Nakamura, H.; Kobayashi, J.; Ohizumi, Y.; Shimonishi, Y. Disulfide pairings in geographutoxin I, a peptide from Conus geographus. FEBS Lett. 1990, 264, 29–32. [CrossRef] 8. Norton, R.S. Mu-conotoxins as leads in the development of new analgesics. Molecules 2010, 15, 2825–2844. [CrossRef][PubMed] 9. Kang, T.S.; Radic, Z.; Talley, T.T.; Jois, S.D.; Taylor, P.; Kini, R.M. Protein folding determinants: Structural features determining alternative disulfide pairing in alpha- and chi/lambda-conotoxins. Biochemistry 2007, 46, 3338–3355. [CrossRef][PubMed] 10. Gongora-Benitez, M.; Tulla-Puche, J.; Albericio, F. Multifaceted roles of disulfide bonds. Peptides as therapeutics. Chem. Rev. 2014, 114, 901–926. [CrossRef][PubMed] 11. Savarin, P.; Romi-Lebrun, R.; Zinn-Justin, S.; Lebrun, B.; Nakajima, T.; Gilquin, B.; Menez, A. Structural and functional consequences of the presence of a fourth disulfide bridge in the short toxins: Solution structure of the inhibitor HsTX1. Protein Sci. 1999, 8, 2672–2685. [CrossRef][PubMed] 12. Zhu, Q.; Liang, S.; Martin, L.; Gasparini, S.; Menez, A.; Vita, C. Role of disulfide bonds in folding and activity of leiurotoxin I: Just two disulfides suffice. Biochemistry 2002, 41, 11488–11494. [CrossRef][PubMed] 13. Han, T.S.; Zhang, M.M.; Walewska, A.; Gruszczynski, P.; Robertson, C.R.; Cheatham, T.E.; Yoshikami, D.; Olivera, B.M.; Bulaj, G. Structurally minimized mu-conotoxin analogues as sodium channel blockers: Implications for designing conopeptide-based therapeutics. ChemMedChem 2009, 4, 406–414. [CrossRef] [PubMed] 14. Price-Carter, M.; Hull, M.S.; Goldenberg, D.P. Roles of individual disulfide bonds in the stability and folding of an omega-conotoxin. Biochemistry 1998, 37, 9851–9861. [CrossRef][PubMed] Mar. Drugs 2016, 14, 213 9 of 9

15. Chen, H.; Gordon, D.; Heinemann, S.H. Modulation of cloned skeletal muscle sodium channels by the scorpion toxins Lqh II, Lqh III, and Lqh alphaIT. Pflugers Arch. 2000, 439, 423–432. [PubMed] 16. Leipold, E.; Markgraf, R.; Miloslavina, A.; Kijas, M.; Schirmeyer, J.; Imhof, D.; Heinemann, S.H. Molecular determinants for the subtype specificity of mu-conotoxin SIIIA targeting neuronal voltage-gated sodium channels. Neuropharmacology 2011, 61, 105–111. [CrossRef][PubMed] 17. Cartier, G.E.; Yoshikami, D.; Gray, W.R.; Luo, S.; Olivera, B.M.; McIntosh, J.M. A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors. J. Biol. Chem. 1996, 271, 7522–7528. [PubMed] 18. Cummins, T.R.; Aglieco, F.; Dib-Hajj, S.D. Critical molecular determinants of voltage-gated sodium channel sensitivity to mu-conotoxins GIIIA/B. Mol. Pharmacol. 2002, 61, 1192–1201. [CrossRef][PubMed] 19. Li, R.A.; Sato, K.; Kodama, K.; Kohno, T.; Xue, T.; Tomaselli, G.F.; Marban, E. Charge conversion enables quantification of the proximity between a normally-neutral mu-conotoxin (GIIIA) site and the Na+ channel pore. FEBS Lett. 2002, 511, 159–164. [CrossRef] 20. Sato, K.; Ishida, Y.; Wakamatsu, K.; Kato, R.; Honda, H.; Ohizumi, Y.; Nakamura, H.; Ohya, M.; Lancelin, J.M.; Kohda, D.; et al. Active site of mu-conotoxin GIIIA, a peptide blocker of muscle sodium channels. J. Biol. Chem. 1991, 266, 16989–16991. [PubMed] 21. Becker, S.; Prusak-Sochaczewski, E.; Zamponi, G.; Beck-Sickinger, A.G.; Gordon, R.D.; French, R.J. Action of derivatives of mu-conotoxin GIIIA on sodium channels. Single amino acid substitutions in the toxin separately affect association and dissociation rates. Biochemistry 1992, 31, 8229–8238. [CrossRef][PubMed] 22. Wakamatsu, K.; Kohda, D.; Hatanaka, H.; Lancelin, J.M.; Ishida, Y.; Oya, M.; Nakamura, H.; Inagaki, F.; Sato, K. Structure-activity relationships of mu-conotoxin GIIIA: Structure determination of active and inactive sodium peptides by NMR and simulated annealing calculations. Biochemistry 1992, 31, 12577–12584. [CrossRef][PubMed] 23. Dudley, S.C., Jr.; Chang, N.; Hall, J.; Lipkind, G.; Fozzard, H.A.; French, R.J. mu-conotoxin GIIIA interactions with the voltage-gated Na(+) channel predict a clockwise arrangement of the domains. J. Gen. Physiol. 2000, 116, 679–690. [CrossRef][PubMed] 24. Choudhary, G.; Aliste, M.P.; Tieleman, D.P.; French, R.J.; Dudley, S.C., Jr. Docking of mu-conotoxin GIIIA in the sodium channel outer vestibule. Channels (Austin) 2007, 1, 344–352. [CrossRef][PubMed] 25. Xue, T.; Ennis, I.L.; Sato, K.; French, R.J.; Li, R.A. Novel interactions identified between micro-Conotoxin and the Na+ channel domain I P-loop: Implications for toxin-pore binding geometry. Biophys. J. 2003, 85, 2299–2310. [CrossRef] 26. Mahdavi, S.; Kuyucak, S. Molecular dynamics study of binding of micro-conotoxin GIIIA to the voltage-gated sodium channel Na(v)1.4. PLoS ONE 2014, 9, e105300. [CrossRef][PubMed] 27. Korkosh, V.S.; Zhorov, B.S.; Tikhonov, D.B. Folding similarity of the outer pore region in prokaryotic and

eukaryotic sodium channels revealed by docking of conotoxins GIIIA, PIIIA, and KIIIA in a NaVAb-based model of NaV1.4. J. Gen. Physiol. 2014, 144, 231–244. [CrossRef][PubMed] 28. Mahdavi, S.; Kuyucak, S. Systematic study of binding of mu-conotoxins to the sodium channel NaV1.4. Toxins (Basel) 2014, 6, 3454–3470. [CrossRef][PubMed] 29. Tietze, A.A.; Tietze, D.; Ohlenschlager, O.; Leipold, E.; Ullrich, F.; Kuhl, T.; Mischo, A.; Buntkowsky, G.; Gorlach, M.; Heinemann, S.H.; et al. Structurally diverse mu-conotoxin PIIIA isomers block sodium channel

NaV1.4. Angew. Chem. Int. Ed. Engl. 2012, 51, 4058–4061. [CrossRef][PubMed] 30. Shon, K.J.; Olivera, B.M.; Watkins, M.; Jacobsen, R.B.; Gray, W.R.; Floresca, C.Z.; Cruz, L.J.; Hillyard, D.R.; Brink, A.; Terlau, H.; et al. mu-Conotoxin PIIIA, a new peptide for discriminating among -sensitive Na channel subtypes. J. Neurosci. 1998, 18, 4473–4481. [PubMed] 31. Green, B.R.; Zhang, M.M.; Chhabra, S.; Robinson, S.D.; Wilson, M.J.; Redding, A.; Olivera, B.M.; Yoshikami, D.; Bulaj, G.; Norton, R.S. Interactions of disulfide-deficient selenocysteine analogs of mu-conotoxin BuIIIB with the alpha-subunit of the voltage-gated sodium channel subtype 1.3. FEBS J. 2014, 281, 2885–2898. [CrossRef][PubMed] 32. Kuang, Z.; Zhang, M.M.; Gupta, K.; Gajewiak, J.; Gulyas, J.; Balaram, P.; Rivier, J.E.; Olivera, B.M.; Yoshikami, D.; Bulaj, G.; et al. Mammalian neuronal mu-conotoxin BuIIIB has a structured N-terminus that influences potency. ACS Chem. Biol. 2013, 8, 1344–1351. [CrossRef][PubMed]

© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).