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• W2050863332 abstract "μ-Conotoxins are peptide inhibitors of voltage-sensitive sodium channels (VSSCs). Synthetic forms of μ-conotoxins PIIIA and PIIIA-(2–22) were found to inhibit tetrodotoxin (TTX)-sensitive VSSC current but had little effect on TTX-resistant VSSC current in sensory ganglion neurons. In rat brain neurons, these peptides preferentially inhibited the persistent over the transient VSSC current. Radioligand binding assays revealed that PIIIA, PIIIA-(2–22), and μ-conotoxin GIIIB discriminated among TTX-sensitive VSSCs in rat brain, that these and GIIIC discriminated among the corresponding VSSCs in human brain, and GIIIA had low affinity for neuronal VSSCs. 1H NMR studies found that PIIIA adopts two conformations in solution due tocis/trans isomerization at hydroxyproline 8. The major trans conformation results in a three-dimensional structure that is significantly different from the previously identified conformation of μ-conotoxins GIIIA and GIIIB that selectively target TTX-sensitive muscle VSSCs. Comparison of the structures and activity of PIIIA to muscle-selective μ-conotoxins provides an insight into the structural requirements for inhibition of different TTX-sensitive sodium channels by μ-conotoxins. μ-Conotoxins are peptide inhibitors of voltage-sensitive sodium channels (VSSCs). Synthetic forms of μ-conotoxins PIIIA and PIIIA-(2–22) were found to inhibit tetrodotoxin (TTX)-sensitive VSSC current but had little effect on TTX-resistant VSSC current in sensory ganglion neurons. In rat brain neurons, these peptides preferentially inhibited the persistent over the transient VSSC current. Radioligand binding assays revealed that PIIIA, PIIIA-(2–22), and μ-conotoxin GIIIB discriminated among TTX-sensitive VSSCs in rat brain, that these and GIIIC discriminated among the corresponding VSSCs in human brain, and GIIIA had low affinity for neuronal VSSCs. 1H NMR studies found that PIIIA adopts two conformations in solution due tocis/trans isomerization at hydroxyproline 8. The major trans conformation results in a three-dimensional structure that is significantly different from the previously identified conformation of μ-conotoxins GIIIA and GIIIB that selectively target TTX-sensitive muscle VSSCs. Comparison of the structures and activity of PIIIA to muscle-selective μ-conotoxins provides an insight into the structural requirements for inhibition of different TTX-sensitive sodium channels by μ-conotoxins. voltage-sensitive sodium channel tetrodotoxin TTX-sensitive TTX-resistant 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid saxitoxin nuclear Overhauser effect NOE spectroscopy hydroxyproline root mean square Voltage-sensitive sodium channels (VSSCs)1 underlie the influx of sodium ions responsible for action potentials in excitable cells (1Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1719) Google Scholar). Based on their susceptibility to block by tetrodotoxin (TTX), VSSCs can be divided into TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) classes. Members of both classes share considerable sequence homology and are closely related structurally (2Goldin A.L. Barchi R.L Caldwell J.H. Hofmann F. Howe J.R. Hunter J.C. Kallen R.G. Mandel G. Meisler M.H. Netter Y.B. Noda M. Tamkun M.M. Waxman S.G. Wood J.N. Catterall W.A. Neuron. 2000; 28: 365-368Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar). These include the neuronal TTX-S type I/Nav1.1, type II/Nav1.2, type III/Nav1.3, PN1/Nav1.7 and PN4/Nav1.6, and the skeletal muscle TTX-S μ1/Nav1.4. The TTX-R sodium channels include the cardiac H1/Nav1.5, which is partially TTX-resistant, and the neuronal TTX-R channels SNS/PN3/Nav1.8 and NaN/PN5/Nav1.9 (2Goldin A.L. Barchi R.L Caldwell J.H. Hofmann F. Howe J.R. Hunter J.C. Kallen R.G. Mandel G. Meisler M.H. Netter Y.B. Noda M. Tamkun M.M. Waxman S.G. Wood J.N. Catterall W.A. Neuron. 2000; 28: 365-368Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar). A number of these VSSC subtypes are implicated in clinical states such as pain (3Akopian A.N. Souslova V. England S. Okuse K. Ogata N. Ure J. Smith A. Kerr B.J. McMahon S.B. Boyce S. Hill R. Stanfa L.C. Dickenson A.H. Wood J.N. Nat. Neurosci. 1999; 6: 541-548Crossref Scopus (722) Google Scholar, 4Eglen R.M. Hunter J.C. Dray A. Trends Pharmacol. Sci. 1999; 20: 337-342Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 5Porreca F. Lai J. Bian D. Wegert S. Ossipov M.H. Eglen R.M. Kassotakis L. Novakovic S. Rabert D.K. Sangameswaran L. Hunter J.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7640-7644Crossref PubMed Scopus (305) Google Scholar, 6Coward K. Plumpton C. Facer P. Birch R. Carlstedt T. Tate S. Bountra C. Anand P. Pain. 2000; 85: 41-50Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), stroke (7Taylor C.P. Meldrum B.S. Trends Pharmacol. Sci. 1995; 16: 309-316Abstract Full Text PDF PubMed Scopus (268) Google Scholar, 8Carter A.J. Amino Acids. 1998; 14: 159-169Crossref PubMed Scopus (34) Google Scholar), and epilepsy (9Ragsdale D.S. Avoli M. Brain Res. Brain Res. Rev. 1998; 26: 16-28Crossref PubMed Scopus (198) Google Scholar, 10Rho J.M. Sankar R. Epilepsia. 1999; 40: 1471-1483Crossref PubMed Scopus (149) Google Scholar). Persistent (noninactivating) forms of the TTX-S sodium channel current that underlie repetitive firing (11Smith M.R. Smith R.D. Plummer N.W. Meisler M.H. Goldin A.L. J. Neurosci. 1998; 18: 6093-6102Crossref PubMed Google Scholar, 12Bevan M.D. Wilson C.J. J. Neurosci. 1999; 19: 7617-7628Crossref PubMed Google Scholar) have less well defined origins but may involve Nav1.3 (13Moorman J.R. Kirsch G.E. Vandongen A.M. Joho R.H. Brown A.M. Neuron. 1990; 4: 243-252Abstract Full Text PDF PubMed Scopus (116) Google Scholar) or Nav1.6 (11Smith M.R. Smith R.D. Plummer N.W. Meisler M.H. Goldin A.L. J. Neurosci. 1998; 18: 6093-6102Crossref PubMed Google Scholar) and are enhanced by hypoxia (14Ju Y. Saint D.A. Gage P.W. Br. J. Pharmacol. 1992; 107: 311-316Crossref PubMed Scopus (56) Google Scholar, 15Hammarström A.K. Gage P.W. J. Physiol. 1998; 510: 735-741Crossref PubMed Scopus (105) Google Scholar, 16Hammarström A.K. Gage P.W. J. Physiol. 2000; 529: 107-118Crossref PubMed Scopus (64) Google Scholar) and nitric oxide (17Hammarström A.K. Gage P.W. J. Physiol. 1999; 520: 451-461Crossref PubMed Scopus (98) Google Scholar). Most TTX-S sodium channels types have a heterogeneous distribution in human brain (18Whitaker W.R. Faull R.L.M. Waldvogel H.J. Plumpton C.J. Emson P.C. Clare J.J. Mol. Brain Res. 2001; 88: 37-53Crossref PubMed Scopus (127) Google Scholar). VSSCs are inhibited by local anesthetics and modulated by toxins that act at one inhibitory site (site 1) and at least four other sites that result in excitatory actions. μ-Conotoxins from the venom of marine cone snails act selectively to occlude the pore of the VSSC by competing with TTX and saxitoxin (STX) for binding to site 1 in the P-loop region of the α subunit. To date, sequences for four members of the three-loop μ-conotoxin class have been published (Table I). GIIIA−GIIIC from Conus geographus venom are potent blockers of skeletal muscle but not neuronal VSSCs. The three-dimensional structures of selected μ-conotoxins (19Lancelin J-M. Kohda D. Tate S. Yanagawa Y. Abe T. Satake M. Inagaki F. Biochemistry. 1991; 30: 6908-6916Crossref PubMed Scopus (90) Google Scholar, 20Hill J.M. Alewood P.F. Craik D.J. Biochemistry. 1996; 35: 8824-8835Crossref PubMed Scopus (98) Google Scholar) have been used to describe the architecture of the outer vestibule of the VSSC (21Chahine M. Chen L-Q. Fotouhi N. Walsky R. Fry D. Santarelli V. Horn R. Kallen R.G. Receptors Channels. 1995; 3: 161-174PubMed Google Scholar, 22Chang N. French R.J. Lipkind G.M. Fozzard H.A. Dudley S., Jr. Biochemistry. 1998; 37: 4407-4419Crossref PubMed Scopus (92) Google Scholar, 23Li R.A. Ennis I.L. Velez P. Tomaselli G.F. Marban E. J. Biol. Chem. 2000; 275: 27551-27558Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 24Lipkind G.M. Fozzard H.A. Biochemistry. 2000; 39: 8161-8170Crossref PubMed Scopus (166) Google Scholar, 25Li R.A. Ennis I.I. French R.J. Dudley S.C. Tomaselli G.F. Marban E. J. Biol. Chem. 2001; 276: 11072-11077Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The most recently described member of this class is μ-conotoxin PIIIA (26Shon 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. Yoshikami D. J. Neurosci. 1998; 18: 4473-4481Crossref PubMed Google Scholar) from C. purpurescens (Fig. 1). PIIIA is notable for its ability to inhibit neuronal as well as muscle TTX-S sodium channels (26Shon 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. Yoshikami D. J. Neurosci. 1998; 18: 4473-4481Crossref PubMed Google Scholar) and to discriminate among VSSCs in rat brain (27Safo P. Rosenbaum T. Shcherbatko A. Choi D.Y. Han E. Toledo-Aral J.J. Olivera B.M. Brehm P. Mandel G. J. Neurosci. 2000; 20: 76-80Crossref PubMed Google Scholar). Thus, PIIIA is the first peptide toxin for investigating the architecture of site 1 of neuronal VSSCs.Table IPotency (pIC50) and extent of inhibition (%) of [3H]STX binding to VSSCs by μ-conotoxins and TTXμ-ConotoxinHuman brainRat brainRat musclePIIIA Potency7.1 (7.6–6.6)6.5 (6.8–6.3)6.8 (7.1–6.5) Inhibition47 (34–61)%79 (68–91)%81 (67–96)%PIIIA-(2–22) Potency6.6 (7.0–6.2)6.4 (6.6–6.3)7.2 (7.4–7.0) Inhibition70 (58–82)%90 (86–94)%81 (73–93)%GIIIA PotencyInactive5.1 (5.5–4.7)7.1 (7.3–6.9) Inhibition40 (28–52)%82 (76–89)%GIIIB Potency5.5 (6.4–4.5)5.9 (6.2–5.7)7.2 (7.5–7.0) Inhibition36 (17–54)%87 (72–101)%85 (78–92)%GIIIC Potency6.0 (6.6–5.4)Inactive7.3 (7.5–7.1) Inhibition30 (20–41)%93 (86–101)%TTX Potency7.6 (7.8–7.4)7.8 (7.9–7.7)7.8 (7.9–7.7) Inhibition98 (91–105)%100 (97–103)%100 (96–104)%95% confidence interval range for pIC50 and percentage of inhibition are given in parenthesis. Open table in a new tab 95% confidence interval range for pIC50 and percentage of inhibition are given in parenthesis. Previous studies on GIIIA (21Chahine M. Chen L-Q. Fotouhi N. Walsky R. Fry D. Santarelli V. Horn R. Kallen R.G. Receptors Channels. 1995; 3: 161-174PubMed Google Scholar, 22Chang N. French R.J. Lipkind G.M. Fozzard H.A. Dudley S., Jr. Biochemistry. 1998; 37: 4407-4419Crossref PubMed Scopus (92) Google Scholar) have revealed that the cationic residues, particularly Arg13, are important for the high potency of this peptide at Nav1.4 (see Fig.1). The high sequence identity and similarities in the three-dimensional structures of GIIIA and GIIIB (19Lancelin J-M. Kohda D. Tate S. Yanagawa Y. Abe T. Satake M. Inagaki F. Biochemistry. 1991; 30: 6908-6916Crossref PubMed Scopus (90) Google Scholar, 20Hill J.M. Alewood P.F. Craik D.J. Biochemistry. 1996; 35: 8824-8835Crossref PubMed Scopus (98) Google Scholar) provide a rational basis for comparison with PIIIA, which also contains a number of conserved residues and the same disulfide connectivities as GIIIA and GIIIB (and GIIIC). However, a number of primary structural differences are apparent between PIIIA and other μ-conotoxins, which may affect the relative position and orientation of backbone loops and their projecting side chains and thus allow PIIIA to interact with both neuronal and muscle forms of TTX-sensitive VSSCs. To further investigate the potential of PIIIA as a probe of VSSCs, we determined its structure by 1H NMR spectroscopy and characterized its mode of action on native tissues using electrophysiological and ligand binding approaches. These studies revealed that PIIIA and PIIIA-(2–22) preferentially inhibited the persistent TTX-S currents in rat hippocampal neurons, whereas in rat DRG the TTX-R current was spared. Comparisons of the three-dimensional structures of PIIIA, GIIIA, and GIIIB revealed important structural differences, including an alternative major conformation accessed by PIIIA, which had not been identified previously in μ-conotoxins. Peptides were prepared by Boc chemistry (28Schnölzer M. Alewood P.F. Jones A. Alewood D. Kent S.B.H. Int. J. Pept. Protein Res. 1992; 40: 180-193Crossref PubMed Scopus (941) Google Scholar) using methods described for ω-conotoxins (29Nielsen K.J. Adams D. Thomas L. Bond T.J. Alewood P.F. Craik D.J. Lewis R.J. J. Mol. Biol. 1999; 289: 1405-1421Crossref PubMed Scopus (69) Google Scholar). The side chain protection chosen was Arg(tos), Asp(OcHex), Lys(CIZ), Ser(Bzl), and Cys(p-MeBzl). The crude reduced peptides were purified by preparative chromatography, using a 1% gradient (100% A to 80% B, 80 min) and UV detection at 230 nm. The reduced peptides were oxidized at a concentration of 0.02 mm in either aqueous 0.33 m NH4OAc, 0.5 m guanidine HCl or aqueous 2 mNH4OH. The solution was stirred for 3–5 days at pH 8.1. Purification of oxidized peptide was completed using preparative reversed phase high pressure liquid chromatography. Whole rat brain (29Nielsen K.J. Adams D. Thomas L. Bond T.J. Alewood P.F. Craik D.J. Lewis R.J. J. Mol. Biol. 1999; 289: 1405-1421Crossref PubMed Scopus (69) Google Scholar), the human frontal cortex (30Maddison J.E. Dodd P.R. Johnston G.A.R. Farrell G.C. Gastroenterology. 1987; 93: 1062-1068Abstract Full Text PDF PubMed Scopus (52) Google Scholar), and rat skeletal muscle (31Yanagawa Y. Abe T. Satake M. Odani S. Suzuki J. Ishikawa K. Biochemistry. 1988; 27: 6256-6262Crossref PubMed Scopus (55) Google Scholar) were homogenized in 50 mm HEPES (pH 7.4), filtered though 100-μm nylon mesh (muscle only), and centrifuged at 28,000 × g (10 min). The pellet was suspended in 50 mm HEPES, 10 mm EDTA (pH 7.4) for 30 min, centrifuged, and resuspended in 50 mm HEPES (pH 7.4). Radioligand binding studies were conducted in assay buffer (130 mm choline chloride, 5.4 mm KCl, 5.5 mm glucose, 0.8 mm MgSO4, 1.8 mm CaCl2, 50 mm HEPES (pH 7.4 with Tris base)). Assays were conducted on rat brain (18 μg of protein, 150-μl total volume), human brain (12 μg of protein, 150 μl), and rat muscle (50 μg of protein, 300 μl) that contained 5.6 nm [3H]STX (14.9 Ci/mmol; AmershamBiosciences) and varying concentrations of μ-conotoxins in assay buffer. Assays were incubated for 1 h at 4 °C and filtered through GFB filters on a Tomtec harvester (brain) or a Millipore manifold (muscle) using wash buffer (163 mm choline chloride, 1.8 mm CaCl2, 0.8 mmMgSO4, 5.0 mm HEPES (pH 7.4 with Tris base)). Filters were dried, scintillant was added, and filters retained radioactivity measured on a Microbeta counter (Wallac). Electrophysiological experiments were conducted to further investigate the effect of PIIIA on TTX-S and TTX-R sodium channels in native tissue. Sensory neurons from rat nodose ganglia and dorsal root ganglia (DRG) were isolated as previously described (32Jeglitsch G. Rein K. Baden D.G. Adams D.J. J. Pharmacol. Exp. Ther. 1998; 284: 516-525PubMed Google Scholar, 33Nicholson G.M. Walsh R. Little M.J. Tyler M.I. Pflugers Arch. 1998; 436: 117-126Crossref PubMed Scopus (76) Google Scholar). Briefly, young rats (10−21 days) were killed by cervical dislocation, and the nodose and DRG were carefully removed. The ganglia were placed in physiological saline solution containing collagenase (∼1.0 mg/ml type 2; Worthington-Biochemical) and incubated for 1 h at 37 °C in 95% air and 5% CO2 for 24–48 h. Neurons from the nodose ganglia that were clear and round were selected for experiments. Small diameter cells (∼20 μm) from the DRG were used, since these have previously been reported to predominantly express TTX-resistant Na+ currents (34Elliott A.A. Elliott J.R. J. Physiol. 1993; 463: 39-56Crossref PubMed Scopus (388) Google Scholar). Young rats (14−21 days) were anesthetized under CO2 and decapitated with an animal guillotine. The brain was removed and transferred to ice-cold artificial cerebrospinal fluid (containing 124 mm NaCl, 26 mm NaH2CO3, 3 mm KCl, 1.3 mm MgSO4, 2.5 mmNaH2PO4, and 20 mm glucose). The brain was mounted in a vibratome and bathed in ice-cold artificial cerebrospinal fluid equilibrated with 95% O2 and 5% CO2 while the 500-μm-thick slices were prepared. Brain slices were incubated for 30 min with 200 units/ml papain (Worthington), 1.1 mm cysteine (Sigma), 0.2 mmEDTA, and 13.4 mm mercaptoethanol at 35 °C. Following incubation, the CA1 region was located, removed, and gently triturated using a fire-polished Pasteur pipette. Neurons of 10–15 μm were used, and cells that were flat, swollen, or grainy in appearance were avoided. Whole cell Na+currents were recorded using the patch clamp technique. Patch pipettes (GC150F; Harvard Apparatus Ltd., Edenbridge, Kent, UK) were prepared that had resistances of between 1 and 2 megaohms (nodose and DRG neurons) and between 6 and 10 megaohms (CA1 neurons) when filled with pipette solution. Whole cell Na+ currents from nodose and DRG neurons were made using a List EPC 7 amplifier (List Medical). Voltage steps were generated by a PC (Dell Pentium) running pClamp (Axon Instruments Inc., Union City, CA). Whole cell Na+currents from CA1 neurons were made using a Axopatch 1D amplifier (Axon Instruments) with voltage steps generated using a PC (Osborne 486-SX) running custom software (14Ju Y. Saint D.A. Gage P.W. Br. J. Pharmacol. 1992; 107: 311-316Crossref PubMed Scopus (56) Google Scholar, 15Hammarström A.K. Gage P.W. J. Physiol. 1998; 510: 735-741Crossref PubMed Scopus (105) Google Scholar, 35Ju Y.K. Saint D.A. Gage P.W. J. Physiol. (Lond.). 1996; 497: 337-347Crossref Scopus (264) Google Scholar). To record Na+ currents from DRG and nodose neurons, patch pipettes were filled with the following solution: 135 mm CsF, 10 mm NaCl, 5 mm HEPES, with pH adjusted to 7.2 with CsOH. The bath solution contained 50 mm NaCl, 3 mm KCl, 90 mm tetraethylammonium chloride, 0.1 mmCdCl2, 7.7 mm glucose, 10 mm HEPES, with pH adjusted to 7.4 with TEA-OH. To record Na+ currents from CA1 neurons, the patch pipette solution contained the following solution: 125 mm CsF, 5 mm NaF, 10 mm KCl, 10 mm TES, with pH adjusted to 7.4 with KOH. The bath solution contained 135 mm NaCl, 5 mm KCl, 3 mm MgCl2, 1 mm CaCl2, 5 mm CoCl2, 5 mm CsCl, 10 mm TES, with pH adjusted to 7.4 with NaOH. Three distinct Na+ currents were measured: a transient TTX sensitive Na+ current (TTX-SINaT), a transient TTX-resistant Na+current (TTX-R INaT), and a persistent TTX-sensitive Na+ current (TTX-SINaP). The amplitude of evoked TTX-SINaT was measured at its peak after subtraction of the current evoked in the presence of TTX (0.5−1 μm). The amplitude of the TTX-R INaT was measured at least 2 min following the addition of 0.5−1 μm TTX. The amplitude of TTX-S INaP was measured at the end of a 400-ms voltage step after subtraction of the current evoked in the presence of TTX (0.5−1 μm). All values are expressed as means ± S.E. with n indicating the number of cells in a given series of experiments. Comparisons of two means were made using Student's two-tailed unpaired t test. All NMR experiments were recorded on a Bruker ARX 500 spectrometer equipped with a z-gradient unit or on a Bruker DMX 750 spectrometer equipped with an x,y,z-gradient unit. Peptide concentrations were ∼2 mm. PIIIA was examined in 95% H2O, 5% D2O (pH 3.0 and 5.5; 275–298 K) and in 50% aqueous CD3CN (260–293 K). 1H NMR experiments recorded were NOESY (36Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4845) Google Scholar, 37Kumar A. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1980; 95: 1-6Crossref PubMed Scopus (2031) Google Scholar) with mixing times of 150, 200, and 400 ms, TOCSY (38Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar) with a mixing time of 80 ms, DQF-COSY (39Rance M. Sørenson O.W. Bodenhausen G. Wagner G. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 177: 479-485Crossref Scopus (2596) Google Scholar), and E-COSY in 100% D2O (40Greisinger C. Sorenson O.W. Ernst R.R. J. Magn. Reson. 1987; 75: 474-492Google Scholar). All spectra were run over 6024 Hz (500 MHz) or 8192 Hz (750 MHz) with 4 K data points, 400–512 free induction decays, 16–64 scans, and a recycle delay of 1 s. The solvent was suppressed using the WATERGATE sequence (41Piotto M. Saudek V. Sklenár V. J. Biolmol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3538) Google Scholar). Spectra were processed using UXNMR as described previously (29Nielsen K.J. Adams D. Thomas L. Bond T.J. Alewood P.F. Craik D.J. Lewis R.J. J. Mol. Biol. 1999; 289: 1405-1421Crossref PubMed Scopus (69) Google Scholar) and using Aurelia; subtraction of background was used to minimize T1noise. Chemical shift values were referenced internally to 4,4-dimethyl-4-silapentane-1-sulfonate at 0.00 ppm. Secondary Hα shifts were measured using random coil shift values of Wishartet al. (42Wishart D.S. Bigam C.G. Yao J. Abildgaard F. Dyason H.J. Oldfield E. Markley J.L. Sykes B.D. J. Biomol. NMR. 1995; 5: 67-81Crossref PubMed Scopus (1426) Google Scholar). 3 JNH-Hαcoupling constants were measured as previously described (29Nielsen K.J. Adams D. Thomas L. Bond T.J. Alewood P.F. Craik D.J. Lewis R.J. J. Mol. Biol. 1999; 289: 1405-1421Crossref PubMed Scopus (69) Google Scholar). Peak volumes in NOESY spectra were classified as strong, medium, weak, and very weak, corresponding to upper bounds on interproton distance of 2.7, 3.5, 5.0, and 6.0 Å, respectively. Lower distance bounds were set to 1.8 Å. Appropriate pseudoatom corrections were made (43Wüthrich K. Billeter M. Braun W. J. Mol. Biol. 1983; 169: 949-961Crossref PubMed Scopus (1007) Google Scholar), and distances of 0.5 and 2.0 Å were added to the upper limits of restraints involving methyl and phenyl protons, respectively.3 JNH-Hα coupling constants were used to determine φ dihedral angle restraints (44Pardi A. Billeter M. Wüthrich K. J. Mol. Biol. 1984; 180: 741-751Crossref PubMed Scopus (940) Google Scholar), and in cases where 3 JNH-Hα was 6−8 Hz and it was clear that a positive dihedral angle was not present, φ was restrained to −100 ± 70°.3 JHα-Hβ coupling constants, together with relevant NOESY peak strengths, were used to determinex1 dihedral angle restraints (45Wagner G. Braun W. Havel T.F. Schaumann T., Go, N. Wüthrich K. J. Mol. Biol. 1987; 196: 611-639Crossref PubMed Scopus (635) Google Scholar). Where there was no diastereospecific assignment for a prochiral pair of protons, the largest upper bound for the two restraints was used. Where stereospecific assignments were established, these distances were specified explicitly. Structures were calculated using the torsion angle dynamics/simulated annealing protocol in X-PLOR (46Brünger A.T. X-PLOR: A System for X-ray Crystallography and NMR, Version 3.1. Yale University, New Haven, CT1992Google Scholar) version 3.8 using a modified geometric force field based on parhdg.pro. Structure refinements were performed using energy minimization (200 steps) under the influence of a full force field derived from Charmm (47Brooks B. Brucoli R. Olafson B.O. States D. Swaminathan S. Karplus M. J. Comput. Chem. 1983; 4: 187-217Crossref Scopus (14017) Google Scholar) parameters. Structure modeling, visualization, and superimpositions were done using InsightII (MSI). Surface calculations, r.m.s. deviations, and hydrogen bond analysis were done using MOLMOL (48Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 51: 29-32Google Scholar). The quality of the structures was analyzed using procheck-NMR (49Laskowski R.A. MacArthur M.W. Thornton J.M. Curr. Opin. Struct. Biol. 1998; 8: 631-639Crossref PubMed Scopus (153) Google Scholar). The effects of μ-conotoxin PIIIA and a truncated analogue, PIIIA-(2–22), were investigated on three distinct VSSCs found in neurons of the peripheral and central nervous system. Rat nodose ganglion neurons were used to investigate the transient TTX-sensitive voltage-dependent Na+ current(TTX-S INaT), DRG neurons were used to investigate the transient TTX-resistant sodium current (TTX-RINaT) (34Elliott A.A. Elliott J.R. J. Physiol. 1993; 463: 39-56Crossref PubMed Scopus (388) Google Scholar), and rat hippocampal neurons in the CA1 region were used to investigate the persistent TTX-sensitive sodium current (TTX-S INaP) (15Hammarström A.K. Gage P.W. J. Physiol. 1998; 510: 735-741Crossref PubMed Scopus (105) Google Scholar, 50French C.R. Gage P.W. Neurosci. Lett. 1985; 56: 289-293Crossref PubMed Scopus (63) Google Scholar, 51French C.R. Sah P. Buckett K.J. Gage P.W. J. Gen. Physiol. 1990; 95: 1139-1157Crossref PubMed Scopus (260) Google Scholar). PIIIA-(2–22) caused a concentration-dependent reduction in the peak amplitude of the TTX-S INaT in rat nodose ganglia neurons (Fig. 2). In contrast, in rat DRG neurons (n = 12), PIIIA-(2–22) produced only a small reduction in the peak amplitude of the TTX-RINaT (Fig. 2). High frequency stimulation can modify the degree of block by some neurotoxins that act in a use-dependent manner. Compared with control, 1 μm PIIIA-(2–22) failed to produce any use-dependent inhibition of peak TTX-SINaT during 20 depolarizing pulses from a holding potential of −80 mV to a test potential of −30 mV for 25 ms delivered at a frequency of 20 Hz (n = 6). In rat hippocampal CA1 neurons, the addition of PIIIA-(2–22) to the bathing solution caused a concentration-dependent reduction in the peak amplitude of the TTX-S INaT and the TTX-SINaP (Fig. 3). Interestingly, PIIIA-(2–22) had a greater effect on the TTX-SINaP than on the TTX-SINaT (Fig. 3, inset). At 1 μm, the peak amplitude of the TTX-SINaT was unaffected, whereas the amplitude of the TTX-S INaP was reduced by ∼70%.Figure 3Effects of PIIIA-(2–22) on the transient and persistent TTX-sensitive Na+ currents recorded from hippocampal CA1 neurons. Na+ currents recorded from a hippocampal CA1 neuron showing the effects of PIIIA-(2–22) on the transient TTX-sensitive Na+ current and the persistent Na+ current. Inset, the differential effects of PIIIA-(2–22) on the amplitude of the transient and persistent Na+ current. Peak amplitudes of the transient and persistent current were normalized to the transient and persistent currents recorded in control solution. Each point represents the mean current amplitude from at least three cells obtained following a voltage step to −30 mV from a holding potential of −80 mV (prepulsed to −130 mV for 300 ms).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The native μ-conotoxin, PIIIA, also reduced the TTX-SINaT in rat nodose (n = 12), DRG (n = 3), and CA1 (n = 3) neurons (data not shown) with a similar potency to PIIIA-(2–22). PIIIA had a preferential effect on the persistent compared with the transient sodium current, being slightly more potent at reducing the amplitude of the TTX-S INaT current in CA1 neurons than PIIIA-(2–22). In preliminary experiments, 10-min bath application of GIIIB (1–10 μm) had no effect on either the TTX-S or TTX-R INaT in rat nodose or DRG neurons, respectively (n = 3; data not shown). The ability of μ-conotoxins to displace [3H]STX from VSSCs in human and rat brain and rat skeletal muscle is shown in Fig. 4. All peptides were more potent at the rat skeletal muscle than rat brain VSSCs, with GIIIA and GIIIC showing most selectivity and PIIIA least selectivity. The pIC50 values and percentage inhibition for these peptides are given in Table I. The data show that PIIIA and PIIIA-(2–22) have greatest potency at rat and human brain VSSCs, GIIIB has intermediate potency, and GIIIA and GIIIC have least potency. These peptides were less potent than TTX, with none able to fully displace [3H]STX from rat or human brain (relative to TTX displacement). PIIIA and PIIIA-(2–22) produced the largest displacement of [3H]STX, and GIIIA and GIIIC produced the least displacement. GIIIB was more effective at displacing [3H]STX from rat compared with human brain (Fig. 4,A and B). All displacement curves were best fitted with a Hill slope of −1. PIIIA was examined by1H NMR spectroscopy in a range of different solvent conditions. In aqueous solution at pH 2.5−5.5 over 275−298 K, it was apparent that two conformations of PIIIA were present in a ∼3:1 ratio. In aqueous solution at low pH over 283−298 K, the NH resonances of several residues, including 4−7, 10−12, 20, and 22, were broad, and that of Cys21 was not observable. At higher pH values and lower temperatures (275 K), these peaks sharpened (residues 4 and 5) or separated into two distinct sets of peaks (residues 6 and 7, 10−12, 20, and 22) so that complete assignment of the major and a partial assignment of the minor conformations was possible. The assignment of PIIIA was improved by the addition of up to 50% CD3CN, where the set of peaks arising from the minor conformation was less evident, and all resonances from the major conformation were present. Chemical shift assignments for PIIIA are given in Table II.Table II1H chemical shifts of PIIIAResidueNHHαHβOtherpGlu1(7.98)(4.29)[4.33]Arg2(8.53)[8.59]3.93 (4.25)[4.19]1.79Hγ 1.52; Hδ 2.92, 2.97; δNH 7.07Leu38.47 (8.45)[8.48]4.40 (4.41)[4.32]1.57Hδ 0.88Cys48.41 (8.43)[8.43]4.47 (4.58)2.34, 2.56Cys57.97 (8.03)4.14 (4.28)3.00, 3.47Gly68.38 (8.65)[7.84]3.83 (3.81, 3.82)[3.63, 4.11]Phe77.38 (7.43)[8.35]4.98 (5.05)[4.72]2.98, 3.13Hδ, Hɛ 7.19; Hζ 7.23Hyp84.41 (4.49)[4.28]2.05, 2.34Hγ 4.67; Hδ 3.75, 3.90Lys98.55 (9.02)[8.87]3.98 (4.07)[4.11]1.93Hγ 1.34; Hδ 1.6; Hɛ 2.90; ɛNH3 7.49Ser107.78 (7.92)[8.63]3.98 (4.05)[4.79]3.82, 3.37Cys118.08 (8.25)[8.34]4.39 (4.51)[4.62]3.12, 2.91Arg127.50 (7.52)[8.43]4.19 (4.28)[4.31]1.84, 1.98Hγ 1.66, 1.72; δNH 7.26Ser137.73 (7.86)[7.90]4.48 (4.53)[4.43]3.99, 4.18(OH 5.72)Arg149.04 (9.19)[9.16]3" @default.
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• W2050863332 date "2002-07-01" @default.
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• W2050863332 title "Solution Structure of μ-Conotoxin PIIIA, a Preferential Inhibitor of Persistent Tetrodotoxin-sensitive Sodium Channels" @default.
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• W2050863332 doi "https://doi.org/10.1074/jbc.m201611200" @default.
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