Molecular Interactions of the Gating Modifier Toxin ProTx-II with Nav1.5
2007; Elsevier BV; Volume: 282; Issue: 17 Linguagem: Inglês
10.1074/jbc.m610462200
ISSN1083-351X
AutoresJaime J. Smith, Theodore Cummins, Sujith Alphy, Kenneth M. Blumenthal,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoVoltage-gated Na+ channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Nav and Cav, but not Kv, channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Nav1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly Kv channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Nav1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Nav channels with potential to shed new light on this process. Voltage-gated Na+ channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Nav and Cav, but not Kv, channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Nav1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly Kv channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Nav1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Nav channels with potential to shed new light on this process. Voltage-gated cation channels are integral membrane proteins that play a critical role in electrical signaling by controlling the flow of Na+, K+, and Ca2+ across the plasma membrane in response to changes in voltage. Nav channel α subunits are composed of four homologous domains, DI-DIV, each having six transmembrane segments, S1-S6. The first four segments of each domain comprise the voltage sensor of these proteins, whereas S5 and S6 form the central ion conducting pore (for review, see Ref. 1Catterall W.A. Goldin A.L. Waxman S.G. Pharmacol. Rev. 2005; 57: 397-409Crossref PubMed Scopus (1115) Google Scholar). The homologous Kv channels are tetramers of four identical subunits, each similar to a Nav channel domain. Site-directed fluorescent labeling has shown that Nav domains I and II move with activation and are unaffected by fast inactivation gating, whereas domains III and IV exhibit kinetic components associated with deactivation and fast inactivation (2Cha A. Ruben P.C. George A.L. Fujimoto E. Bezanilla F. Neuron. 1999; 22: 73-87Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 3Chanda B. Bezanilla F. J. Gen. Physiol. 2002; 120: 629-645Crossref PubMed Scopus (271) Google Scholar). DIV-S4 has a unique role in gating in that its charges only move during inactivation (4Chahine M. George A.L. Zhou M. Ji S. Sun W. Barchi R.L. Horn R. Neuron. 1994; 12: 281-294Abstract Full Text PDF PubMed Scopus (306) Google Scholar, 5Sheets M.F. Kyle J.W. Kallen R.G. Hanck D.A. Biophys. J. 1999; 77: 747-757Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), and there is evidence for strong cooperativity among Nav channel domains throughout the gating process. In contrast, subunit coupling is only seen late in Kv channel gating transitions (6Chanda B. Asamoah O.K. Bezanilla F. J. Gen. Physiol. 2004; 123: 217-230Crossref PubMed Scopus (91) Google Scholar, 7Zagotta W.N. Hoshi T. Aldrich R.W. J. Gen. Physiol. 1994; 103: 321-362Crossref PubMed Scopus (451) Google Scholar, 8Schoppa N.E. Sigworth F.J. J. Gen. Physiol. 1998; 111: 313-342Crossref PubMed Scopus (254) Google Scholar). It has been suggested that this major difference between Nav and Kv channel kinetics underlies the basis of fast electrical transmission, i.e. domain cooperativity is necessary for the rapid upstroke of an action potential (6Chanda B. Asamoah O.K. Bezanilla F. J. Gen. Physiol. 2004; 123: 217-230Crossref PubMed Scopus (91) Google Scholar). The functional differences among Nav channel domains raise the possibility that distinct domain structures might exist as well. Given the many distinctions between Nav and Kv channels, detailed studies of Nav channel gating mechanisms are essential. Studies using neurotoxins that bind Nav channels with high affinity to alter conductance or gating properties have been extremely useful for this purpose. Polypeptide toxins derived from the venom of spiders, sea anemones, scorpions, and snails interact with voltage sensors to modify activation or inactivation and have been tremendously useful probes of gating mechanisms. These gating modifier toxins bind to sites 3 and 4, respectively. Site 3 has been localized to the extracellular S3/S4 linker of domain IV (9Rogers J.C. Qu Y. Tanada T.N. Scheuer T. Catterall W .A. J. Biol. Chem. 1996; 271: 15950-15962Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 10Benzinger G.R. Kyle J.W. Blumenthal K.M. Hanck D.A. J. Biol. Chem. 1998; 273: 80-84Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), whereas residues in domain II S3/S4 make a major contribution to site 4 (11Cestele S. Qu Y. Rogers J.C. Rochat H. Scheuer T. Catterall W.A. Neuron. 1998; 21: 919-931Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 12Cestele S. Scheuer T. Mantegazza M. Rochat H. Catterall W.A. J. Gen. Physiol. 2001; 118: 291-301Crossref PubMed Scopus (68) Google Scholar). Site 3 toxins, such as those from sea anemone venom, delay channel inactivation upon binding, most likely by inhibiting the normal outward movement of gating charges in DIV, resulting in the inability of the inactivation particle to mobilize (5Sheets M.F. Kyle J.W. Kallen R.G. Hanck D.A. Biophys. J. 1999; 77: 747-757Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In contrast, site 4 toxins enhance channel activation and shift the voltage dependence of activation to more hyperpolarized potentials via a voltage sensor-trapping mechanism (11Cestele S. Qu Y. Rogers J.C. Rochat H. Scheuer T. Catterall W.A. Neuron. 1998; 21: 919-931Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 13Cestele S. Yarov-Yarovoy V. Qu Y. Sampieri F. Scheuer T. Catterall W.A. J. Biol. Chem. 2006; 281: 21332-21344Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). It is thought that the open probability of the channel increases because of the toxin locking the channel in its activated conformation after a depolarizing pre-pulse. ProTx-II is a 30-amino acid peptide toxin purified from the venom of the tarantula, Thrixopelma pruriens, that modifies activation of both Nav and Cav, but not Kv, channel isoforms by inhibiting peak current and shifting the voltage dependence of activation to more depolarized potentials (14Middleton R.E. Warren V.A. Kraus R.L. Hwang J.C. Liu C.J. Dai G. Brochu R.M. Kohler M.G. Gao Y.D. Garsky V.M. Bogusky M.J. Mehl J.T. Cohen C.J. Smith M.M. Biochemistry. 2002; 41: 14734-14747Crossref PubMed Scopus (204) Google Scholar, 15Smith J.J. Alphy S. Seibert A.L. Blumenthal K.M. J. Biol. Chem. 2005; 280: 11127-11133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). ProTx-II conforms to the inhibitory cystine knot (ICK) 2The abbreviations used are: ICK, inhibitory cystine knot; HPLC, high performance liquid chromatography; RP, reverse phase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HEK cells, human embryonic kidney cells; SGTx, toxin 1 from the spider Scodra griseipes. motif, a common structural fold among spider toxins targeting ion channels (see The KNOTTIN database online). ICK peptides are defined by a 1-4, 2-5, 3-6 cystine connectivity and often have limited regular secondary structure. Based on its ability to modify activation, but not inactivation kinetics, it has been suggested that ProTx-II binds to site 4, but no direct evidence exists to validate this claim (14Middleton R.E. Warren V.A. Kraus R.L. Hwang J.C. Liu C.J. Dai G. Brochu R.M. Kohler M.G. Gao Y.D. Garsky V.M. Bogusky M.J. Mehl J.T. Cohen C.J. Smith M.M. Biochemistry. 2002; 41: 14734-14747Crossref PubMed Scopus (204) Google Scholar, 15Smith J.J. Alphy S. Seibert A.L. Blumenthal K.M. J. Biol. Chem. 2005; 280: 11127-11133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 16Bosmans F. Rash L. Zhu S. Diochot S. Lazdunski M. Escoubas P. Tytgat J. Mol. Pharmacol. 2006; 69: 419-429Crossref PubMed Scopus (130) Google Scholar, 17Cohen L. Gilles N. Karbat I. Ilan N. Gordon D. Gurevitz M. J. Biol. Chem. 2006; 281: 20673-20679Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The Nav channel isoform-specific actions of other tarantula venom ICK peptide toxins have been characterized. These toxins modify activation by inhibiting sodium current and causing a depolarizing shift in gating (16Bosmans F. Rash L. Zhu S. Diochot S. Lazdunski M. Escoubas P. Tytgat J. Mol. Pharmacol. 2006; 69: 419-429Crossref PubMed Scopus (130) Google Scholar). Like ProTx-II, their receptor sites have not been identified. However, detailed studies on the Kv channel ICK toxins hanatoxin and SGTx have shown that they inhibit potassium current by shifting channel opening to more depolarized potentials via an interaction with a receptor site on the C-terminal end of S3 near the extracellular surface (18Swartz K.J. MacKinnon R. Neuron. 1997; 18: 675-682Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 19Wang J.M. Roh S.H. Kim S. Lee C.W. Kim J.I. Swartz K.J. J. Gen. Physiol. 2004; 123: 455-467Crossref PubMed Scopus (92) Google Scholar). The similar functional effects of ProTx-II and these Kv channel toxins on their targets raise the possibility that their receptor sites are similar as well, although Nav channel asymmetry will likely introduce an additional level of complexity into binding site identification. Because ProTx-II modifies activation in a manner distinct from the previously characterized site 4 toxins, it is important to understand the basis for its activity. In the present study we investigate the bioactive surface of ProTx-II by alanine-scanning the peptide and analyzing the interactions of the mutants with the cardiac isoform, Nav1.5, by whole-cell voltage clamp. In addition, we carried out extensive mutagenesis of Nav1.5 to ascertain whether ProTx-II, like other toxins that modify Nav channel activation, interacts with receptor site 4. Our results indicate that the active face of ProTx-II consists of many hydrophobic as well as cationic residues that likely interact with a receptor site on Nav channels that is separate and distinct from site 4. ProTx-II—The ProTx-II coding sequence and upstream enterokinase site were amplified from a previously constructed expression vector in our laboratory (15Smith J.J. Alphy S. Seibert A.L. Blumenthal K.M. J. Biol. Chem. 2005; 280: 11127-11133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) using standard PCR procedures and cut with EcoRI/HindIII using sites introduced in the primers. The cleaved product was then subcloned into an octahistidine version of the pMALc2x vector (New England Biolabs) between EcoRI and HindIII using standard molecular biology protocols. As described previously, two additional N-terminal amino acids derived from a StuI restriction site remain in the ProTx-II coding sequence (15Smith J.J. Alphy S. Seibert A.L. Blumenthal K.M. J. Biol. Chem. 2005; 280: 11127-11133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Site-directed mutagenesis was performed using the QuikChange method (Stratagene, La Jolla, CA) to create all recombinant toxin mutants described in this paper, and all constructs were verified by sequencing. NaV1.5—The pBluescript plasmid containing the SCN5A gene encoding human Nav1.5 α subunit was used for all channel mutant constructs. We initially sought to swap the extracellular S3/S4 linkers in domains II and IV to assess the functional effects of toxins that are known to bind these regions. To replace DIV S3/S4 with its DII counterpart, five residues (SPTLF) were deleted in DIV S3/S4 using a loop out PCR procedure to obtain a DIV S3/S4 linker that matched the length of DII S3/S4. The remaining linker residues were then mutated to match the sequence of DII-S3/S4 using the QuikChange method. We named this construct II:II. To create the construct in which the DIV S3/S4 linker replaced the corresponding DII linker, splicing by overlap extension (20Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2694) Google Scholar) was used to insert five residues (SPTLF) downstream of the DII S3/S4 linker to match the length of DIV S3/S4. This was followed by mutation of the eight upstream residues in DII S3/S4 to match the complete sequence to that of DIV S3/S4. We called this construct IV:IV. The swapped construct was created by digesting the IV:IV construct with AgeI/NheI to remove the newly created S3/S4 linker region in DII and subcloning that region into the II:II construct digested with AgeI/NheI. This new construct became IV:II. Sites in the remaining extracellular linker regions were targeted by mutating residues in Nav1.5 that differ from Nav1.7, an isoform for which ProTx-II has a 100-fold higher affinity (14Middleton R.E. Warren V.A. Kraus R.L. Hwang J.C. Liu C.J. Dai G. Brochu R.M. Kohler M.G. Gao Y.D. Garsky V.M. Bogusky M.J. Mehl J.T. Cohen C.J. Smith M.M. Biochemistry. 2002; 41: 14734-14747Crossref PubMed Scopus (204) Google Scholar, 21Kraus R.L. Warren V.A. Smith M.M. Middleton R.E. Blumenthal K.M. Cohen C.J. Biophysical Society 46th Annual Meeting, February 23–27, San Francisco. 2002; (Abstract 85c)Google Scholar). 1–7 amino acid mutations were made in a single primer set to account for any binding determinant in that particular linker using an inverse PCR protocol. Transmembrane segment mutations were made as single amino acid replacements. Wild-type and mutant toxins were expressed as fusion proteins containing maltose-binding protein upstream of the toxin in Escherichia coli BL21 (DE3) as described (15Smith J.J. Alphy S. Seibert A.L. Blumenthal K.M. J. Biol. Chem. 2005; 280: 11127-11133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). After lysis in a French press, the supernatants obtained were purified on Ni2+-nitrilotriacetic acid resin and reduced with 10 mm dithiothreitol for 1 h at 37 °C. After diluting the proteins to 0.2 mg/ml, they were dialyzed against 2.5 mm GSH, 50 mm Tris, 100 mm NaCl, pH 8.3. After dialysis, the proteins were oxidized by dropwise addition of GSSG to a final concentration of 0.5 mm and allowed to incubate for 72 h. Fusion proteins were then dialyzed against 50 mm NH4HCO3 and cleaved overnight at room temperature with enterokinase (Novagen/EMD Biosciences). Toxins were purified via RP-HPLC as described (15Smith J.J. Alphy S. Seibert A.L. Blumenthal K.M. J. Biol. Chem. 2005; 280: 11127-11133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Molecular weights of purified toxins were confirmed by MALDI-TOF mass spectroscopy analysis on a Bruker Biflex IV spectrometer. Because of folding difficulties encountered with a subset of mutants, some positions were mutated to an amino acid with a larger side chain to facilitate proper packing. These include K4Q, R13Q, W24L, K26Q, and K27Q. In addition to recombinant mutant toxins, some synthetic mutants were studied, including Y1A, S11A, K14A, E17A, L23A, K28A, L29A, and W30A (GenScript Corp., Piscataway, NJ). Lyophilized peptides were resuspended to a peptide concentration of 5 mg/ml in nitrogen saturated 8 m urea, 50 mm Tris, 50 mm NH4HCO3, 120 mm GSH, pH 7.8. The peptides were then diluted to a concentration of 0.5 mg/ml and a GSH concentration of 12 mm. A final dilution brought the peptide concentration to 0.125 mg/ml and urea to 2 m, whereas GSH remained at 12 mm. GSSG was then added dropwise to a final concentration of 1.2 mm and allowed to incubate at 4 °C for 48 h. Samples were taken at various stages throughout the folding reactions for RP-HPLC analysis. MALDI-TOF analysis of samples confirmed that the peptides were oxidized. To purify folded peptides, we used cation exchange chromatography (HiTrap SP FF, GE Healthcare) followed by RP-HPLC. We were unable to produce significant amounts of the G18A mutant, presumably because of its inability to fold. All cell culture reagents were purchased from Invitrogen. Standard whole-cell voltage-clamp recordings were made from all cells. To analyze the effects of wild-type and mutant ProTx-II on the human cardiac Nav channel, a stable cell line expressing Nav1.5 was constructed in HEK 293 cells as previously described (22Seibert A. Liu J.R. Hanck D.A. Blumenthal K.M. Biochemistry. 2003; 42: 14515-14521Crossref PubMed Scopus (22) Google Scholar). To verify previously reported affinity data for ProTx-II on the peripheral nerve Nav channel, a stable HEK 293 cell line expressing human Nav1.7 was utilized (23Cummins T.R. Howe J.R. Waxman S.G. J. Neurosci. 1998; 18: 9607-9619Crossref PubMed Google Scholar). To study the effects of ProTx-II on neuronal Nav channels, the murine neuroblastoma cell line, N1E-115 was obtained from ATCC (Manassas, VA). Because these cells predominantly express Nav1.2, we ascribe ProTx-II modification to this isoform (24Hirsch J.K. Quandt F.N. Brain Res. 1996; 706: 343-346Crossref PubMed Scopus (23) Google Scholar). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a 5% CO2 atmosphere. 200 μg/ml G418 was used for selection of HEK 293 cells expressing Nav1.5 or Nav1.7 channels. Toxin affinities for Nav1.5 and Nav1.2 channels were measured after introduction by gravity perfusion into a 300-μl bath chamber at a flow rate of ∼3 ml/min. For Nav1.7 measurements, toxin was diluted into a 250-μl recording chamber and mixed by repeatedly pipetting 25 μl over ∼5 s to achieve the specified concentration. All toxin solutions contained 1 mg/ml bovine serum albumin to prevent adsorption to tubing. Single cell recordings were made at room temperature using an Axopatch 200B amplifier with a Digidata 1322A analogue to digital converter and pCLAMP software (Axon Instruments). Pipettes were pulled from borosilicate glass (World Precision Instruments) and fire-polished to a final resistance of 1–3 megaohms when filled with recording solution. Solutions used for sodium current measurements through Nav1.5 channels contained the following: bath solution, 10 mm NaCl, 130 mm CsCl, 2 mm CaCl2, 10 mm HEPES, pH 7.4 with CsOH; pipette solution, 95 mm CsF, 30 mm CsCl, 5 mm NaCl, 10 mm EGTA, 10 mm HEPES, pH 7.0 with CsOH. Solutions used for Nav1.7 channels contained the following: bath solution, 140 mm NaCl, 3 mm KCl, 1 mm MgCl2, 1 mm CaCl2, 10 mm HEPES, pH 7.3; pipette solution, 140 mm CsF, 1 mm EGTA, 1 mm MgCl2, 10 mm NaCl, 10 mm HEPES, pH 7.3. Solutions used for Nav1.2 channels contained: bath solution, 70 mm NaCl, 70 mm CsCl, 2 mm CaCl2, 10 mm HEPES, pH 7.4; pipette solution, 10 mm NaCl, 90 mm CsF, 30 mm CsCl, 10 mm EGTA, 10 mm HEPES, pH 7.0. Recordings were initiated 5–8 min after patch rupture. To measure time courses of modification and dissociation, cells were held hyperpolarized at -130 mV then stepped to either -30 mV (Nav1.5 channels) or -10 mV (Nav1.2 channels) for 10 ms at a frequency of 1 Hz as either toxin-containing or toxin-free solution was introduced into the bath. To generate current-voltage (I-V) relationships, cells were stepped in 5-mV increments from -80 to +20 mV (Nav1.5 channels), from -80 to +70 mV (Nav1.7 channels), or from -50 to +50 mV (Nav1.2 channels) from a holding potential of -120 or -130 mV for 30 ms (Nav1.5 and 1.2) or 50 ms (Nav1.7), and the resulting peak currents were plotted against voltage. Current-voltage relationships were obtained just before toxin application and after steady-state inhibition was achieved. Only cells having a >4.2-mV slope were included. Currents were capacity-corrected using MatLab 6.5 (The MathWorks, Inc., Natick, MA). Only cells with a leak resistance of >750 megaohms were included in analyses. Toxin test concentrations ranged from 250 to 20 μm and were determined empirically for each toxin mutant. Conductance-voltage (g-V) relationships quantitating the voltage dependence of activation were obtained from peak current-voltage (I-V) relationships according to g = INa/V - Vr, where INa is the peak Na+ current at test potential V, and Vr represents reversal potential. To assess the voltage dependence of inactivation, a 2-step protocol was used in which cells were stepped in 5-mV increments from -130 to -30 mV for 300 ms followed by a step to the test potential -30 mV for 20 ms to evaluate channel availability. Normalized activation and inactivation curves were fit to a Boltzmann function y = 1/[1 + e(V - V0.5)/k], where y is normalized gNa or INa, V is membrane potential, V0.5 is the midpoint of activation or inactivation, and k is the slope factor. Data are depicted as ±S.E. The half-blocking concentration (IC50) for ProTx-II on Nav1.7 was calculated based on the single-site Langmuir inhibition isotherm using the following function: (Itoxin/I0) × [toxin]/(1 - Itoxin/I0), where I0 and Itoxin are the peak sodium currents measured with a test pulse to -30 mV before and after application of toxin, respectively, and[toxin] is the concentration of toxin. Mutant channel DNA was transiently transfected into HEK 293 cells followed by whole-cell voltage clamp analysis to assess mutant channel function and interaction with wild-type ProTx-II. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Lipofectamine reagent in combination with PLUS reagent (Invitrogen) was used for transient transfections, and after 24–36 h of incubation cells were trypsinized and moved to coverslips for analysis. Because of lower expression levels of mutant channels, [Na+] was increased in the bath solution to either 70 mm (medium Na+) or 140 mm (high Na+), and [Cs+] was adjusted accordingly. Other components were kept constant as described above. Corresponding pipette solutions contained either 35 mm NaCl, 65 mm CsF (medium Na) or 70 mm NaCl, 30 mm CsF (high Na). The voltage dependence of activation and inactivation of linker swap mutants was analyzed and compared with wild-type Nav1.5 to ensure normal channel function using protocols described above. A wild-type toxin concentration of 1 μm was used to assess modification of mutant channels. We looked for a loss of inhibition during simple step protocols from -130 to -30 mV that would indicate the receptor site on the channel had been disrupted. Peak currents were plotted versus time from the point of toxin wash-in or wash-out. The data were fit to a first order exponential decay equation using Origin 6.1 software (Microcal Software Inc., Northampton, MA). We calculated the kinetic constants for channel modification (kmod) and toxin dissociation (koff) using the inverse of τ of the fit for toxin wash-in and wash-out, respectively. To determine the rate of toxin association, the following equation was used: kon = kmod - koff/[toxin]. The dissociation constant was determined using koff/kon = KD. Data are reported as ±S.E. An energy-minimized molecular model of ProTx-II was created using the Protein Data Bank coordinates for HpTx-2, an ICK motif peptide targeting Kv4 channels (PDB code 1emx; Refs. 25Brahmajothi M.V. Campbell D.L. Rasmusson R.L. Morales M.J. Trimmer J.S. Nerbonne J.M. Strauss H.L. J. Gen. Physiol. 1999; 113: 581-600Crossref PubMed Scopus (157) Google Scholar and 26Bernard C. Legros C. Ferrat G. Bischoff U. Marquardt A. Pongs Francisco O. Darbon H. Protein Sci. 2000; 9: 2059-2067PubMed Google Scholar). Conversion to the ProTx-II sequence was done in the Biopolymer module of InsightII, and the resulting model was then subjected to energy minimization (initially using a steepest descents protocol followed by at least 2500 cycles of conjugate gradients) in Discover to remove steric clashes. After minimization, the total energy of the model structure was ∼300 kcal/mol, and it retained the backbone structure typical of the ICK motif. Functional Characterization of Wild-type and Mutant Forms of ProTx-II—The first aim of the present study was to characterize the effects of ProTx-II mutants on Nav1.5 to isolate the pharmacophore of the toxin molecule. In addition, we hoped to identify the channel receptor site and ultimately establish a mechanism of action for this novel acting peptide toxin. We produced wild-type ProTx-II and several mutated forms either recombinantly or synthetically and purified them to homogeneity using RP-HPLC. To characterize the effects of wild-type or mutated ProTx-II on Nav1.5, we used a whole-cell voltage clamp on HEK 293 cells stably expressing this channel. We evaluated the extent of channel modification by depolarizing the cell membrane to -30 mV as toxin-free solution was replaced with toxin-containing solution. At 1 μm ProTx-II, we observed rapid and near complete inhibition of sodium current (τ ∼ 2.5 s) (Fig. 1A). This inhibition was completely reversible upon toxin wash-out (τ ∼ 40 s) (Fig. 1B). To verify that toxin binding is concentration-dependent, we examined channel modification over the range of 250–1000 nm ProTx-II. Modification decreased accordingly at both concentrations (τ250 nm = 9.3 ± 0.23 s, n = 3; τ500 nm = 5.48 ± 0.17 s, n = 3). In contrast, dissociation remained a zero-order reaction as expected (τ250 nm = 53.3 ± 3.2 s, n = 3; τ500 nm = 41.7 ± 2.2 s, n = 3). ProTx-II also shifts the voltage dependence of gating to more depolarized potentials, indicating that the toxin does not inhibit through a pore-blocking mechanism but, rather, interferes with the energetics of gating (15Smith J.J. Alphy S. Seibert A.L. Blumenthal K.M. J. Biol. Chem. 2005; 280: 11127-11133Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Analysis of ProTx-II on steady-state activation and inactivation kinetics revealed that the midpoint of the activation curve shifted by 23 mV in the depolarizing direction, whereas inactivation remained unaffected by the toxin (Fig. 2, A and B). This mode of channel modification is similar to that of other ICK toxins that target Nav and Kv channels but very different from site 4 toxins targeting Nav channels. Our kinetic analysis yielded an equilibrium dissociation constant for ProTx-II of 93 nm, similar to the value obtained using natural material (14Middleton R.E. Warren V.A. Kraus R.L. Hwang J.C. Liu C.J. Dai G. Brochu R.M. Kohler M.G. Gao Y.D. Garsky V.M. Bogusky M.J. Mehl J.T. Cohen C.J. Smith M.M. Biochemistry. 2002; 41: 14734-14747Crossref PubMed Scopus (204) Google Scholar).FIGURE 2ProTx-II modifies the steady-state voltage dependence of activation, but not inactivation, of NaV1.5 channels. A, ProTx-II shifts the steady-state activation curve 23 mV in the depolarizing direction. Cells (n = 10) stably expressing Nav1.5 were stepped in 5-mV increments from -80 to +20 mV from a holding potential of -130 mV for 30 ms. For control cells (○), the midpoint of activation Va =-42.5 ± 0.57mV, and the slope factor k = 5.91 ± 0.22 mV; for cells treated with 1 μm ProTx-II (•), midpoint of activation Va =-19.6 ± 0.5 mV, and the slope factor k = 7.48 ± 0.32 mV. B, ProTx-II has no effect on steady-state inactivation. Cells (n = 8) were stepped in 5-mV increments from -130 mV to -30 mV for 300 ms followed by a test pulse to -30 mV for 20 ms. For control cells (○), the midpoint of inactivation Vh = -79.2 ± 0.7 mV, and the slope factor k = 4.26 ± 0.20 mV; for cells treated with 1 μm ProTx-II (•), the midpoint of inactivation Vh =-79.5 ± 0.7 mV, and the slope factor k = 3.91 ± 0.44 mV. Normalized conductances (activation) and currents (inactivation) were generated for both data sets, fit to a Boltzmann function, and are shown as data ± S.E.View Large
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