Am J Physiol Cell Physiol Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 293: C783-C789, 2007. First published May 23, 2007; doi:10.1152/ajpcell.00070.2007
0363-6143/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Tables
Right arrow All Versions of this Article:
293/2/C783    most recent
00070.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rosker, C.
Right arrow Articles by Schreibmayer, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rosker, C.
Right arrow Articles by Schreibmayer, W.

RECEPTORS AND SIGNAL TRANSDUCTION

The TTX metabolite 4,9-anhydro-TTX is a highly specific blocker of the Nav1.6 voltage-dependent sodium channel

Christian Rosker,1 Birgit Lohberger,1 Doris Hofer,1 Bibiane Steinecker,1 Stefan Quasthoff,2 and Wolfgang Schreibmayer1

1Institute for Biophysics, Center for Physiological Medicine and 2Department for Neurology, Medical University of Graz, Graz, Austria

Submitted 20 February 2007 ; accepted in final form 16 May 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The blocking efficacy of 4,9-anhydro-TTX (4,9-ah-TTX) and TTX on several isoforms of voltage-dependent sodium channels, expressed in Xenopus laevis oocytes, was tested (Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, and Nav1.8). Generally, TTX was 40–231 times more effective, when compared with 4,9-ah-TTX, on a given isoform. An exception was Nav1.6, where 4,9-ah-TTX in nanomole per liter concentrations sufficed to result in substantial block, indicating that 4,9-ah-TTX acts specifically at this peculiar isoform. The IC50 values for TTX/4,9-ah-TTX were as follows (in nmol/l): 7.8 ± 1.3/1,260 ± 121 (Nav1.2), 2.8 ± 2.3/341 ± 36 (Nav1.3), 4.5 ± 1.0/988 ± 62 (Nav1.4), 1,970 ± 565/78,500 ± 11,600 (Nav1.5), 3.8 ± 1.5/7.8 ± 2.3 (Nav1.6), 5.5 ± 1.4/1,270 ± 251 (Nav1.7), and 1,330 ± 459/>30,000 (Nav1.8). Analysis of approximal half-maximal doses of both compounds revealed minor effects on voltage-dependent activation only, whereas steady-state inactivation was shifted to more negative potentials by both TTX and 4,9-ah-TTX in the case of the Nav1.6 subunit, but not in the case of other TTX-sensitive ones. TTX shifted steady-state inactivation also to more negative potentials in case of the TTX-insensitive Nav1.5 subunit, where it also exerted profound effects on the time course of recovery from inactivation. Isoform-specific interaction of toxins with ion channels is frequently observed in the case of proteinaceous toxins. Although the sensitivity of Nav1.1 to 4,9-ah-TTX is not known, here we report evidence on a highly isoform-specific TTX analog that may well turn out to be an invaluable tool in research for the identification of Nav1.6-mediated function, but also for therapeutic intervention.

sodium channel; tetrodotoxin

TETRODOTOXIN (TTX) IS A POTENT neurotoxin that is abundant in the ovary and liver of puffer fish, but found also in other species, like, e.g., frogs, newts, and octopi (22). Since its potent and selective block of voltage-gated sodium channels was discovered (23), it has become an invaluable tool in ion channel research. It became clear that symbiotic bacteria are the origin of TTX, and subsequently several interesting metabolites of TTX biosynthesis and degradation were discovered (20). Although one of them, the rarely occurring 10-oxotetrodotoxin, turned out to be even more potent in blocking voltage-dependent sodium channels than TTX itself (26), it is generally believed that TTX represents the major toxic principle of TTX action. To test for the toxicity of the abundant TTX-related compound 4,9-anhydro-TTX (4,9-ah-TTX; see Fig. 1), several TTX-sensitive, as well as TTX-insensitive, isoforms of voltage-dependent sodium channel {alpha}-subunits (Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, and Nav1.8) were heterologously expressed in Xenopus laevis oocytes, and the blocking efficacy of 4,9-ah-TTX was tested and compared with TTX.


Figure 1
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 1. Chemical structure of TTX and 4,9-anhydro-TTX.

 

   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid vectors encoding the full-length Nav1.6, Nav1.7, and Nav1.8 {alpha}-subunits of different mammals (rat: Nav1.2, Nav1.3, Nav1.4 and Nav1.8; human: Nav1.5 and Nav1.7; mouse: Nav1.6) were used as previously described (1, 2, 11, 16, 25, 30, 32). Following RNA synthesis (9) and preparation of oocytes from X. laevis (12), 0.05–0.25 ng (Nav1.2), 10–15 ng (Nav1.3), 0.25–1.5 ng (Nav1.4), 0.25–1.5 ng (Nav1.5), 2.5 ng (Nav1.6), 25–50 ng (Nav1.7), and 50 ng (Nav1.8) RNA were injected per oocyte. Oocytes were incubated for 3–5 days at 19°C. Electrophysiological recordings on oocytes, including voltage-jump protocols, drug application, and evaluation and analysis of data, were performed exactly as described recently (12). Chemicals used were reagent grade throughout. TTX and 4,9-ah-TTX were kindly provided by ESTEVE laboratories (Barcelona, Spain). Approximal half-maximal concentrations of TTX/4,9-ah-TTX to investigate effects on channel kinetics were as follows (in nmol/l): 5/1,000 Nav1.2, 5/1,000 Nav1.3, 5/1,000 Nav1.4, 1,000/ND Nav1.5, 5/10 Nav1.6, 5/1,000 Nav1.7, 1,000/ND Nav1.8, where ND indicates not done, because of the extraorbitant amount of 4,9-ah-TTX required. Stock solutions were prepared by dissolving TTX or 4,9-ah-TTX at a concentration of 1 mmol/l in citrate–/Na+ buffer (10 mmol/l), pH 4.8. Small aliquots of these stock solutions were shock-frozen in liquid nitrogen and stored at –20°C until use.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In oocytes, expressing either of the Nav1.x {alpha}-subunit, sodium currents were recorded, and increasing concentrations of TTX or 4,9-ah-TTX were applied (see Fig. 2 for original recordings from the Nav1.6 and Nav1.7 subunits). As expected, TTX blocked both the Nav1.6 and the Nav1.7 subunit at nanomole per liter concentrations. Interestingly, 4,9-ah-TTX blocked the Nav1.6 isoform at nanomole per liter concentrations, quite similar to TTX. In contrast to the Nav1.6 subunit, >200 times higher concentrations of 4,9-ah-TTX were required for block of the Nav1.7 subunit and also the other TTX-sensitive isoforms tested. In contrast to the nanomole per liter concentrations of TTX used to block the TTX-sensitive isoforms, the two TTX-insensitive isoforms, Nav1.5 and Nav1.8, required micromole per liter concentrations of TTX to be blocked. Even higher concentrations of 4,9-ah-TTX were required to block the TTX-insensitive Nav1.5 isoform (78.5 ± 11.6 µmol/l). The Nav1.8 isoform did not even react to concentrations up to 100 µmol/l 4,9-ah-TTX. In Fig. 3, three characteristic dose-response relations for the peculiar behavior of Nav1.6 (Fig. 3A), the TTX-sensitive Nav1.7 (Fig. 3B), and the TTX-insensitive Nav1.5 (Fig. 3C) are shown. The complete statistical analysis of the IC50 values for TTX and 4,9-ah-TTX is shown in Fig. 4. With the exception of Nav1.6, the concentrations of 4,9-ah-TTX required to achieve 50% blockage of sodium currents through a given isoform were ~40 to 230 times higher when compared with TTX.


Figure 2
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 2. Differential action of 4,9-anhydro-TTX (4,9-ah-TTX) on the voltage-dependent sodium channel {alpha}-subunit Nav1.6 and Nav1.7 isoforms. A: original experiment on Nav1.6. Left: peak sodium inward current [Ip(Na+)] resulting from increasing concentrations of TTX are shown vs. time. Insets show original sodium current registrations (scaling shown with the first example applies to all). Right: same as left, but 4,9-ah-TTX was used to block sodium currents. B: same as A, but Nav1.7 was used as drug target.

 

Figure 3
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 3. Cumulative dose-response curves for Nav1.6, Nav1.7, and Nav1.5. A: average cumulative dose-response curve for TTX (N = 7) and 4,9-ah-TTX (N = 19) on the Nav1.6 isoform. Straight and dotted lines represent least-square fit through the data. B: average cumulative dose-response curve for TTX (N = 8) and 4,9-ah-TTX (N = 6) on the Nav1.7 isoform. C: average cumulative dose-response curve for TTX (N = 12) and 4,9-ah-TTX (N = 4) on the Nav1.8 isoform.

 

Figure 4
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 4. Top: mean value of IC50 and the slope of dose-response curves are given as means ± SE; n, no. of experiments. The mean values between TTX and 4,9-ah-TTX differ significantly at the P < 0.05 (*), 0.01 (**), and 0.001 (***) level between identical isoforms. ND, could not be determined. Bottom: IC50 values for TTX (left) or 4,9-ah-TTX (right) were tested for statistically significant differences between the different isoforms tested. The IC50 value differs significantly at the P < 0.05 (*), 0.01 (**), and 0.001 (***) level between the corresponding isoforms. NS, difference was not significant.

 
Effects on channel kinetics. IC50 concentrations, assessed for sodium channel blocking drugs during cumulative dose-response experiments, are the result of both 1) the dynamic interaction of the drug with the channel protein and its conformational equilibrium ("use dependence") and 2) the component of block that does neither depend on time nor on channel conformations ("tonic" component). To discriminate between these two components, half-maximal concentrations of TTX and 4,9-ah-TTX were applied, and their effect on channel kinetics was assessed.

Steady state activation. A typical experiment for the assessment of voltage-dependent activation and a potential influence of TTX and/or 4,9-ah-TTX on this process is shown in Fig. 5 for the Nav1.6 isoform. Although current amplitudes are reduced by both TTX and 4,9-ah-TTX (Fig. 5B), the normalized I/Em relations are practically identical for control conditions and under the influence of TTX or 4,9-ah-TTX (Fig. 5C). Parameters for the Nav1.x subunits tested are summarized in supplemental Table 1 (suplemental data for this article are available online at the American Journal of Physiology: Cell Physiology website.). It was observed that the potentials for half-maximal activation (Ea0.5) and the slopes of the Boltzmann-isotherms (ka) were practically unchanged by both TTX and 4,9-ah-TTX for most of the Nav1.x isoforms tested. The few statistically significant changes observed were minor in their numerical value. In summary, neither TTX nor 4,9-ah-TTX exerted considerable effects on voltage-dependent steady-state activation. On the other side, {Delta}Gmax% values were greatly and significantly reduced in all the instances tested, indicating that the blocking effect of both TTX and 4,9-ah-TTX was mainly the result of tonic, i.e., state-independent, action (see supplemental Table 2, which is available as supplementary material in electronic form).


Figure 5
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 5. Effect on voltage-dependent activation (Nav1.6). A: scheme of voltage-jump protocol: the oocyte membrane was held at –100 mV for 6 s and then stepped to the desired command potential (indicated by arrow) to elicit sodium currents. B: original current traces recorded at control conditions (left) and under the influence of 5 nmol/l TTX (bottom right) and 10 nmol/l 4,9-ah-TTX (bottom right). Command potentials were as follows: 1) –45 mV, 2) –20 mV, 3) –5 mV, and 4) +20 mV. C: average IpNa+/Em relations from 9 (control; TTX) or 10 (4,9-ah-TTX) experiments, normalized to the maximal peak sodium inward current (occurring at –10 mV) ± SE. Solid lines represent a fit through the data according to a Boltzmann isotherm.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Effects of TTX and 4,9-ah-TTX on the recovery from inactivation

 
Steady-state inactivation. The effect of approximately half-maximal doses of TTX and 4,9-ah-TTX on steady-state inactivation was tested. Original current traces and h{infty} vs. Em curves for the Nav1.6 isoform are shown in Fig. 6. Interestingly, the potential for half-maximal inactivation (Ei0.5) was shifted significantly to more negative potentials by 10 nmol/l 4,9-ah-TTX (from –62.4 ± 0.7 to –68.0 ± 1.4 mV; P < 0,001; N = 9) as well as by 5 nmol/l TTX for Nav1.6 (from –62.4 ± 0.7 to –67.6 ± 0.5 mV; P < 0,001; N = 9; see Fig. 6C). Such a negative shift in Ei0.5 was observed also for the TTX-insensitive Nav1.5 subunit, in the case of TTX (from –69.0 ± 1.3 to –77.5 ± 0.8 mV; P < 0,001; N = 9). Because of the extremely high concentrations required, 4,9-ah-TTX could not be tested on the Nav1.5 isoform. In all of the other TTX-sensitive isoforms tested, such a shift in Ei0.5 was not observed (a complete survey of results on all the isoforms tested is given in supplemental Table 3, available as supplementary information in electronic form). To substantiate the negative shift of Ei0.5, induced by the toxins, on the Nav1.6 subunit, increasing concentrations of TTX were tested. It was revealed that the negative shift increased with increasing concentrations of TTX, whereas another isoform taken as a control (Nav1.7) remained unaffected (Fig. 6D).


Figure 6
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 6. Effect on steady-state inactivation of Nav1.6. A: scheme of voltage-jump protocol: the oocyte membrane was held at –120 mV for 6 s to allow recovery from inactivation and then stepped to the desired command potential for the duration of 1 s. Subsequently, sodium currents were elicited (indicated by arrow). B: original current traces recorded at control conditions (left) and under the influence of 5 nmol/l TTX (top right) and 10 nmol/l 4,9-ah-TTX (bottom right). Command potentials were as follows: 1) –100 mV, 2) –80 mV, and 3) –60 mV. C: average steady-state inactivation curve from 9 experiments ± SE. Solid lines represent a fit through the data according to a Boltzmann isotherm. D: effect of increasing concentrations of TTX on half-maximal potential of steady-state inactivation of Nav1.7 (white bars; N = 4) and Nav1.6 (gray bars; N = 4) ± SE. ***Mean values differ significantly at the P < 0.001 level from the control value. §Mean value for 10 nmol/l TTX differs significantly at the P < 0.05 level from the value at 5 nmol/l TTX.

 
Recovery from inactivation. In case of a use-dependent component of channel block, often the time constant of recovery from inactivation is markedly prolonged by drug/toxin action. These time constants (for both the fast and the slow inactivated state) were measured at a membrane potential of –100 mV. Original current traces for the recovery of Nav1.5 at 1 µmol/l TTX and the recovery of Nav1.6 at 10 nmol/l 4,9-ah-TTX are shown in Fig. 7. Both a direct inspection of the original traces, but also the averaged fractional recovery vs. time (Fig. 7C), reveal that the time course of recovery from inactivation is strongly affected by TTX in the case of Nav1.5. Moreover, in the case of Nav1.5, the fraction of channels exerting the slow recovery process at the cost of the fast one is also greatly enhanced by TTX. Hence, TTX exerts classical properties of a classical antiarrhythmic agent on Nav1.5. To the contrary, neither TTX nor 4,9-ah-TTX exerted an influence on the time constants of both the fast and the slow recovery process of Nav1.6. Also the fraction of channels exerting fast and slow recovery was unchanged (see Fig. 7D). In Table 1, a summary of the results obtained on different Nav1.x isoforms with TTX and 4,9-ah-TTX is given.


Figure 7
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 7. Effect of TTX and 4,9-ah-TTX on the recovery from inactivation of Nav1.6 and Nav1.5. A: scheme of voltage-jump protocol: the oocyte membrane was held at –120 mV for 6 s to allow recovery and then steady-state inactivation was allowed for 3 s at 0 mV. Subsequently, recovery at –100 mV took place for various time intervals, and available sodium currents were elicited. B: original current traces recorded from Nav1.5 at control conditions (top left) and at 1 µmol/l TTX (top right). Recovery time intervals were as follows: 1) 50 s, 2) 5 s, 3) 500 ms, and 4) 5 ms. Recordings obtained from Nav1.6 at control conditions (bottom left) and at 10 nmol/l 4,9-ah-TTX (bottom right) are also displayed. Recovery time intervals were as follows: 1) 50 s, 2) 5 s, 3) 500 ms, and 4) 5 ms. C: average fractional recovery of Nav1.5 at control (white circles; N = 7) and at 1 µmol/l TTX (black circles; N = 6) ± SE. Solid lines represent a fit through the data according to a biexponential function. D: average fractional recovery of Nav1.6 at control (white circles; N = 9), at 5 nmol/l TTX (black circles; N = 10), and at 10 nmol/l 4,9-ah-TTX (gray circles; N = 11) ± SE. Solid lines represent a fit through the data according to a biexponential function.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our experiments show clearly that 4,9-ah-TTX represents a highly specific blocker for the Nav1.6 isoform among the ones tested by us (Nav1.2-8). One important site of interaction and the site determining TTX sensitivity of Nav channels can be deduced to a single amino acid, a hydrophobic F/Y within the pore-forming region of the first pseudo-domain of TTX-sensitive Nav channels that is replaced by a polar S/C in TTX-insensitive isoforms (see, e.g., Ref. 29). Both TTX-insensitive isoforms tested by us exerted an even lower susceptibility to 4,9-ah-TTX than to TTX. Whereas ~40 times higher concentrations of 4,9-ah-TTX, compared with TTX, were required to achieve half-maximal block of the Nav1.5 isoform, the Nav1.8 did not even react to 4,9-ah-TTX at the concentrations tested by us. Also, the TTX-sensitive subunits (except Nav1.6) required 162 (Nav1.2), 171 (Nav1.3), 220 (Nav1.4) and 231 (Nav1.7) times higher concentrations of 4,9-ah-TTX, compared with TTX, to achieve half-maximal current inhibition. Hence, 4,9-ah-TTX likely blocks sodium channels via the same site and mechanism as TTX, but generally with lower affinity. The micromole per liter amounts of 4,9-ah-TTX required to block the TTX-sensitive Nav1.x subunits tested are in good agreement with the work of Yotsu-Yamashita et al. (34) who found an equilibrium dissociation constant for the displacement of radioactively labeled saxitoxin (STX) from rat brain membranes that was ~100 times higher for 4,9-ah-TTX, when compared with TTX. Given the vast heterogeneity of Nav isoforms in rat brain membranes together with the fact that Nav1.6 is abundant mainly in peripheral nerves, it is not surprising that the exceptional high-affinity interaction of 4,9-ah-TTX with Nav1.6 was not detected in this study. Apparently, the TTX receptor of Nav1.6 has unique features that makes it a high-affinity target for 4,9-ah-TTX. Because the primary determinant for TTX sensitivity, a Y in the pore region, is well conserved, other likely steric factors must be involved. Such contributions by protein surfaces, not within the actual site of drug/toxin binding but near it and within the access path for the agent, has already been described for the interaction of local anesthetics with Nav1.x subunits, when regions other than the structural determinant responsible for local anesthetic binding considerably contributed to the pharmacokinetics of drug/Nav1.x interaction (27). Starting from the known KcsA crystal structure, Lipkind and Fozzard (18) came up with a model of the sodium ion channel pore, including the guanidinium toxin (TTX and STX) binding sites. Indeed this model shows that the Y in the pore region of the TTX-sensitive channel contributes considerably with strong nonbonded interactions between the aromatic ring of this Y (position 401 in the Nav1.4 sequence) and the nonpolar surface of TTX. Additional polar interactions between the guanidinium toxins and several amino acid residues contributing to the channel's outer vestibule are also predicted. Starting from this model and using thermodynamic mutant cycle analysis, Choudhary et al. (6) found that the C-11 hydroxyl group of TTX contributes considerably to the interaction of TTX with the outer vestibule of the Nav1.4 isoform. Moreover, their modified model predicts considerable contributions of the other hydroxyls of TTX, including those at C-9 and C-4, to the interaction of TTX with the outer vestibule. In another independent study, the unique three-dimensional properties of the outer vestibule of Nav1.6 also emerged; Schiavon et al. (28) observed that the scorpion beta-toxin Cn2 induced a pronounced shift in voltage-dependent activation and transient resurgent currents exclusively in Nav1.6 but not in other sodium channel isoforms. Unfortunately, because the exact three-dimensional structure of the outer vestibule of sodium channels is up to now unknown, the exact structural determinants in the vestibule that account for the specific 4,9-ah-TTX blockage of Nav1.6 remain obscured. In our study, these peculiar properties of the TTX receptor of Nav1.6 are underlined by the effect of both TTX and 4,9-ah-TTX on steady-state inactivation in the case of Nav1.6, but not in the case of the other TTX-sensitive subunits. Such a concentration-dependent shift of the potential required for half-maximal inactivation to more negative potential is frequently observed in the case of organic Nav1.x channel blockers (see, e.g., Ref. 12), but not for TTX. Hence, TTX and 4,9-ah-TTX exert functional properties resembling local anesthetics with respect to their effect on steady-state inactivation of Nav1.6. Only in the TTX-insensitive Nav1.5 isoform are such pronounced effects on channel steady-state inactivation and voltage-dependent gating parameters reported (3, 5). In the current study, we were able to show that not only steady-state inactivation of Nav1.5 is shifted to more negative potentials by TTX and 4,9-ah-TTX, but the time course of recovery from inactivation is also greatly affected by TTX: TTX significantly prolongs the time constant of the fast recovery process. In addition, the fraction of channels exerting fast recovery is substantially and significantly reduced, whereas the fraction of channels exerting slow recovery is greatly increased under the action of the toxin. Such an influence of TTX or 4,9-ah-TTX on the time constant of recovery from inactivation was not observed in the case of Nav1.6. Unfortunately, the complete proteomics of sodium channels is, at present, unknown. An impressive body of work exists, however, that shows Nav1.6 to be an important player in several cellular functions: Nav1.6 is the major isoform in the nodes of Ranvier of myelinated nerve fibers but also abundant in unmyelinated C-fibers (8). Its important role in cerebellar motor function is well documented (17). Besides this, Nav1.6 has been reported to exist in Schwann cells (21) and in T tubules of the myocardium (19), to be involved in the maturation of photoreceptors (7), and to have broad distribution in the enteric nervous system (4) and in the cochlea (14). Nav1.6 channels have also been identified to be involved in several pathophysiological processes like chronic demyelinization accompanying multiple sclerosis (33), diabetic neuropathy (13), epilepsia (15), and neoplastic transformation (10). Although isoform-specific interaction of toxins with ion channels is frequently observed with proteinaceous toxins (see, e.g., Refs. 31 and 24), such specific behavior is rarely observed for other compounds. Although the sensitivity of Nav1.1 to 4,9-ah-TTX is not known, here we report evidence on a highly isoform-specific TTX analog that may well turn out to be an invaluable tool in research for the identification of Nav1.6-mediated function, but also for therapeutic intervention.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. Schreibmayer, Institute for Biophysics, Center for Physiological Medicine, Medical Univ. of Graz, Harrachgasse 21/4, A-8010 Graz, Austria (e-mail: wolfgang.schreibmayer{at}meduni-graz.at)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Akopian AN, Sivilotti L, Wood JN. A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature 379: 257–262, 1996.[CrossRef][Medline]

2. Auld VJ, Goldin AL, Krafte DS, Catterall WA, Lester HA, Davidson N, Dunn RJ. A neutral amino acid change in segment IIS4 dramatically alters the gating properties of the voltage-dependent sodium channel. Proc Natl Acad Sci USA 87: 323–327, 1990.[Abstract/Free Full Text]

3. Baer M, Best PM, Reuter H. Voltage-dependent action of tetrodotoxin in mammalian cardiac muscle. Nature 263: 344–345, 1976.[CrossRef][Medline]

4. Bartoo AC, Sprunger LK, Schneider DA. Expression and distribution of TTX-sensitive sodium channel alpha subunits in the enteric nervous system. J Comp Neurol 486: 117–131, 2005.[CrossRef][ISI][Medline]

5. Carmeliet E. Voltage-dependent block by tetrodotoxin of the sodium channel in rabbit cardiac Purkinje fibers. Biophys J 51: 109–114, 1987.[ISI][Medline]

6. Choudhary G, Yotsu-Yamashita M, Shang L, Yasumoto T, Dudley SC. Interactions of the C-11 hydroxyl of tetrodotoxin with the sodium channel outer vestibule. Biophys J 84: 287–294, 2003.[ISI][Medline]

7. Cote PD, De RY, Coupland SG, Schwab Y, Roux MJ, Levinson SR, Kothary R. Physiological maturation of photoreceptors depends on the voltage-gated sodium channel NaV1.6 (Scn8a). J Neurosci 25: 5046–5050, 2005.[Abstract/Free Full Text]

8. Craner MJ, Newcombe J, Black JA, Hartle C, Cuzner ML, Waxman SG. Molecular changes in neurons in multiple sclerosis: altered axonal expression of Nav1.2 and Nav16 sodium channels and Na+/Ca2+ exchanger. Proc Natl Acad Sci USA 101: 8168–8173, 2004.[Abstract/Free Full Text]

9. Dascal N, Lotan I. Expression of exogenous ion channels and neurotransmitter receptors in RNA-injected Xenopus oocytes. In: Methods in Neurobiology, edited by Longstaff A and Revest P. Totowa, NJ: Humana, 1992, vol. 13, p. 205–225.

10. Fulgenzi G, Graciotti L, Faronato M, Soldovieri MV, Miceli F, Amoroso S, Annunziato L, Procopio A, Taglialatela M. Human neoplastic mesothelial cells express voltage-gated sodium channels involved in cell motility. Int J Biochem Cell Biol 38: 1146–1159, 2006.[CrossRef][ISI][Medline]

11. Gellens ME, George AL Jr, Chen LQ, Chahine M, Horn R, Barchi RL, Kallen RG. Primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel. Proc Natl Acad Sci USA 89: 554–558, 1992.[Abstract/Free Full Text]

12. Hofer D, Lohberger B, Steinecker B, Schmidt K, Quasthoff S, Schreibmayer W. A comparative study of the action of tolperisone on seven different voltage dependent sodium channel isoforms. Eur J Pharmacol 538: 5–14, 2006.[CrossRef][ISI][Medline]

13. Hong SS, Morrow TJ, Paulson PE, Isom LL, Wiley JW. Early painful diabetic neuropathy is associated with differential changes in tetrodotoxin-sensitive and -resistant sodium channels in dorsal root ganglion neurons in the rat. J Biol Chem 279: 29341–29350, 2004.[Abstract/Free Full Text]

14. Hossain WA, Antic SD, Yang Y, Rasband MN, Morest DK. Where is the spike generator of the cochlear nerve? Voltage-gated sodium channels in the mouse cochlea. J Neurosci 25: 6857–6868, 2005.[Abstract/Free Full Text]

15. Klein JP, Khera DS, Nersesyan H, Kimchi EY, Waxman SG, Blumenfeld H. Dysregulation of sodium channel expression in cortical neurons in a rodent model of absence epilepsy. Brain Res 1000: 102–109, 2004.[CrossRef][ISI][Medline]

16. Klugbauer N, Lacinova L, Flockerzi V, Hofmann F. Structure and functional expression of a new member of the tetrodotoxin-sensitive voltage-activated sodium channel family from human neuroendocrine cells. EMBO J 14: 1084–1090, 1995.[ISI][Medline]

17. Levin SI, Khaliq ZM, Aman TK, Grieco TM, Kearney JA, Raman IM, Meisler MH. Impaired motor function in mice with cell-specific knockout of sodium channel Scn8a [Na(V)1.6] in cerebellar Purkinje neurons and granule cells. J Neurophysiol 96: 785–793, 2006.[Abstract/Free Full Text]

18. Lipkind GM, Fozzard HA. KcsA crystal structure as framework for a molecular model of the Na(+) channel pore. Biochemistry 39: 8161–8170, 2000.[CrossRef][Medline]

19. Maier SKG, Westenbroek RE, Schenkman KA, Feigl EO, Scheuer T, Catterall WA. An unexpected role for brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart. Proc Natl Acad Sci USA 99: 4073–4078, 2002.[Abstract/Free Full Text]

20. Miyazawa K, Noguchi T. Distribution and origin of tetrodotoxin. J Toxicol Toxin Rev 20: 11–33, 2001.[CrossRef]

21. Musarella M, Alcaraz G, Caillol G, Boudier JL, Couraud F, Utillo-Touati A. Expression of Nav16 sodium channels by Schwann cells at neuromuscular junctions: role in the motor endplate disease phenotype. Glia 53: 13–23, 2006.[CrossRef][ISI][Medline]

22. Narahashi T. Pharmacology of tetrodotoxin. J Toxicol Toxin Rev 20: 67–84, 2001.

23. Narahashi T, Moore JW, Scott WR. Tetrodotoxin blockage of sodium conductance increase in lobster giant axons. J Gen Physiol 47: 965–974, 1964.[Abstract/Free Full Text]

24. Oliveira JS, Redaelli E, Zaharenko AJ, Cassulini RR, Konno K, Pimenta DC, Freitas JC, Clare JJ, Wanke E. Binding specificity of sea anemone toxins to Nav 1.1–16 sodium channels: unexpected contributions from differences in the IV/S3–S4 outer loop. J Biol Chem 279: 33323–33335, 2004.[Abstract/Free Full Text]

25. Patton DE, Isom LL, Catterall WA, Goldin AL. The adult rat brain beta 1 subunit modifies activation and inactivation gating of multiple sodium channel alpha subunits. J Biol Chem 269: 17649–17655, 1994.[Abstract/Free Full Text]

26. Pires OR, Sebben A, Schwartz EF, Bloch C, Morales RAV, Schwartz CA. The occurrence of 11-oxotetrodotoxin, a rare tetrodotoxin analogue, in the brachycephalidae frog Brachycephalus ephippium. Toxicon 42: 563–566, 2003.[Medline]

27. Sandtner W, Szendroedi J, Zarrabi T, Zebedin E, Hilber K, Glaaser I, Fozzard HA, Dudley SC, Todt H. Lidocaine: a foot in the door of the inner vestibule prevents ultra-slow inactivation of a voltage-gated sodium channel. Mol Pharmacol 66: 648–657, 2004.[Abstract/Free Full Text]

28. Schiavon E, Sacco T, Cassulini RR, Gurrola G, Tempia F, Possani LD, Wanke E. Resurgent current and voltage sensor trapping enhanced activation by a beta-scorpion toxin solely in Na(v)1.6 channel - Significance in mice Purkinje neurons. J Biol Chem 281: 20326–20337, 2006.[Abstract/Free Full Text]

29. Sivilotti L, Okuse K, Akopian AN, Moss S, Wood JN. A single serine residue confers tetrodotoxin insensitivity on the rat sensory-neuron-specific sodium channel SNS. FENS Lett 409: 49–52, 1997.

30. Smith MR, Smith RD, Plummer NW, Meisler MH, Goldin AL. Functional analysis of the mouse Scn8a sodium channel. J Neurosci 18: 6093–6102, 1998.[Abstract/Free Full Text]

31. Terlau H, Olivera BM. Conus venoms: a rich source of novel ion channel-targeted peptides. Physiol Rev 84: 41–68, 2004.[Abstract/Free Full Text]

32. Trimmer JS, Cooperman SS, Tomiko SA, Zhou JY, Crean SM, Boyle MB, Kallen RG, Sheng ZH, Barchi RL, Sigworth FJ, Goodman RH, Agnew WS, Mandel G. Primary structure and functional expression of a mammalian skeletal muscle sodium channel. Neuron 3: 33–49, 1989.[CrossRef][ISI][Medline]

33. Waxman SG, Craner MJ, Black JA. Na+ channel expression along axons in multiple sclerosis and its models. Trends Pharmacol Sci 25: 584–591, 2004.[CrossRef][Medline]

34. Yotsu-Yamashita M, Sugimoto A, Takai A, Yasumoto T. Effects of specific modifications of several hydroxyls of tetrodotoxin on its affinity to rat brain membrane. J Pharmacol Exp Ther 289: 1688–1696, 1999.[Abstract/Free Full Text]





This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Tables
Right arrow All Versions of this Article:
293/2/C783    most recent
00070.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rosker, C.
Right arrow Articles by Schreibmayer, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rosker, C.
Right arrow Articles by Schreibmayer, W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.