Skip to main content
Log in

Modification of cloned brain Na+ channels by batrachotoxin

  • Published:
Pflügers Archiv Aims and scope Submit manuscript

Abstract

The effects of batrachotoxin (BTX) on cloned α-subunit Na+ channels were examined in CHO-K1 cells (a chinese hamster ovary cell line) transfected with rat brain NaIIA cDNA. Under whole-cell patch clamp conditions, BTX shifted the voltage dependence of the activation process by about 45 mV towards the hyperpolarizing direction and eliminated the inactivating phase of Na+ currents. Repetitive depolarizations greatly facilitated the binding of BTX with NaIIA channels while the membrane was held at −100 mV. In chloramine-T-pretreated cells, the association rate of BTX binding with the NaIIA channel was 6.5-fold faster than that in untreated cells. The estimated association rate constant for BTX binding with the open form of NaIIA channel was 1.11×106 mol−1·s−1 at room temperature. BTX-modified NaIIA channels were blocked by tetrodotoxin (TTX) in a complicated manner. First, the TTX binding to the closed state of BTX-modified NaIIA channels was not voltage dependent. The K D value of TTX was measured at 8.9 nM, which was similar to that of unmodified channels (K D=14.2 nM). Second, the block of the open state of BTX-modified NaIIA channels by TTX was voltage dependent; depolarization reduced the potency of TTX block between −20 mV to +50 mV. Below −30 mV, the TTX affinity began to level off, probably because of the increased presence of the closed state. Unexpectedly, steady-state inactivation of BTX-modified NaIIA channels was minimal as measured by the two-pulse protocol, a phenomenon distinctly different from that found in GH3 cells. Neutral local anesthetic benzocaine, however, drastically enhanced the steady-state inactivation of BTX-modified NaIIA channels, with its maximal effect around −60 mV. We conclude that BTX can bind and modify the NaIIA α-subunit. However, a specific subtype of α-subunits and/or an unidentified modulating process may be required for the optimal steady-state inactivation of BTX-modified Na+ channels.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Auld VJ, Goldin AL, Krafte DS, Marshall J, Dunn JM, Catterall WA, Lester HA, Davidson N, Dunn RJ (1988) A rat brain Na+ channel α subunit with novel gating properties. Neuron 1:449–461

    Article  CAS  PubMed  Google Scholar 

  2. Barnes S, Hille B (1988) Veratridine modifies open sodium channels. J Gen Physiol 91:421–443

    Article  CAS  PubMed  Google Scholar 

  3. Caterall WA (1980) Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes. Annu Rev Pharmacol Toxicol 20:15–43

    Google Scholar 

  4. Cota G, Armstrong CM (1989) Sodium channel gating in clonal pituitary cells: the inactivation step is not voltage dependent. J Gen Physiol 94:213–232

    CAS  PubMed  Google Scholar 

  5. Dubois J-M, Coulombe A (1984) Current dependent inactivation induced by sodium depletion in normal and batrachotoxin-treated frog node of Ranvier. J Gen Physiol 84:25–48

    Article  CAS  PubMed  Google Scholar 

  6. Hamill OP, Marty A, Neher ME, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85–100

    Article  CAS  PubMed  Google Scholar 

  7. Hille B (1977) Local anesthetics: hydrophilic and hydrophobic pathways for the drug receptor reaction. J Gen Physiol 69:497–515

    CAS  PubMed  Google Scholar 

  8. Hille B (1992) Modifiers of gating. In: Hille B (ed) Ionic channels of excitable membranes, 2nd edn. Sinauer, Sunderland, pp 445–471

    Google Scholar 

  9. Isom LL, DeJongh KS, Patton DE, Reber BFX, Offord J, Charboneau H, Walsh K, Goldin AL, Catterall WA (1992) Primary structure and functional expression of the β1 subunit of the rat brain sodium channel. Science 256:839–842

    CAS  PubMed  Google Scholar 

  10. Khodorov BI (1978) Chemicals as tools to study nerve fiber sodium channels: effect of batrochotoxin and some local anesthetics. In: Tosteson D, Ovchinnikov Y, Latorre R (eds) Membrane transport processes, vol 2. Raven Press, New York, pp 153–174

    Google Scholar 

  11. Krueger BK, Worley JF, French RJ (1983) Single sodium channels from rat brain incorporated into planar lipid bilayer membranes. Nature 303:172–175

    Article  CAS  PubMed  Google Scholar 

  12. Lönnendonker U (1989) Binding of tetrodotoxin and saxitoxin to Na+ channels at different holding potentials: fluctuation measurements in frog myelinated nerve. Biochim Biophys Acta 985:161–167

    PubMed  Google Scholar 

  13. Moczydlowski E, Hall S, Garber SS, Strichartz GR, Miller C (1984) Voltage-dependent blockade of muscle Na+ channels by guanidinium toxins. Effect of toxin charge. J Gen Physiol 84, 687–704

    CAS  PubMed  Google Scholar 

  14. Rando TA, Strichartz GR (1986) Saxitoxin blocks batrachotoxin-modified sodium channels in the node of Ranvier in a voltage-dependent manner. Biophys J 49:785–794

    CAS  PubMed  Google Scholar 

  15. Strichartz GR, Rando TA, Wang GK (1987) An integrated view of the molecular toxinology of sodium channel gating in excitable cells. Annu Rev Neurosci 10:237–267

    Article  CAS  PubMed  Google Scholar 

  16. Tanguy J, Yeh JZ (1991) BTX modification of Na Channels in squid axons. I. State dependence of BTX action. J Gen Physiol 97:499–519

    Article  CAS  PubMed  Google Scholar 

  17. Ulbricht W (1990) The inactivation of sodium channels in the node of Ranvier and its chemical modification. In: Narahashi T (ed) Ion channels, vol 2. Plenum, New York, pp 123–168

    Google Scholar 

  18. Wang GK (1984) Irreversible modification of sodium channel inactivation in toad myelinated nerve fibers by the oxidant chloramine-T. J Physiol (Lond) 346:127–141

    CAS  Google Scholar 

  19. Wang GK, Wang S-Y (1992) Inactivation of BTX-modified Na+ channels in GH3 cells: characterization and pharmacological modification. J Gen Physiol 99:1–20.

    Article  CAS  PubMed  Google Scholar 

  20. Wang GK, Wang S-Y (1994) Binding of benzocaine in batrachotoxin-modified Na+ channels: state-dependent interactions. J Gen Physiol 103:501–518

    Article  CAS  PubMed  Google Scholar 

  21. West JW, Scheurer T, Maechler L, Catterall WA (1992) Efficient expression of rat brain type IIA Na+ channel α-subunits in a somatic cell line. Neuron 8:59–70

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, G.K., Wang, S.Y. Modification of cloned brain Na+ channels by batrachotoxin. Pflügers Arch 427, 309–316 (1994). https://doi.org/10.1007/BF00374539

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00374539

Key words

Navigation