Anemone toxin II unmasks two conductance states in neuronal sodium channels.

Anemone toxin II (ATX)-modified voltage-dependent neuronal sodium channels were studied in planar lipid bilayers. ATX-modified channels displayed two predominant conducting states: a short-lived (ms-s) high-conductance (approximately 65 pS) state and a long-lived (s-min) low-conductance (approximately 10 pS) state. The high-conductance state underwent brief closures (ms) and the low-conductance state underwent long closures (s). The probability of detecting these states was time- and voltage-dependent. The channel's fractional open time (fo) due to the high-conductance state increased with depolarization and had a midpoint potential (Va) of -36 mV and an apparent gating charge (Za) of 2.8. The channel's fo due to the low-conductance state increased with depolarization and had a Va of +13 mV and a Za of 1.4. At positive potentials, ATX-modified channels slowly (minutes) entered an absorbing non-conducting state. The permeability ratio of Na+/K+ was 2 and 4 for the low- and high-conductance states, respectively. The saxitoxin analog C3 blocked ATX-modified sodium channels with high affinity (Kd(60-90 mV) = 410 nM, 0.5 M NaCl). The data suggest that upon a depolarization step, ATX-modified channels enter rapidly (ms) into a high-conductance state and more slowly (s-min) into a low-conductance state. Also as the membrane potential becomes more positive, the equilibrium is shifted from the high- to the low-conductance state and from the conducting states to an absorbing non-conducting state.

Interactions between anemone toxin II and veratridine on single neuronal sodium channels.

The nature of the known positive cooperativity between alkaloid and alpha-polypeptide toxins on macroscopic sodium currents was studied at the single-channel level. We have previously characterized the single-channel function of veratridine (VTD)-modified and anemone toxin II (ATX)-modified channels from lobster leg nerve. VTD and ATX are known to potentiate each other's effects in stimulating 22Na flux into vesicles containing sodium channels from lobster leg nerve. These channels, therefore, provided an excellent model for further investigation of the interactions between the toxins. A variety of such interactions were found, some of which would contribute to the positive cooperativity between these toxins. These included first, a decrease in the frequency of occurrence, but not in the lifetime, of the long channel closed state (minute range). This effect resulted in a hyperpolarization shift of the voltage dependence of the overall channel fractional open time. The second effect was a decrease in the apparent-unbinding rate of ATX at -60 mV. These interactions, which could not have been predicted by the effects of the individual toxins, were observed at negative but not at positive potentials, and led to increases in sodium channel currents. Some of the observed interactions could not contribute to the positive cooperativity between these toxins. These included the elimination of the high-conductance state of ATX-modified channels, the predominance of the VTD effect on the voltage dependence of the fast-process, the predominance of the ATX effect on the rate of decay of sodium currents at +60 mV, and the resulting intermediate toxin effect on the level of the noisy open state.

Expression of sodium channels with different saxitoxin affinity during rat forebrain development.

This work characterizes the development of the saxitoxin (STX)-sensitive Na+ channels from rat whole forebrain between embryonic day 15 (E15) and postnatal day 90 (P90), both with binding studies and with single channel studies. The Na+ channel total mRNA and the individual mRNAs encoding Na+ channels I, II and III were also determined. The total STX binding rose about 40-fold from E15 to reach a plateau at P30 and its temporal course correlated with the expression of Na+ channel total mRNA. Low affinity and high-affinity STX binding sites, predominant in embryonic and postnatal forebrains, respectively, were found. The single channel studies of batrachotoxin-modified channels also revealed two main populations. In E15 only low-affinity channels (KD = 32.7 nM; 200 mM NaCl) and in P30 only high affinity ones (KD = 1.6 nM) were present. At P0 channels with intermediate affinity (KD range 3-34 nM) were observed. The increase in affinity was due to a gradual increase in the STX association rate.