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.

Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers.

Alkaloid-modified, voltage-dependent sodium channels from lobster walking leg nerves were studied in planar neutral lipid bilayers. In symmetrical 0.5 M NaCl the single channel conductance of veratridine (VTD) (10 pS) was less than that of batrachotoxin (BTX) (16 pS) modified channels. At positive potentials, VTD- but not BTX-modified channels remained open at a flickery substate. VTD-modified channels underwent closures on the order of milliseconds (fast process), seconds (slow process), and minutes. The channel fractional open time (f(o)) due to the fast process, the slow process, and all channel closures (overall f(o)) increased with depolarization. The fast process had a midpoint potential (V(a)) of -122 mV and an apparent gating charge (z(a)) of 2.9, and the slow process had a V(a) of -95 mV and a z(a) of 1.6. The overall f(o) was predominantly determined by closures on the order of minutes, and had a V(a) of about -24 mV and a shallow voltage dependence (z(a) approximately 0.7). Augmenting the VTD concentration increased the overall f(o) without changing the number of detectable channels. However, the occurrence of closures on the order of minutes persisted even at super-saturating concentrations of VTD. The occurrence of these long closures was nonrandom and the level of nonrandomness was usually unaffected by the number of channels, suggesting that channel behavior was nonindependent. BTX-modified channels also underwent closures on the order of milliseconds, seconds, and minutes. Their characterization, however, was complicated by the apparent low BTX binding affinity and by an apparent high binding reversibility (channel disappearance) of BTX to these channels. VTD- but not BTX-modified channels inactivated slowly at high positive potentials (greater than +30 mV). Single channel conductance versus NaCl concentrations saturated at high NaCl concentrations and was non-Langmuirian at low NaCl concentrations. At all NaCl concentrations the conductance of VTD-modified channels was lower than that of BTX-modified channels. However, this difference in conductance decreased as NaCl concentrations neared zero, approaching the same limiting value. The permeability ratio of sodium over potassium obtained under mixed ionic conditions was similar for VTD (2.46)- and BTX (2.48)-modified channels, whereas that obtained under bi-ionic conditions was lower for VTD (1.83)- than for BTX (2.70)-modified channels. Tetrodotoxin blocked these alkaloid-modified channels with an apparent binding affinity in the nanomolar range.