Batrachotoxin-modified, voltage-dependent sodium channels from canine forebrain were incorporated into planar lipid bilayers . Singlechannel conductances were studied for [Na'] ranging between 0.02 and 3.5 M. Typically, the single-channel currents exhibited a simple two-state behavior, with transitions between closed and fully open states . Two other conductance states were observed : a subconductance state, usually seen at [NaCl] >_ 0.5 M, and a flickery state, usually seen at [NaCl] :5 0.5 M. The flickery state became more frequent as [NaCl] was decreased below 0.5 M . The K'/Na' permeability ratio was -0.16 in 0.5 and 2.5 M salt, independent of the Na' mole fraction, which indicates that there are no interactions among permeant ions in the channels . Impermeant and permeant blocking ions (tetraethylammonium, Ca", Zn ++ , and K+) have different effects when added to the extracellular and intracellular solutions, which indicates that the channel is asymmetrical and has at least two cation-binding sites. The conductance vs .[Na+] relation saturated at high concentrations, but could not be described by a Langmuir isotherm, as the conductance at low [NaCl] is higher than predicted from the data at [NaCl] > 1 .0 M. At low [NaCl] (__<0.1 M), increasing the ionic strength by additions of impermeant monovalent and divalent cations reduced the conductance, as if the magnitude of negative electrostatic potentials at the channel entrances were reduced. The conductances were comparable for channels in bilayers that carry a net negative charge and bilayers that carry no net charge . Together, these results lead to the conclusion that negative charges on the channel protein near the channel entrances increase the conductance, while lipid surface charges are less important.

The guanidinium toxin-induced inhibition of the current through voltage-dependent sodium channels was examined for batrachotoxinmodified channels incorporated into planar lipid bilayers that carry no net charge . To ascertain whether a net negative charge exists in the vicinity of the toxin-binding site, we studied the channel closures induced by tetrodotoxin (TTX) and saxitoxin (STX) over a wide range of [Na']. These toxins carry charges of +1 and +2, respectively. The frequency and duration of the toxininduced closures are voltage dependent. The voltage dependence was similar for STX and TTX, independent of [Na+], which indicates that the binding site is located superficially at the extracellular surface of the sodium channel. The toxin dissociation constant, KD, and the rate constant for the toxin-induced closures, kc, varied as a function of [Na+]. The Na' dependence was larger for STX than for TTX. Similarly, the addition of tetraethylammoniurn (TEA') or Zn`increased KD and decreased kc more for STX than for TTX. These differential effects are interpreted to arise fro#n changes in the electrostatic potential near the toxin-binding site. The charges giving rise to this potential must reside on the channel since the bilayers had no net charge . The Na' dependence of the ratios KsD x /KDx and ksTx /kc'x was used to estimate an apparent charge density near the toxin-binding site of about -0.33 e -nm Y . Zn`causes a voltage-dependent block of the single-channel current, as if Znb ound at a site within the permeation path, thereby blocking Na' movement. There was no measurable interaction between Zn`at its blocking site and STX or TTX at their binding site, which suggests that the toxin-binding site is separate from the channel entrance. The separation between the toxin-binding site and the Zn`blocking site was estimated to be at least 1 .5 nm. A model for toxin-induced channel closures is proposed, based on conformational changes in the channel subsequent to toxin binding. Address reprint requests to Dr .

The modulation of gramicidin A single-channel characteristics by the amino acid side chains was investigated using gramicidin A analogues in which the NH2 terminal valine was chemically replaced by other amino acids. The replacements were chosen such that pairs of analogues would have essentially isosteric side chains of different polarities at position 1 (valine vs. trifluorovaline or hexafluorovaline; norvaline vs. S-methyl-cysteine; and norleucine vs. methionine). Even though the side chains are not in direct contact with the permeating ions, the single-channel conductances for Na+ and Cs+ are markedly affected by the changes in the physico-chemical characteristics of the side chains. The maximum single-channel conductance for Na+ is decreased by as much as 10-fold in channels formed by analogues with polar side chains at position 1 compared with their counterparts with nonpolar side chains, while the Na+ affinity is fairly insensitive to these changes. The relative conductance changes seen with Cs+ were less than those seen with Na+; the ion selectivity of the channels with polar side chains at position 1 was increased. Hybrid channels could form between compounds with a polar side chain at position 1 and either valine gramicidin A or their counterparts with a nonpolar side chain at position 1. The structure of channels formed by the modified gramicidins is thus essentially identical to the structure of channels formed by valine gramicidin A. The polarity of the side chain at position 1 is an important determinant of the permeability characteristics of the gramicidin A channel. We discuss the importance of having structural information when interpreting the functional consequences of site-directed amino acid modifications.

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