Structural determinants of quaternary ammonium blockers for batrachotoxin-modified Na+ channels

Amphipathic quaternary ammonium (QA) compounds are potent blockers of batrachotoxin (BTX)-modified Na+ channels incorporated into planar lipid bilayers. To examine the topology of the QA binding site, we selected two series of QA compounds as structural probes. One series contains two separate hydrophobic moieties but with a common hydrophilic dimethyl QA ion. Most of the QAs within this group bind to BTX-modified Na+ channels with relatively high affinities. For example, benzyldimethyldodecyl ammonium ions, when applied internally, block single, muscle, BTX-modified Na+ channels in bilayers with a one-to-one relationship and display an equilibrium dissociation constant (Kd) of 0.2 microM at +50 mV. Furthermore, the QA dwell times appear to correlate with QA hydrophobic interactions with the channel. These results indicate that there are two large hydrophobic binding domains within the QA binding site. The QAs in the second series contain a hydrophilic head group (trialkylammonium) of variable size but with a common dodecyl hydrophobic tail. Tripropyldodecyl QAs block BTX-modified Na+ channels more effectively (Kd = 0.4 microM at +50 mV) than do trimethyl- and triethyldodecyl QAs, suggesting that the internal Na+ permeation pathway is at least 9 A wide. However, tributyl- and tripentyldodecyl QAs show much lower affinities for BTX-modified Na+ channels at comparable concentrations. These drugs are cut off from binding, probably as a result of the size of their hydrophilic heads (> 10 A), which may be too large to fit in the QA binding site and too bulky to travel freely within the internal permeation pathway. Under whole-cell voltage-clamp conditions, we have further found that BTX-modified Na+ currents in clonal GH3 cells can be blocked by these two series of QA ions, albeit only when the activation gate is open. Closed channels at rest do not bind appreciably with these QA ions. Binding of QA ions is reduced by external Na+ ions in GH3 cells in a manner indicating that external Na+ ions can clear the bound QA ions from the Na+ pore. These results from GH3 cells mirror those obtained with QA blockers in K+ channels of squid axons and suggest that the QA binding domains in BTX-modified Na+ channels and K+ channels may be structurally conserved.