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 lip id bilayers. To examine the topology of the QA binding site, we select ed two series of QA compounds as structural probes. One series contain s two separate hydrophobic moieties but with a common hydrophilic dime thyl QA ion. Most of the QAs within this group bind to BTX-modified Na + channels with relatively high affinities. For example, benzyldimethy ldodecyl 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 (K(d)) of 0.2 muM at +50 mV. Furthermore, the QA dwell times appear to correlate with QA hy drophobic interactions with the channel. These results indicate that t here are two large hydrophobic binding domains within the QA binding s ite. The QAs in the second series contain a hydrophilic head group (tr ialkylammonium) of variable size but with a common dodecyl hydrophobic tail. Tripropyldodecyl QAs block BTX-modified Na+ channels more effec tively (K(d) = 0.4 muM at +50 mV) than do trimethyl- and triethyldodec yl QAs, suggesting that the internal Na+ permeation pathway is at leas t 9 angstrom wide. However, tributyl- and tripentyldodecyl QAs show mu ch lower affinities for BTX-modified Na+ channels at comparable concen trations. These drugs are cut off from binding, probably as a result o f the size of their hydrophilic heads (> 10 angstrom), which may be to o large to fit in the QA binding site and too bulky to travel freely w ithin the internal permeation pathway. Under whole-cell voltage-clamp conditions, we have further found that BTX-modified Na+ currents in cl onal GH3 cells can be blocked by these two series of QA ions, albeit o nly when the activation gate is open. Closed channels at rest do not b ind appreciably with these QA ions. Binding of QA ions is reduced by e xternal 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 fro m GH3 cells mirror those obtained with QA blockers in K+ channels of s quid axons and suggest that the QA binding domains in BTX-modified Na channels and K+ channels may be structurally conserved.