Permeation of large tetra-alkylammonium cations through mutant and wild-type voltage-gated sodium channels as revealed by relief of block at high voltage

Many large organic cations are potent blockers of Ki channels and other cat ion-selective channels belonging to the P-region superfamily. However, the mechanism by which large hydrophobic cations enter and exit the narrow pore s of these proteins is obscure. Previous work has shown that a conserved Ly s residue in the DEKA locus of voltage-gated Na+ channels is an important d eterminant of Na+/K+ discrimination, exclusion of Ca2+, and molecular sievi ng of organic cations, In this study, we sought to deter-mine whether die L ys(III) residue of die DEKA locus interacts with internal tetra-alkylammoni um cations (TAA(+)) that block Na+ channels in a voltage-dependent fashion, We investigated block by a series of TAA(+) cations of the wild-type rat m uscle Na+ channel (DEKA) and two different mutants of the DEKA locus, DEAA and DERA, using whole-cell recording. TEA(+) and larger TAA(+) cations bloc k both wild-type and DEAA channels. However, DEAA exhibits dramatic relief of block by large TAA(+) cations as revealed by a positive inflection in th e macroscopic I-V curve at voltages greater than +140 mV. Paradoxically, re lief of block at high positive voltage is observed for large (e.g., tetrape ntylammonium) but not small (e.g,, TEA(+)) symmetrical TAA(+) cations. The DEKA wild-type channel and the DERA mutant exhibit a similar relief-of-bloc k phenomenon superimposed on background current rectification. The results indicate: (a) hydrophobic TAA(+) cations with a molecular diameter as large as 15 Angstrom can permeate Na+ channels from inside to outside when drive n by high positive voltage, and (b) the Lys(III) residue of the DEKA locus is an important determinant of inward rectification and internal block in N a+ channels. From these observations, we suggest that hydrophobic interface s between subunits, pseudosubunits, or packed helices of P-region channel p roteins may function in facilitating blocker access to the pore, and may th us play an important role in the blocking and permeation behavior of large TAA(+) cations and potentially other kinds of local anesthetic molecules.