Interaction between tetraethylammonium and amino acid residues in the pore of cloned voltage-dependent potassium channels

Extracellular tetraethylammonium (TEA) inhibits currents in Xenopus oocytes that have been injected with mRNAs encoding voltage-dependent potassium channels. Concentration-response curves were used to measure the affinity of TEA; this differed up to 700-fold among channels RBK1 (K_D 0.3 mM), RGK5 (K_D 11 mM), and RBK2 (K_D > 200 mM). Studies in which chimeric channels were expressed localized TEA binding to the putative extracellular loop between transmembrane domains S5 and S6. Site-directed mutagenesis of residues in this region identified the residue Tyr³⁷⁹ of RBK1 as a crucial determinant of TEA sensitivity; substitution of Tyr in the equivalent positions of RBK2 (Val³⁸¹) and RGK5 (His⁴⁰¹) made these channels as sensitive to TEA as RBK1. Nonionic forces are involved in TEA binding because (i) substitution of the Phe for Tyr³⁷⁹ in RBK1 increased its affinity, (ii) protonation of His⁴⁰¹ in RGK5 selectively reduced its affinity, and (iii) the affinity of TEA was unaffected by changes in ionic strength. The results suggest an explanation for the marked differences in TEA sensitivity that have been observed among naturally occurring and cloned potassium channels and indicate that the amino acid corresponding to residue 379 in RBK1 lies within the external mouth of the ion channel.