CONTROL OF ION FLUX AND SELECTIVITY BY NEGATIVELY CHARGED RESIDUES INTHE OUTER MOUTH OF RAT SODIUM-CHANNELS

1. The sodium channel has a ring of negatively charged amino acids on its external face. This common structural feature of cation-selective channels has been proposed to optimize conduction by electrostatic att raction of permeant cations into the channel mouth. We tested this ide a by mutagenesis of mu 1 rat skeletal sodium channels expressed in Xen opus oocytes. 2. Replacement of the external glutamate residue in doma in II by cysteine reduces sodium current by decreasing single-channel conductance. While this effect can be reversed by the negatively charg ed sulfhydryl modifying reagent methanethiosulphonate ethylsulphonate (MTSES), the flux saturation behaviour cannot be rationalized simply b y changes in the surface charge. 3. The analogous mutations in domains I, III and IV affect not only conductance but also selectivity. These changes in selectivity are only partially reversed by exposure to MTS ES. 4. Our findings necessitate revision of prevailing concepts regard ing the role of superficial negatively charged residues in the process of ion permeation. These residues do not act solely by electrostatic attraction of permeant ions, but instead may help to form ion-specific binding sites within the pore.