Residues in a jellyfish shaker-like channel involved in modulation by external potassium

Residues in a jellyfish Shaker-like channel involved in modulation by exter nal potassium. J. Neurophysiol. 82: 1740-1747, 1999. The jellyfish gene, jS hak2, coded for a potassium channel that showed increased conductance and a decreased inactivation rate as [K+](out) was increased. The relative modul atory effectiveness of K+, Rb+, Cs+, and Na+ indicated that a weak-field-st rength site is present. Cysteine substituted mutants (L369C and F370C) of a n N-terminal truncated construct, (jShak2 Delta 2-38) which only showed C-t ype inactivation, were used to establish the position and nature of this si te(s). In comparison with jShak2 Delta 2-38 and F370C, L369C showed a great er relative increase in peak current when [K+](out) was increased from 1 to 100 mM because the affinity of this site was reduced at low [K+](out). Inc reasing [K+](out) had little effect on the rate of inactivation of L369C; h owever, the appearance of a second, hyperbolic component to the inactivatio n curve for F370C indicated that this mutation had increased the affinity o f the low-affinity site by bringing the backbone oxygens closer together. M ethanethiosulphonate reagents were used to form positively (MTSET), negativ ely (MTSES), and neutrally (MTSM) charged side groups on the cysteine-subst ituted residues at the purported K+ binding site(s) in the channel mouth an d conductance and inactivation kinetic measurements made. The reduced affin ity of the site produced by the mutation L369C was probably due to the incr eased hydrophobicity of cysteine,which changed the relative positions of ca rbonyl oxygens since MTSES modification did not form a high-held-strength s ite as might be expected if the cysteine residues project into the pore. Ad dition of the side chain -CH2-S-S-CH3 which is similar to the side chain of methionine, a conserved residue in many potassium channels, resulted in an increased peak current and reduced inactivation rate, hence a higher affin ity binding site. Modification of cysleine substituted mutants occurred mor e readily from the inactivated state confirming that side chains probably r otate into the pore from a buried position when no K ions are in the pore. In conclusion we were able to show that, as for certain potassium channels in higher taxonomic groups, the site(s) responsible for modulation by [K+]( out) is situated just outside the selectivity filter and is represented by the residues L-369 and F-370 in thr jellyfish Shaker channel, jShak2.