Residues in a jellyfish shaker-like channel involved in modulation by external potassium.
The jellyfish gene, jShak2, coded for a potassium channel that showed increased conductance and a decreased inactivation rate as [K(+)](out) was increased. The relative modulatory effectiveness of K(+), Rb(+), Cs(+), and Na(+) indicated that a weak-field-strength site is present. Cysteine substituted mutants (L369C and F370C) of an N-terminal truncated construct, (jShak2Delta2-38) which only showed C-type inactivation, were used to establish the position and nature of this site(s). In comparison with jShak2Delta2-38 and F370C, L369C showed a greater 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). Increasing [K(+)](out) had little effect on the rate of inactivation of L369C; however, the appearance of a second, hyperbolic component to the inactivation curve for F370C indicated that this mutation had increased the affinity of the low-affinity site by bringing the backbone oxygens closer together. Methanethiosulphonate reagents were used to form positively (MTSET), negatively (MTSES), and neutrally (MTSM) charged side groups on the cysteine-substituted residues at the purported K(+) binding site(s) in the channel mouth and conductance and inactivation kinetic measurements made. The reduced affinity of the site produced by the mutation L369C was probably due to the increased hydrophobicity of cysteine, which changed the relative positions of carbonyl oxygens since MTSES modification did not form a high-field-strength site as might be expected if the cysteine residues project into the pore. Addition of the side chain -CH(2)-S-S-CH(3), 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 affinity binding site. Modification of cysteine substituted mutants occurred more readily from the inactivated state confirming that side chains probably rotate 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 the jellyfish Shaker channel, jShak2.
Grigoriev
N G
NG
Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, Health Sciences Centre, Calgary, Alberta T2N 4N1, Canada.
Spafford
J D
JD
Spencer
A N
AN
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016384:Consensus Sequence; D005260:Female; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D011188:Potassium / Q000494:pharmacology / Q000502:physiology*; D015221:Potassium Channels / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D037401:Scyphozoa; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D051637:Shaker Superfamily of Potassium Channels; D019789:Tetraethylammonium / Q000494:pharmacology; D014982:Xenopus laevis
D015221:Potassium Channels; D011994:Recombinant Proteins; D051637:Shaker Superfamily of Potassium Channels; D019789:Tetraethylammonium; D011188:Potassium