Tail end of the s6 segment: role in permeation in shaker potassium channels.
The permeation pathway in voltage-gated potassium channels has narrow constrictions at both the extracellular and intracellular ends. These constrictions might limit the flux of cations from one side of the membrane to the other. The extracellular constriction is the selectivity filter, whereas the intracellular bundle crossing is proposed to act as the activation gate that opens in response to a depolarization. This four-helix bundle crossing is composed of S6 transmembrane segments, one contributed by each subunit. Here, we explore the cytoplasmic extension of the S6 transmembrane segment of Shaker potassium channels, just downstream from the bundle crossing. We substituted cysteine for each residue from N482 to T489 and determined the amplitudes of single channel currents and maximum open probability (P(o,max)) at depolarized voltages using nonstationary noise analysis. One mutant, F484C, significantly reduces P(o,max), whereas Y483C, F484C, and most notably Y485C, reduce single channel conductance (gamma). Mutations of residue Y485 have no effect on the Rb(+)/K(+) selectivity, suggesting a local effect on gamma rather than an allosteric effect on the selectivity filter. Y485 mutations also reduce pore block by tetrabutylammonium, apparently by increasing the energy barrier for blocker movement through the open activation gate. Replacing Rb(+) ions for K(+) ions reduces the amplitude of single channel currents and makes gamma insensitive to mutations of Y485. These results suggest that Rb(+) ions increase an extracellular energy barrier, presumably at the selectivity filter, thus making it rate limiting for flux of permeant ions. These results indicate that S6(T) residues have an influence on the conformation of the open activation gate, reflected in both the stability of the open state and the energy barriers it presents to ions.
Ding
Shinghua
S
Department of Physiology, Jefferson Medical College, Philadelphia, PA 19107, USA.
Horn
Richard
R
D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004594:Electrophysiology; D006801:Humans; D008954:Models, Biological; D009154:Mutation / Q000502:physiology; D018408:Patch-Clamp Techniques; D010539:Permeability; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D012413:Rubidium / Q000378:metabolism; D051637:Shaker Superfamily of Potassium Channels; D019789:Tetraethylammonium / Q000494:pharmacology
D026902:Potassium Channel Blockers; D015221:Potassium Channels; D051637:Shaker Superfamily of Potassium Channels; D019789:Tetraethylammonium; D012413:Rubidium; D011188:Potassium