K⁺ conduction and Mg²⁺ blockade in a shaker Kv-channel single point mutant with an unusually high conductance

Biophys J. 2012 Sep 19;103(6):1198-207. doi: 10.1016/j.bpj.2012.08.015.


Potassium channels exhibit a large diversity of single-channel conductances. Shaker is a low-conductance K-channel in which Pro475→Asp, a single-point mutation near the internal pore entrance, promotes 6- to 8-fold higher unitary current. To assess the mechanism for this higher conductance, we measured Shaker-P475D single-channel current in a wide range of symmetrical K(+) concentrations and voltages. Below 300 mM K(+), the current-to-voltage relations (i-V) showed inward rectification that disappeared at 1000 mM K(+). Single-channel conductance reached a maximum of ∼190 pS at saturating [K(+)], a value 4- to 5-fold larger than that estimated for the native channel. Intracellular Mg(2+) blocked this variant with ∼100-fold higher affinity. Near zero voltage, blockade was competitively antagonized by K(+); however, at voltages >100 mV, it was enhanced by K(+). This result is consistent with a lock-in effect in a single-file diffusion regime of Mg(2+) and K(+) along the pore. Molecular-dynamics simulations revealed higher K(+) density in the pore, especially near the Asp-475 side chains, as in the high-conductance MthK bacterial channel. The molecular dynamics also showed that K(+) ions bound distally can coexist with other K(+) or Mg(2+) in the cavity, supporting a lock-in mechanism. The maximal K(+) transport rate and higher occupancy could be due to a decrease in the electrostatic energy profile for K(+) throughout the pore, reducing the energy wells and barriers differentially by ∼0.7 and ∼2 kT, respectively.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Biological Transport / drug effects
  • Electric Conductivity*
  • Intracellular Space / drug effects
  • Intracellular Space / metabolism
  • Magnesium / pharmacology*
  • Molecular Dynamics Simulation
  • Point Mutation*
  • Porosity
  • Potassium / metabolism*
  • Potassium Channel Blockers / pharmacology*
  • Protein Conformation
  • Shaker Superfamily of Potassium Channels / antagonists & inhibitors*
  • Shaker Superfamily of Potassium Channels / chemistry
  • Shaker Superfamily of Potassium Channels / genetics
  • Shaker Superfamily of Potassium Channels / metabolism*
  • Static Electricity
  • Xenopus laevis


  • Potassium Channel Blockers
  • Shaker Superfamily of Potassium Channels
  • Magnesium
  • Potassium