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

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 Mg²⁺ 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 Mg²⁺ 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 Mg²⁺ 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.