K? conduction and Mg²? blockade in a shaker Kv-channel single point mutant with an unusually high conductance.
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ABSTRACT: 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.
SUBMITTER: Moscoso C
PROVIDER: S-EPMC3446664 | biostudies-literature | 2012 Sep
REPOSITORIES: biostudies-literature
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