title: K⁺ conduction and Mg²⁺ blockade in a shaker Kv-channel single point mutant with an unusually high conductance.
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.
authors.affiliation: Centro Interdisciplinario de Neurociencias de Valparaíso, Universidad de Valparaíso, Valparaíso, Chile.
authors.forename: Cristian
authors.initials: C
authors.lastname: Moscoso
authors.forename: Ariela
authors.initials: A
authors.lastname: Vergara-Jaque
authors.forename: Valeria
authors.initials: V
authors.lastname: Márquez-Miranda
authors.forename: Romina V
authors.initials: RV
authors.lastname: Sepúlveda
authors.forename: Ignacio
authors.initials: I
authors.lastname: Valencia
authors.forename: Ignacio
authors.initials: I
authors.lastname: Díaz-Franulic
authors.forename: Fernando
authors.initials: F
authors.lastname: González-Nilo
authors.forename: David
authors.initials: D
authors.lastname: Naranjo
mesh_terms: D000818:Animals; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D004553:Electric Conductivity; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology*; D056004:Molecular Dynamics Simulation; D017354:Point Mutation; D016062:Porosity; D011188:Potassium / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D011487:Protein Conformation; D051637:Shaker Superfamily of Potassium Channels / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D014982:Xenopus laevis
chemical_list: D026902:Potassium Channel Blockers; D051637:Shaker Superfamily of Potassium Channels; D008274:Magnesium; D011188:Potassium