Resumen
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.
| Idioma original | English |
|---|---|
| Páginas (desde-hasta) | 1198-1207 |
| Número de páginas | 10 |
| Publicación | Biophysical Journal |
| Volumen | 103 |
| N.º | 6 |
| DOI | |
| Estado | Published - 19 sep 2012 |
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ASJC Scopus subject areas
- Biophysics
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K+ conduction and Mg2+ blockade in a Shaker Kv-channel single point mutant with an unusually high conductance. / Moscoso, Cristian; Vergara-Jaque, Ariela; Márquez-Miranda, Valeria; Sepúlveda, Romina V.; Valencia, Ignacio; Díaz-Franulic, Ignacio; González-Nilo, Fernando; Naranjo, David.
En: Biophysical Journal, Vol. 103, N.º 6, 19.09.2012, p. 1198-1207.Resultado de la investigación: Article
TY - JOUR
T1 - K+ conduction and Mg2+ blockade in a Shaker Kv-channel single point mutant with an unusually high conductance
AU - Moscoso, Cristian
AU - Vergara-Jaque, Ariela
AU - Márquez-Miranda, Valeria
AU - Sepúlveda, Romina V.
AU - Valencia, Ignacio
AU - Díaz-Franulic, Ignacio
AU - González-Nilo, Fernando
AU - Naranjo, David
PY - 2012/9/19
Y1 - 2012/9/19
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84866506140&partnerID=8YFLogxK
U2 - 10.1016/j.bpj.2012.08.015
DO - 10.1016/j.bpj.2012.08.015
M3 - Article
C2 - 22995492
AN - SCOPUS:84866506140
VL - 103
SP - 1198
EP - 1207
JO - Biophysical Journal
JF - Biophysical Journal
SN - 0006-3495
IS - 6
ER -