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Correspondence to Fernando D. Gonzalez-Nilo: dgonzalez{at}utalca.cl; or Ramon Latorre: ramon.latorre{at}uv.cl
The internal vestibule of large-conductance Ca2+ voltage-activated K+ (BK) channels contains a ring of eight negative charges not present in K+ channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T.I., X. Niu, and K.L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100:9017–9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K+ was 18% lower than the control. Current–voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K+ (30 mM) and vanished at high K+ (1 M K+). We determine the electrostatic potential change, 
, caused by the charge neutralization using TEA+ block for the extracellular charges and Ba2+ for intracellular charges. We measured 13 ± 2 mV for at the TEA+ site when turning off the extracellular charges, and 17 ± 2 mV for the at the Ba2+ site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329.
R. Latorre's present address is Centro de Neurociencias, Universidad de Valparaiso, Valparaiso 2360102, Chile.
Abbreviations used in this paper: AChR, acetylcholine receptor; BK, large conductance Ca2+- and voltage-dependent channel; KcsA, K+ channel from Streptomyces lividans; MD, molecular dynamics; MthK, Ca2+-gated K+ channel from Methanobacterium autotrophicum; PB, Poisson-Boltzmann; P-helix, pore helix; WT, wild-type.
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