title: Intrinsic electrostatic potential in the BK channel pore: role in determining single channel conductance and block.
abstract: The internal vestibule of large-conductance Ca(2+) 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, Deltaphi, caused by the charge neutralization using TEA(+) block for the extracellular charges and Ba(2+) for intracellular charges. We measured 13 +/- 2 mV for Deltaphi at the TEA(+) site when turning off the extracellular charges, and 17 +/- 2 mV for the Deltaphi at the Ba(2+) site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Deltaphi 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.
authors.affiliation: Centro de Estudios Científicos, Casilla, Valdivia, Chile.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Carvacho
authors.forename: Wendy
authors.initials: W
authors.lastname: Gonzalez
authors.forename: Yolima P
authors.initials: YP
authors.lastname: Torres
authors.forename: Sebastian
authors.initials: S
authors.lastname: Brauchi
authors.forename: Osvaldo
authors.initials: O
authors.lastname: Alvarez
authors.forename: Fernando D
authors.initials: FD
authors.lastname: Gonzalez-Nilo
authors.forename: Ramon
authors.initials: R
authors.lastname: Latorre
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001464:Barium / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D018999:Charybdotoxin / Q000494:pharmacology; D003198:Computer Simulation; D005260:Female; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D018075:RNA, Complementary / Q000008:administration & dosage / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D019789:Tetraethylammonium / Q000494:pharmacology; D014982:Xenopus laevis
chemical_list: D002121:Calcium Channel Blockers; C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D018075:RNA, Complementary; D018999:Charybdotoxin; D001464:Barium; D019789:Tetraethylammonium; D011188:Potassium