The Journal of General Physiology
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Published online 11 April 2005 doi:10.1085/jgp.200409175
The Rockefeller University Press, 0022-1295 $8.00
JGP, Volume 125, Number 5, 493-503
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Conduction through the Inward Rectifier Potassium Channel, Kir2.1, Is Increased by Negatively Charged Extracellular Residues

Nazzareno D'Avanzo1, Hee Cheol Cho2, Illya Tolokh3, Roman Pekhletski1, Igor Tolokh3, Chris Gray3, Saul Goldman3, and Peter H. Backx1

1 Department of Physiology and Medicine and Department of Heart and Stroke, Richard Lewar Centre, University of Toronto, Toronto, Ontario M5S 3E2, Canada
2 Institute of Molecular Cardiobiology, Johns Hopkins University, Baltimore, MD 21218
3 Department of Chemistry and Department of Physics, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Correspondence to Peter H. Backx: p.backx{at}utoronto.ca

Ion channel conductance can be influenced by electrostatic effects originating from fixed "surface" charges that are remote from the selectivity filter. To explore whether surface charges contribute to the conductance properties of Kir2.1 channels, unitary conductance was measured in cell-attached recordings of Chinese hamster ovary (CHO) cells transfected with Kir2.1 channels over a range of K+ activities (4.6–293.5 mM) using single-channel measurements as well as nonstationary fluctuation analysis for low K+ activities. K+ ion concentrations were shown to equilibrate across the cell membrane in our studies using the voltage-sensitive dye DiBAC4(5). The dependence of {gamma} on the K+ activity (aK) was fit well by a modified Langmuir binding isotherm, with a nonzero intercept as aK approaches 0 mM, suggesting electrostatic surface charge effects. Following the addition of 100 mM N-methyl-D-glucamine (NMG+), a nonpermeant, nonblocking cation or following pretreatment with 50 mM trimethyloxonium (TMO), a carboxylic acid esterifying agent, the {gamma}–aK relationship did not show nonzero intercepts, suggesting the presence of surface charges formed by glutamate or aspartate residues. Consistent with surface charges in Kir2.1 channels, the rates of current decay induced by Ba2+ block were slowed with the addition of NMG or TMO. Using a molecular model of Kir2.1 channels, three candidate negatively charged residues were identified near the extracellular mouth of the pore and mutated to cysteine (E125C, D152C, and E153C). E153C channels, but not E125C or D152C channels, showed hyperbolic {gamma}–aK relationships going through the origin. Moreover, the addition of MTSES to restore the negative charges in E53C channels reestablished wild-type conductance properties. Our results demonstrate that E153 contributes to the conductance properties of Kir2.1 channels by acting as a surface charge.

Key Words: surface charges • Kir2.1 • conduction • NMG • TMO

N. D'Avanzo and H.C. Cho contributed equally to this project.

Abbreviations used in this paper: CHO, Chinese hamster ovarian; DiBAC4(5), bis-(1,3-dibutylbarbituric acid)pentamethine oxonol; GCS, Gouy-Chapman-Stern; NAChR, nicotinic acetylcholine receptor; NMG, N-methyl-D-glucamine; NSFA, nonstationary fluctuation analysis; TMO, trimethyloxonium; WT, wild-type.


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