Conduction through the inward rectifier potassium channel, Kir2.1, is increased by negatively charged extracellular residues.
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 (a(K)) was fit well by a modified Langmuir binding isotherm, with a nonzero intercept as a(K) 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-a(K) 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-a(K) 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.
D'Avanzo
Nazzareno
N
Department of Physiology and Medicine, Richard Lewar Centre, University of Toronto, Ontario, Canada.
Cho
Hee Cheol
HC
Tolokh
Illya
I
Pekhletski
Roman
R
Tolokh
Igor
I
Gray
Chris
C
Goldman
Saul
S
Backx
Peter H
PH
D000818:Animals; D000838:Anions / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D004553:Electric Conductivity; D005110:Extracellular Space / Q000502:physiology; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D051379:Mice; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000502:physiology*
D000838:Anions; C489461:Kir2.1 channel; D024661:Potassium Channels, Inwardly Rectifying; D011188:Potassium