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Nonindependent K+ movement through the pore in IRK1 potassium channels.
Stampe P
,
Arreola J
,
Pérez-Cornejo P
,
Begenisich T
.
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We measured unidirectional K+ in- and efflux through an inward rectifier K channel (IRK1) expressed in Xenopus oocytes. The ratio of these unidirectional fluxes differed significantly from expectations based on independent ion movement. In an extracellular solution with a K+ concentration of 25 mM, the data were described by a Ussing flux-ratio exponent, n', of approximately 2.2 and was constant over a voltage range from -50 to -25 mV. This result indicates that the pore of IRK1 channels may be simultaneously occupied by at least three ions. The IRK1 n' value of 2.2 is significantly smaller than the value of 3.5 obtained for Shaker K channels under identical conditions. To determine if other permeation properties that reflect multi-ion behavior differed between these two channel types, we measured the conductance (at 0 mV) of single IRK1 channels as a function of symmetrical K+ concentration. The conductance could be fit by a saturating hyperbola with a half-saturation K+ activity of 40 mM, substantially less than the reported value of 300 mM for Shaker K channels. We investigated the ability of simple permeation models based on absolute reaction rate theory to simulate IRK1 current-voltage, conductance, and flux-ratio data. Certain classes of four-barrier, three-site permeation models are inconsistent with the data, but models with high lateral barriers and a deep central well were able to account for the flux-ratio and single channel data. We conclude that while the pore in IRK1 and Shaker channels share important similarities, including K+ selectivity and multi-ion occupancy, they differ in other properties, including the sensitivity of pore conductance to K+ concentration, and may differ in the number of K+ ions that can simultaneously occupy the pore: IRK1 channels may contain three ions, but the pore in Shaker channels can accommodate four or more ions.
Figure 2. IRK1 channel flux-ratio exponent from influx experiments. Flux-ratio values at â50 (âª), â30 (â´), and â25 (â¢) mV as
a function of influx.
Figure 3. Flux-ratio exponent
data from influx and efflux experiments. (inset) 42K efflux determined as the number of
counts in 1-min samples, corrected for radioactive background as described in methods. The oocyte membrane potential was maintained at +20
mV except for the indicated 4-min
period when the potential was
â25 mV. (main figure) Flux-ratio
exponent values at several potentials. Shown are mean flux-ratio
values with SEM limits from IRK1
channel influx measurements
(âª) at â50, â30, and â25 mV,
and the mean value from efflux
measurement at â25 mV (â¢).
The mean of Shaker K channel
flux-ratio values from both influx and efflux measurements
(from Stampe and Begenisich,
1996) at â30 mV is shown (âµ)
for comparison. The number of
measurements are shown in parentheses.
Figure 6. Permeation properties and barrier profiles. Each
column represents a different
permeation model characterized by the zero-voltage free energy barrier profile at the top. In
all cases, the energy minima and
maxima are equally spaced along
the membrane electric field. The
external solution is to the left of
the barriers. Below the barrier
profile in each column are the
computed currentâvoltage relations at 50 (dashed line) and 200
(solid line) mM symmetrical ion
concentration, the ion activity
dependence of the zero-voltage
slope conductance, and the flux-ratio exponent over the â100- to
100-mV voltage range.
Figure 4. IRK1 single channel
current. (A) Sample single channel current measurements at the
indicated potentials recorded
from an inside-out patch with
symmetrical 100 mM K+ solutions. The dotted line represents
the zero current level for each
record. Calibration: 2 pA and 50
ms. (B) Current amplitude histogram from several records from
the same patch obtained at â80
mV. (line) A two-Gaussian function fitted to the data. The nonzero component is centered on a
level of â1.7 pA. (C) Single
channel currents from the same
patch at several potentials. (line)
Fit of a second-order polynomial
to the data from which a zero-current slope of 19 pS was obtained.
Figure 5. IRK1 single channel conductance as a function of symmetrical K+ activity. Mean single channel conductance level and
standard error limits are shown. The number of patches is shown
in parentheses, except at the lowest K activity, where the two symbols represent individual patches. (line) A rectangular hyperbola
fit to the data with a maximum of 32 pS and a half-maximal activity
of 40 mM.
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