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Subunit composition determines the single channel kinetics of the epithelial sodium channel.
Fyfe GK
,
Canessa CM
.
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We have further characterized at the single channel level the properties of epithelial sodium channels formed by coexpression of alpha with either wild-type beta or gamma subunits and alpha with carboxy-terminal truncated beta (betaT) or gamma (gammaT) subunits in Xenopus laevis oocytes. alphabeta and alphabetaT channels (9.6 and 8.7 pS, respectively, with 150 mM Li+) were found to be constitutively open. Only upon inclusion of 1 microM amiloride in the pipette solution could channel activity be resolved; both channel types had short open and closed times. Mean channel open probability (Po) for alphabeta was 0.54 and for alphabetaT was 0.50. In comparison, alphagamma and alphagammaT channels exhibited different kinetics: alphagamma channels (6.7 pS in Li+) had either long open times with short closings, resulting in a high Po (0.78), or short openings with long closed times, resulting in a low Po (0. 16). The mean Po for all alphagamma channels was 0.48. alphagammaT (6.6 pS in Li+) behaved as a single population of channels with distinct kinetics: mean open time of 1.2 s and closed time of 0.4 s, with a mean Po of 0.6, similar to that of alphagamma. Inclusion of 0. 1 microM amiloride in the pipette solution reduced the mean open time of alphagammaT to 151 ms without significantly altering the closed time. We also examined the kinetics of amiloride block of alphabeta, alphabetaT (1 microM amiloride), and alphagammaT (0.1 microM amiloride) channels. alphabeta and alphabetaT had similar blocking and unblocking rate constants, whereas the unblocking rate constant for alphagammaT was 10-fold slower than alphabetaT. Our results indicate that subunit composition of ENaC is a main determinant of Po. In addition, channel kinetics and Po are not altered by carboxy-terminal deletion in the beta subunit, whereas a similar deletion in the gamma subunit affects channel kinetics but not Po.
Figure 2. Single channel records from oocytes expressing αγ
or αγT channels. Pipette solution was 150 mM LiCl, and 0.1
μM amiloride was present where
indicated (+amilâ). Low Po, Po <
0.25; high Po, Po > 0.65. âVp has
the same meaning as Fig. 1. The
time scale and current amplitude
are indicated on the scale bar.
Figure 3. Current (Iâ)âvoltage
(Vâ) relationships for αβ (A), αβT
(B), αγ (C), and αγT (D). Single
channel conductance was estimated by linear regression either
between â40 and â80 mV (A
and B) or â30 and â80 mV (C
and D) and is represented by the
dashed line. The pipette solution
contained 150 mM Li+. Each
point is the mean ± SEM of
three experiments. In this and all
subsequent figures, where no error bars are shown, the error was
within the symbol.
Figure 4. Plot of channel open probability (Po) against pipette
holding potential (âVp) for αβ (âª) and αβT (â¢). Pipette solution
contained 1 μM amiloride. Channel Po was measured from single
channel patches recorded at each of the indicated voltages for several minutes. Each point is the mean ± SEM of three experiments.
Figure 5. Plot of channel Po
against time for αγ (A) and αγT
with and without 0.1 μM
amiloride (B). The data used to
generate A was from a patch that
contained a single high Po αγ
channel. B was generated from
data from two separate patches,
both containing single αγT channels. Po was measured at 30-s intervals in all three of these representative examples. Mean Po refers to Po measurements averaged
over the entire life of the patch.
Figure 6. Examples of open
and closed time histograms for
αβ, αβT, and αγT in the presence
of amiloride. Data was obtained
from single channel patches. Fits
were done using Pstat. All histograms were best fitted with one
exponential. The y ordinate in
all histograms corresponds to the
number of observations and the
x ordinate log dwell-time (milliseconds). Numbers to the right
of each histogram represent the
calculated closed or open times
for the given example.
Figure 7. Block of αβ and αβT by 1 μM amiloride: estimation of
blocking (KON) and unblocking (KOFF) rate constants. Each point
is the mean ± SEM from three single channel patches and corresponds to 20-mV increments in the pipette holding potential. KON
and KOFF were estimated as described in the text. Note that the y
axis is a logarithmic scale. â¡,⪠αβ; and â,⢠αβT.
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