Properties of an inwardly rectifying K+ channel in
the basolateral membrane of mouse TAL
Marc
Paulais,
Stéphane
Lourdel, and
Jacques
Teulon
Institut National de la Santé et de la Recherche
Médicale U.426, Institut Fédératif de Recherche
02, Faculté de Médecine Xavier Bichat, Université
Paris 7, 75018 Paris, France
 |
ABSTRACT |
We investigated the properties of
K+ channels in the basolateral membrane of the cortical
thick ascending limb (CTAL) using the patch-clamp technique.
Approximately 34% of cell-attached patches contained an inwardly
rectifying K+ channel (K+-to-Na+
permeability ratio ~22), having an inward conductance
(Gin) of 44 pS and an outward conductance
(Gout) of ~10 pS
(Gin/Gout ~ 4).
Channel activity (NPo) increased with
depolarization. When the cytosolic sides of inside-out patches were
exposed to an Mg2+-free medium, the channel had a
Gin of 50 pS and was weakly inwardly rectifying
(Gin/Gout ~ 1).
Cytosolic Mg2+ reduced Gout,
yielding a Gin/Gout of
3.8 at 1.3 mM Mg2+. Internal Na+ also yielded a
Gin/Gout of 1.6 at 20 mM
Na+. Spermine reduced NPo on
inside-out membrane patches. Sensitivity to spermine at depolarizing
voltages [half-maximal inhibitory concentration
(Ki) = 0.2 µM] was much greater than at
hyperpolarizing voltages (Ki = 26 µM).
Half-inactivation by 0.5 µM spermine occurred at a clamp potential of
43 mV, with an effective valence of 1.25. A sigmoid relationship
between bath pH and NPo of inside-out membrane patches was observed, with a pK of 7.6 and a Hill
coefficient of 1.8. Intracellular acidification also reduced the
NPo of cell-attached patches. This channel is
probably a major component of K+ conductance in the CTAL
basolateral membrane.
thick ascending limb of Henle's loop; basolateral membrane; potassium channel; patch clamp; mouse
 |
INTRODUCTION |
THE THICK
ASCENDING LIMB (TAL) of Henle's loop of the mammalian nephron
reabsorbs ~30% of the NaCl filtered by the glomerulus and plays a
crucial role in urinary concentrating ability. Basically, Na+ and Cl
ions enter the cell via a
Na+-K+-2Cl
cotransporter at the
apical membrane and, on the basolateral side, Cl
ions
leave the cell through a Cl
conductance and a K-Cl
cotransporter (Ref. 41; see also Fig. 9). In addition, a
basolateral K+ conductance has been detected in the hamster
medullary TAL (MTAL) (54), the rabbit cortical TAL (CTAL)
(3), and the Amphiuma early distal tubule
(13), the diluting segment in amphibians, and plays a key
role in NaCl reabsorption by the TAL. This conductance would maintain
basolateral membrane potential (VBl) difference and thus the driving force for the basolateral diffusion of
Cl
. In addition, by participating in the basolateral exit
of K+ ions and compensating for part of the influx of
K+ ions associated with
Na+/K+-ATPase activity (24), this
conductance is essential for the generation of a chemical gradient of
Na+ ions promoting apical
Na+-K+-2Cl
cotransport activity.
The properties of ion channels underlying the basolateral potassium
conductance of renal epithelial cells have been mainly studied in the
proximal and cortical collecting tubules of various species (see Ref.
45). In contrast, very little is known about the
conductive properties of the basolateral K+ channels in the
TAL. The only patch-clamp study on this subject was restricted to
channel properties in the cell-attached configuration and identified a
35-pS, inwardly rectifying K+ channel in rabbit CTAL
(22).
The present study was undertaken to identify the basolateral potassium
channels of mouse CTAL tubules. We were particularly interested in
characterizing the conductive and regulatory properties of the channel
that would allow a fruitful comparison with potassium channels
described in other nephron segments and with cloned potassium channels.
 |
MATERIALS AND METHODS |
Chemicals.
EDTA (disodium or dipotassium salt, when appropriate), ATP (disodium
salt), and
N,N'-bis[3-aminopropyl]-1,4-butanediamine tetrahydrochloride (spermine; SPM) were from Sigma-Aldrich Chimie (Saint Quentin Fallavier, France). EGTA was from Research Organics (Cleveland, OH) or from Sigma-Aldrich Chimie.
Tubule preparation.
Male 15- to 20-g CD1 (Charles River France, Saint Aubin Lès
Elbeuf, France) or ICR (Harlan France, Gannat, France) mice were killed
by cervical dislocation. CTALs were microdissected as previously described (14). Briefly, the left kidney was first rinsed
with L-15 Leibovitz medium (Eurobio, Les Ulis, France; GIBCO BRL, Life Technologies, Cergy Pontoise, France) containing 300 U/ml collagenase (300 U/ml CLS II; Worthington, Freehold, NJ). Small pieces of cortex
were then incubated at 37°C for 45-75 min in this
collagenase-containing medium, rinsed, and kept at 4°C until
microdissection. No further enzymatic or mechanical treatment of
tubules was necessary for successful seal formation.
Current recordings.
Cell-attached and cell-excised, inside-out variants of the patch-clamp
technique (16) were applied to the basolateral membrane of
CTAL fragments. Single-channel currents were recorded with a
patch-clamp amplifier (LM-EPC 7, List Electronic, Darmstadt, Germany;
RK-400, Bio-Logic, Claix, France) and stored on digital audiotape
(DTR-1205, Bio-Logic). The bath reference was 0.5 M KCl in a 4% agar
bridge connected to an Ag-AgCl pellet. In the cell-attached
configuration, the clamp potential (Vc =
Vbath
Vpipette) is
superimposed on the cell membrane potential
(Vm). In excised inside-out membrane patches,
Vc = Vm. All
Vc values were corrected for liquid-junction
potential, as calculated by a routine of AxoScope software (Axon
Instruments, Foster City, CA). The accuracy of these calculations for
our experimental setup was confirmed by direct measurements utilizing a
procedure described previously (30). Cations flowing from
the inner to the outer face of the membrane patch are positive and
shown as upward deflexions in current tracings. All experiments were
conducted at room temperature.
Data analysis.
Signals were generally low-pass filtered at 500 Hz by an eight-pole
Bessel filter (LPBF-48DG; NPI Electronic, Tamm, Germany) and digitized
at 3 kHz with a Digidata 1200 analog-to-digital converter and AxoScope
software (Axon Instruments). For analysis of conductance substates (see
RESULTS), signals were filtered at 2 KHz and digitized at
10 KHz. In most cases, channel activity was quantified using software
kindly provided by Prof. T. Van den Abbeele (Paris, France). The mean
current (I) passing through N channels was
estimated from current-amplitude histograms and used to calculate the
normalized current (NPo) according to the equation NPo = I/i,
where i is the unit current amplitude. In some cases (see
RESULTS), NPo was measured with
AxoScope software on the basis of visual differentiation between the
fast and noisier K+ channel openings and the slower
kinetics of 9-pS Cl
channels (14, 15).
Channel selectivity.
PK/PNa, the
permeabilities to K+ and Na+ ions,
respectively, was determined from both cell-attached and inside-out
patches. In cell-attached patches, Vm was set
close to 0 mV by superfusing the tubules with a high-K solution. The
shift in reversal potential (
Erev) was then
estimated from i-Vc relationships
obtained with various K+ and Na+ concentrations
in the pipette while the sum of K+ and Na+
concentrations was kept constant.
PK/PNa was calculated
using the Goldman-Hodgkin-Katz voltage equation (17),
taking the Erev in the presence of 144.8 mM
K+ in the pipette
(Erev[144.8K]pip) as a reference
where F is the Faraday constant, R is the
universal gas constant, and T is absolute temperature. The
use of this equation is based on the assumptions that channel anion
permeability is negligible (see RESULTS) and that
intracellular Na+ and K+ concentrations between
cells are constant.
PK/PNa on inside-out membrane patches was determined from Erev values
obtained with opposite Na+ and K+ ion gradients
across the membrane patch, using the equation
|
|
where o and i denote the outside and inside faces of the
membrane patch, respectively.
Solutions.
Tubules were bathed either in a high-Na solution containing (in mM) 140 NaCl, 4.8 KCl, 10 glucose, and 10 HEPES-NaOH, pH 7.4, or in a high-K
solution containing 144.8 KCl, 10 glucose, and 10 HEPES-KOH, pH 7.4. Free Ca2+ and Mg2+ concentrations were adjusted
as required (see below). Unless otherwise stated, pipettes were filled
with the high-K solution. Lower K+/Na+
concentration ratios were obtained by mixing appropriate volumes of
high-K and high-Na solutions, both the sum of K and Na pipette concentrations ([K+]pip + [Na+]pip) and the Cl
pipette
concentration ([Cl
]pip) being kept at 144.8 mM. Higher values of [K+]pip were obtained by
adding the appropriate amount of KCl to the high-K solution. No
correction was made for changes in osmolarity. When necessary (see
RESULTS), ion currents through 9-pS Cl
channels were minimized by using solutions in which all but 4.8 mM
Cl
had been replaced by NO
(14). The pipette solution contained 1.2 mM free
Mg2+. The bath solution typically contained 1.2 mM free
Mg2+ and 1 mM free Ca2+. Various concentrations
of free Mg2+ under low free Ca2+ concentrations
were obtained with either EDTA or EGTA. The proportions of
Mg2+ and EDTA or EGTA were calculated using the absolute
stability constants for Mg2+, Ca2+, and
H+ and EDTA (5) or EGTA (4, 5,
29).
Data presentation and statistics.
Results are given as means ± SE for n patches.
P values <0.05 (paired or unpaired t-test, when
appropriate; SigmaPlot or SigmaStat, SPSS, Erkrath, Germany) were taken
to represent statistically significant differences. Nonlinear
regression analyses were performed with Origin software (Microcal
Software, Northampton, MA). All-points amplitude histograms were
constructed by TAC event detection and analysis software (Bruxton,
Seattle, WA) from current traces after baseline current subtraction.
 |
RESULTS |
K+ channel activity was observed in 34% (52 of 153)
of cell-attached membrane patches of CTAL tubules bathed in the
high-NaCl solution. With no applied potential, the number of active
K+ channels per patch averaged 5.8 ± 0.5 (n = 52), with a mean NPo of
1.9 ± 0.4 (range 0.02-10.49; n = 40) (Fig.
1A). We found no correlation
between the tip resistance of KCl-filled pipettes (4-13 M
) and
the number of active channels (r = 0.017).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1.
K+ channel activity (NPo) in
cell-attached patches. A: no. of patches displaying the
indicated NPo at a clamp potential
(Vc) = 4 mV. Tubules were bathed in high-Na
solution, with the high-K solution in the pipette. B:
typical recordings of K+ channel activity in the conditions
given in A. Recordings were from the same patch at the
Vc values given to the right of each
trace. C , current level corresponding to closure of all
K+ channels. Note the expanded scale for current amplitude
obtained at +104 mV (*).
|
|
General properties of cell-attached patches.
Figure 1B shows recordings of channel activity in a
cell-attached patch from a tubule bathed in the high-NaCl solution, the pipette being filled with the high-KCl solution. The corresponding i-Vc relationship is shown in Fig.
2A. Under these conditions, EK across the membrane patch should be close to
0 mV, and Erev provides an estimate of
Vm. Erev was 75 ± 1 mV (n = 22), in good agreement with the
Vm values of
70/
80 mV reported for isolated perfused rabbit CTAL (12) and mouse MTAL (42)
tubules. The i-Vc relationship for
inward currents was nearly linear, single-channel Gin averaging 43.5 ± 0.6 pS
(n = 22). Measurements of outward currents beyond
Erev were hampered by the openings of previously described small-conductance Cl
channels (14)
(not shown), but the current amplitude, measured on three occasions at
114 and 124 mV, was lower than expected for an ohmic K+
conductance (see Fig. 2A). The use of low-Cl
solutions (see MATERIALS AND METHODS) facilitated
measurements of K+ currents at more depolarizing voltages
(not shown) and yielded an outward chord conductance
[G(chord)out], as measured between Erev and 144 mV, of 11 ± 0.6 pS
(n = 3), whereas Gin was 44 ± 2 pS (n = 9), indicating inward rectification.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
K+ channel properties in cell-attached
patches. A: unit current amplitude
(i)-Vc relationships in conditions of
Fig. 1 ( ) or in tubules bathed in a high-K solution
( ). Each point is the mean of 3-22 measurements,
except at Vc = 124 mV, where
n = 2. SE are error bars when larger than symbols.
Inset: voltage dependence of NPo in
conditions given in A ( ). Each point is the
mean of 5-14 measurements, and error bars are SE. Dotted line,
nonlinear least squares fit of Boltzmann equation (see text) to the
data given by maximum NPo
(NPo max) = 3.75, voltage when
NPo is NPo max/2
(V1/2) = 11.5 mV, and effective valence
(z) = 0.7 (r = 0.846). B:
relationship between channel conductance and K+
concentration in the pipette ([K+]pip). For
each [K+]pip value, the conductance at
Vc = 0 mV
(G0 mV) was taken as the slope of the
i-Vc relationship between 10 and 10 mV in tubules bathed in high-Na solution. Each point is the mean of at
least 3 determinations, and SE values are shown as error bars when
larger than symbols. Dotted line, nonlinear least squares fit to mean
values given by maximal G0 mV
[G0 mV(max)] = 58 pS, dissociation
constant (Km) = 83 mM, and value of
G0 mV when
[K+]pip = 0 [G0 mV(0)] = 7 pS (see
text). C: ionic selectivity. The shift in reversal potential
( Erev) was determined as described in
MATERIALS AND METHODS from
i-Vc relationships established in the
cell-attached configuration in the presence of the indicated
[K+]pip value. Each point is the mean of at
least 4 determinations, and SE values are shown as error bars when
larger than symbols. Dotted line through data points, nonlinear least
squares fit using the Goldman-Hodgkin-Katz voltage equation (see
MATERIALS AND METHODS) and a K-to-Na permeability ratio
(PK/PNa) of 22.
|
|
To precisely establish inward rectification, we determined
i-Vc relationships from tubules
bathed in a high-K solution. With the high-K solution in the pipette,
single-channel currents reversed at 2 ± 0.7 mV (P = 0.02 vs. 0 mV; n = 5), which was consistent with a
fairly constant and nearly zero Vm, and
Gin averaged 44 ± 1 pS (n = 5). As seen in Fig. 2A, it is clear that outward currents were smaller than inward currents. Thus
G(chord)out measured between Erev and +70 mV was 11 ± 1.2 pS
(P = 0.001 vs. Gin,
paired t-test; n = 4). A
Gin/Gout ratio of
4.94 ± 1.45 (n = 4) was obtained. Therefore, the
channel we observed here belongs to the inwardly rectifying
K+ channel family.
NPo of Na-bathed tubules increased with
depolarization up to Vc = 44 mV (Fig.
2A, inset). The relationship between
NPo and Vc was well
described by the Boltzmann distribution equation (17)
where NPo max is the maximum
NPo, V1/2 is the voltage
when NPo is NPo max/2,
and z is the effective valence. Here,
V1/2 =
11.5 mV and z = 0.7. NPo tended to decrease at more depolarizing
voltages, but there were too few observations to permit a quantitative
description of the data.
The effect of [K+]pip on channel properties
was also studied in CTAL tubules bathed in the high-Na solution.
Varying [K+]pip over the 18-400 mM range
had no influence on the number of active channels per patch or on
NPo (P = 0.65, 1-way ANOVA; data not shown) but shifted Erev and altered
Gin. The effects of
[K+]pip on Gin were
quantified by taking the slope of the corresponding i-Vc relationships at 0 mV,
G0 mV. Plotting of
G0 mV as a function of
[K+]pip revealed saturation (Fig.
2B), which was well described by the modified
Michaelis-Menten equation (17) ZZZ, where
G0 mV(max) is the maximal
G0 mV, Km is the
dissociation constant, and
G0 mV(0) is the value of
G0 mV when
[K+]pip = 0. The best fit of
experimental data points was obtained with
G0 mV(max) = 58 pS,
Km = 83 mM, and
G0 mV(0) = 7 pS. Similar
Km values were obtained at
Vc of
20 and 20 mV (111 and 98 mM,
respectively; not shown).
PK/PNa in cell-attached
patches was determined as indicated in MATERIALS AND
METHODS. The relative shift in
Erev (
Erev) (Fig. 2C) on the variance in
[Na+]pip/[K+]pip
yielded a PK/PNa value of
22. Preliminary results also revealed negligible anion permeability
(not shown).
Inspection of K+ channel activity recordings in
cell-attached membrane patches revealed conductance substates (Fig.
3). However, because these partial
openings contributed too little to the total open time and because of
the low occurrence of patches containing only one active K+
channel (see Fig. 1A), the partial openings could not be
discriminated from all-points amplitude histograms. We therefore
manually measured the duration of these partial openings from
stretches of cell-attached recordings where current due to one
active channel was reached at Vc = 0 mV
(Fig. 3A). Three conductance substates, S1,
S2, and S3, emerged (Fig. 3A),
having respective fractional amplitudes of 0.25 ± 0.03 (n = 4), 0.4 ± 0.03 (n = 6), and
0.7 ± 0.05 (n = 6) (Fig. 3B). Within a
burst, S1, S2, and S3 accounted for
3.8 ± 0.6 (n = 4), 7 ± 7 (n = 6), and 9.5 ± 4.6% (n = 6) of the total open
time, respectively (not shown).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 3.
Conductance substates of cortical thick ascending limb of
Henle's loop (CTAL) K+ channels. A: excerpts of
single-channel recordings from the same cell-attached patch. Tubule was
bathed in high-Na solution, and patch was clamped at 0 mV. C,
S1, S2, S3, and O: closed level,
1st, 2nd, and 3rd subconductance levels, and level of complete opening,
respectively. B: histogram of current amplitude of each
conductance substate, given as the fraction of the fully open level.
S1, S2, and S3 were significantly
different from each other (1-way ANOVA).
|
|
Channel conductive properties in cell-excised patches.
As for basolateral K+ channels from rabbit CTAL
(22), the activity of basolateral K+ channels
from mouse CTAL was very rapidly lost on patch excision into
physiological saline. However, as with other K+ channels,
the rundown of which can be partly antagonized by using a
Mg2+-free bath (35), we found that CTAL
K+ channel activity could be maintained for up to 20 min by
carrying out patch excision in a Mg2+- and
Ca2+-free solution (+5 mM of either Na2-EDTA or
K2-EDTA) in ~88% of patches. Results presented
below were obtained according to this procedure.
We confirmed the high
PK/PNa ratio of the
channel in cell-free patches. Thus, with the high-K solution kept in
the pipette, the replacement of all but 39.8 mM KCl by NaCl in the bath
shifted the reversal potential of the
i-Vc curves by 29.5 ± 0.84 mV
(n = 5) (not shown), which corresponds to a
PK/PNa of 33. Channel selectivity to NH
was also measured in the presence
of the 144.8 mM KCl solution on the cytoplasmic side of inside-out
patches (not shown). The substitution of 144.8 mM NH4Cl for
pipette KCl shifted Erev of
i-Vc curves toward a negative value.
Because inward currents could be resolved in only one of four
experiments, Erev was extrapolated from the
average i-Vc curves for outward
currents and was estimated to be approximately
51 ± 3 mV
(n = 4). This yielded a
K+-NH
PK/PNa of ~7.3 ± 0.7.
Mechanisms underlying K+ channel
inward rectification.
Under symmetrical, high-K conditions, the
i-Vc relationships in the absence of
Mg2+ (+5 mM K2-EDTA) (Fig.
4B) were linear. The mean
values from four experiments were as follows:
Gin and Gout were
50.5 ± 2 and 47.9 ± 2 pS, respectively (P = 0.41, paired t-test). The
Gin/Gout ratio was
1.06 ± 0.06 (n = 4). We then investigated whether
inward rectification in cell-attached patches resulted from a
voltage-dependent block of outward currents by intracellular
Mg2+ ions, as reported in other inwardly rectifying
K+ channels (32, 37). Figure 4A
compares the current amplitudes of an inside-out membrane patch, with
its cytosolic side exposed to either an Mg2+-free medium
(no Mg2+ + 5 mM K2-EDTA) or to a medium
containing 1.3 mM Mg2+ (1.5 mM MgNO3 and 2 mM
EGTA), at Vc of +70 mV. Internal
Mg2+ reduced the single-channel amplitude of outward
currents (Fig. 4A) and had little influence on inward
currents (traces not shown). The corresponding
i-Vc relationships are shown in Fig.
4B. Addition of 1.3 mM cytosolic Mg2+ slightly
but significantly (P < 0.01) reduced
Gin (42 ± 1.7 pS, n = 4, vs. 50.5 ± 2 pS, n = 4), but its most
striking effect was a major reduction in
G(chord)out (measured between
Erev and +70 mV), which fell to 12.2 ± 1.58 pS (n = 4). The resulting
Gin/Gout ratio was
3.8 ± 0.2 (n = 4). The
i-Vc relationship for outward currents obtained under these conditions was very similar to that obtained from cell-attached patches on KCl-depolarized cells (compare with Fig. 2), and indeed the
Gin/Gout ratios were not
statistically different (P = 0.29, unpaired
t-test).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of internal Mg2+ and Na+
on i-Vc relationships of basolateral
CTAL K+ channel. A: current traces from an
inside-out membrane patch bathed in symmetrical high-K+ and
the cytosolic side of which was bathed by either a medium containing 0 Mg2+ (0 Mg2++5 mM K2-EDTA;
top trace) or a medium containing 1.3 mM Mg2+
(1.5 mM Mg2++2 mM EGTA; bottom trace). Clamped
potential was +70 mV. C, current level corresponding to closure of all
K+ channels. Also shown are corresponding all-point
amplitude histograms from traces after baseline current subtraction
(bottom). Solid lines are fits with a sum of 5 (0 Mg2+) or 3 (1.3 Mg2+) Gaussian components by
the maximum-likelihood method. Means of components corresponding to
channel opening level were 3.3 ± 0.75, 6.5 ± 0.74, 9.5 ± 0.8, 12.6 ± 0.79, and 16.5 ± 0.45 pA for 0 Mg2+ and 0.9 ± 0.21 and 1.9 ± 0.25 pA for 1.3 Mg2+. B: averaged
i-Vc relationships corresponding to
the conditions given in A for 0 ( ) or 1.3 mM
Mg2+ ( ) or in the presence of a medium
containing 0 Mg2+ (+5 mM K2-EDTA) supplemented
with 20 mM Na+ on the cytosolic side of the patch
( ). Each point is the average of at least 3 determinations, and SE values are shown as error bars when larger than
symbols. Solid line and , best fit to respective data,
as given in RESULTS.
|
|
These data were analyzed according to a model in which Mg2+
moves into the electric field of the pore, as described by Woodhull (17), which assumes a single
Mg2+-binding site located within the pore, using the
equation
where IMg is the current in the presence of
Mg2+, G is the inward conductance, z
is the valence of 2, a is the number of binding sites (here,
a = 1),
is the electrical distance of the binding site from the outside of the pore, and K0 is the
half-maximal inhibitory concentration (Ki) at
Vc = 0 mV. The best fit obtained with 1.3 mM Mg2+ (Fig. 4B) yielded
K0 = 1.58 ± 0.25 mM Mg2+,
= 0.23 ± 0.02 from the inside, and
G = 49.4 ± 2.42 pS.
We also investigated the possible involvement of Na+ during
inward rectification. The i-Vc
relationships established under symmetrical K+, with a
Mg2+-free medium (+5 mM K2-EDTA) supplemented
with 20 mM Na+ on the cytosolic side of the patch (Fig.
4B), showed a slight inward rectification
(Gin/Gout = 1.6 ± 0.14, n = 3). An analysis of these data using the
model given above, but with the assumption of two binding sites for
Na+ [a = 2; (19)],
Gin = 50.5 pS, and
= 0.23, yields
an estimate of K0 of ~40 mM Na+.
This indicates that intracellular Na+ made a significant
contribution to inward rectification in cell-attached patches.
Intracellular SPM induces a voltage-dependent block.
SPM is a naturally occurring polyamine that carries four positive
charges at physiological pH and acts as a gating molecule, inducing a
concentration- and voltage-dependent block of several inwardly
rectifying K+ channels (6, 7, 26). We found
that SPM rapidly and reversibly inhibited channel activity in
cell-excised, inside-out membrane patches without affecting
single-channel current amplitude (Fig. 5,
insets). From Fig. 5, which compares the effects of 5 and
500 µM internal SPM on NPo at both
60 and
+60 mV, it is clear that the inhibitory effect of SPM was concentration
and voltage dependent. This is quantified in Fig.
6A. At both membrane
potentials, increasing SPM concentration reduced
NPo in a sigmoidal fashion. Fitting experimental
data points with the Hill equation (see Fig. 6) revealed a channel
sensitivity to SPM at positive potential
(Ki = 0.2 µM) that was greater by two
orders of magnitude than at negative potential (Ki = 26 µM). In contrast, Hill
coefficients were similar and close to unity at both potentials (0.8 vs. 0.7, respectively). This voltage dependence was further
investigated by observing the effects of 0.5 µM SPM on
NPo over an extended Vc
range. The results of two separate experiments are illustrated in Fig.
6B. The relationship between NPo and
Vc was described by the Boltzmann distribution
(Eq. 2). The best fit of data was given by
NPo max = 1.0, V1/2 = 43 mV, and z = 1.25 (Fig. 6B).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of intracellular spermine (SPM) on basolateral CTAL
K+ channel activity. Traces show recording of
K+ channel activity on the same cell-excised,
inside-out membrane patch clamped at either 60 mV
(top traces) or +60 mV (bottom traces). The patch
was bathed in symmetrical high-K (144.8 mM, Cl - and
Mg2+-free+5 mM Na2-EDTA). C , current level
corresponding to closure of all K+ channels. Each trace was
obtained in the absence (Control; left traces) or presence
of 5 (middle traces) or 500 µM SPM (right
traces) in the bath. Insets: a and b,
excerpts at an expanded time scale (*) taken from the indicated
sections of the traces.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 6.
Concentration and voltage dependence of the effect of
internal SPM on basolateral CTAL K+ channel activity.
A: dose-response relationships for the inhibitory effect of
SPM on NPo at hyperpolarizing ( )
and depolarizing ( ) membrane potentials. The
cytoplasmic side of cell-excised, inside-out membrane patches was
exposed to the indicated SPM concentrations ([SPM]) at either
Vc of 60 or +60 mV under the same conditions
as in Fig. 5. Because of patch-to-patch variability, the effects of a
given [SPM] on NPo were normalized to
NPo for the same patch in the absence of SPM.
Each value is the mean of at least 3 determinations, and SE are shown
as error bars when larger than symbols. At both potentials,
relationships between [SPM] and NPo were
described by the equation NPo = 100/[1 + ([SPM]/Ki)nH],
where Ki is the half-maximal inhibitory
concentration, and nH is the Hill coefficient.
The lines drawn are nonlinear, least squares fits to the
respective data given by Ki = 26 ± 1 µM and nH = 0.8 ± 0.03 for
hyperpolarizing voltages and Ki = 0.2 ± 0.06 µM and nH = 0.7 ± 0.1 for
depolarizing voltages. B: voltage dependence of the effect
of 0.5 µM SPM on NPo. Results obtained from 2 separate patches are shown ( and ).
Within each experiment, NPo at a given
Vc value in the presence of SPM was normalized
to that for the same patch in the absence of SPM (control). Dotted
line, is a nonlinear least squares fit of the Boltzmann equation (see
RESULTS) to the data given by
NPo max = 1.0, Vo = 43 mV, and z = 1.25.
|
|
Modulation by internal pH.
Varying bath pH rapidly reversibly altered the K+ channel
activity in cell-excised, inside-out membrane patches (Fig.
7A). NPo was altered by intracellular pH
(pHi) in a sigmoidal fashion (Fig. 7B), and
fitting data points using a modified Hill equation (Fig. 7) yield an
apparent pK of 7.6. A Hill coefficient of 1.8 suggested that
several protons were acting cooperatively in channel regulation by
pHi. The unit current amplitude through the CTAL K+ channel was not altered by pHi.
i-Vc relationships at pH 6.8 (n = 3) and 7.4 (n = 5) did not show
any pH-dependent change in Gin (48 ± 4 vs.
45 ± 1 pS, respectively; P = 0.32) or
Gout (36 ± 0.7 vs. 37 ± 3 pS,
respectively; P = 0.83).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of variations in intracellular pH
(pHi) on basolateral CTAL K+-channel activity.
A: single-channel recordings from a cell-excised, inside-out
membrane patch at the bath pH (i.e., pHi) values indicated
on the right of each trace. Patch was bathed in symmetrical
high-K (Cl - and Mg2+-free+5 mM
Na2-EDTA) and clamped at Vc = 50 mV. C -, current corresponding to closure of all K+
channels. B: dose-response relationship of the effect of
pHi on K+ channel activity under the same
conditions as in A. Within each experiment,
NPo at a given pHi value was
normalized to NPo at pH = 7.4. Each point
is the mean of at least 4 determinations, and SE values are shown as
error bars when larger than symbols. The relationship between
pHi and NPo is described by the
equation NPo = NPo (max)/ [1 + ([H+]/K)nH],
where NPo(max) is the maximal
NPo, [H+] is the bath proton
concentration, pK = logK, and
nH is the Hill coefficient. The line drawn is a
nonlinear least squares fit to the data given by
NPo(max) = 2.93 ± 0.09, pK = 7.6 ± 0.1, and
nH = 1.8 ± 0.1.
|
|
This suggests that the basolateral CTAL K+ channel may be
regulated by pHi in situ. This was tested using
a previous observation that 5 mM bath NH4Cl rapidly and
tonically acidifies mouse CTAL cells by 0.3-0.4 pH units
(15), an approach used to demonstrate in situ the
pHi sensitivity of the basolateral 9-pS Cl
channel in mouse TAL (15). Here we investigated the
effects of bath NH4Cl on basolateral K+ channel
activity in situ in cell-attached membrane patches, using Cl
-containing solutions but under a nil
Vc, thus minimizing the influence of the
pH-sensitive 9-pS Cl
channel (15). In three
of four attempts, K+ channel activity decreased when the
tubule was exposed to 5 mM NH4Cl. This procedure led to the
complete abolition of activity (Fig. 8)
in two cases. In the third case, NPo fell from
1.96 to 0.87. Channel activity recovered completely when
NH4Cl was removed from the bath (Fig. 8).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 8.
Effects of intracellular acidification on K+
channel activity in situ. The trace is a continuous recording of
K+ channel activity of a cell-attached membrane patch with
the high-Na solution in the bath. Pipette was filled with the high-K
solution, and Vc was set at 0 mV. At the period
indicated by the horizontal bar, 5 mM NH4Cl was added to
the superfusate. C , current corresponding to closure of all
K+ channels.
|
|
Internal ATP does not inhibit the basolateral CTAL
K+ channel.
ATP has no inhibitory effect on K+ channel activity (data
not shown). Adding 1 mM internal ATP (Na salt) without Mg2+
for at least 1 min had no influence on channel activity of inside-out patches at Vc =
50 mV (P = 0.3, paired t-test, n = 3). No change in
channel activity was seen when 5 mM internal ATP was added to the bath
(n = 2).
 |
DISCUSSION |
Inward rectification properties.
There has been very little investigation of rectification of
K+ channels in native renal tissue (45). An
intermediate rectification coefficient
(Gin/Gout) of 4 has been
explicitly determined for the basolateral 25-pS K+ channel
in the Ambystoma proximal tubule (33). A
Gin/Gout ~ 4-6 can also be extrapolated from published data on proximal
tubule basolateral K+ channels (1, 20, 39).
The i-Vc relationship of the
basolateral CTAL K+ channel also displays a
Gin/Gout of ~4.5
in cell-attached patches, indicative of an intermediate-type inward
rectification. This differentiates the channel from strong inward
rectifiers such as Kir 2.3 (CCD-IRK3) (48) and from the
weak rectifier of the apical membrane of the cortical collecting duct
(CCD) (47) and Kir 1.1 (2, 18) for which a
Gin/Gout of 2-2.5
can be calculated. It is worth noting that the rectification would be
hardly apparent in the presence of a physiological K+
concentration: Gin would be then comparable to
Gout (~10 pS).
The K0 for the Mg2+-induced
rectification of the CTAL K+ channel (1.6 mM) is consistent
with a weakly inwardly rectifying K+ channel. This is
similar to the 2 mM for the weakly rectifying ATP-dependent
K+ channel in ventricular cells (19, 31) and
to the 2-29 mM for Kir 1.1 (2, 36, 43). This is
considerably higher than the ~10 µM of the strong inwardly
rectifying IRK1 (31, 43, 52) and slightly lower than the
only published value for native intermediate inwardly rectifying renal
K+ channels (K0 = 7.7 mM)
(33). The electrical distance (
) sensed by
Mg2+ (~0.2) for the CTAL K+ channel also
matches that of weak inward rectifiers (~0.3) (2, 31,
36). Furthermore, the contribution of internal Na+
to inward rectification (K0 = 40 mM
Na+) of the CTAL channel is very similar to the 15 mM
Na+ at 40 mV (~30 mM at 0 mV) for the weakly rectifying
ATP-dependent K+ channel (8, 9, 19).
On the other hand, a high sensitivity to SPM
(Ki = 0.2-25 µM) distinguishes the
CTAL K+ channel from weak inward rectifiers
(37), which have a considerably higher
Ki (~1 mM) (49, 51, 53). Still,
it is 10 times higher than that for strong inward rectifiers, such as
the muscarinic K+ channel in the atrial myocyte or the
cloned Kir 2.1 and Kir 4.1, with a Ki in the 10 nM range (6, 51). On the other hand, the voltage
dependence of the SPM block shows a z of ~1.3,
yielding an
of ~0.3, in accordance with our results for
Mg2+. This disagrees with the usual characteristics of the
SPM block, as
values greater than unity have been reported for
strong inward rectifiers (10, 27). High sensitivity to SPM
usually correlates with strong Mg2+-induced inward
rectification (27), but this was not the case here. Such
discrepancies between the effects of SPM and Mg2+ on the
CTAL channel could arise from a heterotetrameric channel structure. For
instance, coexpression of Kir 1.1 (a mild inward rectifier) and Kir 4.1 (a strong inward rectifier) K+ channels in
Xenopus laevis oocytes produces a channel with
lower sensitivity to SPM than the Kir 4.1 homotetramer
(10).
Channel regulation.
The CTAL K+ channel is also sensitive to pHi. A
decrease in channel activity at acid pH is observed for many renal
K+ channels (45), including the
K+-secreting channel (and ROMK) in the apical membrane of
the CCD and the proximal convoluted tubule basolateral channel
(34, 35, 47). The pK of ROMK channels was
7.0-7.2, and the channel activity peaks at pH >7.4, as a
consequence of a Hill coefficient of 3. A quite different pattern was
observed for the CTAL channel, with a Hill coefficient of ~2 and a
pK of ~7.6. Therefore, in situ channel activity would be
in the lower part of the curve, so that even slight intracellular
acidification would suffice to produce a significant reduction in
NPo. Our experiments indeed showed that
acidifying the intracellular compartment by 0.3 pH unit was sufficient
to inhibit channel activity in cell-attached patches. In view of the
possible role played by this channel in NaCl reabsorption by the TAL
(see below), its pH dependence is of physiological interest. Indeed, in
vitro acute acidosis inhibits NaCl transport by the TAL
(50). Thus, in addition to the basolateral pH-sensitive
Cl
channels (15), the 45-pS K+
channel we describe here appears as another basolateral target of
acidosis in the inhibition of NaCl reabsorption by the TAL.
Comparison with other K+ channels in
renal basolateral membranes.
The only previous patch-clamp study in the basolateral membrane of CTAL
reported the presence of a 35-pS K+ channel in the rabbit
(22), showing inward rectification
(Gout = 7 pS). As in the mouse, activity
increases with depolarization in cell-attached patches. Thus the
channels in rabbit and mouse CTALs seem to be similar, but some
conductive and regulatory properties were not investigated in rabbit
CTAL, which limits further comparisons. Several channels described in
basolateral membranes of other nephron segments share some, but not
all, properties of the CTAL channel. In the basolateral membrane of rat
CCD, an inwardly rectifying K+ channel has been reported,
but it is highly active and voltage independent, and pH insensitive and
has a slightly lower conductance (27 pS) (28, 46). A
45-pS, pH-sensitive K+ channel has also been described in
cell-attached patches of rat CCD (46), but it is activated
by hyperpolarization. Other inwardly rectifying K+ channels
have been studied in the basolateral membrane of the proximal tubule.
Although some of these channels may differ in terms of voltage
dependence, they do share a number of properties: they are all
inhibited by acid pH (1, 34), and their inward conductance
is ~50 pS in mammalian physiological saline (21, 38, 39)
and depends on the external K+ concentration, with a
Km of 65-77 mM (23, 33).
However, on the other hand, regarding inward rectification, the CTAL
channel has a higher affinity toward Mg2+ and, unlike the
PCT channel (33), exhibits high sensitivity to SPM.
Second, the CTAL channel is not inhibited by ATP.
We did not address the nature of the molecular entity underlying the
basolateral K+ channel in the CTAL, but functional
properties allow us to rule out the Kir 2, Kir 6, and Kir 7 channels
families, as well as the tandem of P domains in the weak inward
rectifier K+ channel and related channels (see Refs.
25 and 37 for reviews). Comparative studies
will probably help to ascertain the molecular nature of the channel.
K+ conductance in the CTAL
basolateral membrane.
Studies of microperfused isolated tubules have given rise to
conflicting interpretations on the issue of basolateral K+
conductance in CTAL. Greger and Schlatter (11) initially
found that the basolateral membrane of rabbit CTAL was not conductive to K+ and postulated a basolateral K+ exit
through a K+-Cl
cotransporter (see Fig.
9). However, subsequent studies reported a K+-conductive pathway in the basolateral membranes of
hamster and rabbit TAL (3, 54) and those of the
Amphiuma diluting segment (13). The previous
patch-clamp study in rabbit CTAL (22) and the present work
in the mouse clearly establish that the basolateral membrane of CTAL
cells is endowed with K+ channels.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 9.
Basic model of NaCl reabsorption by the TAL
epithelium. Dotted lines indicate passive ion movements down
electrochemical gradients. Vm, membrane
potential.
|
|
We found an average of six channels per patch displaying K+
channel activity. The dependence of channel conductance on external K+ concentration yielded a unitary conductance of ~10 pS
for the K+ channel under physiological conditions (5 mM
external K+). This corresponds to a maximal K+
conductance of ~60 pS/membrane patch. In previous studies, we observed an average of 20 Cl
channels of 9 pS/patch under
similar experimental conditions (14, 30), giving a
Cl
conductance of 180 pS. Although this is probably an
underestimate, as it does not take into account the contribution of the
less common 45-pS Cl
channel in this membrane
(40), Cl
conductance does seem to be greater
than K+ conductance by a factor of at least three. This is
in qualitative agreement with macroscopic electrophysiological data,
which show that Cl
conductance predominates over
K+ conductance in the basolateral membrane (13,
54). Thus it does not contradict the fact that the basolateral
electromotive force is primarily determined by Cl
conductance, as required for the observed positive transepithelial voltage difference (Fig. 9).
A depolarization-activated K+ channel showing noticeable
activity at resting membrane potential may serve two purposes. First, it may help maintain VBl above
ECl (see Fig. 9) to provide a continuous driving
force for basolateral conductive Cl
exit. On stimulation
of overall NaCl reabsorption, the opening of basolateral
Cl
channels allows Cl
efflux but, in turn,
tends to decrease VBl toward
ECl and thus dissipate the driving force for
Cl
exit. The decrease in VBl could
be the signal upmodulating the depolarization-activated 45-pS
K+ channel, the resulting increase in
VBl restoring the driving force for basolateral
Cl
efflux.
Second, because continuous activity of the basolateral
Na+/K+-ATPase is mandatory to transcellular
NaCl reabsorption by the TAL, basolateral K+ recycling is
necessary to avoid the intracellular accumulation of K+. In
proximal tubular cells, this role is devoted to basolateral ATP-sensitive K+ channels (44), the cell ATP
content being the link between the pump and activities of the these
channels. In the CTAL, the 45-pS K+ channel might have a
similar role, but because this channel is insensitive to ATP, another
factor would have to link Na+/K+-ATPase
activity and basolateral K+ conductance in the CTAL. For
instance, an increase in apical Na+ entry into CCD cells
stimulates both pump rate and nitric oxide production, nitric oxide
then activating an ATP-insensitive, low-conductance basolateral
K+ channel (45).
The K+ channel was present in ~30% of the patches, but
there were usually several channels per patch. This suggests that
K+ channels are arranged in clusters. Alternatively, this
would also be consistent with different cell types in TAL tubules, as suggested for the diluting segment of the Amphiuma
(13) and for hamster MTAL (54). In their
study, Yoshitomi et al. (54) reported the presence of a
first cell type with high basolateral conductance of K+ and
Cl
and then a second with low conductances of these ions.
In this respect, it is worth mentioning that we occasionally recorded Cl
channels in patches with no K+ channel
activity, and, conversely, some patches contained K+
channels, but no Cl
channel (not shown). This finding
does not support the hypothesis of two cell types, although it could be
argued that the K+ channel linked to the Cl
channel may not be the same as the K+ channel reported here
but another, hitherto unidentified, K+ channel.
 |
ACKNOWLEDGEMENTS |
We thank Stéphanie Nelson for technical assistance and
Nadège Hurson, Séverine Layre, and Lydie René-Corail
for secretarial work. The English text was corrected by Monika Ghosh.
 |
FOOTNOTES |
This study was supported by Contrat Prisme Institut National de la
Santé et de la Recherche Médicale. S. Lourdel is a
recipient of a research studentship from the Ministère de la Recherche.
Address for reprint requests and other correspondence: M. Paulais, INSERM U.426, Faculté de Médecine Xavier
Bichat, 16 Rue Henri Huchard, B. P. 416, 75870 Paris Cedex 18, France (E-mail: paulais{at}bichat.inserm.fr).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published November 27, 2001;10.1152/ajprenal.00238.2001
Received 1 August 2001; accepted in final form 23 November 2001.
 |
REFERENCES |
1.
Beck, JS,
Hurst AM,
Lapointe JY,
and
Laprade R.
Regulation of basolateral K channels in proximal tubule studied during continuous microperfusion.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F496-F501,
1993[Abstract/Free Full Text].
2.
Chepilko, S,
Zhou H,
Sackin H,
and
Palmer LG.
Permeation and gating properties of a cloned renal K+ channel.
Am J Physiol Cell Physiol
268:
C389-C401,
1995[Abstract/Free Full Text].
3.
Di Stefano, A,
Greger R,
Desfleurs E,
de Rouffignac C,
and
Wittner M.
A Ba2+-insensitive K+ conductance in the basolateral membrane of rabbit cortical thick ascending limb cells.
Cell Physiol Biochem
8:
89-105,
1998[ISI][Medline].
4.
Fabiato, A.
Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium-induced release from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle.
J Gen Physiol
78:
457-497,
1981[Abstract].
5.
Fabiato, A,
and
Fabiato F.
Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells.
J Physiol Paris
75:
463-505,
1979[Medline].
6.
Fakler, B,
Brändle U,
Bond C,
Glowatzki E,
König C,
Adelman JP,
Zenner HP,
and
Ruppersberg JP.
A structural determinant of differential sensitivity of cloned inward rectifier K+ channels to intracellular spermine.
FEBS Let
356:
199-203,
1994[ISI][Medline].
7.
Ficker, E,
Taglialatela M,
Wible BA,
Henley CM,
and
Brown AM.
Spermine and spermidine as gating molecules for inward rectifier K+ channels.
Science
266:
1068-1072,
1994[ISI][Medline].
8.
Findlay, I.
ATP-sensitive K+ channels in rat ventricular myocytes are blocked and inactivated by internal divalent cations.
Pflügers Arch
410:
313-320,
1987[ISI][Medline].
9.
Findlay, I.
The effects of magnesium upon adenosine triphosphate-sensitive potassium channels in a rat insulin-secreting cell line.
J Physiol (Lond)
391:
611-629,
1987[Abstract].
10.
Glowatzki, E,
Fakler G,
Brandle U,
Rexhausen U,
Zenner HP,
Ruppersberg JP,
and
Fakler B.
Subunit-dependent assembly of inward-rectifier K+ channels.
Proc Royal Soc Lond B Biol Sci
261:
251-261,
1995[ISI][Medline].
11.
Greger, R,
and
Schlatter E.
Properties of the basolateral membrane of the cortical thick ascending limb of Henle's loop of rabbit kidney. A model for secondary active chloride transport.
Pflügers Arch
396:
325-334,
1983[ISI][Medline].
12.
Greger, R,
Weidtke C,
Schlatter E,
Wittner M,
and
Gebler B.
Potassium activity in cells of isolated perfused cortical thick ascending limbs of rabbit kidney.
Pflügers Arch
401:
52-57,
1984[ISI][Medline].
13.
Guggino, WB.
Functional heterogeneity in the early distal tubule of the Amphiuma kidney: evidence for two modes of Cl
and K+ transport across the basolateral cell membrane.
Am J Physiol Renal Fluid Electrolyte Physiol
250:
F430-F440,
1986[ISI][Medline].
14.
Guinamard, R,
Chraïbi A,
and
Teulon J.
A small-conductance Cl
channel in the mouse thick ascending limb that is activated by ATP and proteine kinase A.
J Physiol (Lond)
485:
97-112,
1995[Abstract].
15.
Guinamard, R,
Paulais M,
and
Teulon J.
Inhibition of a small-conductance cAMP-dependent Cl
channel in the mouse thick ascending limb at low internal pH.
J Physiol (Lond)
490:
759-765,
1995[Abstract].
16.
Hamill, OP,
Marty A,
Neher E,
Sakmann B,
and
Sigworth FJ.
Improved patch-clamp technique for high-resolution recording from cells and cell-free membrane patches.
Pflügers Arch
391:
85-100,
1981[ISI][Medline].
17.
Hille, B.
Ionic Channels in Excitable Membranes (2nd ed.). Sunderland, MA: Sinauer, 1992.
18.
Ho, K,
Nichols CG,
Lederer WJ,
Lytton J,
Vassilev PM,
Kanazirska MV,
and
Hebert SC.
Cloning and expression of an inwardly rectifying ATP-regulated potassium channel.
Nature
362:
31-38,
1993[ISI][Medline].
19.
Horie, M,
Irisawa H,
and
Noma A.
Voltage-dependent magnesium block of adenosine-triphosphate-sensitive potassium channel in guinea-pig ventricular cells.
J Physiol (Lond)
387:
251-272,
1987[Abstract].
20.
Hunter, M.
Potassium-selective channels in the basolateral membrane of single proximal tubule cells of frog kidney.
Pflügers Arch
418:
26-34,
1991[ISI][Medline].
21.
Hurst, AM,
Beck JS,
Laprade R,
and
Lapointe JY.
Na+ pump inhibition downregulates an ATP-sensitive K+ channel in rabbit proximal convoluted tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F760-F764,
1993[Abstract/Free Full Text].
22.
Hurst, AM,
Duplain M,
and
Lapointe JY.
Basolateral membrane potassium channels in the rabbit cortical thick ascending limb.
Am J Physiol Renal Fluid Electrolyte Physiol
263:
F262-F267,
1992[Abstract/Free Full Text].
23.
Kawahara, K,
Hunter M,
and
Giebisch G.
Potassium channels in Necturus proximal tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
253:
F488-F494,
1987[Abstract/Free Full Text].
24.
Koefoed-Johnsen, V,
and
Ussing HH.
The nature of the frog skin potential.
Acta Physiol Scand
42:
298-308,
1958[ISI]..
25.
Lesage, F,
and
Lazdunski M.
Molecular and functional properties of two-pore-domain potassium channels.
Am J Physiol Renal Physiol
279:
F793-F801,
2000[Abstract/Free Full Text].
26.
Lopatin, AN,
Makhina EN,
and
Nichols CG.
Potassium channel block by cytoplasmic polyamines as the mechanism of intrinsic rectification.
Nature
372:
366-369,
1994[ISI][Medline].
27.
Lopatin, AN,
Makhina EN,
and
Nichols CG.
The mechanism of inward rectification of potassium channels.
J Gen Physiol
106:
923-955,
1995[Abstract].
28.
Lu, M,
and
Wang WH.
Nitric oxide regulates the low-conductance K+ channel in basolateral membrane of cortical collecting duct.
Am J Physiol Cell Physiol
270:
C1336-C1342,
1996[Abstract/Free Full Text].
29.
Martell, AE,
and
Smith RM.
Critical Stability Constants. New York: Plenum, 1974, vol. 1.
30.
Marvão, P,
de Jesus Fereira MC,
Bailly C,
Paulais M,
Bens M,
Guinamard R,
Moreau R,
Vandewalle A,
and
Teulon J.
Cl
absorption across the thick ascending limb is not altered in cystic fibrosis mice. A role for a pseudo-CFTR Cl
channel.
J Clin Invest
102:
1986-1993,
1998[Abstract/Free Full Text].
31.
Matsuda, H.
Magnesium gating of the inwardly rectifying K+ channel.
Annu Rev Physiol
53:
289-298,
1991[ISI][Medline].
32.
Matsuda, H,
Saigusa A,
and
Irisawa H.
Ohmic conductance through the inwardly rectifying K channel and blocking by internal Mg2+.
Nature
325:
156-159,
1987[ISI][Medline].
33.
Mauerer, UR,
Boulpaep EL,
and
Segal AS.
Properties of an inwardly rectifying ATP-sensitive K+ channel in the basolateral membrane of renal proximal tubule.
J Gen Physiol
111:
139-160,
1998[Abstract/Free Full Text].
34.
Mauerer, UR,
Boulpaep EL,
and
Segal AS.
Regulation of an inwardly rectifying ATP-sensitive K+ channel in the basolateral membrane of renal proximal tubule.
J Gen Physiol
111:
161-180,
1998[Abstract/Free Full Text].
35.
McNicholas, CM,
MacGregor CG,
Islas LD,
Yang Y,
Hebert SC,
and
Giebisch G.
pH-dependent modulation of the cloned renal K+ channel, ROMK.
Am J Physiol Renal Physiol
275:
F972-F981,
1998[Abstract/Free Full Text].
36.
Nichols, CG,
Ho K,
and
Hebert SC.
Mg2+-dependent inward rectification of ROMK1 potassium channels expressed in Xenopus oocytes.
J Physiol (Lond)
476:
399-409,
1994[Abstract].
37.
Nichols, CG,
and
Lopatin AN.
Inward rectifier potassium channels.
Annu Rev Physiol
59:
171-191,
1997[ISI][Medline].
38.
Noulin, JF,
Brochiero E,
Lapointe JY,
and
Laprade R.
Two types of K+ channels at the basolateral membrane of proximal tubule: inhibitory effect of taurine.
Am J Physiol Renal Physiol
277:
F290-F297,
1999[Abstract/Free Full Text].
39.
Parent, L,
Cardinal J,
and
Sauvé R.
Single-channel analysis of a K channel at basolateral membrane of rabbit proximal convoluted tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
254:
F105-F113,
1988[Abstract/Free Full Text].
40.
Paulais, M,
and
Teulon J.
cAMP-activated chloride channel in the basolateral membrane of the thick ascending limb of the mouse kidney.
J Membr Biol
113:
253-260,
1990[ISI][Medline].
41.
Reeves, WB,
and
Andreoli TE.
Sodium chloride transport in the loop of Henle.
In: The Kidney, edited by Seldin DW,
and Giebisch G.. New York: Raven, 2002.
42.
Schlatter, E,
and
Greger R.
cAMP increases the basolateral Cl
conductance in the isolated perfused medullary thick ascending limb of Henle's loop of the mouse.
Pflügers Arch
405:
367-376,
1985[ISI][Medline].
43.
Taglialatela, M,
Ficker F,
Wible BA,
and
Brown AM.
C-terminus determinants for Mg2+ and polyamine block of the inward rectifier K+ channel IRK1.
EMBO J
14:
5532-5541,
1995[Abstract].
44.
Tsuchiya, K,
Wang W,
Giebisch G,
and
Welling PA.
ATP is a coupling modulator of parallel Na, K-ATPase-K channel activity in the renal proximal tubule.
Proc Natl Acad Sci USA
89:
6418-6422,
1992[Abstract].
45.
Wang, WH,
Hebert SC,
and
Giebisch G.
Renal K+ channels: structure and function.
Annu Rev Physiol
59:
413-436,
1997[ISI][Medline].
46.
Wang, WH,
McNicholas C,
Segal AS,
and
Giebisch G.
A novel approach allows identification of K channels in the basolateral membrane of rat CCD.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F813-F822,
1994[Abstract/Free Full Text].
47.
Wang, WH,
Schwab A,
and
Giebisch G.
Regulation of small-conductance K+ channel in apical membrane of rat cortical collecting tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
259:
F494-F502,
1990[Abstract/Free Full Text].
48.
Welling, PA.
Primary structure and functional expression of a cortical collecting duct Kir channel.
Am J Physiol Renal Physiol
273:
F825-F836,
1997[ISI][Medline].
49.
Wible, BA,
Taglialatela M,
Ficker E,
and
Brown AM.
Gating of inwardly rectifying K+ channels localized to a single negatively charged residue.
Nature
371:
246-249,
1994[ISI][Medline].
50.
Wingo, CS.
Effect of acidosis on chloride transport in the cortical thick ascending limb of Henle perfused in vitro.
J Clin Invest,
78:
1324-1330,
1986[ISI][Medline].
51.
Yamada, M,
and
Kurachi Y.
Spermine gates inward-rectifying muscarinic but not ATP-sensitive K+ channels in rabbit atrial myocytes. Intracellular substance-mediated mechanism of inward rectification.
J Biol Chem
270:
9289-9294,
1995[Abstract/Free Full Text].
52.
Yamashita, T,
Horio Y,
Yamada M,
Takahashi N,
Kondo C,
and
Kurachi Y.
Competition between Mg2+ and spermine for a cloned IRK2 channel expressed in a human cell line.
J Physiol (Lond)
493:
143-156,
1996[Abstract].
53.
Yang, J,
Jan YN,
and
Jan LY.
Control of rectification and permeation by residues in two distinct domains in an inward rectifier K+ channel.
Neuron
14:
1047-1054,
1995[ISI][Medline].
54.
Yoshitomi, K,
Koseki C,
Taniguchi J,
and
Imai M.
Functional heterogeneity in the hamster medullary thick ascending limb of Henle's loop.
Pflügers Arch
408:
600-608,
1987[ISI][Medline].
Am J Physiol Renal Fluid Electrolyte Physiol 282(5):F866-F876
0363-6127/02 $5.00
Copyright © 2002 the American Physiological Society