Two types of K+ channels at
the basolateral membrane of proximal tubule: inhibitory effect of
taurine
Jean-François
Noulin,
Emmanuelle
Brochiero,
Jean-Yves
Lapointe, and
Raynald
Laprade
Groupe de Recherche en Transport Membranaire, Université de
Montréal, Montreal, Quebec, Canada H3C 3J7
 |
ABSTRACT |
The cell-attached configuration of the patch-clamp technique was
used to investigate the effects of taurine on the basolateral potassium
channels of rabbit proximal convoluted tubule. In the absence of
taurine, the previously reported ATP-blockable channel, KATP, was observed in 51% of
patches. It is characterized by an inwardly rectifying current-voltage
curve with an inward slope conductance of 49 ± 5 pS
(n = 15) and an outward slope
conductance of 13 ± 6 pS (n = 15).
The KATP channel open probability
(Po) is low,
0.15 ± 0.06 (n = 15) at a
Vp =
100 mV (Vp
is the pipette potential), and increases slightly with depolarization.
The gating kinetics are characterized by one open time constant
(
o = 5.0 ± 1.9 ms,
n = 6) and two closed time constants
(
C1 = 5.2 ± 1.5 ms,
C2 = 140 ± 40 ms;
n = 6). In 34% of patches, a second
type of potassium channel, sK, with distinct properties was recorded. Its current-voltage curve is characterized by a sigmoidal shape, with
an inward slope conductance of 12 ± 2 pS
(n = 4). Its
Po is voltage
independent and averages 0.67 ± 0.03 (n = 4) at
Vp =
80 mV. Both its open time and closed time distributions are described by a single time constant
(
o = 96 ± 19 ms,
C = 10.5 ± 3.6 ms;
n = 4). Extracellular perfusion of 40 mM taurine fails to affect sK channels, whereas
KATP channel
Po decreases by
75% (from 0.17 ± 0.06 to 0.04 ± 0.02, n = 7, P < 0.05). In conclusion, the
absolute basolateral potassium conductance of rabbit proximal tubules
is the resulting combination of, at least, two types of potassium
channels of roughly equal importance: a high-conductance low-open
probability KATP channel and a
low-conductance high-open probability sK channel. The previously
described decrease in the basolateral absolute potassium conductance by
taurine is, however, mediated by a single type of K channel: the
ATP-blockable K channel.
patch clamp; regulatory volume decrease; potassium conductance; adenosine 5'-triphosphate-dependent potassium channel; low-conductance potassium channel
 |
INTRODUCTION |
BASOLATERAL POTASSIUM channels have a very important
role to play in the transepithelial reabsorption mediated by proximal convoluted tubules (PCT). Indeed, the sodium-dependent translocation of
luminal substrates requires the maintenance of a cellular
Na+ electrochemical gradient,
which is controlled by the basolateral Na-K-ATPase, coupled to the
basolateral potassium conductance (3, 14, 17, 29). Sodium ions,
cotransported with the luminal substrates, are extruded by the
basolateral pump, while potassium ions pumped into the cell by the
Na-K-ATPase are recycled by the basolateral potassium conductance and
create the negative intracellular potential.
In rabbit proximal tubules, potassium diffusion across the basolateral
membrane is known to be mainly mediated by an inwardly rectifying
channel with an inward unitary conductance of ~50 pS (11, 16, 19).
This potassium channel is inhibited by raising intracellular ATP (14, 24) and by lowering intracellular
pH (2). In addition to these regulators, we have recently found that an
increase in intracellular taurine concentration inhibits the
basolateral potassium conductance
(GK) by 50%
(4). Very interestingly, the basolateral taurine permeability was shown to be activated by cell swelling (4), which suggests that taurine could
play a significant role in maintaining cell volume and membrane potential during stimulation of transepithelial sodium transport. First, the swelling-activated basolateral taurine efflux would directly
contribute to the volume regulatory decrease. Then, the resulting
decrease of intracellular taurine concentration would increase the
potassium conductance and facilitate further volume and membrane
potential restoration by enhancing the potassium efflux.
In this study, we investigated the effect of taurine on the basolateral
potassium conductance, at the single-channel level, using the patch
clamp technique in the cell-attached configuration. This allowed the
identification of a previously unrecognized potassium channel in the
basolateral membrane of PCT, which is responsible for ~40% of the
membrane conductance. At the single- channel level, only the previously
identified 50-pS channel was proved to be taurine sensitive.
 |
MATERIALS AND METHODS |
Tubule preparation. Female New Zealand
White rabbits were decapitated, and the left kidney was perfused with a
cold (4°C) preservation solution containing (in mM) 56 Na2HPO4,
13 NaH2PO4, and 140 sucrose (5). Thin kidney slices were obtained and placed immediately in the cold preservation fluid. Segments of S1 and S2
cortical PCT were dissected from the midcortical region under ×40
magnification at 4°C in the same solution. Once dissected, PCTs
were transferred to the perfusion chamber and bathed, at room
temperature, in the control solution, buffered at pH 7.4 and containing
(in mM) 30 NaCl, 5 KCl, 1.2 MgSO4,
1.8 CaCl2, 1 NaH2PO4,
3 Na2HPO4,
4 sodium acetate, 1 trisodium citrate, 25 NaHCO3, 10 glucamine chloride, 128 mannitol, 5.5 glucose, and 6 alanine. Tubules were held by
micropipettes and placed in the flux of a perfusion system as
previously described (18). To gain access to the basolateral membrane,
the basal membrane was removed by a 20-min incubation with collagenase
A (Boehringer Mannheim) added to the control solution (final enzymatic
activity of 0.5 U/ml) (2). All experiments were conducted on collapsed tubules.
Electrophysiology. The cell-attached
configuration of the patch-clamp technique was used to record currents
from basolateral membrane channels. Patch pipettes were fabricated from
hematocrit capillary tubes (Fisher) using a Narishige PP-83
two-stage patch pipette puller. Pipettes filled with a solution
containing (in mM) 150 KCl, 2 CaCl2, 1 MgCl2, and 10 HEPES (titrated to
pH 7.4 with 2.4 NaOH) had resistances between 5 and 10 M
when placed in the control solution. Ionic selectivity was studied in inside-out configuration. Micropipette solution contained (in mM) 150 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 2.4 NaOH, and 20 mannitol. In symmetrical potassium gradients, bath solution contained
(in mM) 140 KCl, 2 MgCl2, 5 EGTA,
10 HEPES, 18 NaOH, 5 glutathione, and 35 mannitol. In asymmetrical
potassium gradients, 70 mM NaCl was substituted for 70 mM KCl of the
bath solution.
The pipettes were advanced onto the basolateral membrane of tubules by
means of a hydraulic micromanipulator (model MX 630R; Newport, Irvine,
CA). The contact between microelectrode and membrane was monitored by
detecting change in the membrane shape before applying suction to
obtain a gigaohm seal. Channel currents amplified with a patch-clamp
amplifier (Axopatch-1D; Axon Instruments, Foster City, CA) were
recorded onto videotape through a digital data recorder (model VR-10B
CRC; Instrutech, Great Neck, NY). To analyze data, records were
low-pass filtered with an eight-pole Butterworth filter (model 901;
Frequency Devices, Haverhill, MA) at 500 Hz, digitized at 2.5 kHz with
a TL-1 DMA Labmaster interface (Axon Instruments), and stored in a 486 PC by means of a specific acquisition software (Axotape 1.2.01, Axon
Instruments). Digitized records with very low-amplitude events were
additionally filtered at 300 Hz (with a digital Gaussian filter) to
improve peak discrimination in amplitude histograms. When such a cutoff
frequency was needed, no kinetic analysis was performed. Channel
currents were analyzed with the pClamp 5.5.1 software (Axon
Instruments). Channel conductance was estimated from linear regression
of single-channel current-voltage curve between
40 mV and +20 mV
for inward currents and between +60 mV and +100 mV for outward
currents. Reversal potentials were obtained by interpolation using a
third-order polynomial fit of the current-voltage curves. Neither a
linear, a quadratic, nor a Goldman-Hodgkin-Katz equation could provide
an acceptable fit of these curves. The open probability
(Po) was
calculated from amplitude histograms according to the equation
|
(1)
|
where i is the number of
channels observed at the same time,
Pi is the
probability that i channels are
simultaneously open, and N is the
total number of channels in the patch. In some experiments, open
probability at a given potential was plotted as a function of time. In
this case, recordings at constant potential were divided into 30-s
segments from which one
Po was
calculated. In the text and legends to Figs. 1-8, potentials are
given as
Vp, where
Vp is the
micropipette potential.
Experimental values are expressed as means ± SE. Student's
t-test for unpaired observations is
used for statistical comparisons.
 |
RESULTS |
With control solution bathing the tubule, cell-attached experiments
confirmed the presence of the ATP-dependent potassium channel,
KATP, which was previously
identified at the basolateral membrane of the PCT (2, 14, 19, 24).
Figure
1A shows the typical KATP channel activity,
which was observed in 51% of the patches showing some channel
activity. At a potential of
40 mV, the amplitude of inward
currents was
3.2 ± 0.5 pA
(n = 14). Interestingly, 34% of
recordings performed in the same experimental conditions demonstrated
the presence of a previously unrecognized potassium channel (sK). As
seen in Fig. 1B, this channel is
characterized by smaller inward currents at
40 mV (
1.4 ± 0.2 pA, n = 5, P < 0.05) and by a more frequent
open state. In the remaining 15% of experiments, the two channel types
were observed together in the same patch (Fig.
1C). The fact that one type of
channel could be recorded independently of the other in some patches
and that independent gating of the two channels was observed when they were present in the same patch strongly suggest the presence of distinct channels.

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Fig. 1.
Representative inward current traces recorded at a potential of
40 mV in cell-attached configuration from 3 different patches.
In control solutions, two types of
K+ channels are observed
separately (A,
B) or together
(C), depending on the experiment.
Arrow ("C") indicates the zero current level.
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Characteristics of sK vs.
KATP.
The current-voltage curves were plotted for the two types of channel in
cell-attached configuration when the tubule is bathed in
the control solution (Fig. 2). The
KATP channel demonstrated an
inward rectification, with an inward slope conductance of 49 ± 5 pS
(n = 15) and an outward slope
conductance of 13 ± 6 pS (n = 15).
Concerning the sK channel, currents were characterized by a sigmoidal
current-voltage relation with an inward slope conductance (measured
between
Vp =
40 mV and
Vp = 20 mV) equal to 12 ± 2 pS (n = 4),
which is significantly lower than the
KATP channel inward conductance.
Reversal potentials
(Erev) for
KATP and sK channels were equal to
+53 ± 14 mV (n = 15) and +48 ± 6 mV (n = 4), respectively. As we
previously demonstrated that the potassium-to-sodium permeability ratio
(PK/PNa)
of the KATP channel was greater
than 7 (2), we assume that the reversal potential of
KATP currents corresponds to the
Vp value
for which potassium ions are at their equilibrium. This already
suggests that sK channels display a good selectivity for potassium
ions.

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Fig. 2.
In control solutions, unitary current of both the ATP-dependent
potassium channel, KATP ( ,
n = 15), and the "small"
potassium channel, sK ( , n = 4),
recorded in the cell-attached configuration, are plotted as a function
of Vp,
where Vp is the
micropipette potential.
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A better evidence of the ionic selectivity was brought by experiments
made in inside-out patch that allows the control of both membrane
potential and ionic gradients (Fig. 3).
With nearly symmetrical potassium concentrations in the pipette and in
the bath (140 mM bath/150 mM pipette,
EK = +1.7 mV),
reversal potentials for KATP
currents and sK currents were equal to 1.1 ± 0.6 mV
(n = 12, Fig.
3A) and 1.7 ± 1.5 mV
(n = 6, Fig.
3B), respectively. When equilibrium
potential for K+ ions was
displaced by reducing bath concentration to 70 mM (70 mM bath/150 mM
pipette, EK = +19.3 mV), the reversal potential of
KATP channels shifted to 19.3 ± 0.9 mV (n = 6, Fig.
3A), indicating a channel almost
perfectly selective for potassium. The smallest observed value for
Erev led to a
minimum permeability ratio
PK/PNa of 26. In similar conditions, reversal potential of sK currents shifted
to 18.0 ± 0.7 mV (n = 13, Fig.
3B), corresponding to a minimum
PK/PNa
of 13.

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Fig. 3.
Currents obtained from the inside-out configuration are plotted as a
function of membrane potential for both
KATP
(A) and sK
(B) channels, in both symmetrical
( , ) and asymmetrical ( , ) potassium gradients (indicated
as ratio of millimolar values in bath vs. pipette).
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The KATP and sK channels can also
be distinguished by considering their open probability
(Po), as
illustrated in Fig. 4. The KATP channel was characterized by
a low Po slightly
dependent on membrane potential, going from 0.15 ± 0.06 (n = 15) at
Vp =
100 mV to 0.35 ± 0.09 at
Vp = +80
mV. In contrast, the sK channel open probability was significantly
higher (P < 0.001) over the whole
potential range. It ranged from 0.67 ± 0.03 (n = 4) at
Vp =
80 mV to 0.53 ± 0.08 at
Vp = 0 mV
and did not vary significantly with membrane potential.

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Fig. 4.
In control conditions, open probability
(Po) of both
KATP ( ,
n = 15) and sK ( ,
n = 4) channels, recorded in the
cell-attached configuration, are plotted as a function of
Vp.
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The kinetic parameters of the two types of potassium channels were also
investigated by means of an analysis of the open and closed dwell-time
distributions (see Fig. 5 for a
representative example). For
Vp =
40 mV, the gating of the
KATP channel was characterized by
brief openings between short or long closed periods (Fig.
1A). Open dwell-time histograms
were well fitted by a single exponential function with an average time
constant,
o, of 5.0 ± 1.9 ms (n = 6). On the other hand,
adjustment of the closed dwell-time histogram led to determination of a
fast (
C1) and a slow
(
C2) time constant equal to
5.2 ± 1.5 and 140 ± 40 ms (n = 6), respectively. Contrary to the
KATP channel, recordings of the
small potassium channel (sK) demonstrated that the open state was the
most often observed event (Fig. 1B).
Analysis of the open and closed dwell-time histograms allowed us to
calculate a single time constant for each of the states (Fig. 5).
Moreover, the open time constant,
o = 96 ± 19 ms
(n = 4), was slower than the closed
time constant,
C = 10.5 ± 3.6 ms (n = 4), which explains the
high open probability of the sK channel.

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Fig. 5.
Distributions of open and closed times in control conditions, plotted
from experiments performed at
Vp = 40 mV. Kinetic parameters of the
KATP channel are described by two
closed time constants (A;
C1 and
C2) and one open time
constant (B;
o), whereas kinetic
parameters of the sK channel are defined by one closed time constant
(C;
C) and one open time constant
(D;
o). sqrt,
square root.
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Effects of taurine. The preceding
results demonstrate that two potassium channels, with very distinct
properties, are present in the basolateral membrane of the PCT. As
previous data on the inhibitory effect of taurine were obtained at the
macroscopic level, it became even more interesting to investigate the
inhibitory effect of taurine at the single channel level.
After a 4-min period in control solution, the taurine solution (40 mM
replacing mannitol in the control solution) was perfused on the
basolateral side of the tubule during another 4-min period. Then,
taurine was removed by perfusing the control solution. During the
entire experimental period,
Vp was
held to
40 mV and currents were continuously recorded.
We first tested the effect of taurine on the small potassium channel.
Figure 6A
illustrates typical recording of the sK channel under the three
experimental periods. It appeared from recordings that neither the
channel gating nor the inward current amplitude were affected by the
peritubular taurine perfusion. The sK channel open probability as
averaged for four experiments was plotted as a function of time in Fig.
6B. It confirms that taurine does not
have any effect on the sK channel open probability, as fluctuations ranging from 0.75 to 1 are very likely related to the relatively short
time interval (30 s) used to calculate
Po.

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Fig. 6.
Effects of taurine on the sK channel.
A: representative sK current traces
recorded in cell-attached configuration at
Vp = 40 mV. Downward transitions correspond to channel openings.
Thick solid bars above the traces indicate taurine perfusion; arrows
indicate zero current level. B: sK
channel Po is
plotted as a function of time (n = 4).
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The same type of experiment was performed on the
KATP channel. Figure
7A
illustrates the effect of taurine on the channel activity. When taurine
was perfused, a progressive decrease of channel openings was observed
within the first minute of perfusion. The maximum effect was reached
after 4 min of perfusion. Replacing taurine solution by control did not
restore the KATP channel activity. To quantify the effects of taurine,
KATP channel open probability was
plotted as a function of time for seven experiments (Fig. 7B). In presence of taurine,
KATP channel
Po decreased from
0.17 ± 0.06 (n = 7) at
the beginning of the perfusion (t = 0 min) to 0.04 ± 0.02 (n = 7, P < 0.05) at
t = 4 min. This decrease in
Po was poorly
reversible after returning to control conditions, as one would expect
if taurine was to accumulate inside the cell. The analysis
of gating kinetics demonstrated that taurine acts by increasing the
slow component of the closed time kinetics. Whereas open time
(
o) and fast closed time
(
C1) constants remained unchanged, slow closed time constant
(
C2) increased significantly from 140 ± 40 to 610 ± 190 ms
(n = 6, P < 0.05).

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Fig. 7.
Effects of taurine on the KATP
channel. A: representative
KATP current traces recorded in
cell-attached configuration at
Vp = 40 mV. Downward transitions correspond to channel openings.
B:
KATP channel
Po is plotted as
a function of time (n = 7).
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Estimation of taurine affinity for
KATP.
In a previous report, we have shown that taurine, under identical
experimental conditions, induced a cell volume increase and that this
volume increase was related to the entry of taurine into the cell by
way of a sodium-dependent taurine transporter (6). Therefore, known
changes in cellular volume induced by taurine perfusion allow an
estimation of the amount of taurine flowing into the cell as a function
of time. The cellular osmolarity,
O, is defined as
|
(2)
|
where
N is the mole number of osmotically
active solutes, and V is the cell volume. The intracellular osmolarity
is assumed to be in equilibrium with the osmolarity of the
extracellular solution (300 mosM). Thus, as the cell volume increases
in response to the osmolyte influx to keep
O constant, the variation of
intracellular osmolyte concentration
(
[Osmolytes]in)
with time (see Fig.
8A) is
calculated as
|
(3)
|
where
V(t) is the change in cell volume with
time. Then, the variation of intracellular taurine concentration,
T,
is estimated by
|
(4)
|
where
s is the net number of molecules
entering the cell per taurine molecule. We assumed that the
cotransporter stoichiometry for sodium vs. taurine was
2:1, as demonstrated in rabbit and rat brush-border
membranes (30, 31) and in the apical and basolateral membranes of
LLC-PK1 and MDCK cell lines (15).
Moreover, we assumed that electroneutrality was maintained by the exit
of one potassium ion and the net entry of one bicarbonate anion for each taurine transport cycle. Thus, according to these hypotheses, the
net number of molecules entering the cell for each taurine molecule is
3. In general, at the time the patch-clamp experiment is
performed, tubules have been bathed in taurine-free solution for more
than 1 h. If we assumed that the initial intracellular taurine
concentration is negligible, Eq. 4 and
the time course of taurine-induced cell swelling predict that
intracellular taurine concentration progressively increases
from 0 to 30 mM during the first 4 min of the experiment.
From the curve illustrated in Fig. 8A
and Eq. 4, the time course of
Po (Fig.
7B) was converted into a relation
between Po and
the estimated taurine concentration (Fig.
8B). This relation was fitted
according to the equation
|
(5)
|
where
PMax is the open
probability in the absence of taurine, and
K0.5 is the
taurine concentration for half inhibition. The
K0.5 value could
be estimated to 8.7 mM, and the
PMax to 0.26, which is close to the measured value (0.23 ± 0.06, n = 7) in the absence of external
taurine. Please note that this rough calculation would not be much
affected by a reasonable underestimation of s; if the number of molecules
accompanying taurine were underestimated by 1, then the calculated
K0.5 would
decrease by only 25%.

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Fig. 8.
A: variation of intracellular osmolyte
concentration, induced by perfusion of extracellular taurine is plotted
as a function of time. B:
Po of the
KATP channel is plotted as a
function of intracellular taurine concentration. Time scale of
x-axis has been converted into taurine
concentration by using the curve plotted in
A.
[Taurine]in,
variation of intracellular taurine concentration.
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DISCUSSION |
Two types of potassium channels.
Passive potassium transport across the basolateral membrane of
mammalian proximal tubules was thought to be mediated by a unique
potassium channel regulated by intracellular ATP (14, 24),
intracellular pH (2), and, potentially, by cell volume (2, 7, 14, 16,
19, 24). Although other types of potassium channels, such as
stretch-activated K channels, have been shown in
Necturus proximal cells, no evidence for existence of such channels was provided for the mammalian proximal
tubule (20), and the relationship between cell volume and potassium
channel remained to be understood. In the present study, single-channel
recordings revealed the presence of a second and previously
unrecognized type of potassium channel, sK, with lower current
amplitude than the KATP channel.
As it is extremely difficult to establish a gigaohm seal on the
basolateral membrane of the proximal tubule, our knowledge of the
basolateral K channel(s) characteristics is limited. This may explain
why the presence of this second type of basolateral K channel was
generally overlooked but for one mention in a 1993 abstract (1) from
our laboratory [see also figure 7 in Parent et al. (19)].
Several lines of evidence allow us to conclude that we are in the
presence of two distinct types of potassium channels. First, when the
two channels are recorded together, independent gating can be observed.
Second, the probability of observing both channel types in the same
patch is equal to 0.15. This measured value is consistent with the
estimated probability of 0.17, obtained from the probabilities of
finding the KATP (0.51) and the sK
(0.34) channels individually (0.51 × 0.34 = 0.17), assuming that
these two types of K+ channels are
independently distributed. Lastly, after excising the membrane patch to
reach inside-out configuration, we usually observed a rapid rundown
(within 30 s, data not shown) of the KATP channel, whereas
low-amplitude activity remained steady all along the experiments,
suggesting the existence of two differently regulated channels. Taken
together, these data strongly suggest that two distinct potassium
channels coexist at the basolateral membrane of PCT cells.
In cell-attached configuration, the sK potassium channel
is characterized by a sigmoidal current-voltage curve with
a unitary conductance of 12 pS as measured between
40 mV and +20
mV. Potassium channels with low conductance (10-30 pS) have been
described at the apical membrane of rabbit thick ascending limb of
Henle's loop (25, 28), at both apical and basolateral membrane of rat
cortical collecting tubule (10, 13) and at the apical membrane of inner
medullary collecting duct (21). These low-conductance channels all
display a high open probability in cell-attached configuration and, in
the case of the inner medullary collecting duct, the cortical
collecting tubule, the thick ascending limb of Henle's loop, and the
proximal sK channel,
Po is not
dependent on the membrane potential and is not altered upon membrane
excision (8, 10, 21, 27, 28). Finally, the gating mechanisms of the
proximal sK channel and the cortical collecting tubule low-conductance
channel are both described by one open dwell-time constant and one
closed dwell-time constant (13, 26). Although these channels appear to
share some common properties, conclusions about the precise identity of
proximal sK channel would be premature.
The finding of a second type of potassium channel in the basolateral
membrane of PCTs raises the question of its role and its importance
with respect to the KATP channel.
Concerning the sK channel contribution to the absolute basolateral
potassium conductance, its high open probability appears to fully
compensate its low conductance. Therefore, the relative conductance for
each type of channel strongly depends on the number of active channels. The mean potassium current, I, carried
by a given type of channel, is given by
|
(6)
|
where
I0 is the unitary
current, Po is
the channel open probability,
NC is the total
number of channels. At the resting membrane potential, corresponding to
a micropipette potential of 0 mV, our experimental data allow the
estimation of both sK and KATP mean
currents, which are equal to IsK =
0.66 × 0.53 × NsK, and IKATP =
1.67 × 0.22 × NKATP,
respectively. To determine the
IsK/IKATP ratio, we measured a
NKATP/NsK
ratio of 1.43 from patches where the two types of channel coexist. From
these data, we estimated that the sK conductance would represent 40%
of the total potassium conductance of the PCT basolateral membrane.
Effects of taurine on the basolateral potassium
channels. Our results demonstrate that neither the open
probability nor the number of channels nor the unitary conductance of
the sK channels is modified by taurine. By contrast, the
KATP channel
Po decreased by
76% (from 0.17 ± 0.06 to 0.04 ± 0.02;
n = 7, P < 0.05) within 4 min. Since
current recordings are performed in cell-attached configuration and
taurine is perfused in the bath solution, the Po decrease must
be related to an intracellular effect. In ventricular myocytes, it has
been shown that taurine could inhibit ATP-dependent potassium channels
in inside-out configuration, suggesting a direct interaction process
(12, 22). In agreement with this hypothesis, reversibility of the
inhibitory effect could be observed when taurine was removed (12, 22).
In comparison with inside-out membrane patches, the use of
cell-attached configuration, particularly on a whole proximal tubule,
is more respectful of the cell physiology but does not
allow an accurate control over the intracellular taurine concentration.
It is possible that the removal of taurine under the membrane patch
studied would require more than a few minutes. It is interesting to
note that the effect of taurine on the basolateral conductance was
fully reversible when we were using microelectrodes (4). The possible
factors that differ between the two experimental conditions are the
temperature (25°C in patch-clamp vs. 38°C with microelectrodes)
and the actual geometry of the membrane studied (a microscopic membrane
patch vs. the whole basolateral membrane). It is possible that, at
25°C, more than a few minutes are needed to lower taurine
concentration under the membrane patch studied.
In proximal tubule cells, as in myocytes, the decrease of open
probability is related to the increase of the long closed time. We
estimated that taurine concentration should be equal to 8.7 mM for the
KATP channel half inhibition. This
K0.5 value is
quite similar to those found (13.5 mM) for the
KATP channels of ventricular myocytes (12). Such a
K0.5 value in the
low millimolar range suggests that in the proximal tubule cell in vivo,
KATP channels are under the
constant inhibitory effect of intracellular taurine. In vitro,
intracellular taurine concentrations are expected to reach 30 mM in 4 min upon exposure to 40 mM basolateral taurine. This
should inhibit most of the KATP
channels, leaving the basolateral K conductance solely dependent on sK.
The observed decrease of the basolateral potassium conductance by 50%
(4) is quite consistent with estimation that sK channels are taurine
insensitive and responsible for ~40% of the macroscopic K conductance.
Thus the present results bring a microscopic explanation for the
inhibitory effects of taurine on the macroscopic basolateral potassium
conductance, previously demonstrated with the intracellular microelectrodes technique (4). Although the mechanisms remain to be
determined, our data allow us to definitively conclude that intracellular taurine acts on basolateral ATP-dependent potassium channels by decreasing their open probability. On the other hand, we
also demonstrated the presence of a second type of potassium channel
that is taurine insensitive and responsible for ~40% of the
basolateral potassium conductance. The estimated sensitivity of
KATP to intracellular taurine
versus its physiological concentration and the fact that cell swelling
modulates taurine permeability indicate that taurine could play a very
significant role in maintaining a constant cell volume and membrane
potential in the face of varying rates of apical Na-coupled solute transport.
 |
ACKNOWLEDGEMENTS |
We acknowledge the excellent technical assistance of Mireille Marsolais.
 |
FOOTNOTES |
This work was supported by the Medical Research Council of Canada Grant
MT-10900 to J.-Y. Lapointe and R. Laprade.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J.-F.
Noulin, Université de Montréal, GRTM, C.P. 6128, Succursale
Centre-ville, Montreal, Quebec, Canada H3C 3J7 (E-mail:
lapoinj{at}ere.umontreal.ca).
Received 8 October 1998; accepted in final form 29 April 1999.
 |
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