From the * Laboratory for Cellular and Molecular Physiology, Division of Respiratory Research, Hospital for Sick Children Research Institute, Division of Respirology, Department of Medicine, The Toronto Hospital, § Department of Paediatrics, and
Department of
Medicine, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada M5G 1X8
It is currently believed that a nonselective cation (NSC) channel, which responds to arginine vasotocin (an antidiuretic hormone) and stretch, regulates Na+ absorption in the distal nephron. However, the mechanisms of regulation of this channel remain incompletely characterized. To study the mechanisms of regulation of
this channel, we used renal epithelial cells (A6) cultured on permeable supports. The apical membrane of confluent monolayers of A6 cells expressed a 29-pS channel, which was activated by stretch or by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase. This channel had an identical selectivity for Na+, K+, Li+, and
Cs+, but little selectivity for Ca2+ (PCa/PNa < 0.005) or Cl (PCl/PNa < 0.01), identifying it as an NSC channel.
Stretch had no additional effects on the open probability (Po) of the IBMX-activated channel. This channel had
one open ("O") and two closed (short "CS" and long "CL") states under basal, stretch-, or IBMX-stimulated conditions. Both stretch and IBMX increased the Po of the channel without any detectable changes in the mean open or
closed times. These observations led us to the conclusion that a kinetic model "CL
CS
O" was the most suitable
among three possible linear models. According to this model, IBMX or stretch would decrease the leaving rate of
the channel for CL from CS, resulting in an increase in Po. Cytochalasin D pretreatment abolished the response to
stretch or IBMX without altering the basal activity. H89 (an inhibitor of cAMP-dependent protein kinase) completely abolished the response to both stretch and IBMX, but, unlike cytochalasin D, also diminished the basal activity. We conclude that: (a) the functional properties of the cAMP-activated NSC channel are similar to those of
the stretch-activated one, (b) the actin cytoskeleton plays a crucial role in the activation of the NSC channel induced by stretch and cAMP, and (c) the basal activity of the NSC channel is maintained by PKA-dependent phosphorylation but is not dependent on actin microfilaments.
Antidiuretic hormone (ADH)1 increases the water permeability of the apical membrane of distal nephron
cells leading to a net increase in water absorption. Furthermore, ADH increases Na+ transport in distal nephron cells (Garty and Benos, 1988; Smith and Benos,
1991
; Palmer, 1992
). Investigations using single channel recording (Marunaka and Eaton, 1991
) have demonstrated that ADH also increases the number of 4-pS
amiloride-sensitive, highly Na+-selective channels. These
channels are thought to play an important role in Na+
reabsorption as the entry pathway of Na+ across the apical membrane from the lumen to the intracellular space in aldosterone-treated A6 cells, an amphibian
kidney cell line commonly used as a model of distal
nephron cells (Perkins and Handler, 1981
; Benos et al.,
1986
; Sariban-Sohraby et al., 1988
; Marunaka and Eaton,
1990a
, 1990b
, 1991
; Chalfant et al., 1993
; Marunaka,
1993
; Marunaka and Tohda, 1993
). On the other hand,
a gadolinium-sensitive stretch-activated nonselective cation (NSC) channel with a single channel conductance
of 28 pS has been frequently observed in the apical
membrane of A6 cells untreated with aldosterone, and
arginine vasotocin (AVT; an ADH) activated this NSC
channel (Marunaka et al., 1994
). Further, in aldosterone-untreated A6 cells, an apical membrane gadolinium-sensitive channel has been suggested to contribute
to Na+ absorption (Marunaka, 1996a
; Niisato and
Marunaka, 1997c
). Importantly, this latter channel retains full activity in the presence of 10 µM amiloride,
which completely blocks the 4-pS amiloride-sensitive, highly Na+-selective channel (Palmer, 1992
). Taken together, these observations suggest that this 28-pS NSC
channel plays a role in Na+ absorption in the aldosterone-untreated distal nephron. Despite the potential
importance of this channel, the mechanisms of its regulation, and, in particular, the relationship between
AVT- and stretch-induced activation of this NSC channel, remain unclear.
In an initial attempt to examine this issue, we studied
the effect of stretch on the AVT-activated channel.
However, because AVT-induced activation of the NSC
channel is only transient (Marunaka et al., 1994), we
were unable to clarify the relationship between the
AVT and stretch action. Accordingly, we needed another means to stably activate the NSC channel in place
of AVT. In this study, we report that 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of phosphodiesterase),
which increased cAMP (a second messenger of AVT),
stably activated the NSC channel and that stretch had no effects on the IBMX-activated channel. Additionally,
the single channel gating properties of the stretch-activated NSC channel were identical to those of the
IBMX-activated one.
Solutions
The bathing and pipette solutions used for single-channel recording in cell-attached patches contained (mM): 120 NaCl, 3.5 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4 (standard solution). When the ion selectivity was determined in inside-out patches, the pipette solution contained (mM): 120 NaCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4. The bathing (cytosolic) solution contained (mM): 120 KCl, 120 LiCl, 120 CsCl, 120 NMDG-Cl or 120 K-gluconate, instead of 120 NaCl with 1 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4. To study the Ca2+ permeability, 60 mM CaCl2 with 1 mM MgCl2 and 10 mM HEPES, pH 7.4, was used in the bathing solution.
Cell Line and Culture Methods
A6 cells were purchased from American Type Culture Collection
(Rockville, MD) in the 68th plating. All experiments were carried out on the 71st-83rd platings; there were no discernible differences between cells from different platings. Cells were maintained in plastic tissue culture flasks (Corning Glass Works, Corning, NY) at 26oC in a humidified incubator with 4% CO2 in air.
The culture medium was an NCTC 109 medium (Gibco Laboratories, Grand Island, NY) modified for amphibian cells with 100 mM NaCl, 20 mM NaHCO3, pH 7.4, containing 10% fetal bovine
serum (Gibco Laboratories), 100 µg/ml streptomycin, and 100 U/ml penicillin (Irvine Scientific, Santa Ana, CA) (Doi and
Marunaka, 1995; Nakahari and Marunaka, 1995
, 1996
). No supplemental aldosterone was added to the cell culture media.
Patch Recording
Standard patch-clamp techniques were used as previously described (Marunaka et al., 1992; Marunaka, 1996b
). Patch pipettes were made from LG 16 glass (Dagan Corp., Minneapolis, MN).
Patch pipettes were fire polished to produce tip diameters of
~0.5 µm. Single-channel currents were obtained at 22-23oC with
an Axopatch 1D patch-clamp amplifier (Axon Instruments, Foster City, CA). An Ag-AgCl reference electrode was connected to a
bathing solution via a 3% agar bridge containing 3 M KCl. Current signals were recorded on a digital video recorder (HF860D; Sony Corp., Tokyo, Japan) with pulse code modulation (1-DR-390; Neuro Data Instruments Corp., New York), and then digitized and analyzed by an IBM PC compatible computer with a
continuous data-acquisition program. A 500-Hz low-pass filter using a software Gaussian filter was used to present the actual
traces. To analyze the channel kinetics, a 2,000-Hz low pass Gaussian filter was used. When cells were used for patch-clamp experiments, cells from the flasks were subcultured on a permeable
support filter (Nunc Tissue Culture Inserts; Nunc, Roskilde, Denmark). Cells plated on permeable supports (Nunc filter) formed
polarized monolayers with their apical surfaces upward. The
patch pipette was applied from the luminal side, and a gigaohm
seal (>100 gigaohm) was made on the apical membrane of cells.
Open Probability (Po) of a Single Channel
Channel activity is expressed as open probability (Po):
![]() |
(1) |
where n is the number of channels, Po is the open probability of
an individual channel, i is the number of channels being simultaneously open; Pi is the probability of only i channels being simultaneously open. As previously reported (Marunaka et al., 1994),
the NSC channel studied in the present report was activated by
stretch or depolarization of the membrane. By applying stretch
or depolarization, which increased Po, we could observe the current level of all the channels in the patch membrane that were simultaneously open. Further, without the application of stretch,
the NSC channel demonstrated very little activity (low Po). This
led us to observe the current level of all the channels in the patch
membrane that were simultaneously closed. From the two current levels, with all the channels either simultaneously open or
closed, we could count the real number of the channels in the
patch membrane. Further, to confirm the real number of channels per patch membrane, we calculated the reliability of the estimated number of channels per patch membrane as the real number as previously described (Marunaka and Eaton, 1991
;
Marunaka and Tohda, 1993
). If the probability of the reliability
was >0.95, we used the estimated number of the channel per
patch membrane for calculation of the Po of the channel. Po was
calculated using the estimated number of channels per patch as
determined by the methods described above.
cAMP Assay
The intracellular cAMP was measured in a similar way described
previously (Niisato and Marunaka, 1997a, 1997b
). Before (basal) or after the cells were stimulated with 1 mM IBMX for 3 and 30 min, the medium was aspirated, ice-cold 6% trichloro acetate was added to the cells and incubated on ice for an additional 30 min. After centrifugation at 2,000 g for 15 min at 4oC, the supernatants
were washed four times with water-saturated diethyl ether. The
lower aqueous extracts were lyophilized. For cAMP assay, the
samples (dried extracts) were dissolved with the appropriate volume of assay buffer. The concentrations of cAMP in the samples
were determined with commercially available cAMP enzyme immunoassay kit (Amersham Corp., Arlington Heights, IL).
Chemicals
All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated.
Statistical Analyses
Results are presented as mean ± SD. Statistical significance was tested with Student's t test or ANOVA as appropriate. P < 0.05 was considered significant.
Single Channel Current Traces in a Cell-attached Patch before and after Application of IBMX
Fig. 1 illustrates representative single-channel current
traces from a cell-attached patch formed on the apical
membrane at the resting membrane potentials before
and after application of 1 mM IBMX. Before application of IBMX, the Po of the channel was low (Fig. 1 A).
IBMX stably increased the Po for up to 30 min after its
application (Fig. 1 B). The time course of IBMX-induced increase in Po of the channel is shown in Fig. 2 A. The
Po reached a stable value ~3-4 min after application of
1 mM IBMX and remained elevated throughout the
time period of our measurement (at least 30 min). The
effects of IBMX on Po increased in a dose-dependent manner (Fig. 2 B). IBMX significantly increased intracellular cAMP concentrations from 230 ± 20 fmol/105
cells (basal) to 540 ± 40 at 3 min and 1,180 ± 220 fmol/
105 cells at 30 min after addition of IBMX (1 mM; n = 4).
The Current-Voltage Relationship in Cell-attached Patches
The current-voltage (I-V) relationship was nearly linear in cell-attached patches formed on the apical membrane (Fig. 3 A). Voltages shown in Fig. 3 A indicate the
deviation from the resting membrane potential in cell-attached patches. The single-channel conductance was
29.0 ± 0.3 pS (n = 6; Fig. 3 A, open circles). The reversal
potential was 45.7 ± 0.7 mV (n = 6) more positive than
the resting membrane potential. IBMX shifted the I-V
relationship to the left by 15 mV (reversal potential = 31.1 ± 1.6 mV; n = 4; Fig. 3 A) without any effects on
single-channel conductance (29.8 ± 0.9 pS, n = 4; Fig.
3 A, closed circles).
Ion Selectivity
This channel had an almost identical permeability to
that of Na+, K+, Cs+, and Li+, but very little permeability to Ca2+ or Cl compared with that of Na+ (PCa/PNa < 0.005, PCl/PNa < 0.01; see Fig. 3 B). These observations suggest that the IBMX-induced shift in the I-V relationship might be due to a depolarization of the apical
membrane.
In general, the sum of Na+ and K+ concentrations in
the cytosolic and bathing solutions are identical. This
means that the reversal potential of an NSC channel
(selectivity for Na+ and K+, 1:1) is 0 mV. In the absence
of IBMX, the reversal potential of the NSC channel in a
cell-attached patch formed on the apical membrane
was 45.7 mV more positive than the resting potential of
the apical membrane (see Fig. 3 A, open circles). These
results suggest that the resting potential of the apical
membrane without IBMX would be 45.7 mV. Similarly, we can estimate that the apical membrane potential in the presence of 1 mM IBMX would be
31.1 mV.
Effects of IBMX and Stretch on the Po of the NSC Channels
We explored the relationship between the stretch- and
IBMX-induced activation of the NSC channel in more
detail. Fig. 4 depicts the effect of stretch on channel activity in the presence and absence of 1 mM IBMX in
cell-attached patches. In the case of the IBMX-activated
channel, Po was measured ~10 min after application of 1 mM IBMX. A negative pressure of 60 cm H2O (stretch)
was applied to the pipette. In the absence of IBMX, application of a negative pressure increased the Po (Fig. 4
A, a) proportionately to the magnitude of the negative
pressure (Fig. 4 B, open circles). IBMX increased Po in the
absence of stretch (Fig. 4 A, a and b). However, application of a negative pressure of 60 cm H2O, which had
the maximum effect on the Po of the channel in the absence of IBMX, had no further effect on the IBMX-activated channel (Fig. 4, A, b and B, closed circles), indicating that the mechanical (stretch) stimulation was not
additive to the biochemical (IBMX) stimulation. These
observations suggest that the biochemical activation of the NSC channel might induce some tension (or stress)
on the membrane analogous to that generated by the
mechanical process (stretch).
Single Channel Kinetics
Fig. 5 illustrates open and closed time-interval histograms obtained from cell-attached patches in the absence of applied potential. The open time-interval histogram obtained from an NSC channel without IBMX
treatment or application of stretch (control) was fitted
by one exponential function (Fig. 5 A). The mean
open time was 3.9 ms (Table I). In contrast, the closed
time-interval histogram without IBMX treatment or application of stretch (control) was fitted by two exponential functions (Fig. 5 A). The mean closed times
were 2.0 and 32.3 ms (Table I). The open time interval histogram obtained from an NSC channel with IBMX
treatment was also fitted by one exponential function
(Fig. 5 B). The mean open time was 4.2 ms (Table I).
Similar to the control experiment, the closed time-
interval histogram with IBMX treatment was fitted by
two exponential functions (Fig. 5 B). The mean closed
times were 2.3 and 34.9 ms (Table I). These results indicate that although IBMX increases the Po, the mean
values of open or closed times do not change significantly with application of IBMX. To study how IBMX
could increase the Po without any detectable changes in
open and closed times, we compared the frequency of
events staying at each state. IBMX increased the frequency of the short closed events and decreased the
frequency of the long closed events (Table I) leading to
an increase in the Po of the channel (see models of
channel kinetics in discussion for details). The effects of stretch on the single channel kinetics and other
channel properties were the same as those of IBMX
(Fig. 5 and Table I).
Table I. The Mean Open and Closed Times, the Number of Events in Each State, and the Fractional Time of Each State of the Basal (Control), IBMX-, and Stretch-activated NSC Channels |
Effects of Cytochalasin D on the Po of the Basal, IBMX-, and Stretch-activated NSC Channels
The actin cytoskeleton has been demonstrated to modulate the activity of ion channels (Prat et al., 1993a,
1993b
) and may also participate in sensing mechanical
deformation (Sadoshima et al., 1993
). As cytochalasin
D (CD) is known to depolymerize actin filaments in A6
cells (Verrey et al., 1995
), we studied the effects of this
cytoskeletal disrupting agent on the channel activity.
CD decreased the Po of the IBMX- or stretch-activated channel to the basal level (Fig. 6) consistent with the
concept that disruption of the actin cytoskeleton abrogated the response of the NSC channel to IBMX or
stretch. However, CD had no significant effects on the
Po of the basal channel (Fig. 6 C, Control). These observations imply that while the IBMX- and stretch-induced
activation of the NSC channel was dependent on an intact actin cytoskeleton, the basal channel activity (Po)
was not.
Effects of H89 on the Po of the Basal, IBMX-, and Stretch-activated NSC Channels
Pretreatment of cells with H89 (0.5 µM, 2 h), an inhibitor of cAMP-dependent protein kinase A, significantly
decreased the Po of the NSC channel (Fig. 7, A and B,
Control), and abolished the response of the channel to
IBMX or stretch (Fig. 7, A and B, IBMX and Stretch).
These observations indicate that all basal activity, and
IBMX- and stretch-induced activation of the NSC channel, may be mediated by PKA-dependent phosphorylation, either of the channel itself or of associated regulatory proteins.
We have previously reported the presence of a stretch-activated NSC channel that is also transiently activated
by AVT (Marunaka et al., 1994). However, the relationship between the AVT- and stretch-induced activation
of this channel was incompletely understood. Since the
AVT-induced activation was found to be transient, we were unable to characterize this relationship in detail.
In the present study, we found that IBMX stably activated the NSC channel, thus enabling us to develop a
model system in which we could investigate the relationship between the cAMP (a second messenger of
AVT)-induced and mechanical (stretch) activation of
the NSC channel.
The activity of the stretch-activated channels is known
to be dependent, at least in part, on actin filaments
(Mills et al., 1994). However, the link between cAMP-dependent activation, stretch-induced activation, and
the cytoskeleton was unclear. Recently, Prat et al. (1993a
,
1993b
) have reported that the catalytic subunit of PKA
activates a 9-pS Na+-selective channel (PNa/PK
4) in
A6 cells cultured on impermeable supports (glass cover
slips), and that activation of the channel by PKA requires actin filaments. However, no information about the action of stretch on the 9-pS Na+-selective channel
was available (indeed, this channel may not be activated
by stretch). Therefore, although the actin filaments are
purportedly required for PKA catalytic subunit-induced
activation of the channel, the relationship between activation by PKA, stretch, and the cytoskeleton was unclear.
In the present study, we have demonstrated that the
mechanism of stretch-induced activation is very similar
to that of cAMP-induced (PKA-mediated) activation of
the channel and that both are contingent on intact actin microfilaments. Moreover, the basal activity of the
NSC channel was found to be dependent on PKA-mediated phosphorylation but apparently independent of
actin filaments.
In the present study, we suggest that the cAMP-activated NSC channel has the same characteristics in single channel kinetics and sensitivity to cytochalasin D as
the stretch-activated NSC channel, and their stimulation depends upon phosphorylation and actin filaments. Possible explanations for these results include: (a) that phosphorylation of the actin filaments (Prat et al., 1993b) produces cortical tension similar to that caused by
stretch, and this tension activates the NSC channel, or
(b) that stretch stimulates the phosphorylation of actin
filaments, which in turn activate the NSC channel. To
test the latter possibility, we endeavored to determine
whether the stretch of the patch membrane induced the phosphorylation of actin filaments. However, given
the limitations of our current system, we were not able
to detect phosphorylation of actin filaments induced by
this force, likely because the magnitude of stretch by
the patch pipette was too small or localized to allow detection of phosphorylation of small numbers of actin
filaments. Thus, it remains to be determined whether
stretch stimulates phosphorylation of actin filaments.
Further, one would need to develop methods suitable
for the measurement of membrane tension that are
sensitive enough to allow detection of the effects of
cAMP and stretch in order to test the former possibility
and reach firm conclusions.
We have previously reported that depolarization increases the Po of the NSC channel (Marunaka et al.,
1994). One interpretation of this observation is that the
IBMX-induced increase in the Po of the NSC channel is
consequent to depolarization of the apical membrane,
since IBMX induced a shift of the I-V relationship by 15 mV to the left without affecting the ion selectivity. Accordingly, we studied the voltage dependency of the
IBMX-stimulated NSC channel and found that the
IBMX-stimulated NSC channel did not show any voltage dependency within the range of ±40 mV in cell-
attached patches; i.e., Po = 0.26 ± 0.04 at
40 mV, 0.25 ± 0.04 at
20 mV, 0.28 ± 0.05 at 0 mV, 0.26 ± 0.04 at 20 mV, and 0.29± 0.03 at 40 mV (voltages indicate the deviation from the resting membrane potential in cell-
attached patches; n = 4). We interpret these data to
mean that the IBMX-induced increase in the Po of the
NSC channel was not due to the depolarization of the
apical membrane but rather due to phosphorylation of
the channel and/or channel-associated proteins including actin filaments.
In the present study, we demonstrated that either IBMX or stretch increases the Po of the NSC channel without changing the mean open or closed time. As an explanation for these observations, we considered three types of linear models of channel gating kinetics designated I, II, and III, since three possible kinetic schemes as linear models of channel gating kinetics are compatible with the observation that the channel has one open and two closed states, irrespective of application of IBMX or stretch. In the ensuing discussion, each model will be discussed separately.
Model I.Eq. A shows a model of the kinetics of the
NSC channel.CS O
CL
where O is the open state; CS, the short closed state; CL,
the long closed state; k, the leaving rate from each
state. Initially, we estimated each rate under the basal
(control) condition. Since the mean value of the short
closed time was 2.0 ms, k1 was 500 s1; since the mean
value of the long closed time was 32.3 ms, k
2 was 31 s
1. The relationship between the mean open time
(
O), k2, and k-1 is as follows:
(B)
Further, the event numbers of the channel staying in
the short (NCS) and long (NCL) closed states depend
upon k1 and k2, respectively (see Eq. A). Namely, NCLand NCS are respectively expressed as follows:
(C)
(D)
where NO is the event number of the channel staying in the open state. From these Eqs. C and D, the following equation is derived.
(E)
From the Eqs. B and E, k1 and k2 were estimated (Table II). Similar to the control condition, the kinetics of
the IBMX-activated NSC channel were estimated (Table II). As shown in Table II, IBMX increases the Po by
decreasing k2 (the leaving rate for the long closed state
from the open state) and increasing k
1 (the leaving
rate for the short closed state from the open state) with little effect on k1 or k
2 (the leaving rate from each
closed state). In other words, without affecting the
closed states, IBMX treatment favors the channel leaving the open state for the short closed state as compared with the long closed state. Further, as a result of
a decrease in the leaving rate for the long closed state
and an increase in the leaving rate for the short closed state, IBMX (presumably via PKA-mediated phosphorylation) did not change the mean open time. According
to this model, phosphorylation would affect the channel only when it remained in the open state. In the case
of the stretch-induced activation of the channel, the
same phenomenon would occur as with IBMX-induced
activation.
Table II. The Rates Leaving from Each State in the Basal (Control), IBMX-, and Stretch-activated NSC Channels in Model I |
Eq. F illustrates a model of the kinetics of
the NSC channel.CL CS
O
where O is the open state, CS is the short closed state, CL
is the long closed state, k is the leaving rate from each
state. In the initial analysis, each rate was estimated under the basal (control) condition. Since the mean value
of the short closed time was 2.0 ms, k4 was 500 s
1;
since the mean value of the open time was 4.0 ms, k
4 was 250 s
1. The mean long closed time (
CL) is expressed as follows:
(G)
Further, the event numbers of the channel staying in
the short (NCS) and long (NCL) closed states depend
upon k4 and k3, respectively. In other words, the relationship between NCL and NCS can be expressed as
follows:
(H)
From the Eqs. G and H, k3 and k3 were estimated (Table III). In an analogous manner, the kinetics of the
IBMX-activated NSC channel was estimated (Table III).
As shown in Table III, IBMX increases the Po by only
decreasing k
3 (the leaving rate for the long closed
state from the short closed state) without any changes in
other rates. In other words, without affecting the open
or long closed state, IBMX decreases the chance for the channel to leave the short closed state for the long
closed state. Although IBMX decreased the leaving rate
for the long closed state from the short closed state, we
could not detect any significant changes in the mean
long closed time induced by IBMX (via PKA-dependent phosphorylation). Since k
3 and k4 are much larger than k3 (Table III), the mean long closed time is dependent
mainly upon the leaving rate from the long closed state
for the short closed state (k3; see Eqs. F and G). The
IBMX-induced change in k
3 (the leaving rate for the
long closed state from the short closed state) would be
too small to be detected as a change in the mean long
closed time, accounting for the lack of significant
change observed in our study. In this model, we suggest
that phosphorylation affects the channel only when it remains in the short closed state. In the case of the stretch-induced activation of the channel, the same phenomenon occurred as with IBMX-induced activation.
Table III. The Rates Leaving from Each State in the Basal (Control), IBMX-, and Stretch-activated NSC Channels in Model II |
A third model was considered as follows:CS CL
O
In the case of Model III, each rate was estimated as described for Model II. Specifically, in control, k5, k5, and k6 were estimated to be 40 ± 10 s
1, 2,090 ± 1,060 s
1,
and 590 ± 310 s
1, respectively. The residency time of
the channel in "CS" (1/k5) is much longer than that in
"CL" (1/(k
5 + k6)). This is inconsistent with the definition of CS and CL. Accordingly, we can eliminate Model
III as a valid explanation for our observations.
Further, a nonlinear model merits consideration as follows:
[View Larger Version of this Image (45K GIF file)]
Unfortunately, in the absence of an experimental method for determining individual rates from one state to another (i.e., specific blocker for communication between each state), such a nonlinear model cannot be evaluated.
Among three linear models, we consider Model II to be the most suitable for the following reasons. As discussed above, Model III can be excluded. If Model I is chosen, the rate of entering the long closed state should decrease just enough to compensate for an increase in the rate of entering the short closed state when IBMX or stretch activates the channel. It is difficult to envision a physical coupling mechanism of the channel that is able to compensate exactly for the changes in gating kinetics of the other. On the other hand, in Model II, if IBMX or stretch decreases the chance of the channel leaving the short closed state for the long closed state, it is sufficient to explain the observations outlined in the current study. For these reasons, we suggest that Model II is the most suitable one.
Unlike the NSC channel studied here, most NSC
channels have some permeability for Ca2+ in addition
to Na+ and K+ (Hoyer et al., 1994; Sackin, 1995
), including another NSC channel in A6 cells (Kawahara
and Matsuzaki, 1993
). In general, the NSC channel
that responds to stretch contributes to an increase in the cytosolic Ca2+ concentration. The NSC channel reported here, however, does not contribute to a change
in the cytosolic Ca2+ concentration, but only to Na+
and K+ transport.
In conclusion, we have demonstrated that: (a) IBMX
stably activates a 29-pS NSC channel, unlike vasotocin
(an antidiuretic hormone); (b) the basal NSC channel
responds to stretch while the IBMX-activated channel
does not; (c) IBMX-induced activation is very similar to
the stretch-induced activation; (d) IBMX- and stretch-induced activation of the channel requires polymerized actin filaments, which appear to make no contribution
to the basal channel activity; (e) phosphorylation maintains the basal, IBMX-, and stretch-activated channel
activity; (f) a linear gating kinetic model "CL CS
O"
is the most suitable of the three possible linear models;
(g) according to Model "CL
CS
O," only the leaving
rate of the channel for CL from CS is decreased by application of IBMX or stretch, resulting in an increase in
the Po of the channel.
Address correspondence to Dr. Yoshinori Marunaka, Division of Respiratory Research, Hospital for Sick Children Research Institute, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Fax: 416-813-5771; E-mail: marunaka{at}sickkids.on.ca
Received for publication 29 November 1996 and accepted in revised form 10 July 1997.
1 Abbreviations used in this paper: ADH, antidiuretic hormone; AVT, arginine vasotocin; CD, cytochalasin D; I-V, current-voltage; IBMX, 3-isobutyl-1-methylxanthine; NSC, nonselective cation.The authors thank Dr. C.E. Bear for critically reading the manuscript.
This work was supported by a Grant-in-Aid from the Kidney Foundation of Canada to Y. Marunaka, and partly supported by Grants-in-Aid from the Medical Research Council of Canada to G.P. Downey and Y. Marunaka (Group Grants and MA13494), the Ontario Thoracic Society (Block Term Grant), and MPG Research Fund to Y. Marunaka. Dr. Marunaka is a Scholar of the Medical Research Council of Canada. Dr. Downey is a Clinician Scientist of the Ontario Ministry of Health.
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