Departments of 1 Medicine and 2 Pharmacology, University of Vermont College of Medicine, Burlington, Vermont 05405
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ion channels that are gated in response
to membrane deformation or "stretch" are empirically designated
stretch-activated channels. Here we describe a stretch-activated
nonselective cation channel in the basolateral membrane (BLM) of the
proximal tubule (PT) that is nucleotide sensitive. Single channels were
studied in cell-intact and cell-free patches from the BLM of PT cells that maintain their epithelial polarity. The limiting inward
Cs+ conductance is ~28 pS, and channel activity persists
after excision into a Ca2+- and ATP-free bath. The
stretch-dose response is sigmoidal, with half-maximal activation of
about 19 mmHg at
40 mV, and the channel is activated by
depolarization. The inward conductance sequence is:
NH
mechanosensitive; stretch-activated; nonselective cation channels; adenosine 5'-trisphosphate sensitive; patch clamp; pressure; proximal tubule; kidney
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
STRETCH-ACTIVATED (SA) channels, gated by changes in hydrostatic pressure and/or cellular swelling, have been found in many tissues (25). Their function is perhaps best understood in sensory organs, where they appear to subserve electromechanical transduction. However, the specific physiological role of SA channels in epithelia such as the kidney remains largely unknown.
The proximal tubule (PT) performs the critical task of bulk solute and water reabsorption for extracellular volume balance. PT cells must also tightly regulate their own volume status because each minute the epithelium transports three times the amount of Na+ contained in its cells (34). To do so requires a reliable feedback system that acts to maintain cell volume when there are transient discrepancies between apical Na+ influx and basolateral Na+ efflux. Accordingly, most evidence to date regarding a possible physiological role of SA channels in kidney pertains to their mechanogating as a result of changes in cell volume. SA channels in the plasma membrane may sense and respond to changes in cell volume and act as part of this feedback system, as has been shown after cellular swelling induced by either hypotonic shock (9, 31) or more physiological substrate-induced Na+ uptake (3).
Here, we describe the properties of a stretch- and swelling-activated channel in the basolateral membrane (BLM) of PT that exhibits nucleotide sensitivity. We conclude that this channel may function as a Ca2+ entry pathway and/or be involved in regulation of cell volume. Furthermore, the coupling of SA channel function to cellular metabolism in renal epithelia would provide evidence that this channel may be important when intracellular ATP concentration ([ATP]i) is depleted, as occurs during increased transepithelial transport or with ischemic cellular injury.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Solutions and drugs. Experiments were performed using bath solutions that were either nominally Ca2+ free or that contained 1.8 mM Ca2+. The composition of the standard NaCl bath recording solution was (in mM): 95 NaCl, 2.5 KCl, 1 MgCl2, 1 EGTA, and 10 HEPES. Unless otherwise noted, patch pipettes were filled with the standard CsCl pipette solution (in mM): 95 CsCl, 2.5 KCl, 1 MgCl2, 1 EGTA, and 10 HEPES. The rationale for using Cs+ in the pipette solution is 1) the SA-nonselective cation (NSC) is the only NSC channel in the BLM that conducts Cs+, 2) Cs+ tends to block BLM K+ channels that might otherwise contaminate recording of the SA-NSC, and 3) the single channel conductance for Cs+ is greater than that for Na+, providing a better signal-to-noise ratio. After titration to pH 7.5 (model 710A; Orion, Boston, MA), sucrose was added to adjust the osmolality (Vapro model 5520; Wescor, Logan, UT) of the solutions to 200 mosmol/kgH2O. In bi-ionic experiments, the pipette was filled with the chloride or gluconate salt of the test cation so that pipette test cation concentration was equal to bath Na+ concentration. Hypotonic swelling experiments were done at constant ionic strength beginning in an isotonic NaCl bath solution containing (in mM) 58.8 NaCl, 2.5 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, and 76 sucrose. Cells were then exposed to a 38% decrease in osmolality (124 mosmol/kgH2O) by changing to the sucrose-free solution. To test the spider venom from Grammostola spatulata (Spider Pharm, Yarnell, AZ) as a candidate specific blocker of the channel in cell-attached patches, pipettes were backfilled with a solution containing 10 µl whole venom per milliliter of buffer (the tip was initially venom free). In solutions containing ATP, the nucleotide was added as the magnesium salt. Nucleotides were prepared fresh daily in bath solution. Chemicals used were obtained from Sigma (St. Louis, MO), Calbiochem (La Jolla, CA), or Biomol (Plymouth Meeting, PA).
Cell preparation. Dissociated PT cells were isolated from amphibian kidneys as previously described (35). Briefly, aquatic phase Ambystoma tigrinum kept at 4°C were killed by submersion in 0.2% tricaine methanesulfonate. The kidneys were rapidly removed and placed in iced HEPES-buffered NaCl (in mM: 90 NaCl, 2.5 KCl, 1 MgCl2, 1 EGTA, and 10 HEPES) at pH 7.5. Renal tissue was cut into 1- to 2-mm3 pieces and incubated in collagenase-dispase (0.2 U/ml of collagenase; Boehringer-Mannheim, Indianapolis, IN) on a shaker for 30 min at room temperature. The cells were then mechanically dispersed into suspension by repeated trituration and resuspended in NaCl and stored at 4°C until use. The dissociated PT cells can retain their epithelial polarity for up to 14 days (35). Cells were used for experiments from 1 to 10 days after dissociation.
Electrophysiology.
A 5-µl aliquot of Ambystoma cell suspension in NaCl was
placed in a recording chamber mounted on an inverted microscope
(Olympus IX-70; Optical Analysis, Nashua, NH). PT cells were readily
recognized by their characteristic asymmetric bilobated morphology. The
standard configurations for the single-channel patch-clamp technique
(11) were used to record channel currents from the BLM.
Patch pipettes were pulled from Corning 7052 borosilicate glass
capillaries (Warner Instrument, Hamden, CT) on a two-step puller
(Narishige PP-83), coated with wax to within 200 µm of the tip, and
fire polished just before use. The open-tip pipette resistance was
3-8 M when placed in the initial bath solution. A motorized
micromanipulator (MP-285; Sutter Instrument, Novato, CA) was used to
guide the patch microelectrode to the cell. The success rate of
forming gigaohm seals on the BLM was ~90%. Data have not been
corrected for liquid-junction potentials, which were all <3.25 mV.
Application of pressure. Negative pressure was applied to the membrane patch through the side port of the pipette holder and measured with an integrated piezoresistive pressure transducer (Fujikura XFPN-03PGVR; Servoflo, Lexington, MA) placed in parallel with the pipette. Steady-state pressure was applied using a pressure transducer tester (DPM-1B; Biotek Instruments, Winooski, VT). For the application of pressure ramps, a syringe pump (YA-12, Yale Apparatus, Wantagh, NY) under negative feedback control (based on the output voltage from the pressure transducer) was connected to the pressure port. For the application of pressure steps, a multivalve pressure system of our own design (14) was connected to the pipette holder. With this system, up to seven different pressure steps (in addition to atmospheric pressure) can be made, typically with a 20-80% step response time of <1 ms. Pressure step protocols were triggered by PULSE and achieved using a parallel port interface (LPTek, Westbury, NY) controlled by a Visual BASIC program written in our laboratory. The interface includes a digital input/output board for valve selection and a counter board used for timing voltage pulses (spike, hold, off) from one channel of the digital-to-analog board to high-current DS3658 chips that drive the valves. During pressure protocols, both the channel currents and pressure data are recorded simultaneously by PULSE, facilitating the analysis of the temporal relationship between pressure stimulus and channel response.
Data analysis.
PULSE datafiles were read directly by Origin 6.0 (OriginLab,
Northampton, MA) using DataAccess 6.0 (Bruxton, Seattle, WA). Custom software for data analysis was written in our laboratory using Matlab 5.2 (The Mathworks, Natick, MA), Lab Talk 6.0 (OriginLab), and Visual BASIC 6.0 (Microsoft, Seattle, WA). Channel activity (NPo, where N is the minimum number
of channels in the patch and Po is the channel
open probability) was calculated by applying a graphical method to
binned data from amplitude histograms representing all points from at
least 30 s of continuous recording as follows. Based on mouse
clicking on the peak corresponding to the all channels closed level
(ic) and then on two adjacent peaks (to
calculate the single-channel current, isc), our
program bins and numerically integrates the histogram to get the total
area under the curve. The all channels closed current is subtracted
from the current of a given bin, and the number of events in that bin
is multiplied by the difference in current. The sum of these products
yields the open channel area of the histogram.
NPo is then given by dividing the open channel
area by the total area under the histogram. That is
![]() |
(1) |
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An NSC channel activated by membrane tension is highly expressed in
the BLM.
More than 75% (425 out of 550) of seals made on the BLM contained at
least one NSC channel that could be activated by negative pressure,
suggesting that the SA-NSC channel protein is highly expressed in this
membrane. After formation of a cell-attached patch, there is typically
no channel activity in the absence of applied pressure at 0-mV pipette
potential, although channel openings will be evident in a minority
(10-15%) of patches. Figure
1A shows that a ramp pressure
protocol reversibly activates an NSC channel. In this representative
excised inside-out membrane patch, rare channel openings are evident at
a command potential of 40 mV before application of pressure, and
channel activity increases with negative pressure. Conversely, during
the ramp back to 0 mmHg, channel activity progressively declines.
|
SA channel inwardly rectifies and is cation nonselective.
When BLM membrane patches are excised in the inside-out configuration
under symmetrical conditions, measurements of the single channel
current demonstrate that the SA-NSC channel is a true weak inward
rectifier (Fig. 2, A and B) with a rectification
index (gin/gout) of 1.4. Kinetic analyses of one channel patches held at 40 mV and
50 mmHg
show that the SA-NSC channel has two open states (6 ms, 75% weight; 25 ms, 25% weight) and three closed states (~0.6 ms, 62% weight; ~16
ms, 22% weight; and ~180 ms, 16% weight), as shown in Fig.
2C. The channel is permeable
to a wide variety of cations. Representative channel records from patches excised under bi-ionic conditions (pipette cation concentration equal to bath Na+ concentration) are shown in Fig.
3A, and the summarized
current-voltage relationships are plotted in Fig. 3B. Note
that the reversal potential is about the same for all conditions, so
although the conductances differ, the permeabilities of the conducting
cations are virtually identical. The single channel conductance for the
outward flow of Na+ is the same (~21 pS) regardless of
the pipette cation. The inward conductance is divided into groups that
can be correlated to the hydrated radius (or the dehydration energy) of
the cation. Larger cations (Rb+, Cs+, and
NH
|
|
BLM SA-NSC channel is depolarization activated.
The open probability of the channel is voltage dependent and increases
with depolarization (Fig. 4A).
The NPo was measured from continuous current
records at least 30 s long from patches held at constant negative
pressure. Because channel activity varies among patches,
NPo was normalized to the maximum
NPo in each patch. Channel activity increases
e-fold for every 49.5-mV depolarization. The experiment
shown in Fig. 4B was designed to examine the instantaneous effects of voltage on channel gating. The pressure system was set to
make a step from 0 to 40 mmHg and back to 0 mmHg over a duration of 5 ms, and channel recordings were made at ±100 mV. Note that the opening
latency time is shorter, and the closing latency time is longer, at
+100 mV compared with those at
100 mV. Pooled latency time data from
seven experiments are summarized in Fig. 4C. This result
suggests that depolarization shortens a closed-state lifetime and
prolongs an open-state lifetime, both of which favor a higher open
probability.
|
This SA channel is Gd3+ insensitive
but is blocked by spider venom.
Sensitivity to the lanthanide gadolinium (Gd3+) is a
common feature of many mechanosensitive channels, especially those that are cation nonselective (29, 45). The SA-NSC channel in
the BLM appears to be insensitive to Gd3+, as detailed in
Fig. 5. Activation of the channel by
negative pressure is unaffected by 100 µM Gd3+ in the
pipette and/or bath of excised inside-out (Fig. 5A) or outside-out (Fig. 5B) patches. The experiment in Fig.
5C shows that addition of 100 µM Gd3+ to an
outside-out patch conducting Na+ has no effect on channel
activity. Other typical inhibitors of mechanogated or NSC channels,
including amiloride, flufenamic acid, and niflumic acid, also have no
significant effect on the SA-NSC channel (data not shown).
|
|
Cellular swelling reversibly activates the SA-NSC channel.
It is reasonable to hypothesize that the SA-NSC channel is involved in
cellular volume regulation, and the experiment shown in Fig.
7A is consistent with such a
role. After baseline channel activity is established in a cell-attached
patch under isotonic conditions, the cell is exposed to hypotonic
conditions by removal of sucrose at a constant ionic strength. The
response of the channel is biphasic, increasing significantly within a
few seconds, reaching a peak (a 70% increase over baseline
NPo) within a minute, and then decreasing over
the next 2 min. Channel activity decreases or abruptly ceases if
cellular swelling is stopped or reversed, respectively. Such behavior
suggests that the channel may be involved in the regulatory volume
decrease (RVD) response of the cell. A summary of six similar swelling
experiments is shown in Fig. 7B.
|
Sensitivity to adenine nucleotides.
Nucleotides have been shown to inhibit some NSC channels
(17). The SA channel is sensitive to ADP and ATP, as shown
in Fig. 8. In this experiment, a constant
negative pressure of 17 mmHg activates the channel. Addition of 5 mM
UDP to the cytoplasmic face of the inside-out patch does not affect
channel activity, but application of 5 mM ADP significantly and
reversibly inhibits the channel. Exposure to 5 mM ATP demonstrates a
more potent inhibition that also reverses (indeed, greater than that
before ATP) after washout. That ADP also blocks the channel indicates
that inhibition by adenine nucleotides neither involves a typical
protein kinase nor requires hydrolysis by an ATPase. Therefore, channel
inhibition most likely occurs when an appropriate adenine nucleotide
binds to a cytoplasmic domain of the channel protein. The dose-response curve for ATP is sigmoidal and has an inhibition constant
(Ki) of 0.48 mM (Fig. 8B). The
increase of NPo upon washout suggests that ATP
refreshes some channels during the period of inhibition. The degree of
inhibition by ATP does not appear to be dependent on the magnitude of
negative pressure applied to the patch. The block achieved using 2 mM
ATP was compared at two levels of applied negative pressure in the same
patch; one at which the baseline Po was ~0.20
and another at which baseline Po was ~0.70.
There was no significant difference in the potency of block (88 ± 3.8 vs. 87 ± 1.1%, n = 3) at the two levels of
negative pressure and open probability (Fig. 8C).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The primary objective of this study was to characterize the electrophysiological properties of an SA-NSC channel found at a very high frequency in the BLM of Ambystoma PT cells. Previous work on SA channels in PT has demonstrated regulation by cell volume changes induced by hypotonic shock and uptake of substrate (3, 9, 16, 30). Our data demonstrate that a Ca2+-independent, mechanogated NSC channel in the BLM of PT is sensitive to ATP. The likelihood of finding at least one SA-NSC channel in a membrane patch is nearly 80%, suggesting that this integral membrane protein is important to the cellular and molecular physiology of the PT.
Mechanosensitivity.
Of the cell surface proteins involved in "mechanotransduction"
(e.g., integrins and ion channels), mechanosensitive ion channels can
theoretically provide a rapid and reversible signal in response to
membrane deformation. The application of steady-state negative pressure
to membrane patches has been shown to both inhibit (40) and activate (30) mechanosensitive channels. Given the
sigmoidal relationship between negative pressure and channel open
probability shown in Fig. 1, the NSC channel described in this study is
clearly a SA channel. Across multiple species, stretch sensitivity of basolateral SA channels is similar. For example, the half-maximal activation of our channel (19.3 mmHg at
40 mV) correlates well with
that reported for SA channels in Xenopus PT cells
(16) and is slightly less stretch sensitive than that
described for the BLM SA channels in Rana temporaria
(12) and Necturus (30), where the
half-maximal activation occurs around
11 mmHg. An osmolal gradient of
1 mosmol/kgH2O results in a hydrostatic pressure of ~17
mmHg, suggesting these channels operate over a physiologically relevant
range. Although there is some variation in the mechanosensitivity of
the channel in some of our cell preparations, the majority of patches
fall into the range detailed in Fig. 1, B and C.
It is unclear what factors are responsible for occasional variations in
the mechanosensitivity between preparations.
Ion conductance, rectification, and selectivity. SA channels in amphibian kidney demonstrate a fairly consistent conductance of ~25-30 pS (12, 13, 16), consistent with that of the SA channel in the present study. Higher-conductance SA channels have been described in Necturus (9) and R. pipiens (3). Irrespective of the cation tested (including TEA+), under symmetrical conditions the SA-NSC channel is a weak inward rectifier. In the present study, Mg2+ was always present on the cytosolic side of the patch; therefore, the possibility that the inward rectification is the result of Mg2+ block, as is the case for some K+ channels (20), cannot be excluded.
The inward conductance sequence suggests grouping correlated to the hydrated radius (or the dehydration energy) of the cations. Therefore, the selectivity sequence favors ions like Cs+ and Rb+ because of the relatively low energy required to shed their waters (Voltage dependence. Of the SA channels described in renal epithelia, those found in Necturus and Xenopus are K+ selective and have uniformly demonstrated hyperpolarization activation (16, 33). The SA-NSC channel described here is depolarization activated, which is similar to the SA-NSC channel in R. temporaria (12). The observation that depolarization increases NPo even at 0 mmHg suggests that the stretch-sensitive closed state is voltage dependent. Using our high-speed pressure step system to investigate the relationship between voltage and latency times, the depolarization activation is seen from a novel perspective. That is, rather than the steady-state measurement of NPo, we see an instantaneous reflection of the depolarization activation (on the order of ms) in the form of a shortened opening latency and a longer closing latency, both of which favor an increased open probability. This important finding is in sharp contrast to the very slow depolarization activation found in the SA channels of Xenopus oocytes (10) and leech central neurons (23), which occurs on the order of seconds to tens of seconds.
Mechanistically, voltage dependence can be the result of voltage-induced changes in 1) channel protein(s), 2) surrounding structures, or 3) membrane deformation. Gil et al. (10) hypothesize that voltage across the patch influences membrane tension and gates SA channels. Because depolarization activation did not occur with soft glass pipettes and was absent in outside-out patches, they concluded that voltage dependence was an artifact resulting from specific interactions between the glass pipette and membrane patch in Xenopus oocytes. In contrast, the SA channel described here is activated by depolarization regardless of the excised-patch configuration, similar to that described for mechanogated channels in leech neurons (23). If the same process of voltage-induced membrane deformation were occurring in Ambystoma epithelia, Xenopus oocytes, and leech neurons, such drastic differences in times to activation would not be expected.Blockade. Since first being described to inhibit the beating of a perfused frog heart (24), lanthanides have become widely used in the study of cellular physiology. Sensitivity to the lanthanide Gd3+ is a common feature of many mechanosensitive channels and is reported in a number of different preparations, including yeast MID1 (15), Necturus PT cells (8), renal A6 cells (43), Xenopus oocytes (45), and human neurons (28). The SA channel described in this paper is unaffected by 100 µM Gd3+ in the pipette and/or bath of excised inside-out or outside-out patches. However, given the broad range of cross-reactivity, Gd3+ inhibition is currently viewed as a less reliable marker for SA channels (29). Other typical inhibitors of mechanogated or NSC channels, including amiloride, flufenamic acid, and niflumic acid, have no significant effect on this SA-NSC channel. Unfortunately, specific pharmacological inhibitors of SA channels are not readily available, but a natural toxin found in the venom of the common Chilean tarantula, G. spatulata, has been shown to block SA channels in GH3 pituitary cells (4). Our results indicate that this venom completely blocks channel activity in cell-attached patches containing SA-NSCs. The block may be because of a 35-amino-acid peptide (GsMTx-4) in the venom recently identified by Suchyna and coworkers (39). GsMTx-4 is the first peptide toxin that specifically blocks SA channels. They showed that 5 µM GsMTx-4 completely blocks mechanosensitive cation channels in astrocytes and cardiac myocytes. Based on a molecular weight of 4093.9 and a concentration of 1.95 mM GsMTx-4 in whole venom (39), channels in our cell-attached patches were exposed to ~20 µM GsMTx-4 toxin. To our knowledge, the SA-NSC in the PT is the first epithelial channel blocked by this venom.
Swelling activation. Swelling-activated mechanogated channels have been described in many tissues, including the PT (3, 30). The SA channel in the BLM of Ambystoma PT cells is also activated by hypotonic challenge. Unlike activation induced by stretch, swelling activation of the SA-NSC results in a biphasic response. Based on studies implementing simultaneous cell volume and patch recording techniques, it appears that the biphasic response of SA channels during swelling can be directly related to the cell volume changes during RVD (9). Whether or not the SA channel in Ambystoma is actually a component of the RVD remains unknown. From a physiological standpoint, hypotonic swelling likely does not reflect cellular volume changes, as they occur during actual transepithelial transport. That is, hypotonic stress may influence a number of cellular signaling systems (e.g., the cytoskeleton and intracellular messengers), making interpretation of these data more complex (29).
Nucleotide sensitivity and possible physiological role. An important feature of the channel is inhibition by cytoplasmic ATP. That the nucleotide diphosphate ADP also blocks the SA-NSC channel indicates that inhibition by adenine nucleotides probably does not require a typical protein kinase or hydrolysis by an ATPase. Therefore, channel inhibition most likely occurs when an appropriate adenine (but not uridine) nucleotide binds to a cytoplasmic domain of the channel protein. Although ATP (41) or cyclic nucleotides (7) regulate certain members of the NSC channel superfamily, to our knowledge the SA-NSC channel is the only mechanogated channel that is also nucleotide sensitive. Reminiscent of ATP-sensitive K+ channels (26), the tendency of NPo to increase upon washout of ATP (see Fig. 8) suggests that some degree of channel refreshment by ATP occurs at a second site, most likely resulting from phosphorylation of the channel or an associated protein.
As is the case with many mechanosensitive channels, its precise physiological function remains uncertain. Indeed, only within the past 2 yr have data been presented to support the hypothesis that epithelial SA channels in cell-attached patches underlie the whole cell currents elicited by cellular swelling (44). One mechanism that may be involved in the RVD of epithelial cells exposed to hypotonicity involves Ca2+ entry via SA-NSC channels, which in turn activates Ca2+-activated K+ channels in the same membrane (5). The BLM of PT cells appears to contain at least two K+ channels (27), one of which is an ATP-dependent K+ (KATP) channel that is inhibited by Ca2+ (21, 22). If the other K+ channel is also regulated by Ca2+, then Ca2+ entry via the SA-NSC channel may contribute to RVD through secondary effects on K+ channels also in the BLM (18). Recently cloned mammalian transient receptor potential (TRP)-like Ca2+-permeable NSC channels [OTRPC4, the osmosensitive transient receptor channel 4 (38) and VR-OAC, the vanilloid receptor-related osmotically activated channel (19)] are both activated by hypotonicity and found at high levels in the renal tubules. Interestingly, TRP-like channels have not been reported in patch-clamp experiments in native kidney, raising the possibility that the properties of recombinant channels expressed in vitro differ from those in situ. If so, it is possible that the SA-NSC channel is related to one of these TRP-like channels. Nucleotide sensitivity is a property that may provide important new insight to the physiological role of this channel because of the possibility that SA channel function is linked to cellular metabolism in the PT. However, care must be exercised regarding interpretation of the physiological significance within the possible range for the changes in ATP and ADP concentrations because we do not yet know what these ranges are, whether the channel responds to the absolute level of these nucleotides or their ratio, and/or if key modifiers are lost after patch excision. On the one hand, the Ki of 0.48 mM for ATP inhibition in excised patches suggests that the channel may not be open given PT [ATP]i levels in the range of 2-5 mM under healthy conditions (2, 42). Rather, it is possible that the channel becomes important when intracellular ATP is depleted, as occurs during ischemic cell injury. During such a stress, SA-NSC channels would open, providing a cation entry pathway for Ca2+ or Na+. In the case of the latter, as opposed to Na+ entry across the apical membrane, Na+ entering across the BLM would decrease net Na+ reabsorption and induce further cellular swelling. This raises the possibility that, if the channel conducts Na+ in vivo, it may be to promote nonspecific cell death, as has been proposed for liver cells (1). On the other hand, as has been shown for KATP channels, protein-lipid interactions may modify ATP sensitivity, essentially permitting channel function in the presence of what would otherwise be inhibitory concentrations of ATP (36). For this reason, the SA-NSC channel described may play a role during transepithelial transport under healthy conditions. In this case, the SA-NSC channel could become active in response to both cellular swelling (44) and reductions in [ATP]i, allowing Na+ or Ca2+ to enter or (less likely) K+ to exit. We have previously shown that intracellular Ca2+ is an important regulator of the hyperpolarization-activated KATP channel in the same membrane (22). With these considerations, it is possible that Ca2+ entry or a depolarizing Na+ current may be important to optimize the pump-leak coupling that occurs between the Na+-K+-ATPase and KATP channels in the BLM of PT. We conclude that an ATP-sensitive mechanogated NSC channel is highly expressed in the BLM of PT. This mechanogated channel could be functionally coupled to the metabolic state of the cell and may be important for cellular volume regulation and/or as a Ca2+ entry pathway during transepithelial transport or in the response to ischemic injury. ![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. J. Brayden, Dr. S. Lidofsky and the reviewers for valuable suggestions and comments on the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported by the Department of Medicine at the University of Vermont. Dr. C. Hurwitz was a recipient of an Individual National Research Service Award from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-10061).
Address for reprint requests and other correspondence: A. S. Segal, 208 South Park Dr., Suite 2, Colchester, VT 05446 (E-mail: asegal{at}zoo.uvm.edu).
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 February 5, 2002;10.1152/ajprenal.00239.2001
Received 1 August 2001; accepted in final form 31 January 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barros, LF,
Stutzin A,
Calixto A,
Catalan M,
Castro J,
Hetz C,
and
Hermosilla T.
Nonselective cation channels as effectors of free radical-induced rat liver cell necrosis.
Hepatology
33:
114-122,
2001[ISI][Medline].
2.
Beck, JS,
Breton S,
Mairbaurl H,
Laprade R,
and
Giebisch G.
Relationship between sodium transport and intracellular ATP in isolated perfused rabbit proximal convoluted tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
261:
F634-F639,
1991
3.
Cemerikic, D,
and
Sackin H.
Substrate activation of mechanosensitive, whole cell currents in renal proximal tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F697-F714,
1993
4.
Chen, Y,
Simasko SM,
Niggel J,
Sigurdson WJ,
and
Sachs F.
Ca2+ uptake in GH3 cells during hypotonic swelling: the sensory role of stretch-activated ion channels.
Am J Physiol Cell Physiol
270:
C1790-C1798,
1996
5.
Christensen, O.
Mediation of cell volume regulation by Ca2+ influx through stretch-activated channels.
Nature
330:
66-68,
1987[ISI][Medline].
6.
Eisenman, G,
and
Dani JA.
An introduction to molecular architecture and permeability of ion channels.
Annu Rev Biophys Biophys Chem
16:
205-226,
1987[ISI][Medline].
7.
Fesenko, EE,
Kolesnikov SS,
and
Lyubarsky AL.
Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment.
Nature
313:
310-313,
1985[ISI][Medline].
8.
Filipovic, D,
and
Sackin H.
A calcium-permeable stretch-activated cation channel in renal proximal tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
260:
F119-F129,
1991
9.
Filipovic, D,
and
Sackin H.
Stretch- and volume-activated channels in isolated proximal tubule cells.
Am J Physiol Renal Fluid Electrolyte Physiol
262:
F857-F870,
1992
10.
Gil, Z,
Magleby KL,
and
Silberberg SD.
Membrane-pipette interactions underlie delayed voltage activation of mechanosensitive channels in Xenopus oocytes.
Biophys J
76:
3118-3127,
1999
11.
Hamill, OP,
Marty A,
Neher E,
Sakmann B,
and
Sigworth FJ.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch
391:
85-100,
1981[ISI][Medline].
12.
Hunter, M.
Stretch-activated channels in the basolateral membrane of single proximal cells of frog kidney.
Pflügers Arch
416:
448-453,
1990[ISI][Medline].
13.
Hurst, AM,
and
Hunter M.
Stretch-activated channels in single early distal tubule cells of the frog.
J Physiol (Lond)
430:
13-24,
1990[Abstract].
14.
Hurwitz, CG,
and
Segal AS.
Application of pressure steps to mechanosensitive channels in membrane patches: a simple, economical, and fast system.
Pflügers Arch
442:
150-156,
2001[ISI][Medline].
15.
Kanzaki, M,
Nagasawa M,
Kojima I,
Sato C,
Naruse K,
Sokabe M,
and
Iida H.
Molecular identification of a eukaryotic, stretch-activated nonselective cation channel.
Science
285:
882-886,
1999
16.
Kawahara, K.
A stretch-activated K+ channel in the basolateral membrane of Xenopus kidney proximal tubule cells.
Pflügers Arch
415:
624-629,
1990[ISI][Medline].
17.
Korbmacher, C,
Volk T,
Segal AS,
Boulpaep EL,
and
Fromter E.
A calcium-activated and nucleotide-sensitive nonselective cation channel in M-1 mouse cortical collecting duct cells.
J Membr Biol
146:
29-45,
1995[ISI][Medline].
18.
Lang, F,
Busch GL,
Ritter M,
Volkl H,
Waldegger S,
Gulbins E,
and
Haussinger D.
Functional significance of cell volume regulatory mechanisms.
Physiol Rev
78:
247-306,
1998
19.
Liedtke, W,
Choe Y,
Marti-Renom MA,
Bell AM,
Denis CS,
Sali A,
Hudspeth AJ,
Friedman JM,
and
Heller S.
Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor.
Cell
103:
525-535,
2000[ISI][Medline].
20.
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].
21.
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,
1998a
22.
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,
1998b
23.
Menconi, MC,
Pellegrini M,
and
Pellegrino M.
Voltage-induced activation of mechanosensitive cation channels of leech neurons.
J Membr Biol
180:
65-72,
2001[ISI][Medline].
24.
Mines, GR.
The action of beryllium, lanthanum, yttrium and cerium on the frog's heart.
J Physiol
40:
327-345,
1910.
25.
Morris, CE.
Mechanosensitive ion channels.
J Membr Biol
113:
93-107,
1990[ISI][Medline].
26.
Noma, A.
ATP-regulated K+ channels in cardiac muscle.
Nature
305:
147-148,
1983[ISI][Medline].
27.
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
28.
Quasthoff, S.
A mechanosensitive K+ channel with fast-gating kinetics on human axons blocked by gadolinium ions.
Neurosci Lett
169:
39-42,
1994[ISI][Medline].
29.
Sachs, F,
and
Morris CE.
Mechanosensitive ion channels in nonspecialized cells.
Rev Physiol Biochem Pharmacol
132:
1-77,
1998[ISI][Medline].
30.
Sackin, H.
Stretch-activated potassium channels in renal proximal tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
253:
F1253-F1262,
1987
31.
Sackin, H.
A stretch-activated K+ channel sensitive to cell volume.
Proc Natl Acad Sci USA
86:
1731-1735,
1989[Abstract].
32.
Sackin, H.
Mechanosensitive channels.
Annu Rev Physiol
57:
333-353,
1995[ISI][Medline].
33.
Sackin, H,
and
Palmer LG.
Basolateral potassium channels in renal proximal tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
253:
F476-F487,
1987
34.
Schultz, SG.
Membrane cross-talk in sodium-absorbing epithelial cells.
In: The Kidney: Physiology and Pathophysiology (2nd ed.), edited by Seldin DW,
and Giebisch G.. New York: Raven, 1992, p. 287-299.
35.
Segal, AS,
Boulpaep EL,
and
Maunsbach AB.
A novel preparation of dissociated renal proximal tubule cells that maintain epithelial polarity in suspension.
Am J Physiol Cell Physiol
270:
C1843-C1863,
1996
36.
Shyng, SL,
and
Nichols CG.
Membrane phospholipid control of nucleotide sensitivity of KATP channels.
Science
282:
1138-1141,
1998
37.
Sigworth, FJ,
and
Sine SM.
Data transformations for improved display and fitting of single-channel dwell time histograms.
Biophys J
52:
1047-1054,
1987[Abstract].
38.
Strotmann, R,
Harteneck C,
Nunnenmacher K,
Schultz G,
and
Plant TD.
OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity.
Nat Cell Biol
2:
695-702,
2000[ISI][Medline].
39.
Suchyna, TM,
Johnson JH,
Hamer K,
Leykam JF,
Gage DA,
Clemo HF,
Baumgarten CM,
and
Sachs F.
Identification of a peptide toxin from Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels.
J Gen Physiol
115:
583-598,
2000
40.
Suzuki, M,
Sato J,
Kutsuwada K,
Ooki G,
and
Imai M.
Cloning of a stretch-inhibitable nonselective cation channel.
J Biol Chem
274:
6330-6335,
1999
41.
Thorn, P,
and
Petersen OH.
Activation of nonselective cation channels by physiological cholecystokinin concentrations in mouse pancreatic acinar cells.
J Gen Physiol
100:
11-25,
1992[Abstract].
42.
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].
43.
Urbach, V,
Leguen I,
O'Kelly I,
and
Harvey BJ.
Mechanosensitive calcium entry and mobilization in renal A6 cells.
J Membr Biol
168:
29-37,
1999[ISI][Medline].
44.
Vanoye, CG,
and
Reuss L.
Stretch-activated single K+ channels account for whole-cell currents elicited by swelling.
Proc Natl Acad Sci USA
96:
6511-6516,
1999
45.
Yang, XC,
and
Sachs F.
Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions.
Science
243:
1068-1071,
1989[ISI][Medline].
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |