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Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch.

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.


INTRODUCTION
Epithelial Na channels in such tissues as the rat cortical collecting tubule (CCT), toad urinary bladder and in amphibian cells (A6) in culture are characterized by a high selectivity for Na over K, single-channel conductance of 4-5 pS at room temperature (8-9 pS at 37~ and long mean open and closed times (Eaton and Hamilton, 1988;Garty and Benos, 1988;Smith and Benos, 1991;Palmer, 1992). The open probability of these channels is, however, quite variable. In the rat the cell membrane activates epithelial Na channels while depolarization deactivates them (Palmer and Frindt, 1988;Frindt et al., 1993). The response to voltage was weak compared with classical voltage-gated channels, with Po increasing by ~2% per mV (Palmer and Frindt, 1988). The voltage dependence of the channels may be important in mediating feedback control of channel activity, especially during changes in the rate of Na entry into the cells . These effects have not been studied in detail.
Recently epithelial Na channels have been cloned from the rat colon (Canessa et al., 1993;Lingueglia et al., 1993;Canessa et al., 1994). These clones called ~x, [3, and ~/rENaC's, show no significant homology to other known vertebrate ion channels. They do share, however, homology of both nucleotide and protein sequences as well as similarity of apparent membrane topology with several genes from the nematode Caenorhabditus elegans which are expressed in sensory neurons and are thought to be involved in the transduction of mechanical stimuli (Driscoll and Chalfie, 1991;Chalfie et al., 1993;Huang and Chalfie, 1994). This raises the possibility that the epithelial Na channels are related to mechanosensory channels. The sensitivity of the Na channels to mechanical perturbations of the membrane, e.g., membrane stretch, has not yet been examined.
In this paper we describe the kinetic properties of the epithelial Na channel from the rat renal cortical collecting tubule, and the effects of changes in membrane voltage and transmembrane pressures on channel gating.

Biological Preparations
Sprague-Dawley rats of either sex (100-150 g) raised free of viral infections (Charles River Laboratories, Kingston, NY) were fed a low Na diet for at least one week to enhance Na channel activity. Animals were killed by cervical dislocation, the kidneys removed, and the CCT dissected free and opened manually to expose the luminal surface. The split tubules were attached to a small plastic rectangle coated with Cell-Tak (Collaborative Research, Bedford, MA) and placed in a perfusion chamber mounted on an inverted microscope. The chamber was continuously perfused with solution which was prewarmed to 37~ by a miniature waterzjacketed glass coil (Radnotti Glass Technology, Monrovia, CA). Principal cells of the tubule were identified visually as described previously (Pficha et al., 1991). To apply pressure across the patch, suction was applied to the back of the pipette through the pipette holder using a micrometer syringe. The pressure was monitored using a pressure transducer (model DPM1-B; Bio-Tek).

Solutions
Tubules were superfused with a solution consisting of (millimolar): 140 NaC1, 5 KC1, 2 CaC12, 1 MgCI2, 2 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) adjusted to pH 7.4 with NaOH. Modifications of the solution are described in the text. The patch-clamp pipettes were filled with solutions containing (millimolar): 140 LiCI, 3 MgCI2, and 10 Hepes, adjusted to pH 7.5 with LiOH. Li was used as the primary cation in the pipette because the single-channel conductance is higher to Li than to Na, giving a better signal-to-noise ratio .

Electrical
Basic patch-clamp methods were as described previously Silver et al., 1993). Recording of currents and analysis of data were carried out with an Atari 1040 ST computer equipped with interface and data acquisition software (Instrutech, Mineola, NY). Current records were stored on video tape using a pulse-code modulator (Instrutech). Computation of the mean number of open channels (NPo), open probability (P,,) and mean open and closed times was carried out using the TAC program (Instrutech). In patches containing a single channel the open-and closed-time distributions were plotted and analyzed according to the method of Sigworth and Sine (Sigworth and Sine, 1987). Fitting of the distributions was carried out using the maximum likelihood method with the TAC program.

Kinetics of Na Channels in Single-Channel Patches
It is rare to find single Na channels in patches from the rat CCT. When the rat is on a low-Na diet, most patches contain many channels, with the average around five (Pficha et al., 1993). When the animal is on a normal diet, there is almost no Na channel activity at all. Intermediate conditions have been difficult to establish. Nevertheless over a period of about four years a reasonable number of patches which did contain just one active channel were observed. In such patches, only two current levels, corresponding to the open and closed state of the channels, were observed over a period of 3 rain or more. These channels had conductances (12-13 pS) similar to those reported previously for the same conditions . This value is higher than the 5 pS value associated with this channel (Palmer, 1992) because Li rather than Na was the major permeant ion and the measurements were made at 37~ rather than room temperature. The kinetics of these channels are described in Figs. 1-3. A striking finding was the variability in the mean open and closed times and the open probability within this group. Fig. 1 A shows an example of a channel with a low P,,. The activity of the channel was recorded for 11.5 min. The Po, which averaged 0.093, varied somewhat over this time period but there was no systematic change  Sigworth and Sine (1987). The solid line represents a fit to two exponentials with time constants 123 ms (51% of total events) and 24 ms (49%). (D) Closed-time histogram. The solid line represents a fit to two exponentials with time constants 1,580 (27%) and 454 ms (73%).
( Fig. 1 B). It was necessary to establish that the Po was constant to use the entire record for assessing the kinetic properties of the channel. The time interval was selected arbitrarily. The mean open time was 77 ms, and the mean closed time was 760 ms. Open and closed-time distributions are shown in Fig. 1, C and D. The open-time histogram could be fitted by two exponentials, as described in the figure legend. The closedtime histogram could be described fairly well with a single exponential, although a better fit was obtained with two exponentials. Five out of six of the channels within this group with Po < 0.22 required two exponentials to fit the open-time distribution. In all of these cases the closed times could be reasonably well described with a single exponential. Fig. 2 A shows a record from a patch with a channel of intermediate Po. The total recording time was 7 min and Po was stable with an average value of 0.55 (Fig. 2  B). The mean open time was 350 ms. As in the case above, the open-time distribution indicated two time constants (Fig. 2 C). The mean closed time was 286 ms 37 PALMER AND FRINDT and was fairly well described by a single exponential ( Fig. 2 D). There were only three channels in the group with intermediate Po values between 0.25 and 0.75. All indicated two open times. One of the three also required two time constants to fit the closed-time distributions. In this patch the pipette potential was zero, compared with +20 mV for Figs. 1 and 3. No comparable record at +20 mV was available for this group. Fig. 3 A shows a record from a patch with a single channel with a high Po. This recording lasted for 10 min. The average Po was 0.87 and again was relatively constant over the recording period ( Fig. 3 B). The  Both the mean open times (25-60 ms) and mean closed times (120-190 ms) were short. All three of these patches were obtained within a three day period on the same batch of rats. The channels had a normal i-V relationship characteristic of the Na channels, with a slope conductance for inward current of 12 pS. We do not know why they behaved differently.
In previous studies using mainly multichannel patches the mean values of Po were given as 0.4-0.5 (Palmer and Frindt, 1988;Pficha et al., 1993). Curi-ously, there were very few single channels which had a Po in this range. It is possible that the criterion of having a single channel selects for conditions with extreme values of Po. Another possibility is that multichannel patches contain two populations of channels with high and low Po which tend to average. Indeed the average Po of the 19 single-channel patches was 0.50 ---0.08. This suggests the presence of at least two gating "modes" for the channels, one with high Po and one with low Po. Multichannel patches may then have a mix of these modes.

Effects of Voltage on Channel Gating
Further evidence for gating modes, and for inter conversion between them, was obtained from observations of the effects of voltage on channel gating. We have previously reported that hyperpolarization can activate the epithelial Na channel (Palmer, 1988;. In the present study we examined this effect in more detail using patches in which the initial P,, was low, such  as that shown in Fig. 1. Most (>80%) channels in this group could be activated by voltage, although the extent of the hyperpolarization required for activation was variable. The time course of the effect of voltage for five such patches is shown in Fig. 5. Three of these had channels with a low Po at the resting potential of the cell (Vpipeue = 0) and activated after a hyperpolarization of 40 mV. The other two had channels which maintained a low Po for more than 1 rain at Vpip~u~ = 40 mV, but activated with a further 40 mV hyperpolarization. There were no consistent differences in the two data sets which are therefore combined. NP o increased within 30 s and continued to rise, on the average, for 3 rain. We did not study more prolonged hyperpolarizations. Upon returning the patches to the initial level NPo decreased but remained above the original level for 1-2 rain. The kinetics of the channels before and after voltage activation are shown in Fig. 6. This summarizes experiments similar to those shown in Fig. 5, but where there was a single active channel in the patch. The initial volt-39 PALMER AND FRINDT age was 0 to 40 mV, and the patch was hyperpolarized by 40 to 80 inV. The increase in Po is shown in A. B and C show the increase in mean open times and the decrease in mean closed times, respectively. In these cases the recording time at any one voltage was not sufficient to resolve multiple open or closed states. Clearly, however, changes in both opening and closing rates contribute to the increased Po.
As indicated in Fig. 5, the effects of voltage on channel behavior often involved substantial delays. An example of an individual record illustrating delayed activation is shown in Fig. 7. Here the pipette voltage was initially 0 mV and NPo was quite low. Hyperpolarization of the patch by 80 mV (changing Vpipette to +80 mV) had little immediate effect on Po but after ~o16 s the channel suddenly activated. The increased NPo was sustained for at least 20 s but was reversed by returning Vpipe,t ~ to 0. A time course of NPo for this patch is shown in Fig.   8. In this case the channel was activated three times in succession. In each case there was a significant delay between the hyperpolarization and the activation. In other cases long delays in the deactivation of the channels by depolarization were observed. One striking example is shown in Fig. 9. Here again the channel activity was low but was increased by hyperpolarization. In this case the activation was sustained for at least two minutes despite the return of the voltage to its original value. Po eventually returned to low levels after a further depolarization of the membrane.
The delays in activation and deactivation are summarized in Fig. 10 for a series of patches in which the original Po was < 0.25. This required a somewhat arbitrary definition of activation. We considered the channels to be activated if the relative value NPo increased by a fac- FIGURE 5. Activation of channels by hyperpolarization. Channel activity was measured for 1 min at Vpipett~ = 0 (three patches) or +40 mV (two patches), during which time NPo remained low. The voltage was then increased by 40 mV for 3 rain and decreased to the initial level for an additional 3 rain. NP o was monitored continuously. Values were averaged over 15 (initial period) or 30 s (final two periods) intervals, and are reported as means + SEM for the five patches.
time. In the other patches activation and deactivation involved delays of up to 30 s.
The possibility that the delayed activation of the channels by hyperpolarization might be due to a voltage-dependent biochemical reaction was tested using excised, inside-out patches. An example is shown in Fig. 11. The Po was initially stable at a low value. Hyperpolarization of the excised patch increased Po after a delay that was similar to that observed in cell-attached patches. Similar results were seen in two other insideout patches. This does not rule out a voltage-dependent chemical reaction, but does imply that the factors involved in such an effect must be retained with the patch after excision.
The complicated, history-dependent effects of voltage on the kinetics of the channel make it difficult to define a precise relationship between open probability . Time course of Po for a patch containing a single channel in which deactivation by depolarization was delayed. Vpipeue was originally +40 mV, and Po was low. Hyperpolarization to Vpipett e = + 100 mV activated the channel after a delay of "'20 s. Po remained high for ~1 min after returning the potential to its original level. Further depolarization to Vpipett e = 0 mV resulted in the eventual deactivation of the channel. Vpipett ~ was switched between 0 and +80 mV three successive times. and voltage at the single-channel level. Therefore, the effects of voltage on Na channels were further assessed using the whole-cell clamp configuration of the patchclamp technique. Previous studies showed that principal cells in the rat CCT are not electrically coupled, so that individual cells within an intact epithelium can be clamped (Frindt et al., 1990). Cells were held at 0 mV and hyperpolarized to various voltages up to 100 mV for 10 s. The protocols were then repeated in the presence of 10 IxM amiloride. A typical response is shown in Fig. 12 A. "Instantaneous" currents were measured after the decay of the capacitative transient, within a few milliseconds of the voltage change. The inward current then increased rapidly, over the course of several hundred milliseconds, and then more slowly during the next 5-10 s. Neither the rapid nor the slow rise were seen in the presence of amiloride. The average initial and steady state levels of amiloride-sensitive current are plotted vs voltage in Fig. 12 B. The difference in the two currents presumably represents the voltage-dependent activation of the channels. There was an 80% increase in current at -100 inV. Substantial, although somewhat smaller increases were seen in the physiological voltage range ~-60 to -80 inV.

Effects of Stretch on Channel Gating
It has been suggested that the epithelial Na channel is related through evolution to mechanosensitive channels found in nematodes. We therefore examined whether mechanical perturbation could alter the gating kinetics or gating mode of the channels in the CCT.
To test this possibility we used the protocol of applying suction to the patch-clamp pipette during recording from a cell-attached patch (Sachs, 1992). To optimize the chances of seeing an effect of mechanical perturbation, patches with a low basal Po were selected. In most cases negative pressures of up to 80 mm Hg had no obvious effect on the channels (Fig. 13 A). However, in six cases there was a substantial, reversible activation of the channels (Fig. 13 B). One of these positive results is illustrated in Fig. 14. In this case the negative pressure was increased in three steps up to 24 mm Hg. The first step to 8 mm Hg had no significant effect on channel gating. The second step to 16 mm Hg elicited only a brief burst of channel openings. Increasing the pressure to 24 mm Hg brought about a prompt, sustained activation of the channels. The increase in Po was reversed after releasing the pressure. This deactivation was not immediate, being complete 20 s after the pressure changed. In the six responsive patches Po was increased by application of between 10 and 30 mm Hg. The mean value of NP o in these patches increased from 0.048 _+ 0.04 to 0.22 _+ 0.09. In one other patch (not shown), Po was reduced by negative pressure and increased again when the pressure was released.

Gating Modes
The main purpose of this study was to examine the variations in open probability of the epithelial Na channel and to identify some of the mechanisms underlying this variability. The extent of the variation is illustrated in Fig. 4. Basal values of Po in single-channel patches ranged from < 0.05 to greater than 0.9. This variability confirms previous studies showing similar ranges of apparent Po in multichannel patches from the rat CCT (Palmer and Frindt, 1988;P~icha et al., 1993). A large variability of Po has also been reported for Na channels in A6 cells, a culture model derived from the amphibian distal tubule (Ling and Eaton, 1989;Ohara et al., 1993). What was somewhat surprising was the clustering of these Po values toward the low and high end of the spectrum. The records with high and low Po appear to represent the same basic channel type. All of the channels reported in this study had similar single-channel conductances of 12-13 pS, and strongly positive reversal potentials. More importantly, at least some of the channels with low Po could be converted to a high Po by membrane hyperpolarization or stretch. This suggests that the kinetic variations represent different gating modes, rather than different channel molecules. However, the data do not rule out a contribution of structural heterogeneity of channels to the variation in kinetic properties.
A gating mode is defined here as a set of kinetic states (open and closed states) governing channel gating which can undergo a shift to a second set of kinetic states, or a second set of rate constants for transitions between states. This shift occurs on a time scale which is slow relative to the lifetimes of open and closed states within a mode. Other ion channels have been shown to undergo such changes in gating mode. Voltage-dependent Ca channels, for example, can undergo changes in gating patterns which are influenced by dihydropyridines (Hess et al., 1984). Voltage-dependent Na channels can exhibit spontaneous shifts in their Po-V relationship even after reconstitution in planar lipid bilayers (O'Connell, 1992).
Previous studies had indicated that the basic Na channel kinetics in the absence of amiloride could be described in terms of a single open state and a single closed state (Palmer and Frindt, 1986;Eaton and Marunaka, 1990) In the case of the CCT, this conclusion was inferred primarily from recordings from multichannel patches. The relatively long single-channel recordings shown here reveal a more complex kinetic pattern. The presence of two time constants in the open-time and closed-time histograms suggest the existence of at least two open and two closed states. It is not yet clear, however, if the channel has access to all of these states at the same time. It is possible, for instance, that the different open and closed time constants arise from relatively short, reversible shifts in gating mode. This would predict that the distribution of open and closed times of sufficiently short records should be describable with single exponentials. Unfortunately, recordings of < 5 rain do not contain enough events to rigorously test this prediction.

Voltage Activation
The gating of the channels is influenced by the membrane voltage. Hyperpolarization increased Po as described previously (Palmer and Frindt, 1988;. A more complete examination of the effect of voltage revealed that the effect on Po involved considerable delays. These delays are not accounted for by a simple two-state model of channel gating in which the rates of opening and closing involve movement of charge through the electric field of the membrane and are therefore directly influenced by voltage. In this case the effect of changing the transmembrane voltage on the rates of channel opening and closure would be instantaneous, and the rate of change in Po would be de- termined by the sum of the new opening and closing rates. The observations are consistent with the idea that the channels have two or more gating modes, with hyperpolarization favoring the high Po mode and depolarization favoring the low Po mode. This mechanism can account for the long delays and the hysteresis effects seen when the voltage was changed. The observation of shifts in channel kinetics in excised patches suggests that the channels themselves, or a closely associated membrane component, is responding to the transmembrane voltage. This does not rule out the possibility that other events, such as biochemical modification of the channel, might also influence the gating mode. In A6 cells, for example, aldosterone was shown to shift channels from a low Po to a high Po state (Kemendy et al., 1992). In contrast, the Po of channels in the rat CCT was not influenced by mineralocorticoid status (P~icha et al., 1993). Increases in cytoplasmic Ca z+  and activators of protein kinase C (Ling and Eaton, 1989) reduce channel activity, possibly by favoring the low Po state. Insulin, on the other hand, increased Po of Na channels (Marunaka et al., 1992). Activation of G-proteins can also have a strong effect on channel gating. Both Po and mean open time of Na channels in A6 cells were increased by GDP and decreased by GTP in excised patches (Ohara et al., 1993).
The voltage dependence of the channels could also be observed under whole-cell conditions. This indicates that the sensitivity of the channels to voltage is not an artifact of the mechanical isolation of the channels within the patch. These data showed that switching the voltage from 0 to -100 mV could activate the channels by 80%. Assuming that this activation involved only an increase in open probability, without a concomitant insertion of new channels into the membrane, it can be concluded that the average Po with a holding potential of zero was at most 0.56. Since the voltage activation showed no signs of saturation as the voltage was increased to -100 mV, the actual initial Po was probably considerably lower.

Mechanical Activation
Recent cloning of the epithelial Na channel from the rat colon (Canessa et al., 1993;Lingueglia et al., 1993;Canessa et al., 1994) indicated homology between the subunits of this channel and certain genes identified in the nematode C. elegans involved in touch-avoidance responses (Driscoll and Chalfie, 1991;Chalfie et al., 1993;Huang and Chalfle, 1994). The homology, together with the similarity of the predicted membrane topology of the two families, suggests that the C. elegans genes may encode a mechanosensitive ion channel. This raises the question of whether the epithelial Na channels may also be influenced by mechanical perturbations of the membrane. We were unable to elicit a consistent response of the channels in the CCT to changes in transmembrane pressure, although in a minority of cases channel activation was observed. The variability in the response could reflect differences in the mechanical deformations of the apical membrane within the tip of the pipette. Mternatively, the Na channels may not respond to pressure in the same way as do the well characterized stretch-sensitive channels (Sachs, 1992;Sackin, 1994). It is possible that the protocol used to test for mechanosensitivity was not the most appropriate one for these channels. It has been shown, for example, that cell swelling can activate Na channels in A6 cells (Wills et al., 1991). Whether this reflects a direct effect on the channels is not yet clear. Substantial delays have been observed between the change in cell volume and the increase in channel activity. As shown here, however, this does not necessarily preclude a direct influence on the gating mode.
This work was supported by grant DK27847 from the National Institutes of Health.
Original version received 3 May 1995 and accepted version received 28July 1995.