INTRODUCTION

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BASOLATERAL POTASSIUM channels have a very important role to play in the transepithelial reabsorption mediated by proximal convoluted tubules (PCT). Indeed, the sodium-dependent translocation of luminal substrates requires the maintenance of a cellular Na⁺ electrochemical gradient, which is controlled by the basolateral Na-K-ATPase, coupled to the basolateral potassium conductance (3, 14, 17, 29). Sodium ions, cotransported with the luminal substrates, are extruded by the basolateral pump, while potassium ions pumped into the cell by the Na-K-ATPase are recycled by the basolateral potassium conductance and create the negative intracellular potential.

In rabbit proximal tubules, potassium diffusion across the basolateral membrane is known to be mainly mediated by an inwardly rectifying channel with an inward unitary conductance of ~50 pS (11, 16, 19). This potassium channel is inhibited by raising intracellular ATP (14, 24) and by lowering intracellular pH (2). In addition to these regulators, we have recently found that an increase in intracellular taurine concentration inhibits the basolateral potassium conductance (G_K) by 50% (4). Very interestingly, the basolateral taurine permeability was shown to be activated by cell swelling (4), which suggests that taurine could play a significant role in maintaining cell volume and membrane potential during stimulation of transepithelial sodium transport. First, the swelling-activated basolateral taurine efflux would directly contribute to the volume regulatory decrease. Then, the resulting decrease of intracellular taurine concentration would increase the potassium conductance and facilitate further volume and membrane potential restoration by enhancing the potassium efflux.

In this study, we investigated the effect of taurine on the basolateral potassium conductance, at the single-channel level, using the patch clamp technique in the cell-attached configuration. This allowed the identification of a previously unrecognized potassium channel in the basolateral membrane of PCT, which is responsible for ~40% of the membrane conductance. At the single- channel level, only the previously identified 50-pS channel was proved to be taurine sensitive.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Tubule preparation. Female New Zealand White rabbits were decapitated, and the left kidney was perfused with a cold (4°C) preservation solution containing (in mM) 56 Na₂HPO₄, 13 NaH₂PO₄, and 140 sucrose (5). Thin kidney slices were obtained and placed immediately in the cold preservation fluid. Segments of S1 and S2 cortical PCT were dissected from the midcortical region under ×40 magnification at 4°C in the same solution. Once dissected, PCTs were transferred to the perfusion chamber and bathed, at room temperature, in the control solution, buffered at pH 7.4 and containing (in mM) 30 NaCl, 5 KCl, 1.2 MgSO₄, 1.8 CaCl₂, 1 NaH₂PO₄, 3 Na₂HPO₄, 4 sodium acetate, 1 trisodium citrate, 25 NaHCO₃, 10 glucamine chloride, 128 mannitol, 5.5 glucose, and 6 alanine. Tubules were held by micropipettes and placed in the flux of a perfusion system as previously described (18). To gain access to the basolateral membrane, the basal membrane was removed by a 20-min incubation with collagenase A (Boehringer Mannheim) added to the control solution (final enzymatic activity of 0.5 U/ml) (2). All experiments were conducted on collapsed tubules.

Electrophysiology. The cell-attached configuration of the patch-clamp technique was used to record currents from basolateral membrane channels. Patch pipettes were fabricated from hematocrit capillary tubes (Fisher) using a Narishige PP-83 two-stage patch pipette puller. Pipettes filled with a solution containing (in mM) 150 KCl, 2 CaCl₂, 1 MgCl₂, and 10 HEPES (titrated to pH 7.4 with 2.4 NaOH) had resistances between 5 and 10 M when placed in the control solution. Ionic selectivity was studied in inside-out configuration. Micropipette solution contained (in mM) 150 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, 2.4 NaOH, and 20 mannitol. In symmetrical potassium gradients, bath solution contained (in mM) 140 KCl, 2 MgCl₂, 5 EGTA, 10 HEPES, 18 NaOH, 5 glutathione, and 35 mannitol. In asymmetrical potassium gradients, 70 mM NaCl was substituted for 70 mM KCl of the bath solution.

The pipettes were advanced onto the basolateral membrane of tubules by means of a hydraulic micromanipulator (model MX 630R; Newport, Irvine, CA). The contact between microelectrode and membrane was monitored by detecting change in the membrane shape before applying suction to obtain a gigaohm seal. Channel currents amplified with a patch-clamp amplifier (Axopatch-1D; Axon Instruments, Foster City, CA) were recorded onto videotape through a digital data recorder (model VR-10B CRC; Instrutech, Great Neck, NY). To analyze data, records were low-pass filtered with an eight-pole Butterworth filter (model 901; Frequency Devices, Haverhill, MA) at 500 Hz, digitized at 2.5 kHz with a TL-1 DMA Labmaster interface (Axon Instruments), and stored in a 486 PC by means of a specific acquisition software (Axotape 1.2.01, Axon Instruments). Digitized records with very low-amplitude events were additionally filtered at 300 Hz (with a digital Gaussian filter) to improve peak discrimination in amplitude histograms. When such a cutoff frequency was needed, no kinetic analysis was performed. Channel currents were analyzed with the pClamp 5.5.1 software (Axon Instruments). Channel conductance was estimated from linear regression of single-channel current-voltage curve between 40 mV and +20 mV for inward currents and between +60 mV and +100 mV for outward currents. Reversal potentials were obtained by interpolation using a third-order polynomial fit of the current-voltage curves. Neither a linear, a quadratic, nor a Goldman-Hodgkin-Katz equation could provide an acceptable fit of these curves. The open probability (P_o) was calculated from amplitude histograms according to the equation

(1)

where i is the number of channels observed at the same time, P_i is the probability that i channels are simultaneously open, and N is the total number of channels in the patch. In some experiments, open probability at a given potential was plotted as a function of time. In this case, recordings at constant potential were divided into 30-s segments from which one P_o was calculated. In the text and legends to Figs. 1-8, potentials are given as V_p, where V_p is the micropipette potential.

Experimental values are expressed as means ± SE. Student's t-test for unpaired observations is used for statistical comparisons.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

With control solution bathing the tubule, cell-attached experiments confirmed the presence of the ATP-dependent potassium channel, K_ATP, which was previously identified at the basolateral membrane of the PCT (2, 14, 19, 24). Figure 1A shows the typical K_ATP channel activity, which was observed in 51% of the patches showing some channel activity. At a potential of 40 mV, the amplitude of inward currents was 3.2 ± 0.5 pA (n = 14). Interestingly, 34% of recordings performed in the same experimental conditions demonstrated the presence of a previously unrecognized potassium channel (sK). As seen in Fig. 1B, this channel is characterized by smaller inward currents at 40 mV (1.4 ± 0.2 pA, n = 5, P < 0.05) and by a more frequent open state. In the remaining 15% of experiments, the two channel types were observed together in the same patch (Fig. 1C). The fact that one type of channel could be recorded independently of the other in some patches and that independent gating of the two channels was observed when they were present in the same patch strongly suggest the presence of distinct channels.

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Representative inward current traces recorded at a potential of 40 mV in cell-attached configuration from 3 different patches. In control solutions, two types of K+ channels are observed separately (A, B) or together (C), depending on the experiment. Arrow ("C") indicates the zero current level.

Characteristics of sK vs. K_ATP. The current-voltage curves were plotted for the two types of channel in cell-attached configuration when the tubule is bathed in the control solution (Fig. 2). The K_ATP channel demonstrated an inward rectification, with an inward slope conductance of 49 ± 5 pS (n = 15) and an outward slope conductance of 13 ± 6 pS (n = 15). Concerning the sK channel, currents were characterized by a sigmoidal current-voltage relation with an inward slope conductance (measured between V_p = 40 mV and V_p = 20 mV) equal to 12 ± 2 pS (n = 4), which is significantly lower than the K_ATP channel inward conductance. Reversal potentials (E_rev) for K_ATP and sK channels were equal to +53 ± 14 mV (n = 15) and +48 ± 6 mV (n = 4), respectively. As we previously demonstrated that the potassium-to-sodium permeability ratio (P_K/P_Na) of the K_ATP channel was greater than 7 (2), we assume that the reversal potential of K_ATP currents corresponds to the V_p value for which potassium ions are at their equilibrium. This already suggests that sK channels display a good selectivity for potassium ions.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   In control solutions, unitary current of both the ATP-dependent potassium channel, KATP (, n = 15), and the "small" potassium channel, sK (, n = 4), recorded in the cell-attached configuration, are plotted as a function of Vp, where Vp is the micropipette potential.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Currents obtained from the inside-out configuration are plotted as a function of membrane potential for both KATP (A) and sK (B) channels, in both symmetrical (, ) and asymmetrical (, ) potassium gradients (indicated as ratio of millimolar values in bath vs. pipette).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   In control conditions, open probability (Po) of both KATP (, n = 15) and sK (, n = 4) channels, recorded in the cell-attached configuration, are plotted as a function of Vp.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Distributions of open and closed times in control conditions, plotted from experiments performed at Vp = 40 mV. Kinetic parameters of the KATP channel are described by two closed time constants (A; C1 and C2) and one open time constant (B; o), whereas kinetic parameters of the sK channel are defined by one closed time constant (C; C) and one open time constant (D; o). sqrt, square root.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effects of taurine on the sK channel. A: representative sK current traces recorded in cell-attached configuration at Vp = 40 mV. Downward transitions correspond to channel openings. Thick solid bars above the traces indicate taurine perfusion; arrows indicate zero current level. B: sK channel Po is plotted as a function of time (n = 4).

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Effects of taurine on the KATP channel. A: representative KATP current traces recorded in cell-attached configuration at Vp = 40 mV. Downward transitions correspond to channel openings. B: KATP channel Po is plotted as a function of time (n = 7).

Estimation of taurine affinity for K_ATP. In a previous report, we have shown that taurine, under identical experimental conditions, induced a cell volume increase and that this volume increase was related to the entry of taurine into the cell by way of a sodium-dependent taurine transporter (6). Therefore, known changes in cellular volume induced by taurine perfusion allow an estimation of the amount of taurine flowing into the cell as a function of time. The cellular osmolarity, _O, is defined as

(2)

where N is the mole number of osmotically active solutes, and V is the cell volume. The intracellular osmolarity is assumed to be in equilibrium with the osmolarity of the extracellular solution (300 mosM). Thus, as the cell volume increases in response to the osmolyte influx to keep _O constant, the variation of intracellular osmolyte concentration ([Osmolytes]_in) with time (see Fig. 8A) is calculated as

(3)

where V_(t) is the change in cell volume with time. Then, the variation of intracellular taurine concentration, T, is estimated by

(4)

where s is the net number of molecules entering the cell per taurine molecule. We assumed that the cotransporter stoichiometry for sodium vs. taurine was 2:1, as demonstrated in rabbit and rat brush-border membranes (30, 31) and in the apical and basolateral membranes of LLC-PK₁ and MDCK cell lines (15). Moreover, we assumed that electroneutrality was maintained by the exit of one potassium ion and the net entry of one bicarbonate anion for each taurine transport cycle. Thus, according to these hypotheses, the net number of molecules entering the cell for each taurine molecule is 3. In general, at the time the patch-clamp experiment is performed, tubules have been bathed in taurine-free solution for more than 1 h. If we assumed that the initial intracellular taurine concentration is negligible, Eq. 4 and the time course of taurine-induced cell swelling predict that intracellular taurine concentration progressively increases from 0 to 30 mM during the first 4 min of the experiment. From the curve illustrated in Fig. 8A and Eq. 4, the time course of P_o (Fig. 7B) was converted into a relation between P_o and the estimated taurine concentration (Fig. 8B). This relation was fitted according to the equation

(5)

where P_Max is the open probability in the absence of taurine, and K_0.5 is the taurine concentration for half inhibition. The K_0.5 value could be estimated to 8.7 mM, and the P_Max to 0.26, which is close to the measured value (0.23 ± 0.06, n = 7) in the absence of external taurine. Please note that this rough calculation would not be much affected by a reasonable underestimation of s; if the number of molecules accompanying taurine were underestimated by 1, then the calculated K_0.5 would decrease by only 25%.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   A: variation of intracellular osmolyte concentration, induced by perfusion of extracellular taurine is plotted as a function of time. B: Po of the KATP channel is plotted as a function of intracellular taurine concentration. Time scale of x-axis has been converted into taurine concentration by using the curve plotted in A. [Taurine]in, variation of intracellular taurine concentration.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Two types of potassium channels. Passive potassium transport across the basolateral membrane of mammalian proximal tubules was thought to be mediated by a unique potassium channel regulated by intracellular ATP (14, 24), intracellular pH (2), and, potentially, by cell volume (2, 7, 14, 16, 19, 24). Although other types of potassium channels, such as stretch-activated K channels, have been shown in Necturus proximal cells, no evidence for existence of such channels was provided for the mammalian proximal tubule (20), and the relationship between cell volume and potassium channel remained to be understood. In the present study, single-channel recordings revealed the presence of a second and previously unrecognized type of potassium channel, sK, with lower current amplitude than the K_ATP channel. As it is extremely difficult to establish a gigaohm seal on the basolateral membrane of the proximal tubule, our knowledge of the basolateral K channel(s) characteristics is limited. This may explain why the presence of this second type of basolateral K channel was generally overlooked but for one mention in a 1993 abstract (1) from our laboratory [see also figure 7 in Parent et al. (19)]. Several lines of evidence allow us to conclude that we are in the presence of two distinct types of potassium channels. First, when the two channels are recorded together, independent gating can be observed. Second, the probability of observing both channel types in the same patch is equal to 0.15. This measured value is consistent with the estimated probability of 0.17, obtained from the probabilities of finding the K_ATP (0.51) and the sK (0.34) channels individually (0.51 × 0.34 = 0.17), assuming that these two types of K⁺ channels are independently distributed. Lastly, after excising the membrane patch to reach inside-out configuration, we usually observed a rapid rundown (within 30 s, data not shown) of the K_ATP channel, whereas low-amplitude activity remained steady all along the experiments, suggesting the existence of two differently regulated channels. Taken together, these data strongly suggest that two distinct potassium channels coexist at the basolateral membrane of PCT cells.

In cell-attached configuration, the sK potassium channel is characterized by a sigmoidal current-voltage curve with a unitary conductance of 12 pS as measured between 40 mV and +20 mV. Potassium channels with low conductance (10-30 pS) have been described at the apical membrane of rabbit thick ascending limb of Henle's loop (25, 28), at both apical and basolateral membrane of rat cortical collecting tubule (10, 13) and at the apical membrane of inner medullary collecting duct (21). These low-conductance channels all display a high open probability in cell-attached configuration and, in the case of the inner medullary collecting duct, the cortical collecting tubule, the thick ascending limb of Henle's loop, and the proximal sK channel, P_o is not dependent on the membrane potential and is not altered upon membrane excision (8, 10, 21, 27, 28). Finally, the gating mechanisms of the proximal sK channel and the cortical collecting tubule low-conductance channel are both described by one open dwell-time constant and one closed dwell-time constant (13, 26). Although these channels appear to share some common properties, conclusions about the precise identity of proximal sK channel would be premature.

The finding of a second type of potassium channel in the basolateral membrane of PCTs raises the question of its role and its importance with respect to the K_ATP channel. Concerning the sK channel contribution to the absolute basolateral potassium conductance, its high open probability appears to fully compensate its low conductance. Therefore, the relative conductance for each type of channel strongly depends on the number of active channels. The mean potassium current, I, carried by a given type of channel, is given by

(6)

where I₀ is the unitary current, P_o is the channel open probability, N_C is the total number of channels. At the resting membrane potential, corresponding to a micropipette potential of 0 mV, our experimental data allow the estimation of both sK and K_ATP mean currents, which are equal to I_sK = 0.66 × 0.53 × N_sK, and I_KATP = 1.67 × 0.22 × N_KATP, respectively. To determine the I_sK/I_KATP ratio, we measured a N_KATP/N_sK ratio of 1.43 from patches where the two types of channel coexist. From these data, we estimated that the sK conductance would represent 40% of the total potassium conductance of the PCT basolateral membrane.

Effects of taurine on the basolateral potassium channels. Our results demonstrate that neither the open probability nor the number of channels nor the unitary conductance of the sK channels is modified by taurine. By contrast, the K_ATP channel P_o decreased by 76% (from 0.17 ± 0.06 to 0.04 ± 0.02; n = 7, P < 0.05) within 4 min. Since current recordings are performed in cell-attached configuration and taurine is perfused in the bath solution, the P_o decrease must be related to an intracellular effect. In ventricular myocytes, it has been shown that taurine could inhibit ATP-dependent potassium channels in inside-out configuration, suggesting a direct interaction process (12, 22). In agreement with this hypothesis, reversibility of the inhibitory effect could be observed when taurine was removed (12, 22). In comparison with inside-out membrane patches, the use of cell-attached configuration, particularly on a whole proximal tubule, is more respectful of the cell physiology but does not allow an accurate control over the intracellular taurine concentration. It is possible that the removal of taurine under the membrane patch studied would require more than a few minutes. It is interesting to note that the effect of taurine on the basolateral conductance was fully reversible when we were using microelectrodes (4). The possible factors that differ between the two experimental conditions are the temperature (25°C in patch-clamp vs. 38°C with microelectrodes) and the actual geometry of the membrane studied (a microscopic membrane patch vs. the whole basolateral membrane). It is possible that, at 25°C, more than a few minutes are needed to lower taurine concentration under the membrane patch studied.

In proximal tubule cells, as in myocytes, the decrease of open probability is related to the increase of the long closed time. We estimated that taurine concentration should be equal to 8.7 mM for the K_ATP channel half inhibition. This K_0.5 value is quite similar to those found (13.5 mM) for the K_ATP channels of ventricular myocytes (12). Such a K_0.5 value in the low millimolar range suggests that in the proximal tubule cell in vivo, K_ATP channels are under the constant inhibitory effect of intracellular taurine. In vitro, intracellular taurine concentrations are expected to reach 30 mM in 4 min upon exposure to 40 mM basolateral taurine. This should inhibit most of the K_ATP channels, leaving the basolateral K conductance solely dependent on sK. The observed decrease of the basolateral potassium conductance by 50% (4) is quite consistent with estimation that sK channels are taurine insensitive and responsible for ~40% of the macroscopic K conductance.

Thus the present results bring a microscopic explanation for the inhibitory effects of taurine on the macroscopic basolateral potassium conductance, previously demonstrated with the intracellular microelectrodes technique (4). Although the mechanisms remain to be determined, our data allow us to definitively conclude that intracellular taurine acts on basolateral ATP-dependent potassium channels by decreasing their open probability. On the other hand, we also demonstrated the presence of a second type of potassium channel that is taurine insensitive and responsible for ~40% of the basolateral potassium conductance. The estimated sensitivity of K_ATP to intracellular taurine versus its physiological concentration and the fact that cell swelling modulates taurine permeability indicate that taurine could play a very significant role in maintaining a constant cell volume and membrane potential in the face of varying rates of apical Na-coupled solute transport.