Basolateral K conductance in principal cells of rat CCD

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Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York* u& Y; Z0 P9 C2 q

* W  T) C& d0 s) }! @* S. x( hABSTRACT
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" E# F* {' e4 U3 D' H1 LWhole cell K  current was measured by forming seals on the luminal membrane of principal cells in split-open rat cortical collecting ducts. The mean inward, Ba2 -sensitive conductance, with 40 mM extracellular K , was 76 ± 12 and 141 ± 22 nS/cell for animals on control and high-K  diets, respectively. The apical contribution to this was estimated to be 3 and 16 nS/cell on control and high-K  diets, respectively. To isolate the basolateral component of whole cell current, we blocked ROMK channels with either tertiapin-Q or intracellular acidification to pH 6.6. The current was weakly inward rectifying when bath K  was 40 mM but became more strongly rectified when bath K  was lowered into the physiological range. Including 1 mM spermine in the pipette moderately increased rectification, but most of the outward current remained. The K  current did not require intracellular Ca2  and was not inhibited by 3 mM ATP in the pipette. The negative log of the acidic dissociation constant (pKa) was 6.5. Block by extracellular Ba2  was voltage dependent with apparent Ki at –40 and –80 mV of 160 and 80 μM, respectively. The conductance was TEA insensitive. Substitution of Rb  or NH4  for K  led to permeability ratios of 0.65 ± 0.07 and 0.15 ± 0.02 and inward conductance ratios of 0.17 ± 0.03 and 0.57 ± 0.09, respectively. Analysis of Ba2 -induced noise, with 40 mM extracellular K , yielded single-channel currents of 0.39 ± 0.04 and –0.28 ± 0.04 pA at voltages of 0 and –40 mV, respectively, and a single-channel conductance of 17 ± 1 pS.' ~6 R2 I% h6 h( K4 E) \" {
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renal K  channels; renal K  transport; noise analysis; tertiapin; K  adaptation8 i3 C+ m7 M( I. B% y& S2 {

  Q$ J! t$ z& @; u8 XTHE BASOLATERAL K  CONDUCTANCE in cortical collecting duct (CCD) principal cells plays a critical role in Na  and K  homeostasis by setting the resting potential of the cell. K  flux across the basolateral membrane can be in either direction depending on the physiological setting (22, 33). In states where K  conservation is required, for example on a low-K  diet or during neonatal growth, K  entering the cell by the active Na -K -ATPase is recycled across the basolateral membrane to prevent losses through apical K  channels. In states of K  excess or in the setting of increased aldosterone, the direction of net K  flux across the basolateral membrane reverses (22, 33) such that K  now enters the cell through the basolateral channels and is then secreted into the urine across the apical membrane. Finally, the basolateral K  conductance influences Na  reabsorption by helping to establish the apical membrane potential.
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Despite its importance, our knowledge of the molecular determinants that underlie the basolateral K  conductance in principal cells is limited. From perfusion studies of isolated rat CCDs, it has been shown that the basolateral K  conductance is Ba2  sensitive but TEA insensitive (35) and that the fractional apical resistance with amiloride in the lumen is 0.9 (36). The permeability sequence is K  > Rb  > NH4  (35). Single-channel recordings from K  channels on the basolateral membrane are technically challenging as the basement membrane presents a barrier to formation of giga-ohm seals on the basolateral surface of these cells. Two approaches have been used to circumvent this problem: enzymatic digestion of the basement membrane (11) and mechanical removal of the adjacent intercalated cells permitting access to the lateral principal cell membrane (50). After collagenase digestion of the basement membrane, a 148-pS channel (cell attached) that decreased to 85 pS when excised into a low-K  bath and a 67-pS channel (cell attached) that decreased to 28 pS when excised were observed (12). The larger conductance channel was inhibited by intracellular acidification with a negative log of the acidic dissociation constant (pKa) of 7.0. Two channels were also reported from the lateral principal cell membrane following mechanical removal of an adjacent intercalated cell: a 45-pS conductance (cell attached) with an open probability of 0.2, which increased with hyperpolarization, and a 27-pS (cell attached) conductance that remained 18–30 pS when excised into symmetric K , with an open probability of 0.8 (50). The lower conductance channel was minimally sensitive to intracellular acidification to pH 6.7, insensitive to TEA, but activated by cGMP (only when cell attached) (21, 49, 50). It is difficult to determine the quantitative significance to overall basolateral conductance of the various channels that have been described.) C2 _! P" |' L8 |" r' v

4 `( R: @8 a7 e' K* ^We developed an alternative approach to characterize the properties of basolateral K  conductance. We recorded whole cell Ba2 -sensitive currents from principal cells of split-open rat CCDs by forming giga-ohm seals on the apical surface under conditions that minimized Na  and Cl– currents. To isolate the basolateral component of this K  conductance, we blocked the apical ROMK channels either with the modified bee venom tertiapin-Q (TPNQ) or by intracellular acidification. TPNQ is a high-affinity ROMK antagonist with a Ki in the low nanomolar range (14). ROMK is very sensitive to intracellular pH with a pKa in the 6.8–7.0 range (23, 45, 51). The sensitivity of basolateral conductance to either TPNQ or intracellular acidification was not known a priori. However, most of the whole cell current remained with either method, suggesting that the apical and basolateral conductances could indeed be separated on the basis of their different sensitivities to TPNQ and pHi. The biophysical properties of this Ba2 -sensitive but TPNQ- and intracellular pH (pHi)-resistant basolateral conductance were then characterized with the goal of determining its underlying molecular identity.. q2 ]' [6 _1 l1 a5 v

$ ~1 _% e/ |: `; Q8 b; zMATERIALS AND METHODS
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Animals. Sprague-Dawley rats (Charles River Laboratories, Kingston, NY) weighing 150–200 g were fed normal (0.97% K , 0.33% Na , 0.52% Cl–, 0.23% Mg2 , 0.8% Ca2 ) or high-K  (5.2% K , 0.4% Na , 5.1% Cl–, 0.2% Mg2 , 0.9% Ca2 ) chow (Harlan Teklad, Madison, WI) for 1–2 wk before experiments. In general, rats were on high-K  chow to maximize K  currents, except where indicated.1 i! A+ W) {& K  S: X' S
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Renal tubule preparation. Under Inactin (Sigma, St. Louis, MO) anesthesia (150 mg/kg ip), kidneys were excised and thin sections were cut with a razor blade. CCDs were isolated with forceps under a dissecting microscope and split open with a fine needle. They were then attached, apical side up, to a small coverslip using Cell-Tak (Collaborative Biomedical Products, Bedford, MA), placed in a glass-bottom chamber on an inverted microscope, and superfused with bath solution at 37°C.
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/ U, o# h' }) ]% U: D" QROMK expression in Xenopus laevis oocytes. pSPORT plasmids (Life Technologies, Rockville, MD) containing either ROMK1 (Kir1.1a) or ROMK2 (Kir1.1b) were linearized with NotI restriction enzyme (New England BioLabs, Beverly, MA) and transcribed in vitro with T7 RNA polymerase using an mMESSAGE mMACHINE kit (Ambion, Austin, TX). Stage V-VI oocytes were harvested from X. laevis after anesthesia with Tricaine (1.5 g/l, adjusted to pH 7.0 with NaOH). Oocytes were defolliculated by incubation in OR2 solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES adjusted to pH 7.5 with NaOH) containing 2 mg/ml of collagenase type II (Sigma) for 60 min at room temperature. Oocytes were then injected with ROMK1 or ROMK2 RNA and incubated at 19°C in 2x diluted Leibovitz's medium (Life Technologies) for 1–3 days before recording.
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6 W) ^6 }; M6 E- K$ V7 tTwo-electrode voltage clamp. Whole cell current-voltage (I-V) relationships were obtained using a two-electrode voltage clamp (OC725C, Warner Instrument, Hamden, CT) with a ITC-16 interface (Instrutech, Long Island, NY) running Pulse software (Heka Elektronik, Lambrecht, Germany). Pipettes were made from hematocrit capillary tubes (15401–650, VWR International, West Chester, PA) with three pulls from a vertical pipette puller (700C, David Kopf Instruments, Tujunga, CA) and filled with 1 M KCl. Pipette resistances were less than 1 M. The bath solution contained (in mM) 100 NaCl, 10 KCl, 2 CaCl2, 1 MgCl2, and 5 HEPES with pH adjusted to 7.4 with NaOH with or without 5 mM BaCl2 to block K  currents. Currents were recorded in response to a voltage protocol consisting of 10-mV steps from –120 to  50 mV from a holding potential of –50 mV.2 B6 F1 \0 \& I4 T, _

4 a3 y' S9 a6 Z- F& k( DPatch clamp. Principal cells in split-open rat CCDs were identified morphologically and a giga-ohm seal was formed on the luminal surface (3). In the CCD, two-thirds of the cells are principal cells whose large, polygonal and flat appearance contrasts with the small, round and raised morphology of intercalated cells. The presence of a large, whole cell K  current confirmed the identification of principal cells, as intercalated cells have a large Cl– conductance but no significant K  conductance under these conditions (L. G. Palmer and G. Frindt, unpublished observations). Pipettes, made from hematocrit capillary tubes (15401–650, VWR International) with three pulls from a vertical pipette puller (700C, David Kopf Instruments), were coated with Sylgard (Dow Corning, Midland, MI) and fire polished to yield tip resistances of 2–7 M. Negative pressure was used to break into the whole cell configuration. Evidence that a principal cell under whole cell clamp is electrically isolated from its neighbors has been presented previously (6). Currents were recorded in response to voltage steps applied in 10-mV increments from –100 to  60 mV from a holding potential of –20 mV. Currents were recorded with an EPC-7 patch-clamp amplifier (Heka Elektronik) and digitized with a Digidata 1332A interface (Axon Instruments, Union City, CA). Data were filtered at 1 kHz and analyzed with pCLAMP8 software (Axon Instruments).) {1 Q, Y  O6 d* [0 x$ E4 T
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Solutions and reagents. The pipette solution contained (in mM) 7 KCl, 123 aspartic acid, 5 EGTA, 10 HEPES, and 3 MgATP, with the pH adjusted to either 6.6 or 7.4 with KOH. This yields a K  concentration ([K ]) of 147 mM at pH 7.4 due to titration of aspartic acid, EGTA, and buffers. (In some experiments, we increased the buffering to better clamp the intracellular pH, by using a solution identical to the above but with 20 PIPES instead of HEPES and with aspartic acid lowered to 116 mM to compensate for the increased KOH needed to titrate the increased buffer. No difference between the two solutions was observed.) Where indicated, 1 mM spermine tetrahydrochloride (Sigma) was added to the pipette solution. The bath solution contained (in mM) 40 K  methanesulfonate, 100 Na  methanesulfonate, 2 CaCl2, 1 MgCl2, 10 HEPES, 2 glucose, and 10 μM amiloride, with pH adjusted to 7.4 with NaOH. When bath K  was lowered or raised, it was replaced by equimolar amounts of Na . In some experiments, bath K  was replaced by Rb  or NH4 . Barium acetate, TEA acetate, and TPNQ were added to the bath solution where indicated. TPNQ was obtained from either Sigma or from Dr. Zhe Lu (Department of Physiology, University of Pennsylvania, Philadelphia, PA). Toxin from both sources gave similar results. Aliquots of TPNQ, stored at –80°C, were thawed, diluted in bath solution to the indicated concentrations, and used within 3 h.- S* v; l- N8 @. a

! A, K2 [& w5 T9 F2 p+ _" ?# o4 ?2 D- XIntracellular acidification. By choosing a ratio of extracellular (bath) to intracellular (pipette) acid (9), the intracellular pH was set to the desired value:1 |) r' T% Y  C( n! H4 G6 |
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The pHs predicted by this approach agreed well with independent fluorescent measurements of pHi (9). The pipette solution contained (in mM) 25 trimethylammonium, 126 aspartic acid, 7 KCl, 5 EGTA, 5 PIPES, 3 MgATP with pH adjusted to 7.4 with KOH (final K  concentration 122 mM). This solution was lightly buffered to allow the desired pH to be established by equilibration of the weak base across the cell membrane with the heavily buffered bath solution. To achieve the desired pHis of 7.4, 7.0, 6.6, 6.2, and 5.8, the bath solutions contained, respectively, 25, 10, 4, 1.6, and 0.6 mM of trimethylammonium methanesulfonate along with (in mM) 80 HEPES, 40 KOH, 2 CaCl2, 1 MgCl2, and 2 glucose, as well as 10 μM amiloride adjusted to pH 7.4 with NaOH. Decreasing trimethylammonium was replaced with Na  while K  was maintained at 40 mM.$ E4 l& _& R- P3 t0 @2 t
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Correction for series resistance and liquid junction potential. Except where indicated, all currents were corrected a posteriori for series resistance, which was assumed to equal the resistance of the pipette before formation of the seal. The pipette resistance was subtracted from the reciprocal of the apparent whole cell slope conductance, yielding the corrected whole cell resistance, which was then inverted to give the corrected whole cell conductance. A liquid junction potential of 10 ± 1 mV was present throughout these experiments. This arose because the pipette was nulled before the start of each experiment in bath solution. The differing ionic composition of the bath and pipette solutions as well as the potentials of the ground and pipette electrodes themselves result in this offset. Ultimately, under whole cell patch configuration, the pipette dialyzes the cell and thus the solutions within and outside the pipette are essentially the same. At this point, the liquid junction potential vanishes but the applied compensating voltage remains. The liquid junction potential was determined a posteriori by nulling the pipette in standard bath solution and then changing the bath solution to pipette solution and measuring the offset. The net effect of this potential was to shift all reported voltages and I-V curves to the right by 10 mV. We did not correct for this offset.
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Statistical analysis. Data are presented as means ± SE. The two-tailed Student's t-test was used to determine whether differences between groups were significant (P 2 M; V+ Z) M7 m1 M, M2 A7 b8 {

* l5 |: U1 F  {3 kCalculation of electrical distance for Ba2  block. From semilog plots of fractional Ba2  block vs. Ba2  concentration, Kis were determined at –40, –60, and –80 mV with a fit to a Hill equation. The Kis were then plotted as a function of voltage using the equation:7 g7 v6 d4 L. f  _) D4 z

3 w/ C) F) ?6 w% i4 lwhere  is the electrical distance, Ki (0) is the apparent Ki when V = 0, z is the valence of the blocker, and F, R, and T have the standard thermodynamic definitions.  and Ki (0) were then determined from the slope and y-intercept of a least-squares linear fit to this data.. j5 Y: s+ t$ t( I) J/ Q0 H
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Noise analysis. Single-channel current was calculated using the method of Lindemann and Van Driessche (20, 47). Briefly, an intermediate barium concentration was used to produce blocking events. Whole cell current was recorded under these conditions and the power spectrum was determined with pCLAMP8 software (Axon Instruments) in the 2- to 1,000-Hz range. The power spectra were fit with a single Lorentzian term of the form:
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" M, p! M, F( OThis provided estimates for the plateau amplitude So and the corner frequency fc. These values were then used to calculate single-channel current with the following expression (20):
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# z& ?3 I6 S7 \* S1 J- o/ W2 ^6 `where IK and FK are the macroscopic Ba2 -sensitive K  current and the fractional Ba2  block for the given [Ba2 ]. For first-order block kinetics, fc is related to [Ba2 ] by the equation:
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where kon is the on-rate and koff the off-rate for block. The apparent Ki for Ba2  can then be calculated as:
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RESULTS) L! Q" I" q. w% A- a* k& V

: x3 G/ i' k- W0 H$ hPrincipal cells of rat CCD have a large Ba2 -sensitive whole cell K  conductance, most of which arises from the basolateral membrane. Whole cell currents were recorded from principal cells of split-open rat CCDs with amiloride in the bath. Figure 1A, left, shows typical whole cell current in response to the family of voltage steps depicted in the inset. The small, time-dependent decay in outward current seen at large positive voltages probably arises from slow voltage-dependent block by intracellular components. Figure 1A, right, shows almost complete block of inward current when 5 mM Ba2  is added to the bath. The time-dependent outward currents arise from partial relief of Ba2  block at positive voltages. Steady-state I-V relationships derived from the traces in Fig. 1A are shown in Fig. 1B. The whole cell current is weakly inward rectifying, reverses near EK (–35 mV for the 147 mM K  in the pipette), and is almost all Ba2  sensitive. The deviation of the reversal potential from EK did not arise primarily from lack of K  selectivity as a semilog plot of the reversal potential, Erev, vs. K ext shown below (see Fig. 7B) was fit by a line of slope 58 mV/decade, which is very close to the ideal value of 61.5 mV/decade. Rather, it was due mainly to a liquid junction potential (see MATERIALS AND METHODS). The inward, Ba2 -sensitive slope conductance, corrected for series resistance, is 130 nS. The apical contribution, estimated to be 16 nS (see APPENDIX), represents a small fraction of this.# V3 G0 M- d" j3 s- w
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High-K  diet increases whole cell K  conductance. The mean Ba2 -sensitive conductance, with 40 mM K  in the bath, was 141 ± 22 nS after 1–2 wk on a high-K  diet vs. 76 ± 12 nS on a control diet under identical recording conditions. The slope conductance was determined between –40 and –80 mV from I-V curves as in Fig. 2. This difference in conductance was significant (P = 0.02) and was based on 3–10 cells from each of 8 rats in each group.
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Isolation of the basolateral component of whole cell K  current. Whole cell K  current includes contributions from both apical and basolateral channels. To isolate the basolateral component, we sought ways to preferentially block the apical K  channels. ROMK (SK) forms the predominant apical K  conductance. It is the channel most frequently encountered in patch-clamp studies of the apical membrane (4, 51), and its conductance alone can account for the K  secretion measured in isolated, perfused CCDs (8). Although the BK channel is encountered in 22% of apical patches of principal cells in the CCD (26), our pipette contains 5 mM EGTA, which should completely inhibit this Ca2 -dependent channel. Two approaches were used to eliminate the small apical ROMK current from whole cell current: block by the modified bee toxin TPNQ and intracellular acidification.
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* t. g" |" m3 i3 Q7 oIsolation of basolateral K  current with TPNQ. TPNQ is a high-affinity antagonist of ROMK with a Ki in the low nanomolar range (14). After a saturating dose of TPNQ, any remaining whole cell K  current should arise from the basolateral membrane. We first tested TPNQ in X. laevis oocytes expressing ROMK using two-electrode voltage clamp. A typical experiment is shown in Fig. 3A where I-V curves at baseline and with 1, 10, and 100 nM of TPNQ in the bath are indicated. Progressively increased inhibition is seen along with complete recovery after washout of the 100-nM dose. Almost all the inward current is inhibited by 5 mM Ba2 . From these experiments, the fraction of remaining current (I/Imax) as a function of TPNQ concentration was plotted (Fig. 3B) and fit to a simple binding isotherm with Ki = 13 ± 2 nM.
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' p' ~! G, L( I: S1 e4 kWhole cell K  currents in principal cells were then tested for TPNQ sensitivity using a concentration over 100x the Ki measured in the oocyte system. After baseline whole cell K  current was established, 2 μM TPNQ was added to the bath and the time course of inhibition was monitored at –80 and  30 mV (Fig. 3C). TPNQ was then washed out and the sensitivity to Ba2  was tested. A small but clear reduction in current in response to the toxin was evident here. In other experiments, only slow increases or decreases in current were detected. There was generally minimal recovery with washoff of this high dose of TPNQ. The mean Ba2 -sensitive whole cell current at –80 mV before and after TPNQ is shown in Fig. 3D. The mean decrease in current, 0.52 ± 0.22 nA, was not quite statistically significant (P = 0.06) but is consistent with our estimation of the ROMK current per principal cell under these conditions of 0.72 nA (see APPENDIX) and the notion that most of the whole cell K  conductance arises from the basolateral membrane. Although ROMK is clearly TPNQ sensitive in the oocyte system, it is conceivable that it has altered sensitivity in native tissue where it forms the SK channel. We therefore turned to intracellular acidification, which has been shown to block both ROMK and native SK channels with similar pKa, as a second independent way to block the apical ROMK conductance.+ V: t' I( N! }( A4 h; _- ]( s" f

+ M9 s: a: u4 bIsolation of basolateral K  current by intracellular acidification. ROMK is very sensitive to intracellular acidification with a pKa in the 6.8 to 7.0 range (23, 45, 51). As mentioned above, ROMK underlies the predominant apical K  conductance. If the basolateral conductance is less acid sensitive than ROMK, lowering pHi would provide a means of isolating the basolateral component of whole cell current. Indeed, the activity of the small-conductance K  channel reported from the principal cell basolateral membrane was minimally affected by excision into a bath of pH 6.7 (50). We therefore measured whole cell K  current as pHi was incrementally lowered by reducing the ratio of bath to pipette trimethylammonium concentration (see MATERIALS AND METHODS). Whole cell current in response to voltage steps was recorded at predicted pHis from 5.8 to 7.4 (Fig. 4A). Changes in current were rapid (within 1–2 min) and reversible. The extent of block at each pH was then determined (Fig. 4B). Essentially, all current was blocked by pH 5.8. These data were fit by a Hill equation with a pKa of 6.5 and a Hill coefficient of 1.5. Because, as discussed above, most of the whole cell K  current arises from the basolateral membrane, the pH dependence of the current shown in Fig. 4B reflects primarily the acid sensitivity of the basolateral K  conductance. The pKa of 6.5, however, is an upper estimate of the sensitivity of the basolateral conductance as at least some component of the total current comes from ROMK channels with their higher pKa. At pHi 6.6, most of the ROMK current is blocked, but most of the current across the basolateral membrane remains and this approach may be used to isolate the basolateral K  current. Because the decrease in whole cell current at pHi 6.6 is 40% (see Fig. 4B) and the expected contribution of ROMK to whole cell current, based on both calculations (see APPENDIX) and the size of the TPNQ-sensitive current (see Fig. 3D) is / n5 [5 t3 w# N- D) ?9 J8 E

0 ~* U" K  I. I) h( X6 RWe also clamped pHi to 6.6 directly by lowering the pH of the pipette solution to 6.6. Because the volume of the pipette is much greater than that of the cell, the pipette solution is heavily buffered and there is a low-resistance pathway between the pipette and the cell, the pH of the intracellular solution should be close to that of the pipette. Figure 5A shows a representative time course of inward and outward current from the moment of breakthrough to a whole cell configuration. Over 90 s, there is a modest decrease in current but most remains. The corresponding I-V curves, recorded immediately on breakthrough and then 90 s later, are shown in Fig. 5B. Most of the current is Ba2  sensitive. From these experiments, the mean Ba2 -sensitive whole cell current before and after acidification was determined (Fig. 5C, left). Most, 65%, of the current remained. This is consistent with the 60% of current that remained when trimethylammonium was used to acidify to a presumed pHi of 6.6 (Fig. 4B). No significant changes in whole cell K  current occurred over the same time course at pHi 7.4 (Fig. 5C, right). These findings strongly support the idea that the acid sensitivity of the basolateral K  conductance is significantly less than that of the apical membrane.+ p" j+ z2 c; `8 {, `) k& g3 a9 P/ f
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Additional biophysical characterization of the basolateral K  conductance. For these experiments, pipette solution of pH 6.6 was used as, at this pH, most of the apical K  conductance should be blocked but most of the basolateral conductance should remain. This is below the physiological cell pH and could, in principle, alter properties of the K  channels. However, we made preliminary measurements of these properties with pipette pH 7.4, which is above the physiological pH, and the results were not markedly different.2 E0 K/ w1 W: b: K* X# y2 q; v/ b
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Ba2  and TEA sensitivity. I-V curves were recorded at baseline and then with progressively increasing bath Ba2  concentrations between 0.05 and 5 mM (Fig. 6A). The fractional block of inward current at –40, –60, and –80 mV was quantified at each Ba2  concentration, assuming maximal block at 5 mM, and these data were fit to simple binding isotherms. Kis of 165 ± 13, 122 ± 9, and 83 ± 11 μM were obtained at –40, –60, and –80 mV, respectively, reflecting the voltage dependence of Ba2  block (Fig. 6B). Compensation for series resistance was not performed due to the nonlinearity of the I-V curves at the intermediate Ba2  concentrations. Thus these apparent Kis represent upper limits on the actual values. The electrical distance, calculated from this data (see MATERIALS AND METHODS), was 0.23 ± 0.02 and the apparent Ki at 0 mV was 333 ± 1 μM. Whole cell K  currents were insensitive to 10 mM TEA in the bath (data not shown).& ?! o3 F/ {( ~* v9 T" `
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Dependence on extracellular [K ]. The sensitivity of the basolateral K  conductance to extracellular [K ] was also quantified. Whole cell K  current was measured with 2.5, 10, 40, and 140 mM K  in the bath (Fig. 7A). As the bath [K ] was increased, the reversal potential shifted to progressively less negative voltages at a rate of 58 mV/decade (Fig. 7B). In addition, the inward slope conductance increased with increasing bath [K ]. A plot of normalized inward slope conductance vs. bath [K ] is shown in Fig. 7C and was fit by a hyperbola with Gmax of 2.3 ± 0.2 and Km of 48 ± 10 mM. The outward current, however, has a more complex behavior. As extracellular [K ] is lowered, the current becomes progressively more inwardly rectified and the outward slope conductance decreases steadily. Simple Goldman-Hodgkin-Katz behavior predicts outward conductance to be independent of extracellular [K ] at large positive voltages. The decrease in outward conductance as bath [K ] is lowered occurs rapidly (at a similar rate as the shift in reversal potential) and is reversible. It could arise either from decreased permeation or from a rapid block or gating process.! M( `+ V/ z3 m- }8 m( n. m

$ N+ h  a3 T) X7 XSpermine sensitivity. To examine the possibility that rapid diffusion of intracellular polycations out of the cell and into the pipette was eliminating strong rectification, we added 1 mM spermine to the pipette solution and recorded I-V curves immediately on breakthrough to a whole cell configuration and over the subsequent several minutes during which time the currents reached a new steady state (Fig. 8). A modest decrease in outward current occurred within 2–3 min but more than half of the outward current remained. We assume that most of this remaining current was through the K  channels, even though it was not blocked by 5 mM Ba2 , as positive cell potentials relieve Ba2  block (see Fig. 1A). To quantify the degree of rectification before and after entry of spermine, we calculated the ratio of outward to inward current (at  30 and –80 mV, respectively) symmetrically about the reversal potential, on breakthrough and then 3 min later. The mean ratio decreased from 0.63 ± 0.09 on breakthrough to 0.39 ± 0.05 (P = 0.004) with the addition of spermine. In addition to its role in K  channel rectification, spermine metabolism has been implicated in cell signaling and proliferation, in protein synthesis and folding, in apoptosis as well as in affecting other ion channels and receptors (39, 44). Given the brief ( 3 min) duration of spermine exposure and the fact that the reversal potential remained stable, it is unlikely that any of these other processes are at play here.4 Z) d2 E4 B- ?, f

" w; b+ j- r0 MIon selectivity. Representative I-V curves with 40 mM NH4  or Rb  replacing K  in the bath are shown in Fig. 9, A and B. This replacement causes a shift in reversal potential, reflecting the altered permeability of the substituted ion relative to K , which was quantified with the equation:
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The results are shown in Fig. 9C where the permeability ratios for Rb  and NH4  were 0.65 ± 0.07 and 0.15 ± 0.02, respectively. Slope conductance also changes with ion substitution. The relative inward slope conductance of Rb  and NH4  to that for K , derived from I-V curves of the type shown in Fig. 9, A and B, were 0.17 ± 0.03 and 0.57 ± 0.09, respectively (Fig. 9D). Thus Rb  is more permeable than NH4  but less conductive. With ion substitution, the outward current is still carried by K  ions leaving the cell but now there is no K  in the bath. Given the dependence of outward current on bath K  (Fig. 7A), ion substitution may affect outward conductance as well. Indeed, while Rb  substitution has minimal effect on outward conductance, substitution by NH4  leads to a decrease in outward slope conductance. This decrease is not as great as that seen, for example, when bath K  was lowered to 2.5 mM, suggesting that NH4  can partially compensate for the lack of K  in the bath in supporting outward K  current.
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; ?; |3 m; |9 k/ wSingle-channel currents. Noise analysis, using the method of Lindemann and Van Driessche (20, 47), was employed to estimate the size of the single-channel conductance underlying the basolateral current (see MATERIALS AND METHODS). Whole cell currents were recorded with 40 mM K  in the bath, and Ba2  was added to produce blocking events. Figure 10A shows current at 0 and –20 mV. The noise amplitude increases in going from –20 to 0 mV. If the noise arose from the leak current of the seal, one would expect the noise to be minimal at 0 mV and increase as the voltage moved to –20 mV. On the other hand, if the noise arises from the channels themselves, one would expect the noise to be minimal at –20 mV, which is near the reversal potential for this bath [K ], and increase as one moved to 0 mV. That the noise amplitude indeed increases in going from –20 to 0 mV supports the latter conclusion. Further evidence that the noise arises from Ba2  block of the channels is seen in Fig. 10B. Here, at –40 mV, we see a progressive decrease in the noise amplitude as the Ba2  concentration in the bath increases.
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' q. I1 e6 R. ~$ q; |' Q! xTo determine outward and inward single-channel currents, whole cell currents at 0 and –40 mV were recorded with a range of intermediate concentrations of Ba2  in the bath. Power spectra were generated from these currents and fit with single Lorenztian functions. Representative spectra are shown in Fig. 11, A and B, top. The corner frequency (fc) and the plateau power (So) were determined from these fits. For a simple first-order blocking reaction, the corner frequency should be a linear function of the Ba2  concentration with the slope corresponding to the Ba2  on-rate, the y-intercept corresponding to the Ba2  off-rate, and the Ki for Ba2  determined by koff/kon (see MATERIALS AND METHODS). Such plots are shown in Fig. 11, A and B, bottom. The kon rates were 0.96 and 2.32 μmol–1﹞s–1 and the koff rates were 413 and 211 s–1 for 0 and –40 mV, respectively. This yielded Kis for Ba2  of 432 μM at 0 mV and 91 μM at –40 mV, values in reasonable agreement with macroscopic Ba2  sensitivity data presented in Fig. 6B and related text (333 μM at 0 mV and 165 μM at –40 mV).2 Q4 q" u0 k/ ?# U
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Single-channel currents were estimated using So and fc values derived from the Lorentzian fit, along with measurement of the Ba2 -sensitive current and the fractional block for the given Ba2  concentration (see MATERIALS AND METHODS). By this approach, single-channel currents of 0.39 ± 0.04 pA at 0 mV and –0.28 ± 0.04 pA at –40 mV were calculated. An I-V curve of these values, along with the mean measured reversal potential of –24.3 ± 0.7 mV, is shown in Fig. 11C. The slope of a least-squares fit through these points yields a single-channel conductance of 17 ± 1 pS.
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DISCUSSION1 j' r, S* v; }. |2 S
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The basolateral principal cell K  conductance sets the resting potential of the basolateral membrane and plays an important role in K  recycling, K  secretion, and Na  reabsorption. The basement membrane presents a barrier to formation of giga-ohm seals on the basolateral membrane and while several channels have been reported from this surface using enzymatic or mechanical methods to circumvent the basement membrane problem, their quantitative significance to whole cell current is not known. To address these issues, we formed giga-ohm seals on the luminal surface of split-open rat CCDs and obtained stable, whole cell K  currents on breaking through the apical patch. We isolated the basolateral component of whole cell K  current using either TPNQ or intracellular acidification to block ROMK, the predominant apical channel. The basolateral conductance was then characterized biophysically with the goal of determining its molecular identity.
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( W8 }- a+ d  y$ |, P  J! NPrincipal cells of rat CCD have a large whole cell K  conductance, most of which arises from the basolateral membrane. This conclusion is consistent with previous studies. Voltage divider experiments in perfused rat CCDs yielded a fractional apical resistance, with amiloride in the lumen, of 0.9 under control conditions (36). In the presence of amiloride, almost all of the current is presumably carried by K  as no significant Cl– conductance was observed in these experiments. This implies that 90% of the whole cell K  conductance arises from the basolateral membrane. We found that the majority of whole cell K  current remained despite exposure to TPNQ and intracellular acidification, maneuvers known to block ROMK (14, 23, 45, 51), the predominant apical K  channel. Specifically, following 2 μM TPNQ, a concentration over 100x the Ki for ROMK, 90% of whole cell K  current remained (Fig. 3D) and after pHi was lowered to 6.6, 60% remained (Figs. 4B and 5C) despite the fact that at pHi 6.6, a significant amount of basolateral current will be inhibited along with most of the ROMK current. Our pipette solution contained 5 mM EGTA, which precluded any apical contribution from the Ca2 -dependent BK channel. Finally, the calculated contribution of ROMK to whole cell K  conductance is small. The K  conductance through ROMK for a single principal cell using measured values of single-channel conductance, open probability, and channel density along with estimates of apical surface area is 16 nS on a high-K  diet with 40 mM K  in the lumen (APPENDIX). The whole cell K  conductance under these conditions is 140 nS (see High-K  diet studies above), which again supports the claim that most of the whole cell conductance arises from the basolateral membrane.) l  r  ?2 w9 |. P8 _: L

! v+ U$ v) k- ~' b& ^Basolateral K  conductance is under dietary regulation. Mean (inward) whole cell K  conductance was 76 ± 12 nS in rats on a control diet and 141 ± 22 nS in rats on a high-K  diet for 1–2 wk. Apical ROMK channel density has been reported to increase about three- to fivefold on a high-K  diet (27, 28). Despite this increase, the apical conductance on a high-K  diet was estimated to be only 16 nS as discussed above. This increase cannot account for the overall increase in whole cell conductance. This suggests that the basolateral conductance significantly increases in response to a high-K  diet. This increase is consistent with morphological measurements of the basolateral membrane following chronic K  loading that indicated a 50% increase in basolateral surface density (40).
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9 k2 |# N( c* k6 z  |* x* ]Biophysical characterization of the basolateral K  conductance. The basolateral component of whole cell K  conductance was isolated by taking advantage of the differential sensitivity of the basolateral and apical membranes to intracellular acidification. At pHi 6.6, most ROMK current is blocked given its pKa of 6.9. However, most of the basolateral current remains. The channels underlying the basolateral K  conductance were weakly inward rectifying at high external [K ] but became more strongly rectified as the bath [K ] was reduced (Fig. 7A). Addition of 1 mM spermine to the pipette increased rectification modestly but substantial outward current remained (Fig. 8). The channels were blocked by external Ba2  in a voltage-sensitive manner with an apparent Ki of 160 and 80 μM at –40 and –80 mV, respectively (Fig. 6B). The corresponding electrical distance was 0.23. They were insensitive to 10 mM TEA and 2 μM TPNQ in the bath. They had a permeability sequence of K  > Rb  > NH4  and an inward conductance sequence of K  > NH4  > Rb  (Fig. 9). They were not voltage activated, did not require intracellular Ca2  (EGTA was in the pipette), and were not inhibited by 3 mM intracellular ATP. Analysis of the Ba2 -induced noise yielded a single-channel conductance of 17 pS with 40 mM external K  (Fig. 11). These properties are summarized in Table 1.1 K- j, v2 N# ~+ K9 I

4 X9 T" }  _3 z/ S1 w# w* vView this table:. O- q: \) A$ X) R

9 K! E" h5 \, D7 q: `5 yHomogeneity of the underlying channels. Although several different channels have been reported from single-channel recordings of the basolateral membrane (11, 50) and, as will be discussed below, immunolocalization indicates that several cloned K  channels are present on this surface, there is also evidence that a single predominant channel may exist here. Of the two channels reported by Wang (48) from the basolateral membrane, both the open probability (0.8) and the open probability times the number of open channels (NPo) (2.5 ± 0.3) of the low-conductance (28 pS) channel were significantly greater than those of the intermediate-conductance (50 pS) channel (open probability = 0.2 and NPo = 0.4 ± 0.1). Our data are also consistent with the notion that a single predominant channel may underlie the basolateral conductance. Inhibition by internal pH (Fig. 4B) and block by Ba2  (Fig. 6B) were both described by a single binding isotherm. In addition, Ba2 -induced fluctuations were fit with a single Lorentzian component over the frequency range that we could study (Fig. 11). However, if two or more channels have similar sensitivities to pH and kinetics of Ba2  block, they would be indistinguishable with our analysis. The various parameters we have measured would then represent an average of the parameters of the individual channel(s).- ^/ ]/ \2 y- Y; B: ?

  f1 [1 u2 q! y; HComparison of our results with published single-channel measurements. We suggest that the low-conductance K  channels described by Hirsch et al. (11, 34) and Wang et al. (49, 50) represent the main K  conductance, at least under our recording conditions. This 27- to 28-pS channel reported by both groups is consistent with the 17-pS channel we described after correcting for the higher bath K  used in their recordings. In other words, because basolateral K  conductance increases by a factor of 1.7 in going from a bath K  of 40 to 140 mM (Fig. 7C), one would expect the single-channel conductance to increase from 17 to 29 pS as the bath K  is increased from 40 to 140 mM, assuming the increased conductance arises from increased permeation.
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8 N& p+ ^' d- J( j7 EMolecular candidates for the basolateral K  conductance. K  channels can be classified into voltage-gated, Ca2 -dependent, cyclic nucleotide-gated, two-pore (2P) domain, and inward-rectifying families (10). The lack of voltage activation and the robust currents in the setting of very low intracellular Ca2  suggest that the basolateral channels do not belong to either of the first two groups. No cyclic nucleotides were present in the bath. In addition, most cyclic nucleotide-gated channels lack high selectivity for K  over Na , whereas the basolateral channel is highly K  selective as evidenced by reversal potentials near the predicted Nernst potential for K  (Fig. 7B). A cGMP-activated K  channel was identified in human collecting duct and proposed as a candidate for the basolateral principal cell K  conductance (16). However, this channel is voltage gated and does not pass inward current, properties quite inconsistent with the basolateral conductance in our study.
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Recently, a PCR strategy revealed that 4 of the 14 known human 2P domain members are expressed at the mRNA level in collecting ducts: TASK-1, TASK-2, TWIK-1, and THIK-1 (19). TASK-1 (14 pS) and TASK-2 (60 pS) have Goldman-Hodgkin-Katz type rectification (18), displaying outward rectification at low extracellular/high intracellular [K ], in contrast to the increased inward rectification of the basolateral K  conductance with low bath [K ] (Fig. 7A). TWIK-1 is a 34-pS inward-rectifying channel sensitive to intracellular acidification in intact cells but not in excised patches (17, 18). It is present in the CCD by immunofluorescence but is restricted to intercalated cells (2). THIK-1 is weakly inward rectifying in symmetric K  but is outward rectifying at low bath [K ] (32). In addition, it was insensitive to intracellular acidification to approximately pH 6.4.
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Members of the Kir4 subfamily of inward rectifiers share several features of the basolateral principal cell K  conductance. Most of the seven subfamilies of inward rectifiers are unlikely candidates based on their biophysical properties and immunolocalization. ROMK (Kir1.1) underlies the predominant apical K  conductance but is distinguished from the basolateral conductance by its sensitivity to TPNQ and intracellular acidification in the physiological range. In addition, although a small amount of basolateral expression cannot be completely ruled out, most ROMK protein is localized to intracellular and apical compartments by immunofluorescence (24, 55). Kir2.3 has been proposed as a candidate for the predominant basolateral channel based on its basolateral localization in the CCD and similar permeability and conductivity sequences (52). However, the Kir2 (IRK) as well as Kir3 (GIRK) subfamilies have much stronger rectification than that seen with the basolateral principal cell K  current even with 1 mM spermine in the pipette (Fig. 8). In addition, the rectification we observe decreases markedly as bath [K ] is increased (Fig. 7). This phenomenon is not seen with Kir2.3 (52). Kir6.1 and Kir6.2 form channels with SUR or CFTR, which would be inhibited by the 3 mM ATP in our pipette (1). In addition, the single-channel conductances are 60–80 pS (1), significantly greater than that estimated for the basolateral conductance. Kir7.1 is a unique weak inward rectifier with single-channel conductance, from noise analysis, in the 50- to 200-fS range (15, 38). It has been localized to the basolateral membrane of CCD principal cells with immunogold labeling (25) but has a Rb  conductance almost 10x greater than that for K  (38), a property quite distinct from the basolateral K  conductance.
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Members of the Kir4 subfamily of inward rectifiers, Kir4.1 and Kir4.2, along with heteromultimeric channels formed by these channels in combination with Kir5.1 (43, 46), share several features of the basolateral principal cell K  conductance. These channels have weak-to-moderate inward rectification with single-channel conductances in symmetric K  of 20–27 (31, 41–43, 56), 25 (30), and 30–60 pS (43) for Kir4.1, Kir4.2, and Kir4.1/5.1, respectively. As discussed above, our measurement of the single-channel conductance underlying the basolateral K  current, extrapolated to symmetric bath K , is 29 pS, consistent with these values. These channels are all expressed in kidney and Kir4.1 and Kir4.1/5.1 have been shown by immunofluorescence to be present in the basolateral membrane of collecting ducts (13, 46).! ~* j' r  g  v3 ]( l; Y

/ e4 e: q6 j6 vSome discrepancies exist, however, between these cloned channels and the native K  conductance. Although Kir4.1, Kir4.2, and Kir4.1/5.1 are all acid sensitive, their pKas do not precisely correspond to the value of 6.5 measured for the basolateral K  conductance. Kir4.1 has a pKa of 6.0–6.1 (30, 37, 46, 53, 54, 56), Kir4.2 has a pKa of 6.7 (whole cell configuration) and 7.1 (excised patches) (30), and Kir4.1/5.1 a pKa of 6.8–6.9 in intact cells (30, 46) and 7.35 in inside-out patches (30). Also, while Kir4.1 is insensitive to TEA, Kir4.2 displays weak TEA sensitivity, with a Ki of 10 mM at –60 mV (29). Finally, all outward K  current is abolished when patches from X. laevis oocytes expressing Kir4.1 are excised into 10 μM spermine (7, 56). With 1 mM spermine in the pipette, we observed a modest decrease in outward whole cell K  current but most of this current remained (Fig. 8).2 {4 B3 E% G, l' O
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In conclusion, using intracellular acidification, we isolated the basolateral component of whole cell K  current in principal cells of the CCD and characterized it electrophysiologically. The basolateral current arises from a 17-pS, acid-sensitive K  channel whose inward rectification increases with decreasing bath [K ]. The basolateral K  conductance exhibits features of inward rectifiers in general, including (mild) inward rectification, pH sensitivity, and activation of outward currents by extracellular K . Several properties of the conductance are shared by the Kir4 subfamily of channels, but a complete match with a cloned K  channel cannot be made. This suggests that the native conductance may arise from heteromultimers or association with accessory proteins.
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: D; @! C! i( V8 `. W* l# nAPPENDIX
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Estimate of Apical K  Conductance Through a Single Principal Cell$ y# h% ]6 _& {7 R; @/ r& h
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The single-channel ROMK conductance (inward direction) with 40 mM K  in the lumen is 45 pS and the open probability is 0.9 (Ref. 8 and D. A. Gray, G. Frindt, and L. G. Palmer, unpublished measurements). ROMK channel density ranges from 0.4 channels/μm2 on a control diet to 2.1 channels/μm2 on a high-K  diet (28). Thus the apical K  conductance on a high-K  diet is 85 pS/μm2. To estimate the apical surface area of a principal cell, we consider the CCD luminal surface to be a cylinder of radius 15 μm. Then, for a 1-mm length of tubule, the apical surface area is 94,248 μm2. There are 509 cells/mm of CCD (5), two-thirds of which are principal cells. If all cells had the same apical surface area, then the apical surface area per cell would be 185 μm2. This yields apical conductances of 3 and 16 nS per principal cell on control and high-K  diets, respectively. The corresponding inward current at a given voltage can be calculated from these conductances and the driving force as the I-V curve is fairly linear here (Ref. 8 and D. A. Gray, G. Frindt, and L. G. Palmer, unpublished observations). Thus, for example, at –80 mV, with 40 mM K  in the bath, the expected reversal potential is –35 mV, resulting in a driving force of –45 mV. This yields ROMK currents of 0.14 and 0.72 nA/cell on control diet and high-K  diets, respectively.
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GRANTS; `5 v, ~$ {& x7 N

+ j4 n' |$ [+ z& l: U0 r* IThis work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-27847 and a postdoctoral fellowship award from the Howard Hughes Medical Institute (to D. Gray).  S6 j5 H) G5 Y* F: y

0 `; F- L- G* J6 c4 CFOOTNOTES
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8 R- L3 M" t; c# y; g. NThe 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.
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awen

自己知道了  

yukun

原来这样也可以  

泡泡鱼

不要等到人人都说你丑时才发现自己真的丑。  

兔兔

长时间没来看了 ～～  

命运的宠儿

支持一下吧  

tuanzi

干细胞存储  

awen

厉害！强~~~~没的说了！  

昕昕

老大,我好崇拜你哟  

s06806

朕要休息了..............