INTRODUCTION

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The ability of the renal proximal tubule to maintain homeostatic electrolyte and water reabsorption in the face of drastic changes in dietary solute and water intake and renal hemodynamics implies that transtubular ion transport is tightly regulated. Proximal tubule potassium channels, particularly in the basolateral membrane (BLM),¹ play pivotal physiologic roles in the regulation of membrane voltage, potassium recycling, and ultimately in transepithelial solute and water reabsorption. That the basolateral membrane potential of a proximal tubule cell is dominated by the BLM K conductance is well established (Sackin and Boulpaep, 1983). The Na⁺,K⁺-ATPase pump in the BLM provides the energy that makes ion transport thermodynamically favorable, but continuous operation of the pump requires there be a K⁺ exit pathway. The BLM K conductance provides such a pathway and thus steady state intracellular K activity can be maintained in the face of large transcellular fluxes of salt and water (Sackin and Boulpaep, 1983). Since a major portion of the transcellular Na⁺ flux is reabsorbed across the BLM by the action of the Na⁺,K⁺-ATPase, a population of BLM K⁺ channels working in concert with the pump would allow K⁺ to recycle in a regulated fashion. Moreover, hyperpolarization secondary to the opening of BLM K⁺ channels enhances the driving force for electrogenic apical Na⁺ entry and basolateral Cl efflux, resulting in net NaCl reabsorption.

The Ambystoma proximal tubule exhibits a large K⁺ conductance in the basolateral membrane (Siebens and Boron, 1987; Sackin and Boulpaep, 1981) that has been shown macroscopically to be sensitive to barium and pH. However, previous studies of the "macroscopic" BLM K⁺ conductance (Boulpaep, 1976) lack the resolution to distinguish whether there is one population of imperfectly selective K⁺ channels or a set of highly selective K⁺ channels coexisting with a population of nonselective cation channels. More recent "microscopic" single-channel patch-clamp studies of BLM K⁺ channels have shown diversity in both experimental design and findings (Tsuchiya et al., 1992; Hunter, 1991; Parent et al., 1988; Kawahara et al., 1987; Sackin and Palmer, 1987; Gögelein and Greger, 1987a, 1987b), so a clear consensus has been elusive and details of the properties and regulation are lacking.

We have now characterized the properties and regulation (see Mauerer et al., 1998) of the principal K⁺ channel in the BLM in a preparation of dissociated yet polarized Ambystoma proximal tubule cells (Segal et al., 1996). Inwardly rectifying, ATP-sensitive K⁺ channels were present in >95% of recordings from the BLM, each containing from 2 to >25 K_ATP channels/patch. Although the regulation of this proximal tubule BLM K⁺ channel is similar to that of recently cloned K_ATP channels in the apical membrane of the distal nephron (ROMK1 and ROMK2), there are important differences, and ROMK has not been found in the proximal tubule (Chepilko et al., 1995; Zhou et al., 1994; Lee and Hebert, 1995; Boim et al., 1995; Ho et al., 1993). The studies reported in this paper and the companion paper elucidate the properties and the regulation, respectively, of the major K⁺ channel underlying the BLM K conductance that is coupled to transport in the proximal tubule.

	MATERIALS AND METHODS

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Solutions and Drugs

The compositions of the solutions used are summarized in Table I. After titration to pH 7.5 (710A; Orion Research, Boston, MA), sucrose was added to adjust the osmolality of the solutions (3MO; Advanced Instruments Inc., Needham Heights, MA). To determine channel conductance as a function of [K⁺], sucrose was added to the standard pipette KCl to maintain osmolality. Chemicals used were of the highest quality and obtained from Sigma Chemical Co. (St. Louis, MO), except thallium acetate (Aldrich Chemical Co., Milwaukee, WI), diazoxide (Calbiochem Corp., La Jolla, CA), ATPS, and ADP (Boehringer-Mannheim Biochemicals, Indianapolis, IN). Nucleotides were prepared fresh daily as 20-50-mM stocks in bath solution. Glibenclamide was dissolved in DMSO (100 mM stock).

Cell Preparation

Dissociated proximal tubule cells were isolated from amphibian kidneys as previously described (Segal et al., 1996). Briefly, aquatic phase Ambystoma tigrinum kept at 4°C were killed by submersion in 0.2% tricaine methanesulfonate. The kidneys were rapidly removed and placed in iced HEPES-buffered NaCl at pH 7.5 (solution a). The adventitial tissue was removed by hand dissection, and the renal tissue was cut into 1-2 mm³ pieces and incubated in collagenase-dispase (0.2 U/ml of collagenase; Boehringer-Mannheim Biochemicals) on a gyratory shaker for 60 min at 22°C. The enzyme reaction was stopped by washing with Ca²⁺- and Mg²⁺-free NaCl (solution b). The cells were then mechanically dispersed into suspension by repeated trituration, and a pellet was obtained by centrifugation at ~1,600 rpm for 3 min. Finally, the cells were resuspended in 2.5 ml NaCl (solution a) in a 35-mm culture dish, and stored at 4°C until use. The dissociated proximal tubule cells can retain their epithelial polarity for up to 14 d (Segal et al., 1996). Cells were used for experiments from 2 to 12 d after dissociation. Representative cells as seen under light microscopy (Fig. 1 A) and scanning electron microscopy (Fig. 1 B) are shown (for details of methods see Segal et al., 1996).

View larger version (106K): [in this window] [in a new window]   Fig. 1.   Dissociated Ambystoma proximal tubule cells retain epithelial cell polarity. (A) Light photomicrograph of a single dissociated Ambystoma proximal tubule cell shows distinct apical and basolateral membrane surfaces. The apical surface of these bilobated cells is the smaller lobe with a microvillar brush border. The robust cytoskeleton of these cells includes an actin-rich "waist-band" (arrowheads) between the two membrane domains that is important in retention of epithelial polarity. Giga-ohm seals can be made on both surfaces. Scale bar, 10 µm. (B and C) Scanning electron micrographs showing the sharp transition between the membrane domains and the detailed topology of the apical surface invested with its microvillar brush border, and the basolateral surface with its folds and projections. Scale bars: 10 µm in B, 1 µm in C.

Electrophysiology

A 5-µl aliquot of cell suspension in NaCl storage solution (solution a) was placed on a Cell-Tak^TM-coated glass coverslip in a recording chamber of our design (RC-5/25; Warner Instruments, Hamden, CT) mounted on an inverted microscope (Olympus IM; Olympus America, Inc., Melville, NY). The chamber has a bath volume of 500 µl, and solutions are perfused directly into an input multiplexer on the chamber at a gravity-driven flow rate of ~10 ml/min.

Nonadherent cells were washed off the coverslip with the recording solution (either Ca²⁺-free NaCl, solution c, or KCl, solution d, unless otherwise noted) and the cells were visualized under Hoffman modulation optics (Modulation Optics, Greenvale, NY). Proximal tubule cells were readily recognized by their characteristic morphology (see Fig. 1; Segal et al., 1996). An individual proximal tubule cell was selected for an experiment only if it fulfilled the following criteria (Segal et al., 1996): (a) distinctly bilobated structure, (b) clearly defined brush border sharply delimited on the apical surface, (c) relatively smooth appearing basolateral membrane, and (d) absence of large vacuoles.

Patch clamp. The standard configurations for single-channel and whole-cell tight seal patch-clamp technique (Hamill et al., 1981) were used to record channel currents from the BLM. Patch pipettes were fabricated from borosilicate glass capillaries (Warner Instruments, Hamden, CT) on a two-step puller (PP-83; Narishige Co., Ltd, Tokyo, Japan), coated with Sylgard 184^TM (Dow-Corning Corp., Midland, MI) to within 200 µm of the tip, and fire-polished just before use. When filled with KCl, the open tip pipette resistance was 3-8 M when placed in the initial bath solution. A hydraulic micromanipulator (Narishige) was used to guide the patch microelectrode to the BLM of the cell. High resistance giga-ohm seals (up to 50 G) were obtained on the BLM in ~75% of attempts by applying gentle suction to the pipette just after it touched the cell membrane. To achieve the whole-cell configuration, further suction was applied to rupture the cell-attached patch. Data have not been corrected for liquid-junction potentials since for most solutions they were <4 mV when measured as follows: the bath Ag-AgCl ground electrode was connected to the control KCl bath through a 3% agar bridge made of KCl pipette solution. A low resistance (<1 M) pipette filled with 3 M KCl was placed in a KCl bath and the DC offset was adjusted to 0 mV in zero current clamp. The liquid-junction potentials were measured as the voltage offset resulting when the control KCl bath was replaced by the test solution. Low [Cl] solutions in which 90% of Cl was replaced by aspartate had a liquid-junction potential of 13.3 mV.

Data Analysis

Current data were played back and low pass filtered at 400 Hz (902LPF eight-pole Bessel filter; Frequency Devices Inc., Haverhill, MA), digitized at 1,000 samples/s, and stored on the PDP-11/ 23. In some cases, currents were filtered at 40 Hz and digitized at 100 samples/s for current binning and averaging analysis. Datafiles were transferred to a Pentium computer (Gateway 2000, North Sioux City, SD) via Kermit (Columbia University, NY) for analysis. Custom software for data acquisition and analysis was written in our laboratory using BASIC-23, AxoBASIC 1.0 (Axon Instruments, Foster City, CA), and Matlab 4.0 (The Mathworks, Natick, MA).

Channel activity (nP_o) was calculated over periods of 60-500 s as follows. The closed current level (i_c) was taken as the mode of the distribution around closed events. This current was subtracted from the current of a given bin, and the difference was multiplied by the number of events in that bin. The sum of these products yields the open channel area of the histogram. nP_o is given by dividing the open channel area by the single-channel current, i_sc. That is,

(1)

For kinetic analysis, currents were filtered at a corner frequency (f_c) of 2 kHz and sampled at 5,000 s¹. An event (transition) was counted each time a data point crossed the 50% level of the unitary channel current. For our recording system, the patch-clamp has a 5-kHz step response, a 5-kHz tape bandwidth, and a 2-kHz eight-pole Bessel filter, yielding an effective bandwidth (3 dB point, f_ceff) of 1.74 kHz. With these settings, the "dead time" of the recording system is given by Colquhoun and Sigworth (1983), T_dead = 0.179/f_ceff. The "50% delay time" of the Bessel filter is T_50% = 0.506/f_c. For f_ceff = 1.74 kHz and f_c = 2 kHz, T_dead = 102.8 µs and T_50% = 253 µs. Therefore, events in time histogram bins <500 s were cut off. Since an event lasting 253 µs would be the margin of detection, all dwell lifetimes <253 µs would be missed events.

Open and closed dwell-time kinetics were fit to a probability density function expressed as a sum of exponentials,

(2)

where n is the number of open or closed states, the A_i are the relative amplitudes, and the i are time constants. This function was transformed according to x = ln(t) and logarithmically binned (Sigworth and Sine, 1987), such that

(3)

This function was used to fit the data using the Levenberg-Marquardt nonlinear least-squares fitting algorithm (Origin 4.0; Microcal Software, Inc., Northampton, MA) to find the appropriate set of 's. Note that if the errors follow a Gaussian distribution, this nonlinear least-squares method is equivalent to the method of maximum likelihood (Colquhoun and Sigworth, 1983).

Dose-response relations for a drug (D) were fitted to the Hill equation as

(4)

where I/I_max is the fractional inhibition, K_i is the concentration of drug giving 50% inhibition, [D] is the concentration of the drug, and n_H is the Hill coefficient. When n_H = 1, this equation reduces to the Langmuir adsorption isotherm.

In the text, the number of observations or experiments is reported, whereas n in the analysis denotes either the whole data set or the subset of total experiments in which precise quantitation could be reliably applied. In some figures, a running average (using a specified window width) of current versus time is displayed. Statistical values for the n elements are given as mean ± SEM. Student's t test was applied where appropriate.

	RESULTS

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Properties of the BLM K⁺ Channel

Overview. With [K⁺] = 95 mM in the patch pipette, we found the K⁺ channel in 551 of 559 seals made on the BLM (98.6%). This K⁺ channel was never detected in seals made on the apical membrane (0 of 16, 0%), consistent with our previous finding that dissociated Ambystoma proximal tubule cells retain epithelial cell polarity (Segal et al., 1996). Seals on the BLM typically contained from 2 to >25 K⁺ channels. Despite numerous attempts to minimize patch area using small-tipped pipettes (resistance  30 M), patches appearing to have one and only one channel were very infrequent (n = 4, only 0.7%).

Cell-attached patches. When cell-attached (c/a) patches were made in NaCl bath, spontaneous inward K⁺ currents were usually observed (95%) at 0 mV (V_pip) command potential. By briefly switching to zero current clamp mode, the resting membrane potential (V_m) of the cell can sometimes be estimated if R_seal >> R_patch. Using this method under these conditions, V_m averaged 37.2 ± 2.1 mV (n = 16), in good agreement with V_m = 40 mV as measured by conventional impalement (Segal et al., 1996). The V_m of the dissociated cells is ~15-20 mV less than that for a cell in the intact tubule (Sackin and Boulpaep, 1983), suggesting an anion conductance exists at either the apical or basolateral membrane. Indeed, we have characterized a cAMP-activated Cl channel in the BLM of these cells, which often appears in the same membrane patch as the K⁺ channel (not shown). Alternatively, the isolated cells may have acquired a nonspecific leak pathway that shunts the normally high K diffusion potential. Since the K⁺ and Cl activity of the pipette solution (aP_K = 0.80*[95] = 76 mM and aP_Cl = 0.78*[92] = 71.8 mM) is greater than the intracellular K⁺ and Cl activity (aⁱ_K = 0.80*[68] = 54.4 mM and aⁱ_Cl = 0.78*[20.5] = 16 mM (Sackin and Boulpaep, 1983), yielding a reversal potential (E_rev) of 28.7 mV. Thus, at V_pip = 0 mV, the inward current must be carried by K⁺ moving down its electrochemical gradient from the pipette into the cell.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   The BLM K+ channel appears to be an inward rectifier. (A) Representative current records at various command potentials (Vpip) from a cell-attached basolateral membrane patch containing at least two K+ channels. Note the increase in channel activity with hyperpolarization. The patch pipette contains 95 mM K+ (solution d) and the bath is NaCl (solution c). The dashed line represents the all channels closed (leak) current at each potential. Each open channel level is denoted by a dotted line. (B) Current-voltage relation for the BLM K+ channel for the conditions described in A. The limiting inward slope conductance is 22.2 ± 1.4 pS (n = 8) and the outward chord conductance 3.5 ± 0.1 pS (between 120 and 180 mV, n = 5). Symbols represent mean () ± SEM (bars).

Excised patches. When BLM membrane patches were excised in the inside-out (i/o) configuration into the standard bath solutions (solution c or d), channel activity typically began to decline and then disappear. Addition of 0.2 mM ATP to the bath before or just after patch excision prevented rundown and maintained channel activity indefinitely.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   The BLM K+ channel is a true inward rectifier. Representative current records at various command potentials (Vpip) from an inside-out basolateral membrane patch in symmetrical [K] containing at least three K+ channels. The patch pipette and bath each contain 95 mM K+ (solution d) with 0.2 mM ATP added to the bath. The dashed line represents the all channels closed (leak) current at each potential. Each open channel level is denoted by a dotted line. (B) Effect of [Mg2+]i on the I-V relation of the BLM K+ channel. The inward rectification evident in 1 mM [Mg2+]i () is relieved when [Mg2+]i is lowered to 200 nM (). (inset) Slope conductance-voltage (g-V) relation for the BLM K+ channel in 1 mM [Mg2+]i () and 200 nM [Mg2+]i (). Symbols represent mean ± SEM. The K+ channel prefers Tl+ over K+. The I-V relation from inside-out patches with Tl-acetate (solution g) in the pipette and K-acetate (solution h) in the bath is also shown (). The limiting inward slope conductance is 29.0 ± 1.0 pS (n = 4) for Tl+ compared with 24.5 ± 0.6 pS (n = 8) for K+. (C) Channel activity (nPo) increases with hyperpolarization. Data from four inside-out membrane patches in symmetrical [K] are plotted. Channel activity at command potentials of 120, 100, 80, 60, and 40 mV was normalized to that at 100 mV for comparison. Solid line is a single exponential fit with a voltage constant of ~83 mV. Symbols represent mean () ± SEM (bars).

Voltage dependence of nP_o. Since BLM membrane patches almost always contained more than one K⁺ channel, nP_o (channel activity) was used to assess the relative voltage dependence of P_o. This assumes that the number of active channel proteins in an excised patch remains constant as voltage varies. (We take n as the maximum number of simultaneously open channels observed, which places a minimum on the actual number of channel proteins in the patch.) As was the case for cell-attached patches, channel activity with KCl on both sides of the excised membrane patch increases with increasing hyperpolarization (Fig. 3 C). Although the absolute values of nP_o differed significantly among patches, relative nP_o increased e-fold per ~83-mV hyperpolarization between 40 and 120 mV, reflecting a 27% increase of nP_o for every 20 mV of hyperpolarization.

Role of magnesium. It has been shown that [Mg²⁺]_i mediates at least part of the inward rectification in other inwardly rectifying K⁺ channels (Matsuda et al., 1987; Horie et al., 1987; Ficker et al., 1994) by blocking outward currents in a voltage-dependent manner. When Mg²⁺_i was removed from the "cytosolic" side of i/o patches (solution e plus 0.2 mM Na₂ATP), we observed flickering, and then rundown of the channel. This is in sharp contrast to the behavior of ROMK1 channels, in which channel rundown is slowed in a Mg²⁺-free bath (McNicholas et al., 1994). Since rundown occurs in the absence of free Mg²⁺_i and the presence of 0.2 mM Na₂ATP (n = 4), it appears that at least the complex of Mg-ATP is required to prevent rundown. Interestingly, it has been shown that both Mg-ATP and free Mg²⁺_i are required to sustain channel activity for an ATP-insensitive inward rectifier K⁺ channel (Fakler et al., 1994). However, since higher levels of ATP block the BLM K⁺ channel (see below), it is not possible for us to dissociate the role of free Mg²⁺_i from that of the Mg-ATP complex. Millimolar concentrations of ATP (or indeed, any nucleotide) block the BLM K⁺ channel, and this effect is independent of both free Mg²⁺_i and Mg-ATP.

(5)

(6)

Concentration and voltage dependence of i_sc. To further investigate the biophysical properties of this channel, we asked the question of how varying extracellular [K⁺] would affect the current carried by the channel. To isolate the change in [K⁺] from any change in driving force across the patch, the same [KCl] was used in the pipette and bath, thus clamping E_rev to 0 mV. This approach allowed us to measure the change in absolute conductance while maintaining a constant relative permeability (i.e., we varied the Goldman-Hodgkin-Katz current equation while holding the result of the Goldman-Hodgkin-Katz voltage equation constant). Under these conditions, the command potential is the only driving force for net K⁺ movement across the membrane patch. Pipette and bath [KCl] ranged from 5 to 205 mM while osmolality was kept at 400 mosm/kg using sucrose as necessary. Since i_sc for [KCl] = 95 mM was the same in both standard KCl solution (solution d, 200 mosm/kg) or 400 mosm/kg KCl solution, the tonicity change itself does not significantly alter the conducting properties of the channel.

(7)

View larger version (12K): [in this window] [in a new window]   Fig. 4.   The operating surface of the BLM K+ channel. (A) The single channel conductance () at a command potential of 100 mV plotted as a function of [K+] and fit to the Hill equation according to is the maximal value of the conductance, Kd is the apparent dissociation constant, and nH is the Hill coefficient. For the chord conductance at 100 mV () and its least-squares fit (solid line), max,slope = 34.3 pS, Kd = 77 mM, and nH = 0.94. (B) The plot of Kd versus Vpip shows that the apparent binding-unbinding rate of K+ to the channel is voltage dependent. The data () were fit with a single exponential (solid line) as follows: Kd = 73 + 141 · exp(Vpip/30).

(8)

d,

d,

Cation selectivity. The cation to anion preference of the total BLM conductance was determined by salt dilution experiments using i/o membrane patches. Starting in symmetrical 95 mM K⁺ and 94.5 mM Cl (E_K = E_Cl = 0 mV) and holding at V_pip = 0 mV, the bath was changed to a 14 mM K⁺ and 13.5 mM Cl solution (solution i, E_K = +48.6 mV, E_Cl = 49.4 mV) plus sucrose to maintain isoosmolality. This maneuver resulted in large inward currents (n = 3), reflecting the cation (K⁺) moving down its chemical gradient. The reversal potential for this membrane current (including leak) was at least +40 mV (n = 3). While holding at this E_rev, outward current developed when the 10% KCl bath was replaced with 100% KCl, due to K⁺ moving along its electrical gradient. Thus, the BLM conductance is cation selective.

(9)

1

1

1

Selectivity among cations. Two approaches were used to determine the selectivity of this inwardly rectifying K⁺ conductance on the BLM. (a) Using c/a and i/o patches, the K⁺ in the patch pipette was replaced with the chloride salt of Na⁺ (n = 48), Rb⁺ (n = 4), Li⁺ (n = 4), Cs⁺ (n = 2), or NH¹₄ (n = 7). Each solution was adjusted to pH 7.5 with the respective hydroxide salt. In all cases, c/a and i/o patches failed to show inward channel currents. These results strongly suggest that the BLM K⁺ channel is highly selective for K⁺ and excludes these cations, since channel activity is seen in >98% of seals made on the BLM when K⁺ is in the pipette. (b) Outside-out patches were made to exclude the remote possibility that the c/a and i/o patches used above did not contain any channels. The pipette was filled with KCl, and 0.2 mM Mg-ATP was included to prevent channel rundown. After recording in a KCl bath, the test cation was introduced into the bath (as the chloride salt) and the voltage protocol was repeated. KCl bath exchanges were interposed between test cations.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   The BLM K+ conductance is highly selective for K+. The cationic selectivity of the BLM is demonstrated in this outside-out patch with KCl (solution d) + 0.2 mM ATP in the pipette and Cl-salt of the test cation in the bath. The running average of current at 40 mV shows that cationic selectivity is K+ >> Rb+  Cs+  NH4+ > Na+  Li+. The dashed line is the zero-current line, and the dotted line is the all channels closed (leak) current at a command potential of 40 mV. The voltage protocol is indicated below the current data.

Thallium. Many types of K⁺ channels have been shown not only to conduct Tl⁺, but often better than they conduct K⁺ (Hille, 1992). To assess the Tl⁺ conductance of the BLM K⁺ channel, the patch pipette was filled with 90 mM Tl-acetate (solution g ; the Cl salt of Tl⁺ was not used due to its low aqueous permeability) and the bath was filled with K-acetate (solution h). Under these conditions, inward Tl⁺ currents were observed in both c/a and i/o patches. The kinetic behavior of these channel events was notably different from those seen when the channel conducts K⁺. When conducting Tl⁺, the channel openings displayed more bursting, with each opening interrupted by fast flickery closures. The probability that this is actually a different channel is low since (a) the frequency of finding the K⁺ channel exceeds 98%, (b) other cation-selective channels were rarely observed, (c) the disparate kinetics were only seen when Tl⁺ was in the pipette, and (d) Tl⁺ currents were sensitive to glibenclamide (see below).

Kinetics. Due to the high density of this K⁺ channel in the BLM, a patch apparently containing only one channel is extremely rare. In over 550 seals, only four membrane patches appeared to contain only one channel (0.7%). Since the open probability (P_o) of the channel is only 0.05 ± 0.01 (n = 4), long recordings were required to accumulate enough transitions for meaningful analysis of the long closed state. Kinetic analyses from such patches show that under resting state conditions at V_pip = 60 mV, the BLM K⁺ channel has two apparent open states and two apparent closed states. Parameters from one c/a patch and one i/o patch show that the open dwell lifetimes are (ms): _{o1 =} 0.78 (c/a), 1.21 (i/o), and _o2 = 4.7 (c/a), 6.6 (i/o). The closed dwell lifetimes are (ms): _c1 = 1.27 (c/a), 0.72 (i/o), and _c2 = 397 (c/a), 502 (i/o). Fig. 6 shows the open and closed time histograms for a c/a patch.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Kinetics of the BLM K+ channel. (A) Open-time histogram for the BLM K+ channel in a cell-attached patch at 60 mV with the time intervals logarithmically binned (Sigworth and Sine, 1987). The data were fitted with two probability density functions (dashed lines) to give the overall fit (solid line), yielding time constants of o1 = 0.78 ms (78%) and o2 = 4.7 ms (22%). Based on the bandwidth of the recording system, the data and the fit were cutoff at 500 µs (vertical dotted line). (B) Closed-time histogram for the BLM K+ channel in a cell-attached patch at 60 mV with the time intervals logarithmically binned. The data were fitted with two probability density functions (dashed lines) to give the overall fit (solid line), yielding time constants of c1 = 1.27 ms (74%) and c2 = 397 ms (26%). Based on the bandwidth of the recording system, the data and the fit were cutoff at 500 µs (dotted vertical line).

Inhibitor Profile

The BLM K⁺ channel is inhibited by barium and glibenclamide. The channel is insensitive to tetraethylammonium (up to 10 mM) applied either to the extracellular or cytoplasmic side.

Barium. We have previously shown by recording single-cell membrane potential that the whole cell conductance is dominated by a barium-sensitive K⁺ conductance (Segal et al., 1996). Perforated patch whole-cell recordings show that the barium-sensitive whole cell conductance inwardly rectifies. When 2 mM Ba²⁺ was included in the KCl pipette solution (unpaired experiments), channel openings were rare and a flickery state was noted. In contrast to the voltage dependence of channel activity without Ba²⁺ in the pipette (see Fig. 3 C), steady depolarization now has the effect of increasing nP_o, and subsequent hyperpolarization reduced activity. This is presumably due to the voltage dependence of the Ba²⁺ block of the channel (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Inhibitors of the BLM K+ channel. (A) Dose- response curve for inhibition of the BLM K+ channel by external Ba2+. Relative nPo (nPo/nPo,control) determined from outside-out patches at a command potential of 40 mV with KCl in the pipette and bath (solution d) is plotted versus [Ba2+]. The data at 40 mV were fitted with the Hill equation (solid curve), yielding a Ki = 460 µM and nH = 0.90. The inhibition by Ba2+ is fully reversible. (B) Glibenclamide inhibits the BLM KATP channel. (top) A running average (window width 16 ms) of current versus time. Glibenclamide (500 µM) is added to the bath where indicated. (bottom) Sample traces from a representative experiment at 60 mV with KCl in the pipette and bath (solution d). Exposure of this inside-out patch to 500 µM glibenclamide reversibly decreases nPo by ~50%. The dashed line denotes the all channels closed (leak) current level and the dotted lines indicate each open channel level.

Glibenclamide. This sulfonylurea is known to inhibit ATP-sensitive K⁺ channels in a number of epithelial tissues by binding to the sulfonylurea receptor (SUR). SUR1 has recently been cloned (Aguilar-Bryan et al., 1995) and is thought to associate with the K_ATP channel, thereby conferring sulfonylurea sensitivity. However, SUR1 may not be present in the kidney (Inagaki et al., 1995), which may explain in part the much higher dose of glibenclamide required to inhibit renal K_ATP channels (Hebert and Ho, 1994). In this context, the BLM K⁺ channel is glibenclamide sensitive, albeit at "renal doses." We treated 22 patches with glibenclamide; 16 excised inside-out patches were exposed to 500 µM, while 6 cell-attached patches were exposed to low (0.01-10 µM) concentrations. In 7 of 16 inside-out patches, 500 µM glibenclamide inhibited activity by 42.3 ± 5.6% (Fig. 7 B). Remarkably, the inhibition was much more potent in the cell-attached patches: 10 µM exerted an 83 ± 2% inhibition in three patches, and 100 nM exerted a 70 ± 3% inhibition in three other patches (data not shown).

ATP Sensitivity

Similar to other K_ATP channels, low doses of ATP are required to prevent Ambystoma BLM K⁺ channel rundown, whereas millimolar doses inhibit channel activity. At a dose of 5 mM ATP, >90% of channel activity is inhibited. This effect is reversible as nP_o returns to baseline when the bath [ATP] is returned to 0.2 mM (Fig. 8 A). The dose-response curve for ATP has a K_i ~ 2.4 mM (Fig. 8 B). The Hill coefficient of ~4 may suggest that the channel has a tetrameric structure, with each subunit possessing an ATP binding site. Thus, in the Ambystoma proximal tubule, the BLM K⁺ channel that appears to be the major K⁺ conductance of the cell is ATP sensitive.

View larger version (39K): [in this window] [in a new window]   Fig. 8.   Nucleotides reversibly inhibit the BLM K+ channel. (A) ATPi reversibly inhibits the BLM K+ channel in inside-out patches. In the experiment depicted, up to 13 channel open levels are seen under control conditions (top) at 60 mV with KCl (solution d) in the pipette and NaCl (solution c) plus 0.2 mM ATP in the bath. (middle) Addition of 5 mM ATP to the cytoplasmic side almost completely blocks channel activity (98% decrease) with only rare openings to one open level. (bottom) The inhibition is readily reversed upon returning to 0.2 mM ATPi. (B) Dose-response curve for inhibition by ATPi. The inhibitory effect of ATPi was determined in inside-out patches under the conditions described in A. Relative nPo (nPo/nPo,control) is plotted versus [ATP]i. The data () was fitted with the Hill equation (solid line) yielding a Ki ~ 2.4 mM and nH = 3.95. Adenosine nucleotides and nucleoside triphosphates (each at 5 mM) inhibit the BLM K+ channel. The inhibitory effect of ATP, ADP, AMP, and adenosine was determined under the conditions described in A. Values plotted are average (nPo,test/nPo,control) SEM (n = 3-9). The rank order of inhibition is ATP (93.3%) > ADP (65.6%) > AMP (38.7%). Adenosine has no inhibitory effect on nPo of the BLM K+ channel. Although all the nucleoside triphosphates inhibit the channel, ATP exerts a significantly stronger block than the other NTPs tested (P < 0.02). There is no significant difference among the other NTPs.

Among the nucleoside diphosphates, ADP is less potent than ATP (ADP inhibits by 65.6 ± 8.1%, n = 4, P < 0.01), but more potent than CDP, GDP, IDP, TDP, or UDP (data not shown). This suggests that the putative nucleotide binding site(s) recognize NDPs as well as NTPs, and that nucleotide hydrolysis is probably not occurring at this site. Indeed, even nucleoside monophosphates have a moderate inhibitory effect, although nucleosides themselves are without effect. The relative potency of the adenosine nucleosides (at 5 mM) in inhibiting the BLM K_ATP channel is ATP (93.3 ± 1.9%) > ADP (65.6 ± 8.1%) > AMP (38.7 ± 3.7%) > adenosine (1 ± 2%) (Fig. 8 C, n = 3-9).

Other nucleotides. The effect of nucleotides was tested in excised i/o patches. All the NTPs tested reversibly inhibited BLM K⁺ channel activity at a dose of 5 mM (ATP 93.3 ± 1.9%, n = 9; CTP 70.3 ± 10.0%, n = 5; GTP 62.3 ± 7.5%, n = 3; ITP 61.1 ± 0.8%, n = 2; TTP 53.7 ± 6.7%, n = 2; UTP 71.7 ± 5.7%, n = 2) (Fig. 8 C). These results suggest that each compound probably interacts with common cytoplasmic nucleotide binding site(s). Note that ATP is significantly more potent than the other nucleoside triphosphates (P < 0.02), but there is no significant difference among the nonadenosine nucleotides.

Rundown of the BLM K⁺ Channel

One characteristic of K_ATP channels is "rundown," a gradual loss of activity when the membrane patch is deprived of cytosolic ATP (Findlay and Dunne, 1986). Typically, both Mg²⁺ and ATP are required to prevent rundown in K_ATP channels (Ashcroft and Ashcroft, 1990). Likewise, the BLM K⁺ channel runs down in the absence of either Mg²⁺ or ATP (or both). Lower concentrations of ATP (100-200 µM) will prevent or "rescue" channel rundown. The experiment shown in Fig. 9 A summarizes the characteristics of BLM K⁺ channel rundown. Channel activity typically begins to decrease (rundown) upon excision of the membrane patch into a nucleotide-free bath. If this process is allowed to continue, channel activity will cease, usually irreversibly. When 0.2 mM of ATP is added back, channel activity can be restored. When ATP is removed, all channels rapidly close. In the continued presence of ATP-S, readdition of ATP is again able to rescue rundown, and activity returns to baseline upon washout of the ATP-S. Frequently (but not invariably), ATP-S has an inhibitory effect on single channel activity when added in the presence of ATP, which is reversible as long as the exposure is not prolonged (n = 6), as shown in Fig. 9 B. When ATP-S is added in the absence of ATP, channel activity runs down very quickly, usually irreversibly (n = 4, data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 9.   ATP but not ATP-S prevents and rescues channel rundown. Hydrolyzable nucleoside triphosphates prevent and rescue BLM K+ channel rundown. A running average (current versus time, window width 768 ms) of a representative experiment is shown. The pipette is KCl (solution d), the bath is NaCl (solution c), the command potential is 60 mV. Upon excision in a nucleotide-free bath, channel activity decreases (channel rundown). After the addition of 0.2 mM ATP to the bath, channel activity slowly recovers. The addition of 0.2 mM ATP-S (a poorly hydrolyzable ATP analogue) in the continued presence of 0.2 mM ATP has no effect. However, when ATP is removed, ATP-S is not able to support channel activity, which rapidly declines and runs down. Readdition of ATP leads to full recovery of channel activity. Single-channel traces showing that ATP-S has an inhibitory effect on KATP channel activity in excised inside-out BLM patches. When compared with control conditions (top), the addition of ATP-S (middle) reduces nPo. This inhibition is reversible as long as the exposure to ATP-S is not prolonged (bottom). (C) Removal of Mg2+ does not prevent channel rundown in an ATP-free bath. The top panel shows that rundown of the BLM KATP channel upon excision into an ATP-free bath proceeds despite removal of bath Mg2+. Representative traces from the regions marked by , , and are shown at bottom.

Thus, ATP-S cannot substitute for ATP in sustaining channel activity, suggesting that phosphorylation itself is not sufficient to prevent rundown. Although phosphorylation may be necessary, it appears that the nucleoside triphosphate must be hydrolyzable to maintain channel activity. This hypothesis is supported by the finding that CTP, GTP, ITP, TTP, and UTP could all prevent or rescue rundown, but the corresponding NDPs could not. Rescue does not appear to require the cAMP-dependent protein kinase, since channel activity can be restored even in the presence of a high concentration of protein kinase inhibitor (PKI, 1 µg/ml, P-0300; Sigma Chemical Co.).

Since it has been reported that removal of free Mg²⁺ nearly abolishes rundown of K_ATP in cultured CRI-G1 insulin-secreting cells (Kozlowski and Ashford, 1990) and partially inhibits rundown of ATP-regulated ROMK1 channels excised in an ATP-free bath (McNicholas et al., 1994), we assessed BLM K_ATP channel activity under these conditions. The representative experiment shown in Fig. 9 C shows that rundown of the BLM K_ATP channel still occurs in the absence of ATP despite excision of the patch into a Mg²⁺-free bath.

Diazoxide. The synthetic K_ATP channel opener diazoxide was applied to the cytoplasmic side of i/o patches. It has been shown that this benzothiadiazine can open K_ATP channels in the presence of Mg-ATP, but it may have an inhibitory effect in the absence of Mg-ATP (Kozlowski et al., 1989). Initial excision of the patch into an ATP-free bath leads to channel rundown as discussed above, and 200 µM diazoxide alone does not rescue rundown. However, addition of 0.2 mM Mg-ATP in the continued presence of, or after exposure to, diazoxide increases channel activity well in excess of that before rundown (n = 3, Fig. 10). The inhibitory effect of 5 mM ATP is not diminished in the presence of, or by previous exposure to, diazoxide (n = 5, data not shown).

View larger version (52K): [in this window] [in a new window]   Fig. 10.   The K channel opener diazoxide activates the BLM K+ channel. Diazoxide activates the BLM K+ channel in the presence of ATP. In the experiment depicted, the pipette contains KCl (solution d), the bath contains NaCl (solution c), and the command potential is 80 mV. (top) A running average (window width 16 ms) of current versus time. (bottom) Sample current traces from the same experiment. With diazoxide alone, the channel opens infrequently (nPo = 0.15) and no more than two channels are open at a time. Diazoxide combined with 0.2 mM ATP, however, promptly increases channel activity and within 5 min, up to 13 simultaneously open channels are evident (nPo = 5.27). The all channels closed level (dashed line) and open channel levels (dotted lines) are indicated.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

The proximal tubule (a leaky epithelium) can be considered to function within the general scheme of the epithelial transport model first proposed by Koefoed-Johnsen and Ussing (1958) (KJU), in which the apical membrane is primarily Na⁺ selective and the BLM is primarily K⁺ selective. Although this model was first applied to tight epithelia such as frog skin (Koefoed Johnsen and Ussing, 1958) and urinary bladder (Davis and Finn, 1982), its essence holds for leaky epithelia such as small intestine (Gunter-Smith et al., 1982) and the proximal tubule (Matsumura et al., 1984). In the KJU model, maintenance of unidirectional Na transport requires that K moves in a closed circuit (recycling) across the BLM. That is, barring intracellular accumulation of K (or K⁺ secretion), the K⁺ pumped into the cell by the pump must be matched by an outward K⁺ current across the BLM.

Steady state vectorial transport in the proximal tubule thus requires continuous activity of the basolateral Na⁺,K⁺-ATPase pump, which consumes ATP and obligates intracellular accumulation of K⁺. A conductance for K⁺ is necessary both to allow this K⁺ to recycle and to maintain V_bl. Experiments performed by Matsumura et al. (1984) on perfused Necturus proximal tubules first demonstrated that the BLM G_K varies as a function of pump activity, and they suggested that the regulation of BLM G_K was linked to cellular metabolism, as had been previously proposed for red cells (Romero, 1978) and suspensions of rabbit cortical tubules (Balaban et al., 1980).

This hypothesis was bolstered when the first single-channel records of an ATP-sensitive K⁺ channel (K_i = 0.1 mM) from cardiac muscle were published (Noma, 1983). Similar ATP-sensitive K⁺ (K_ATP) channels were subsequently found in pancreatic -islet cells (Cook and Hales, 1984), skeletal muscle (Spruce et al., 1985), and smooth muscle (Standen et al., 1989). The K_i for ATP is 10-100 µM for all these Type I K_ATP channels (Ashcroft and Ashcroft, 1990), and they are inhibited by sulfonylurea agents (Edwards and Weston, 1993). A K_ATP channel with thes