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Cross-talk and the role of KATP channels in the proximal tubule
Kidney International, Vol. 48 (1995), pp. 1017—1023
Cross-talk and the role of KATP channels in the proximal tubule PAUL A. WELLING Department of Physiology, University of Maiyland School of Medicine, Baltimore, Ma,yland, USA
Faithfully shuttling scores of different solutes between various bodily compartments, epithelia are strategically equipped with a plethora of diverse ion channels, cotransporters, exchangers and active pumps. Despite their functional eccentricities, however, these transport molecules don't toil away as renegade individualists. Rather, by continuously talking to their transporting companions through multiple and overlapping signaling pathways [1, 21, they consort to preserve intracellular homeostasis in the face of physiologic alterations in transcellular solute transport.
elevation of intracellular K and cell volume. Neither happens. Instead, the basolateral K conductive pathway, comprised of K-selective channels [reviewed in 22, 23], is summoned to augment its activity in parallel with the increase in Na,K-ATPase turnover. The synchronized cross-talk response insures that the
filtrate, the proximal tubule amazingly reabsorbs about ten times its own weight in intracellular Na about once eveiy minute [3, 4].
dumping when the activity of the Na,K-ATPase is reduced [15, 25,
The cellular volume and intracellular potassium content are
Although the tight and parallel coupling between the activity of the Na,K-ATPase and the magnitude of the K conductance at the basolateral membrane is a fundamental and essential property of nearly all salt translocating epithelial [2], the underlying transduction mechanism had proved to be rather elusive. Only in the last few years, with the application of the patch-clamp technique and
obligate active influx of potassium through the Na,K-ATPase will be efficiently recycled back across the basolateral membrane. In
this way, intracellular K activity [7, 12], cell volume [5] and membrane potential [11, 24] are preserved during physiological The renal proximal tubule is no exception to the cross-talk surges in electrogenic Na transport. An analogous response phenomena. While recapturing the bulk of the glomerular ultra- reduces the extent passive K efflux and prevents deleterious K
turned over at similar pace. Obviously, under the pressure of these herculean transcellular transport rates, intracellular homeostasis is constantly threatened. Even small physiological alterations in
the filtered solute load and subsequent changes in transport through the cell would have dramatic and deleterious consequences on the intracellular milieu if it wasn't for the cross-talk signaling pathways that balance the activities of transporters carrying solutes into the cell with those that affect solute exit. For instance, consider the homeostatic conversation between the Na,K-ATPase and the potassium conductive pathway on the basolateral membrane [2, 5—13] observed during a substrateevoked boost in transcellular Na reabsorption (Fig. 1). In the proximal tubule and other leaky epithelial, like the small intestine [3, 4, 14—16], the vectorial transport of Na from lumen to blood is
governed by the in-series operation of a number of different,
26].
the ability to mointor intracellular levels of potential coupling modulators, have the pieces of the pump-leak coupling puzzle begun to fit together. The role variety of putative signaling modulators, such as membrane stretch [6, 27—29], changes in Ca
[10, 30] and pH1 [11, 31 321 have now been clarified. Several laboratories have conclusively implicated the role of ATP-sensitive K channels and changes in intracellular ATP levels [13, 25, 26]. The present review focuses on these recent developments and the role of KATP in the cellular physiology of the renal proximal tubule.
Na-coupled solute carriers at the apical membrane and an active Role of cell volume and membrane-stretch as a Na-translocation step, the Na,K-ATPase, on the basolateral membrane. By design, the passage of Na into the cell is dependent on coupling modulator the availability of its cotransported partners [4, 14—18]. SubseThe small degree of cell swelling caused by the stimulation of quently, when substrates like glucose or amino acids are added to Na-nutrient cotransport and subsequent gain of osmotically active the lumenal compartment, apical Na entry is turned on [14, 17, 19]. Transcellular sodium reabsorption then accelerates as the solutes [5] has been a prime suspect for the initial stimulus that active effiux step across the basolateral membrane, the Na,K- evokes the pump-K leak coupling response [5, 6, 15, 28, 29]. This ATPase, increases to match passive Na entry by mechanisms line of thinking logically followed from unraveling the mystery of involving a Nat-dependent alteration in the turnover rate [16] and hypotonic volume regulatory decrease (VRD). Using video-optipossibility an increase in functional pump number or change in cal analysis of cell volume and/or conventional microelectrode Lopes and Guggino [34], Kirk, DiBona and Schafer kinetic properties [20]. Remembering, that the Na,K-ATPase techniques, [35], Welling and O'Neil [36] and Beck et al [31] independently
actively translocates three sodium ions in exchange for two established that volume regulatory decrease in the proximal potassium ions per cycle [21], first principals predict that the tubule is, like many other cell types [37], critically dependent on a increase in pump rate would occur at the expense of a harmful cell swelling-activated K conductance at the basolateral mem© 1995 by the International Society of Nephrology
brane. Accordingly, it seemed quite plausible that the solute-load induced cell swelling might also increase the basolateral potassium conductance [5, 6, 371. Lau et al [33, 38] provided direct
1017
1018
Welling: Cross talk and KArp channels
S
—®*-
Na
Na
Na
K
Fig. 1. Homeostatic coupling between the Na,KA TPase and the K recycling pathway in the renal proximal tubule observed during a substrateevoked boost transcellular Na reabsorption: (1) apical Na entry via Na-dependent substrate cotransporters is stimulated in the presence of substrates (S). (2) The Na,K-ATPase subsequently reves-up so that active efflux of Na into the interstitium matches passive apical
sodium entry. (3) Insuring that the increased active uptake of K via the Na,K-ATPase will be efficiently recycled back across the basolateral membrane, K channel activation ensues.
the Xenopus proximal tubule. Employing the whole cell patch configuration to evade the squabble about artifactual stretchsensitization in the excised-patch configuration [39], Sackin and Cermerikic proved that the phenylalanine-evoked stimulation of Na-cotransport and the subsequent increase in cell volume was accompanied by an increase in the basolateral SAK channel lular osmolarity. While the cell swelling-K channel activation courtship has been macroscopic current. Since the chemical composition of cytosupport for this hypothesis in a protypical leaky epithelium, the Necturus small intestine. These investigators found they could inhibit the increase in basolateral K conductance that faithfully accompanies Na-nutient co-transport activatation if they countered the solute-induced cell swelling by increasing the extracelwell documented [37], the underlying transduction pathways have remained a major question. Could cell swelling induce a cascade
plasm was effectively clamped by the pipette solution, they argued that membrane stress itself, rather than a cytoplasmic intermediof signals that eventually activates the basolateral K channels ate, must trigger the increase in K conductance. However intrigusimilar to way that a hormone or neurotransmitter might stimu- ing these observations are, they cannot explain pump-K leak late a second-messenger activated ion channel following ligand- coupling in the mammalian proximal tubule. In surprising contrast receptor interaction? Instead, might cell swelling activate a baso- to the amphibian proximal tubule, exhaustive characterization of lateral K channel directly by means of membrane stress? Support these K channels in the mammalian preparation have failed to for the latter mechanism, at least in the amphibian proximal show any evidence of stretch-activation [23, 26, 40]. The lack of tubule, began to mount as patch clamp techniques were employed direct machanotransduction, predicting a signaling cascade beto characterize the individual proximal tubule basolateral mem- tween the Na,K-ATPase and the K channel, ignited a tremendous brane K channels. search for the underlying intermediate coupling messenger(s). As would be predicted for a direct machanotransduction model, Negligible role of Ca as the coupling modulator Sackin observed that the basolateral membrane K channel in the Necturus proximal tubule could be rapidly activated when negative Sparked by observations in tight epithelia that intracellular Ca pressure was applied to the patch pipette and deactivated when apparently modulates the coupling between apical and basolateral membrane suction was released [28, 29]. A similar observation by membrane sodium permeabilities [41], high hopes were placed on Kawahara in the Xenopus proximal tubule suggested a common Ca as a pump-K leak coupling modulator in leaky epithelia [10, mechanism for basolateral K channel regulation 1271 and a 30, 42]. Even more alluring, McCarthy and O'Neil observed that unifying model for pump-leak coupling was put forward. It was cell volume regulation in the rabbit proximal tubule is dependent generally presumed that increased cellular uptake of substrates on transient, swelling-evoked increase in intracellular Ca [43, 44].
via Na-coupled substrate cotransporters would swell the cells Because the parallel activation of both K and Cl channels is enough to change either membrane tension, cytoskeletal-mem- generally assumed to be the central and final common step brane linkages or channel conformation itself to directly increase required for an appropriate VRD response [37, 45], the observation made one wonder if the proximal tubule exploits the same channel activity [6, 27—29]. Despite the attractive speculation based on the salient feature Ca-dependent VRD machinery for more general K channel of the SAK channel, the model was challenged. The physiological role of stretch activation was brought under fire when Morris and Horn discovered that cell swelling, an insult assumed to increase membrane tension, failed to augment the macroscopic activity of neuronal channels previously shown to be machanosensitive at the microscopic level [39]. Cermerikic and Sackin [6] recently provided compelling evidence that this definitely not case for SAK in
activation processes like pump-leak coupling. In a logical progression from McCarthy's result, a number of laboratories began to determine if Ca could activate the basolat-
eral K channel. On this note, the Ca-coupling hypothesis fell apart. First, excised-patch clamp studies repeatedly demonstrated
that the mammalian proximal tubule basolateral membrane K
channel exhibited no direct sensitivity to cytoplasmic Ca [40, 46].
Welling: Cross talk and KATP channels
1-
1019
a.
I
Fig. 2. ATP is a coupling modulator of parallel Na,K-A TPase-K channel activity in the renal proximal tubule. KATP channels, the major
determinant of the basolateral membrane K recycling pathway [13], are gated according to the availability of intracellular ATP: an increase in ATP1 closes the channel whereas a decrease in ATP1 opens it. As the largest energy consumer in the renal proximal tubule, an increase in Na,K ATPase activity can cause ATP levels to fall and increase upon pump inhibition [13, 25]. Subsequently, extent of basolateral membrane K recycling changes in parallel with pump consumption of the negative channel modulator.
Second, efforts to address whether the apparent Ca-dependent and Duplain observed a biphasic pH1 response upon transcellular regulatory process might be orchestrated indirectly and upstream Na transport stimulation in the rabbit proximal tubule [111; the from the channel, as might be imagined if a Ca-dependent protein initial depolarization resulting from the sudden jolt of inwardlykinase or phosphatase were involved, also failed the test of directed Na movement across the apical membrane was accomexperiment. Using a conventional microelectrode strategy in the panied by transient intracellular alkalization, as might be preisolated perfused preparation and ionomycin to change Ca, in a dicted based on the rheogenic properties of the major alkaline predicable fashion, Beck et al [30] showed that elevation of extruder in the proximal tubule, the basolateral membrane intracellular Ca to levels comparable to that seen with hypotonic Na(HCO2)3 cotransporter [50]. In contrast, however, to what shock had no effect on the relative macroscopic basolateral K would be anticipated if pH1 were the coupling modulator, the conductance. Although Beck et al could not rule out the role of a transient alkalization was followed by a sustained phase of swelling-induced sensitization of the Ca-dependent signaling intracellular acidification during the same interval that GK was pathway as proposed earlier by McCarthy and O'Neil [44], the increasing. The conflicting results of Beck et a! and Lapointe et al, result cast doubt on Ca as the coupling agent. Finally and most while not yet resolved, illustrate that changes in pH1 cannot damaging to the Ca-coupling hypothesis, Beck et al confirmed an universally account for the pump-K leak coupling response. earlier observation of LaPointe et al [10] that lumenal addition of
glucose and alanine stimulated transcellular Na transport and Obligatory role of the KATP in pump-leak coupling activated the basolateral K conductance without a concomitant increase in intracellular calcium. Although the results of McAs the role of the classical coupling candidates, namely memCarthy and O'Neil suggest that Ca might be a prerequisite for brane stretch, intracellular Ca1 and pH1, became less certain, VRD, these subsequent studies make Ca as the pump-K leak epithelialogists began to ask what other first-order transcellular Na transport sequelae could be exploited as the pump-leak coupling modulator quite unlikely. transducer. Bucking the conventional wisdom that intracellular Conflicting roles of pH as the coupling modulator ATP levels remain constant over a wide range cellular work loads As had been shown by Harvey, Thomas and Ehrenfeld in frog [51], several of us naively envisioned a role of intracellular ATP skin [47], intracellular pH (pH1) was regarded to be a possible [25, 26, 13]. At the heart of our model was the largest energy candidate for the pump-leak coupling modulator in the proximal consumer in the proximal tubule, the Na/K-ATPase [51]; an tubule. The idea was particularly attractive because, unlike the increase in its hydrolytic activity would tend to decrease intracelrole of Ca1, microelectrode and patch-clamp studies firmly estab- lular ATP levels and, in turn, would somehow activate the lished that both the macroscopic basolateral membrane K con- basolateral K channel (Fig. 2). The theory was particularly ductance (GK) and single channel activity were exquisitely sensi- enticing because Wang et a! [52—54] and Bleich et a! [55] tive to changes in intracellular pH [22, 31, 32, 48]. Acidifying the discovered a particular class of K channels in other segments of cell caused a reduction in K channel activity while cellular the mammalian nephron which would be ideally suited for sensing alkalization increased it. Furthermore, a swelling-induced intra- changes in intracellular ATP. Like the ATP-sensitive K channels cellular alkalization was shown to be one of several factors (KATP) first identified by Noma in the cardiac myocyte [56], the responsible for the activation of °K in the VRD response [31]. physiologically-active apical membrane K channels in the thick Curiously, however, measurements of pH in the mammalian ascending limb [52, 55] and cortical collecting duct principal cell proximal tubule during the pump-leak response have yielded [53, 54] are characterized by channel closure upon exposure to rather ambiguous results. While Beck et al demonstrated that cytoplasmic ATP. Over the last few years, each of the predictions of the ATP rabbit proximal tubule exhibits a modest degree of alkalization in response to increased sodium transport [31], the response has not coupling hypothesis have been borne out by experiment. The first been universally observed. Somewhat more reminiscent of earlier clue in favor of the theory advanced as several groups began to observations in the amphibian proximal tubule [15, 49], Lapointe probe into the inner workings of the mammalian proximal tubule
1020
Welling: Cross talk and KATP channels
basolateral membrane K channel. Using the patch-clamp tech- and basolateral K channel activity can be evoked with confidence nique, several different laboratories [26, 32, 40, 461 including our and monitored with precision. Casting doubt on the notion that own [13] detected consistently, if not exclusively, the presence of cellular energy production and consumption are so efficiently a slightly inwardly recti'ing, voltage-insensitive, 50 to 6OpS, K coupled that ATP1 remains constant [51] while advancing the role selective channel on the rabbit proximal tubule (S1-S2 segment) of ATP1 as a pump-leak coupling modulator, both groups dembasolateral membrane. The finding that the channel was sponta- onstrated that ATP changes in response to alterations in Na,Kneously active in the cell-attached configuration provided reassur- ATPase activity. Upon substrate-induced Na transport stimulaance that the channel was doing something meaningful. Most tion, ATP decreased at a rate that is compatible with the importantly, however, and just as would be predicted if ATP was increased degree of Na,K-ATPase activity [13]. An opposite the language that the pump-K leak response was speaking, change in ATP1 occurred when the Na,K-ATPase was inhibited channel activity instantly and reversibly diminished when a baso- with strophatidin [25]. Furthermore, as anticipated from the lateral membrane patch was excised and a physiological dose of microscopic characteristics of KATP the glibenclamide-sensitive ATP was applied to the cytoplasmic surface of the channel [13]. In macroscopic K conductance increased during the Na,K-pump an elegant series of papers, Beck et al [321 and Hurst et al [261 mediated fall in ATP, [13] and fell during the stophanathinwent a step further after confirming our initial observation of K induced decrease in ATP [27]. Both observations provided circhannel ATP-sensitivity [131. Performing a heroic feat, these cumstantial evidence that the two events were related. Tsuchiya et investigators were able to patch the hasolateral membrane of al went on to establish a more definitive causal relationship continuously microperfused rabbit proximal tubules so that the between the fall in ATP1 and increase in GK. In these studies, behavior of the basolateral K channel could be investigated fully proximal tubule cells were first loaded with ATP and provoked during physiological variations in transcellular transport and with lumenal glucose and amino acids. Demonstrating that parpump activity. Establishing a definitive relationship between the allel Na,K-ATPase activity-basolateral K recycling can be uncouactivity of the Na,K-ATPase and the KATP channel, they demon- pled by preventing a critical ATP1 decline, transcellular Na strated that KATP activity increased upon substrate-induced stimulation of transcellular sodium transport [32] and decreased upon Na,K-ATPase inhibition with the cardiac glycoside, strophanthi-
din [26]. Furthermore, the result that channel activity could be restored in the presence of strophanthidin simply by excising the patch from the cell provided compelling evidence that the pumpleak transduction pathway involved a cytoplasmic rather than membrane delimited intermediate as would be imagined if ATP
transport and pump activity increased as before, but the K
conductance declined to reciprocate under the ATP-clamp conditions.
In summary, the major tenets of the ATP coupling modulator hypothesis have directly confirmed by several independent lines of
evidence. First, the activity of KATP channels on the proximal tubule basolateral membrane [13] change in parallel with the
activity of the Na,K-ATPase [26, 32]. Second, as the major
determinant of the basolateral membrane macroscopic K conductance, the activity of the KATp channel dictates the extent of K didn't tell the entire story. Macroscopic conductance measure- recycling across the basolateral membrane [13]. Third, variations ments by conventional microelectrode analysis of Tsuchiya et al in the hydrolytic activity of the Na,K-ATPase cause predictable provided the pivotal link between the single channel observations changes in ATP1 levels [13, 25]. Finally, the parallel pump-leak and the basolateral membrane K recycling pathway [13]. First, we response can be uncoupled by preventing a critical ATP decline established that sulfonylurea agents like glibenclamide or tolbut- during Na,K-ATPase stimulation [131. At a more quantitative level, however, a central issue about the amide, touted to be fairly specific KATP channel antagonists [211, blocked nearly all of the total basolateral membrane K conduc- regulation of the proximal tubule KA1.p channel remains a puzzle. tance under both basal and Na-transport stimulated conditions. Crude estimates of ATP-dependent gating (Ki 4 to 5 mM) from Even more compelling, a near complete inhibition of the macro- macroscopic K conductance analysis indicate that the channel scopic K conductance could be evoked by loading the proximal normally operates in situ within the physiologic range of measured tubule cell with endogenous ATP (2.5 times above endogenous ATP1 [13]. However, like other epithelial KATP channels, the levels). The series of studies demonstrated that KATP channels do proximal tubule channel is surprisingly more susceptible to ATP indeed comprise a significant fraction of the total basolateral block (Ki 0.2 to 1 mM, [131) in the excised patch configuration membrane K conductance, a result suggesting that ATP mediated than would be presumed from its rather robust activity in the changes in KArp activity might alter the extent of K recycling in intact cell at basal ATP levels (2 to 4 mM) [13, 26, 32]. Two general explanations for this dilemma have been suggested. First, concert with the activity of the Na,K-ATPase [13]. While the studies above demonstrated that the K recycling since estimates of the intracellular ATP concentration are based machinery has all the requisite parts to shift its activity in response on measurements of the total cellular ATP content and the to varying intracellular ATP levels, a central prerequisite of the inaccurate assumption that ATP is uniformally distributed within ATP-coupling hypothesis remained to be proven. That is, do ATP the cell [591, the reported ATP level may not reflect the concenlevels ever change when the rate of activity of the Na,K-ATPase is tration actually sensed by the channel. For instance, a steep spatial augmented or curtailed? Unfortunately, conflicting studies [51, cytoplasmic gradient of ATP due to the activity of integral 57, 581 with homogenous tubule suspensions provided no clear membrane ATPases [60] been proposed to reduce the ATP picture. To resolve the issue, Beck et al [25] and Tsuchiya et al available to the channel [61]. Not regarded as a mutually exclusive [13] independently measured ATP1 in the isolated perfused prox- alterative to the ATP compartmentalization scheme, cytoplasmic imal tubule, a preparation that offers the clear advantage over regulatory factors might also adjust ATP affinity closer to meaothers because ATP1 can be accurately measured while substrate- sured basal ATP1 levels [21, 611. Unquestionably and often cited induced changes in transepithelial Na transport, pump activity as evidence for rectification of ATP sensitivity, ADP reduces the where the coupling agent [261. Such observations at the single channel level, while provocative,
Welling: Cross talk and KATP channels
extent of ATP interaction by both competition and allosteric action in most other KATP channels [21, 53, 54, 61, 62]. The major
caveat with the ADP argument, however, is that the reliable measurements of intracellular ADP indicate that the concentration is far too low for ADP [58] to have any tangible impact on ATP-dependent gating under physiologic conditions. In this light, other factors proposed to modify both the gating properties and
1021
channels predicts a similar protein topology that dramatically deviates from the common structural motif shared by the superfamily of voltage-gated (I) and second messenger-gated ion channels. Hydrophobicity analysis of the IRK channel family predicts two membrane-spanning regions that flank a IK-pore like structure [68, 69]. A long and highly divergent hydrophilic endoplasmic region, postulated to form a ATP [68], G-protein [71, 72]
the ATP affinity of various KATP channels, such as channel or kinase regulated inactivation gate [68], follows the second phosphorylation [21, 54], deserve further exploration. If such membrane segment. Although the basic body plan of the IRK regulatory factors are identified, it will be interesting to determine channel is quite different than that exhibited by the Ic, several whether these elements collude with the changes in ATP to parallels predict a oligomeric IRK channel. Because of the regulate the extent of K recycling in concert with Na,K-ATPase
biophysical similarities in K permeation between the IRK and Ic channels, the conservation of the pore region, and the tetrameric nature of Shaker-type channels [73], it seems reasonable to believe that the IRK channel polypeptides also assemble as tetrameres Future perspectives on the proximal tubule KATP channel [67—691. In this regard, KATP might be generated by mixing, Molecular biology matching and appropriate assembly of separate subunits. CerIn addition to the critical role that KATP plays in the renal tainly the extensive sequence similarity between the presently proximal tubule, the same class of channels are thought to be cloned inwardly-rectifying channels predicts a new superfamily of involved in a wide range of physiological and pathophysiological K channels that presumably includes other functionally comparaprocesses [21, 61]. For instance, similar but pharmacologically and ble K channels like KATP. Where does the proximal tubule basolateral membrane KATP fit biophysically distinct KATP channels perform obligatory roles in mediating and regulating urinary dilution [52, 55], extracellular K into the picture? Based on preliminary RT-PCR expression homeostasis [53], pancreatic beta cell insulin secretion [63], mapping [74] and in situ hybridization studies [75], it seems likely vascular smooth muscle tone [61], cardiac electrical activity in that the ROMK-subfamily of IRK genes encode for subunits of ischemia [64, 65] and suppressing neuronal excitability in anoxia the TAL and CCD apical KATP channels rather the proximal activity.
[66]. Despite the major breakthroughs in demonstrating and tubule basolateral membrane channel. The tremendous funccharacterizing the repertoire of diverse performances played by KATP channels, definitive answers to the precise mechanisms of gating, regulation, and diversity have awaited the isolation of the cDNA(s) encoding these ATP-sensitive channels.
In these regards, an exciting recent development in the K channel field has provided some important hints about the molecular structure of renal epithelial KATP [45, 67—70]. In the last two years, several groups lead by Ho et al [68] have independently employed an expression cloning strategy to isolate cDNAs encoding different renal epithelial KATP-like channels. Ho et al [68] isolated a cDNA they call ROMK1 from the inner strip of the
tional similarities shared between the KATP channels expressed along the length of the nephron do, however, predict a common structural ancestry. With the present pace of field, the question will likely be answered in the near future. Furthermore, as the physiological role of the members of IRK gene family in epithelial KATP function becomes more certain, the stage is set for determining the molecular basis for KATP operation. Possible roles of proximal tubule KATP in pathophysiology Although the KATP channel performs a vital homeostatic role in
a typical day in-the-life of a proximal tubule cell, might KATP,
outer medulla of the rat kidney (principally medullary thick under certain circumstances, play the villain? Consider an ischascending limb, mTAL). Zhou, Tate and Palmer isolated a splice emic insult, a common cause of acute renal failure and tubular variant of ROMK1, ROMK2 [70], while Welling, Tsuchiya and necrosis. Strangled by a compromised energy supply, ATP1 can Giebisch isolated a different, but related, cDNA from the mouse plummet to less that a fourth of basal levels in the nephron cortical collecting duct cell line, Ml [45]. In Xenopus oocytes, each
segment that is especially susceptible to ischemic injury, the
of the channels exhibit many properties characteristic of KATP
proximal tubule [76—78j. Interestingly, the magnitude of the drop
with several important exceptions. Unlike the TAL KATP channel, ROMK1 is activated rather than inhibited by ATP [68] and unlike
is a predictive index of the extent of postischemic renal failure [77]. Moreover, various maneuvers that maintain ATP at basal
the CCD KATP channel, the channel encoded by the Ml cell cDNA is insensitive to sulfonylurea agents [45]. Based on the observation that these gene products are normally expressed in nephron segments where KATP is the major determinant of the macroscopic K conductance [23], it seems plausible that the
levels offer an intriguing degree of protection against the irrëversible effects of ischemia [76, 78]. Observations such as these have suggested that the drop in ATP lies near the top of the hierarchy
of ischemic sequela which can ultimately lead to cell death.
Despite these captivating observations, surprisingly little is known cloned structures encode for parts of a more complex heteroligo- about the most direct and consequent effectors of the ATP meric KATP structure [45, 68]. In this respect, expression of a depletion. Given the ATP-dependent gating properties of the single subunit gives rise to a homologmeric structure with many basolateral membrane proximal tubule K channel, this transport comparable, but not identical, properties of the native KATP element is an obvious candidate. Confronted by an inadequate intracellular ATP level to safechannel. The supposition is partially supported by the primary structure of these channels and their kinship to other inwardly- guard the efficient coupling between the Na,K-ATPase and passive K recycling process, the homeostatic communication would rectifying K, IRK, channels [67, 69, 71, 72]. Defining a new class of channel proteins [67], the deduced be expected to grow mute. Since the KATP channel is normally amino acid sequence of each of these recently cloned IRK released from varying degrees of inhibition upon a fall in ATP1
1022
Welling: Cross talk and KAIP channels
[13, 21], KATP should exist an inappropriately active state in the
face of an energy-compromised Na,K-ATPase at the low ATP levels of ischemia. Indeed, just as expected for the predicament when passive K efflux exceeds active uptake, microprobe analysis demonstrated that ischemia precipitates deleterious dumping of
intracellular K from the proximal tubule cell [79]. Considering that intracellular K homeostasis is essential for numerous cellular processes, the response might be a decisive blow to the proximal tubule in the pathogenesis of acute tubular necrosis. Obviously, it presently remains to be seen whether specific inhibition of the proximal tubule KATP channel can prevent disruption of intracellular K homeostasis and progression of renal failure in ischemia. Summary Over the last few years it has become evident that an assortment of functionally-related, but diverse, KATP channels provide an
important and physiologically-regulated determinant of the K conductive pathways in many, if not all, epithelial cells expressed along the nephron. As such, KATP plays central roles in regulating and maintaining a number of transport processes in concert with physiological demands of the kidney. In the renal proximal tubule,
KATP channels and changes in the hydrolytic activity of the Na,K-ATPase permit ATP to act as a coupling modulator of parallel Na,K-ATPase-K recycling. The response insures that cell membrane potential, intracellular K activity and cell volume are protected in the face of physiological variations in transcellular
ion transport. In addition to demonstrating the physiological
9. LAPOINTE J-Y, LAPRADE R, CARDINAL J: Transepithelial and cell
membrane electrical resistances of the rabbit proximal convoluted tubule. Am J Physiol 247:F637—F649, 1984 10. LAPOINTE J-Y, GAREAU L, BELL DP, CARDINAL J: Membrane
crosstalk in the mammalian proximal tubule during alterations in transepithelial sodium transport. Am J Physiol 258:F339—F345, 1990 11. LAPOINTE J-Y, DUPI.AIN M: Regulation of basolateral membrane
potential after stimulation of Na transport in Proximal tubules. I Memhr Biol 120:165—172, 1991 12. LAPRADE R, LAPOINTE J-Y, DUPLAIN M, CARDINAL J: Intracellular
potassium activity in mammalian proximal tubule: Effect of perturbations in transepithelial sodium transport. J Membr Biol 121:249—259, 1991 13. TSUCHIYA K, WANG W, GIEaIsCH G, WELLING PA: ATP is a coupling
modulator of parallel Na/K ATPase-K channel activity in the renal proximal tubule. Proc Nati Acad Sci USA 89:6418—6422, 1992
14. FROMTER E: Electrophysiological Analysis of rat renal sugar and amino acid transport. I Basic phenomena. Pflugers Arch 393:179—189, 1982 15. LANG F, MESSNER 0, REHWALD W: Electrophysiolgy of sodium coupled transport in proximal renal tubules. Am J Physiol 250:F953— F962, 1986 16. MORGUNOV N, BOULPAEP EL: Electroehemical analysis of renal Na-glucose eotransport in salamander proximal tubules. Am J Physiol 252:F154—F169, 1987 17. ARONSON PS, SAKTOR B: The Na-gradient dependent transport of D-glucose in renal brush border membranes. J Biol Chem 250:6032— 6039, 1975 18. SILBERNAGL S, FOULKES EC, DEETJEN P: Renal transport of amino acid. Rev Physiol Bioch Pharmacol 74:105—167, 1975 19. BURG M, PATALACK C, GREEN N, VILLEY D: Organic solutes in fluid absorption by renal proximal convoluted tubules. Am J Physiol 231: 627—637, 1976
relevance of KATP in renal epithelial, these studies have provided 20. HUDSON RL, SCHULTZ SG: Sodium-coupled sugar transport: Effects on intracellular sodium activities and sodium-pump activity. Science a long awaited answer to the underlying mechanism of pump-leak 234:1237-1239, 1994 coupling, a universal and essential homeostatic mechanism ob21. ASHCROFT FM: Adenosine 5' triphosphate-sensitive potassium chanserved in nearly all salt translocating epithelia. nels. Annu Rev Neurosci 11:97—118, 1988 22. KUWAHARA M, ISHIBASHI K, KRAVF R, RECTOR R, BERRY C: Effect of
Acknowledgment This work was supported by a grant from the American Heart Association, Maryland Affiliate.
Reprint requests to Paul A. Welling, M.D., Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
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Cross-talk and the role of KATP channels in the proximal tubule PAUL A. WELLING Department of Physiology, University of Maiyland School of Medicine, Baltimore, Ma,yland, USA
Faithfully shuttling scores of different solutes between various bodily compartments, epithelia are strategically equipped with a plethora of diverse ion channels, cotransporters, exchangers and active pumps. Despite their functional eccentricities, however, these transport molecules don't toil away as renegade individualists. Rather, by continuously talking to their transporting companions through multiple and overlapping signaling pathways [1, 21, they consort to preserve intracellular homeostasis in the face of physiologic alterations in transcellular solute transport.
elevation of intracellular K and cell volume. Neither happens. Instead, the basolateral K conductive pathway, comprised of K-selective channels [reviewed in 22, 23], is summoned to augment its activity in parallel with the increase in Na,K-ATPase turnover. The synchronized cross-talk response insures that the
filtrate, the proximal tubule amazingly reabsorbs about ten times its own weight in intracellular Na about once eveiy minute [3, 4].
dumping when the activity of the Na,K-ATPase is reduced [15, 25,
The cellular volume and intracellular potassium content are
Although the tight and parallel coupling between the activity of the Na,K-ATPase and the magnitude of the K conductance at the basolateral membrane is a fundamental and essential property of nearly all salt translocating epithelial [2], the underlying transduction mechanism had proved to be rather elusive. Only in the last few years, with the application of the patch-clamp technique and
obligate active influx of potassium through the Na,K-ATPase will be efficiently recycled back across the basolateral membrane. In
this way, intracellular K activity [7, 12], cell volume [5] and membrane potential [11, 24] are preserved during physiological The renal proximal tubule is no exception to the cross-talk surges in electrogenic Na transport. An analogous response phenomena. While recapturing the bulk of the glomerular ultra- reduces the extent passive K efflux and prevents deleterious K
turned over at similar pace. Obviously, under the pressure of these herculean transcellular transport rates, intracellular homeostasis is constantly threatened. Even small physiological alterations in
the filtered solute load and subsequent changes in transport through the cell would have dramatic and deleterious consequences on the intracellular milieu if it wasn't for the cross-talk signaling pathways that balance the activities of transporters carrying solutes into the cell with those that affect solute exit. For instance, consider the homeostatic conversation between the Na,K-ATPase and the potassium conductive pathway on the basolateral membrane [2, 5—13] observed during a substrateevoked boost in transcellular Na reabsorption (Fig. 1). In the proximal tubule and other leaky epithelial, like the small intestine [3, 4, 14—16], the vectorial transport of Na from lumen to blood is
governed by the in-series operation of a number of different,
26].
the ability to mointor intracellular levels of potential coupling modulators, have the pieces of the pump-leak coupling puzzle begun to fit together. The role variety of putative signaling modulators, such as membrane stretch [6, 27—29], changes in Ca
[10, 30] and pH1 [11, 31 321 have now been clarified. Several laboratories have conclusively implicated the role of ATP-sensitive K channels and changes in intracellular ATP levels [13, 25, 26]. The present review focuses on these recent developments and the role of KATP in the cellular physiology of the renal proximal tubule.
Na-coupled solute carriers at the apical membrane and an active Role of cell volume and membrane-stretch as a Na-translocation step, the Na,K-ATPase, on the basolateral membrane. By design, the passage of Na into the cell is dependent on coupling modulator the availability of its cotransported partners [4, 14—18]. SubseThe small degree of cell swelling caused by the stimulation of quently, when substrates like glucose or amino acids are added to Na-nutrient cotransport and subsequent gain of osmotically active the lumenal compartment, apical Na entry is turned on [14, 17, 19]. Transcellular sodium reabsorption then accelerates as the solutes [5] has been a prime suspect for the initial stimulus that active effiux step across the basolateral membrane, the Na,K- evokes the pump-K leak coupling response [5, 6, 15, 28, 29]. This ATPase, increases to match passive Na entry by mechanisms line of thinking logically followed from unraveling the mystery of involving a Nat-dependent alteration in the turnover rate [16] and hypotonic volume regulatory decrease (VRD). Using video-optipossibility an increase in functional pump number or change in cal analysis of cell volume and/or conventional microelectrode Lopes and Guggino [34], Kirk, DiBona and Schafer kinetic properties [20]. Remembering, that the Na,K-ATPase techniques, [35], Welling and O'Neil [36] and Beck et al [31] independently
actively translocates three sodium ions in exchange for two established that volume regulatory decrease in the proximal potassium ions per cycle [21], first principals predict that the tubule is, like many other cell types [37], critically dependent on a increase in pump rate would occur at the expense of a harmful cell swelling-activated K conductance at the basolateral mem© 1995 by the International Society of Nephrology
brane. Accordingly, it seemed quite plausible that the solute-load induced cell swelling might also increase the basolateral potassium conductance [5, 6, 371. Lau et al [33, 38] provided direct
1017
1018
Welling: Cross talk and KArp channels
S
—®*-
Na
Na
Na
K
Fig. 1. Homeostatic coupling between the Na,KA TPase and the K recycling pathway in the renal proximal tubule observed during a substrateevoked boost transcellular Na reabsorption: (1) apical Na entry via Na-dependent substrate cotransporters is stimulated in the presence of substrates (S). (2) The Na,K-ATPase subsequently reves-up so that active efflux of Na into the interstitium matches passive apical
sodium entry. (3) Insuring that the increased active uptake of K via the Na,K-ATPase will be efficiently recycled back across the basolateral membrane, K channel activation ensues.
the Xenopus proximal tubule. Employing the whole cell patch configuration to evade the squabble about artifactual stretchsensitization in the excised-patch configuration [39], Sackin and Cermerikic proved that the phenylalanine-evoked stimulation of Na-cotransport and the subsequent increase in cell volume was accompanied by an increase in the basolateral SAK channel lular osmolarity. While the cell swelling-K channel activation courtship has been macroscopic current. Since the chemical composition of cytosupport for this hypothesis in a protypical leaky epithelium, the Necturus small intestine. These investigators found they could inhibit the increase in basolateral K conductance that faithfully accompanies Na-nutient co-transport activatation if they countered the solute-induced cell swelling by increasing the extracelwell documented [37], the underlying transduction pathways have remained a major question. Could cell swelling induce a cascade
plasm was effectively clamped by the pipette solution, they argued that membrane stress itself, rather than a cytoplasmic intermediof signals that eventually activates the basolateral K channels ate, must trigger the increase in K conductance. However intrigusimilar to way that a hormone or neurotransmitter might stimu- ing these observations are, they cannot explain pump-K leak late a second-messenger activated ion channel following ligand- coupling in the mammalian proximal tubule. In surprising contrast receptor interaction? Instead, might cell swelling activate a baso- to the amphibian proximal tubule, exhaustive characterization of lateral K channel directly by means of membrane stress? Support these K channels in the mammalian preparation have failed to for the latter mechanism, at least in the amphibian proximal show any evidence of stretch-activation [23, 26, 40]. The lack of tubule, began to mount as patch clamp techniques were employed direct machanotransduction, predicting a signaling cascade beto characterize the individual proximal tubule basolateral mem- tween the Na,K-ATPase and the K channel, ignited a tremendous brane K channels. search for the underlying intermediate coupling messenger(s). As would be predicted for a direct machanotransduction model, Negligible role of Ca as the coupling modulator Sackin observed that the basolateral membrane K channel in the Necturus proximal tubule could be rapidly activated when negative Sparked by observations in tight epithelia that intracellular Ca pressure was applied to the patch pipette and deactivated when apparently modulates the coupling between apical and basolateral membrane suction was released [28, 29]. A similar observation by membrane sodium permeabilities [41], high hopes were placed on Kawahara in the Xenopus proximal tubule suggested a common Ca as a pump-K leak coupling modulator in leaky epithelia [10, mechanism for basolateral K channel regulation 1271 and a 30, 42]. Even more alluring, McCarthy and O'Neil observed that unifying model for pump-leak coupling was put forward. It was cell volume regulation in the rabbit proximal tubule is dependent generally presumed that increased cellular uptake of substrates on transient, swelling-evoked increase in intracellular Ca [43, 44].
via Na-coupled substrate cotransporters would swell the cells Because the parallel activation of both K and Cl channels is enough to change either membrane tension, cytoskeletal-mem- generally assumed to be the central and final common step brane linkages or channel conformation itself to directly increase required for an appropriate VRD response [37, 45], the observation made one wonder if the proximal tubule exploits the same channel activity [6, 27—29]. Despite the attractive speculation based on the salient feature Ca-dependent VRD machinery for more general K channel of the SAK channel, the model was challenged. The physiological role of stretch activation was brought under fire when Morris and Horn discovered that cell swelling, an insult assumed to increase membrane tension, failed to augment the macroscopic activity of neuronal channels previously shown to be machanosensitive at the microscopic level [39]. Cermerikic and Sackin [6] recently provided compelling evidence that this definitely not case for SAK in
activation processes like pump-leak coupling. In a logical progression from McCarthy's result, a number of laboratories began to determine if Ca could activate the basolat-
eral K channel. On this note, the Ca-coupling hypothesis fell apart. First, excised-patch clamp studies repeatedly demonstrated
that the mammalian proximal tubule basolateral membrane K
channel exhibited no direct sensitivity to cytoplasmic Ca [40, 46].
Welling: Cross talk and KATP channels
1-
1019
a.
I
Fig. 2. ATP is a coupling modulator of parallel Na,K-A TPase-K channel activity in the renal proximal tubule. KATP channels, the major
determinant of the basolateral membrane K recycling pathway [13], are gated according to the availability of intracellular ATP: an increase in ATP1 closes the channel whereas a decrease in ATP1 opens it. As the largest energy consumer in the renal proximal tubule, an increase in Na,K ATPase activity can cause ATP levels to fall and increase upon pump inhibition [13, 25]. Subsequently, extent of basolateral membrane K recycling changes in parallel with pump consumption of the negative channel modulator.
Second, efforts to address whether the apparent Ca-dependent and Duplain observed a biphasic pH1 response upon transcellular regulatory process might be orchestrated indirectly and upstream Na transport stimulation in the rabbit proximal tubule [111; the from the channel, as might be imagined if a Ca-dependent protein initial depolarization resulting from the sudden jolt of inwardlykinase or phosphatase were involved, also failed the test of directed Na movement across the apical membrane was accomexperiment. Using a conventional microelectrode strategy in the panied by transient intracellular alkalization, as might be preisolated perfused preparation and ionomycin to change Ca, in a dicted based on the rheogenic properties of the major alkaline predicable fashion, Beck et al [30] showed that elevation of extruder in the proximal tubule, the basolateral membrane intracellular Ca to levels comparable to that seen with hypotonic Na(HCO2)3 cotransporter [50]. In contrast, however, to what shock had no effect on the relative macroscopic basolateral K would be anticipated if pH1 were the coupling modulator, the conductance. Although Beck et al could not rule out the role of a transient alkalization was followed by a sustained phase of swelling-induced sensitization of the Ca-dependent signaling intracellular acidification during the same interval that GK was pathway as proposed earlier by McCarthy and O'Neil [44], the increasing. The conflicting results of Beck et a! and Lapointe et al, result cast doubt on Ca as the coupling agent. Finally and most while not yet resolved, illustrate that changes in pH1 cannot damaging to the Ca-coupling hypothesis, Beck et al confirmed an universally account for the pump-K leak coupling response. earlier observation of LaPointe et al [10] that lumenal addition of
glucose and alanine stimulated transcellular Na transport and Obligatory role of the KATP in pump-leak coupling activated the basolateral K conductance without a concomitant increase in intracellular calcium. Although the results of McAs the role of the classical coupling candidates, namely memCarthy and O'Neil suggest that Ca might be a prerequisite for brane stretch, intracellular Ca1 and pH1, became less certain, VRD, these subsequent studies make Ca as the pump-K leak epithelialogists began to ask what other first-order transcellular Na transport sequelae could be exploited as the pump-leak coupling modulator quite unlikely. transducer. Bucking the conventional wisdom that intracellular Conflicting roles of pH as the coupling modulator ATP levels remain constant over a wide range cellular work loads As had been shown by Harvey, Thomas and Ehrenfeld in frog [51], several of us naively envisioned a role of intracellular ATP skin [47], intracellular pH (pH1) was regarded to be a possible [25, 26, 13]. At the heart of our model was the largest energy candidate for the pump-leak coupling modulator in the proximal consumer in the proximal tubule, the Na/K-ATPase [51]; an tubule. The idea was particularly attractive because, unlike the increase in its hydrolytic activity would tend to decrease intracelrole of Ca1, microelectrode and patch-clamp studies firmly estab- lular ATP levels and, in turn, would somehow activate the lished that both the macroscopic basolateral membrane K con- basolateral K channel (Fig. 2). The theory was particularly ductance (GK) and single channel activity were exquisitely sensi- enticing because Wang et a! [52—54] and Bleich et a! [55] tive to changes in intracellular pH [22, 31, 32, 48]. Acidifying the discovered a particular class of K channels in other segments of cell caused a reduction in K channel activity while cellular the mammalian nephron which would be ideally suited for sensing alkalization increased it. Furthermore, a swelling-induced intra- changes in intracellular ATP. Like the ATP-sensitive K channels cellular alkalization was shown to be one of several factors (KATP) first identified by Noma in the cardiac myocyte [56], the responsible for the activation of °K in the VRD response [31]. physiologically-active apical membrane K channels in the thick Curiously, however, measurements of pH in the mammalian ascending limb [52, 55] and cortical collecting duct principal cell proximal tubule during the pump-leak response have yielded [53, 54] are characterized by channel closure upon exposure to rather ambiguous results. While Beck et al demonstrated that cytoplasmic ATP. Over the last few years, each of the predictions of the ATP rabbit proximal tubule exhibits a modest degree of alkalization in response to increased sodium transport [31], the response has not coupling hypothesis have been borne out by experiment. The first been universally observed. Somewhat more reminiscent of earlier clue in favor of the theory advanced as several groups began to observations in the amphibian proximal tubule [15, 49], Lapointe probe into the inner workings of the mammalian proximal tubule
1020
Welling: Cross talk and KATP channels
basolateral membrane K channel. Using the patch-clamp tech- and basolateral K channel activity can be evoked with confidence nique, several different laboratories [26, 32, 40, 461 including our and monitored with precision. Casting doubt on the notion that own [13] detected consistently, if not exclusively, the presence of cellular energy production and consumption are so efficiently a slightly inwardly recti'ing, voltage-insensitive, 50 to 6OpS, K coupled that ATP1 remains constant [51] while advancing the role selective channel on the rabbit proximal tubule (S1-S2 segment) of ATP1 as a pump-leak coupling modulator, both groups dembasolateral membrane. The finding that the channel was sponta- onstrated that ATP changes in response to alterations in Na,Kneously active in the cell-attached configuration provided reassur- ATPase activity. Upon substrate-induced Na transport stimulaance that the channel was doing something meaningful. Most tion, ATP decreased at a rate that is compatible with the importantly, however, and just as would be predicted if ATP was increased degree of Na,K-ATPase activity [13]. An opposite the language that the pump-K leak response was speaking, change in ATP1 occurred when the Na,K-ATPase was inhibited channel activity instantly and reversibly diminished when a baso- with strophatidin [25]. Furthermore, as anticipated from the lateral membrane patch was excised and a physiological dose of microscopic characteristics of KATP the glibenclamide-sensitive ATP was applied to the cytoplasmic surface of the channel [13]. In macroscopic K conductance increased during the Na,K-pump an elegant series of papers, Beck et al [321 and Hurst et al [261 mediated fall in ATP, [13] and fell during the stophanathinwent a step further after confirming our initial observation of K induced decrease in ATP [27]. Both observations provided circhannel ATP-sensitivity [131. Performing a heroic feat, these cumstantial evidence that the two events were related. Tsuchiya et investigators were able to patch the hasolateral membrane of al went on to establish a more definitive causal relationship continuously microperfused rabbit proximal tubules so that the between the fall in ATP1 and increase in GK. In these studies, behavior of the basolateral K channel could be investigated fully proximal tubule cells were first loaded with ATP and provoked during physiological variations in transcellular transport and with lumenal glucose and amino acids. Demonstrating that parpump activity. Establishing a definitive relationship between the allel Na,K-ATPase activity-basolateral K recycling can be uncouactivity of the Na,K-ATPase and the KATP channel, they demon- pled by preventing a critical ATP1 decline, transcellular Na strated that KATP activity increased upon substrate-induced stimulation of transcellular sodium transport [32] and decreased upon Na,K-ATPase inhibition with the cardiac glycoside, strophanthi-
din [26]. Furthermore, the result that channel activity could be restored in the presence of strophanthidin simply by excising the patch from the cell provided compelling evidence that the pumpleak transduction pathway involved a cytoplasmic rather than membrane delimited intermediate as would be imagined if ATP
transport and pump activity increased as before, but the K
conductance declined to reciprocate under the ATP-clamp conditions.
In summary, the major tenets of the ATP coupling modulator hypothesis have directly confirmed by several independent lines of
evidence. First, the activity of KATP channels on the proximal tubule basolateral membrane [13] change in parallel with the
activity of the Na,K-ATPase [26, 32]. Second, as the major
determinant of the basolateral membrane macroscopic K conductance, the activity of the KATp channel dictates the extent of K didn't tell the entire story. Macroscopic conductance measure- recycling across the basolateral membrane [13]. Third, variations ments by conventional microelectrode analysis of Tsuchiya et al in the hydrolytic activity of the Na,K-ATPase cause predictable provided the pivotal link between the single channel observations changes in ATP1 levels [13, 25]. Finally, the parallel pump-leak and the basolateral membrane K recycling pathway [13]. First, we response can be uncoupled by preventing a critical ATP decline established that sulfonylurea agents like glibenclamide or tolbut- during Na,K-ATPase stimulation [131. At a more quantitative level, however, a central issue about the amide, touted to be fairly specific KATP channel antagonists [211, blocked nearly all of the total basolateral membrane K conduc- regulation of the proximal tubule KA1.p channel remains a puzzle. tance under both basal and Na-transport stimulated conditions. Crude estimates of ATP-dependent gating (Ki 4 to 5 mM) from Even more compelling, a near complete inhibition of the macro- macroscopic K conductance analysis indicate that the channel scopic K conductance could be evoked by loading the proximal normally operates in situ within the physiologic range of measured tubule cell with endogenous ATP (2.5 times above endogenous ATP1 [13]. However, like other epithelial KATP channels, the levels). The series of studies demonstrated that KATP channels do proximal tubule channel is surprisingly more susceptible to ATP indeed comprise a significant fraction of the total basolateral block (Ki 0.2 to 1 mM, [131) in the excised patch configuration membrane K conductance, a result suggesting that ATP mediated than would be presumed from its rather robust activity in the changes in KArp activity might alter the extent of K recycling in intact cell at basal ATP levels (2 to 4 mM) [13, 26, 32]. Two general explanations for this dilemma have been suggested. First, concert with the activity of the Na,K-ATPase [13]. While the studies above demonstrated that the K recycling since estimates of the intracellular ATP concentration are based machinery has all the requisite parts to shift its activity in response on measurements of the total cellular ATP content and the to varying intracellular ATP levels, a central prerequisite of the inaccurate assumption that ATP is uniformally distributed within ATP-coupling hypothesis remained to be proven. That is, do ATP the cell [591, the reported ATP level may not reflect the concenlevels ever change when the rate of activity of the Na,K-ATPase is tration actually sensed by the channel. For instance, a steep spatial augmented or curtailed? Unfortunately, conflicting studies [51, cytoplasmic gradient of ATP due to the activity of integral 57, 581 with homogenous tubule suspensions provided no clear membrane ATPases [60] been proposed to reduce the ATP picture. To resolve the issue, Beck et al [25] and Tsuchiya et al available to the channel [61]. Not regarded as a mutually exclusive [13] independently measured ATP1 in the isolated perfused prox- alterative to the ATP compartmentalization scheme, cytoplasmic imal tubule, a preparation that offers the clear advantage over regulatory factors might also adjust ATP affinity closer to meaothers because ATP1 can be accurately measured while substrate- sured basal ATP1 levels [21, 611. Unquestionably and often cited induced changes in transepithelial Na transport, pump activity as evidence for rectification of ATP sensitivity, ADP reduces the where the coupling agent [261. Such observations at the single channel level, while provocative,
Welling: Cross talk and KATP channels
extent of ATP interaction by both competition and allosteric action in most other KATP channels [21, 53, 54, 61, 62]. The major
caveat with the ADP argument, however, is that the reliable measurements of intracellular ADP indicate that the concentration is far too low for ADP [58] to have any tangible impact on ATP-dependent gating under physiologic conditions. In this light, other factors proposed to modify both the gating properties and
1021
channels predicts a similar protein topology that dramatically deviates from the common structural motif shared by the superfamily of voltage-gated (I) and second messenger-gated ion channels. Hydrophobicity analysis of the IRK channel family predicts two membrane-spanning regions that flank a IK-pore like structure [68, 69]. A long and highly divergent hydrophilic endoplasmic region, postulated to form a ATP [68], G-protein [71, 72]
the ATP affinity of various KATP channels, such as channel or kinase regulated inactivation gate [68], follows the second phosphorylation [21, 54], deserve further exploration. If such membrane segment. Although the basic body plan of the IRK regulatory factors are identified, it will be interesting to determine channel is quite different than that exhibited by the Ic, several whether these elements collude with the changes in ATP to parallels predict a oligomeric IRK channel. Because of the regulate the extent of K recycling in concert with Na,K-ATPase
biophysical similarities in K permeation between the IRK and Ic channels, the conservation of the pore region, and the tetrameric nature of Shaker-type channels [73], it seems reasonable to believe that the IRK channel polypeptides also assemble as tetrameres Future perspectives on the proximal tubule KATP channel [67—691. In this regard, KATP might be generated by mixing, Molecular biology matching and appropriate assembly of separate subunits. CerIn addition to the critical role that KATP plays in the renal tainly the extensive sequence similarity between the presently proximal tubule, the same class of channels are thought to be cloned inwardly-rectifying channels predicts a new superfamily of involved in a wide range of physiological and pathophysiological K channels that presumably includes other functionally comparaprocesses [21, 61]. For instance, similar but pharmacologically and ble K channels like KATP. Where does the proximal tubule basolateral membrane KATP fit biophysically distinct KATP channels perform obligatory roles in mediating and regulating urinary dilution [52, 55], extracellular K into the picture? Based on preliminary RT-PCR expression homeostasis [53], pancreatic beta cell insulin secretion [63], mapping [74] and in situ hybridization studies [75], it seems likely vascular smooth muscle tone [61], cardiac electrical activity in that the ROMK-subfamily of IRK genes encode for subunits of ischemia [64, 65] and suppressing neuronal excitability in anoxia the TAL and CCD apical KATP channels rather the proximal activity.
[66]. Despite the major breakthroughs in demonstrating and tubule basolateral membrane channel. The tremendous funccharacterizing the repertoire of diverse performances played by KATP channels, definitive answers to the precise mechanisms of gating, regulation, and diversity have awaited the isolation of the cDNA(s) encoding these ATP-sensitive channels.
In these regards, an exciting recent development in the K channel field has provided some important hints about the molecular structure of renal epithelial KATP [45, 67—70]. In the last two years, several groups lead by Ho et al [68] have independently employed an expression cloning strategy to isolate cDNAs encoding different renal epithelial KATP-like channels. Ho et al [68] isolated a cDNA they call ROMK1 from the inner strip of the
tional similarities shared between the KATP channels expressed along the length of the nephron do, however, predict a common structural ancestry. With the present pace of field, the question will likely be answered in the near future. Furthermore, as the physiological role of the members of IRK gene family in epithelial KATP function becomes more certain, the stage is set for determining the molecular basis for KATP operation. Possible roles of proximal tubule KATP in pathophysiology Although the KATP channel performs a vital homeostatic role in
a typical day in-the-life of a proximal tubule cell, might KATP,
outer medulla of the rat kidney (principally medullary thick under certain circumstances, play the villain? Consider an ischascending limb, mTAL). Zhou, Tate and Palmer isolated a splice emic insult, a common cause of acute renal failure and tubular variant of ROMK1, ROMK2 [70], while Welling, Tsuchiya and necrosis. Strangled by a compromised energy supply, ATP1 can Giebisch isolated a different, but related, cDNA from the mouse plummet to less that a fourth of basal levels in the nephron cortical collecting duct cell line, Ml [45]. In Xenopus oocytes, each
segment that is especially susceptible to ischemic injury, the
of the channels exhibit many properties characteristic of KATP
proximal tubule [76—78j. Interestingly, the magnitude of the drop
with several important exceptions. Unlike the TAL KATP channel, ROMK1 is activated rather than inhibited by ATP [68] and unlike
is a predictive index of the extent of postischemic renal failure [77]. Moreover, various maneuvers that maintain ATP at basal
the CCD KATP channel, the channel encoded by the Ml cell cDNA is insensitive to sulfonylurea agents [45]. Based on the observation that these gene products are normally expressed in nephron segments where KATP is the major determinant of the macroscopic K conductance [23], it seems plausible that the
levels offer an intriguing degree of protection against the irrëversible effects of ischemia [76, 78]. Observations such as these have suggested that the drop in ATP lies near the top of the hierarchy
of ischemic sequela which can ultimately lead to cell death.
Despite these captivating observations, surprisingly little is known cloned structures encode for parts of a more complex heteroligo- about the most direct and consequent effectors of the ATP meric KATP structure [45, 68]. In this respect, expression of a depletion. Given the ATP-dependent gating properties of the single subunit gives rise to a homologmeric structure with many basolateral membrane proximal tubule K channel, this transport comparable, but not identical, properties of the native KATP element is an obvious candidate. Confronted by an inadequate intracellular ATP level to safechannel. The supposition is partially supported by the primary structure of these channels and their kinship to other inwardly- guard the efficient coupling between the Na,K-ATPase and passive K recycling process, the homeostatic communication would rectifying K, IRK, channels [67, 69, 71, 72]. Defining a new class of channel proteins [67], the deduced be expected to grow mute. Since the KATP channel is normally amino acid sequence of each of these recently cloned IRK released from varying degrees of inhibition upon a fall in ATP1
1022
Welling: Cross talk and KAIP channels
[13, 21], KATP should exist an inappropriately active state in the
face of an energy-compromised Na,K-ATPase at the low ATP levels of ischemia. Indeed, just as expected for the predicament when passive K efflux exceeds active uptake, microprobe analysis demonstrated that ischemia precipitates deleterious dumping of
intracellular K from the proximal tubule cell [79]. Considering that intracellular K homeostasis is essential for numerous cellular processes, the response might be a decisive blow to the proximal tubule in the pathogenesis of acute tubular necrosis. Obviously, it presently remains to be seen whether specific inhibition of the proximal tubule KATP channel can prevent disruption of intracellular K homeostasis and progression of renal failure in ischemia. Summary Over the last few years it has become evident that an assortment of functionally-related, but diverse, KATP channels provide an
important and physiologically-regulated determinant of the K conductive pathways in many, if not all, epithelial cells expressed along the nephron. As such, KATP plays central roles in regulating and maintaining a number of transport processes in concert with physiological demands of the kidney. In the renal proximal tubule,
KATP channels and changes in the hydrolytic activity of the Na,K-ATPase permit ATP to act as a coupling modulator of parallel Na,K-ATPase-K recycling. The response insures that cell membrane potential, intracellular K activity and cell volume are protected in the face of physiological variations in transcellular
ion transport. In addition to demonstrating the physiological
9. LAPOINTE J-Y, LAPRADE R, CARDINAL J: Transepithelial and cell
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Acknowledgment This work was supported by a grant from the American Heart Association, Maryland Affiliate.
Reprint requests to Paul A. Welling, M.D., Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
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