Kidney International, Vol. 36 (1989), pp. 392—402

The electrogenic Na/HCO3 cotransporter WALTER F. BORON and EMILE L. BOULPAEP

Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut, USA

Bicarbonate transport systems are important pathways forHowever, the Na-dependent Cl-HCO exchanger differs from acid-base movement in a wide variety of cell types [1]. HC03 the Na-independent one in three key respects. First, the Na- transporters participate in the regulation of cell pH (pH1) anddependent Cl-HCO3 exchanger has an absolute requirement for volume, in the movement of acid-base equivalents acrossNat Second, because it is driven by the Na gradient, the epithelia, and in the carriage of CO2 between the peripheralNa-dependent Cl-HCO3 exchanger generally moves HC03 tissues and the lungs. As of ten years ago, only two HC03 into the cell, thereby raising pH1. This direction of acid-base transporters had been identified in animal cells, the Cl-HCO3transport is known generically as "acid extrusion." Finally, the exchanger and the Na-dependent Cl-HCO3 exchanger. In addi-acid-extrusion rate is very low at alkaline pH1 values, but rises tion, the existence of a HC03 channel had been hypothesizedas pH1 falls below normal. Thus, in many cells, the Na- for the kidney. dependent Cl-HCO3 exchanger is the major mechanism of resisting intracellular acid loads. In renal mesangial cells, Cl-HCO3 exchangers Na-dependent Cl-HCO3 exchange is stimulated nearly 100% by The Cl-HCO3 exchanger or, more accurately, the Na-inde-the growth factors arginine vasopressin [5] and epidermal pendent Cl-HCO3 exchanger (Fig. 1B) has been studied exten-growth factor (Ganz and Boron, unpublished data). sively in erythrocytes [2], where it is responsible for the "C1 shift" that plays a central role in CO2 carriage. Similar exchang- Channels ers are widely distributed, and have been identified in cells of the kidney and other epithelia. In these nonerythroid cells, the In addition to the Na-independent and -dependent Cl-HCO3 Cl-HCO3 exchanger normally mediates a net HC03 efflux,exchangers, a HC03 channel (Fig. IC) had long been postu- thereby tending to acidify or "acid load" the cytoplasm. Thelated to exist, particularly at the basolateral (or blood-side) acid loding rate is rather low at low pH1 values, but risesmembrane of renal tubule cells. Recent work on crayfish muscle steeply as pH1 increases above the normal range [3, 4]. Thus, infibers [14] has demonstrated a GABA-activated Ci channel many cells, the Na-independent Cl-HCO3 exchanger is thethat also shows selectivity for HC03. Inasmuch as the HCO major mechanism of resisting intracellular alkali loads. In renalelectrochemical gradient is outward (that is, EHOC3 is more mesangial cells, Cl-HCO3 is stimulated more than 100% by thepositive than Vm), a HC03 flux through this C1 channel tends growth factors arginine vasopressin [5] and epidermal growthto depolarize and acidify the cell. This channel is also perme- factor (Ganz and Boron, unpublished data). able to formate and acetate, but not to lactate [15]. However, no A characteristic of all Cl-HCO3 exchangers is that they aresupport has been mustered for a HC03 channel in any cell, nor blocked by the stilbene derivatives 4,4'-diisothiocyano-2,2'-has evidence emerged in epithelia even for a HC03-permeable stilbene disulfonate (DIDS) and 4-acetamido-4'-isothiocyano-Cl channel. 2,2'-stilbene disulfonate (SITS). The protein responsible for Cl-HCOj exchange in erythrocytes is known as the band-3 protein because of its position in SDS-polyacrylamide gel The missing link electrophoresis. The cDNA for the band-3 protein of mouse Given the lack of a channel, an explanation was not forth- erythroçytes has been cloned and sequenced [6] and relatedcoming ten years ago for the data suggesting the existence of an cDNA's have been prepared from other cell types [7]. electrogenic pathway for HC03 at the basolateral membrane The Na-dependent Cl-HCO3 exchanger of the renal proximal tubule. Also problematic was another seemingly unrelated observation: replacing basolateral Na The second HC03 transporter identified as of ten years agowith an organic cation depolarized the proximal-tubule cell. The is the Na-dependent Cl-HCO3 exchanger (Fig. 1A), first char-conventional explanation for this observation, that Na is less acterized in invertebrate cells [8—111, and more recently identi- permeant than bulky organic cations, was counterintuitive. It fied in a variety of mammalian cells [4, 12, 13]. Like thenow appears that the most likely explanation for the conductive Na-independent variety, the Na-dependent Cl-HCO3 exchangermovements of both HC03 and Na across the basolateral requires Cl and HC03, and is blocked by SITS and DIDS.membrane is an unconventional one. That is, a novel, stilbene- sensitive transporter mediates the isodirectional movement of one Na , two or three HC03 and net negative charge © 1989 by the International Society of Nephrology (Fig. 1D).

392 Boron and Boulpaep:NaIHCO3 cotransporter 393

Cell Both pH 7.5 pH 7.5 Lumen: I

Na pH 6.8 pH6.8 pH6.8 HCO Bath: L.__J Sits

Cl-

Cl 0

HCO

C 5 mm HCO Fig. 2. Effect on pH1 and Vb, of reducing bath pH (i.e., bath [HCO3j). During the three indicated periods, [HCO3]b was reduced from 10 to 2 m at a fixed "CO2 of 1.5%,causingpHb to fall from 7.5 to 6.8. In the absence of SITS, the reduction in HC03 caused a large and rapid fall in pH1 as well as a large and rapid depolarization. Both changes were substantially reduced by a 0.5 mrt SITS. Taken from ref. [16], repro- Na duced by permission of Rockefeller University Press.

removing bath (that is, blood-side) C1 should lead to the n HCO uptake of HC03 and therefore a rapid increase in pH1. Conversely, reducing [HC03]b should lead to the uptake of C1, and therefore a rapid increase in a1c. However, removing bath C1 caused a slow fall in pH1, whereas reducing [HCO3Ib caused only a slow and small rise in a' [16]. These observa- Fig.1. Four demonstrated mechanisms by which HC03 can perme- ate animal cell membranes. (A) The Nat-dependent Cl-HCO3 ex-tions (not shown) made it unlikely that that substantial Na- changer. (B) The Nat-independent Cl-HCO3 exchanger. (C) Theindependent Cl-HCO3 exchange activity existed at the basolat- GABA-activated C1 channel, which also admits HCO3. (D) Theeral membrane. Experiments on basolateral membrane vesicles electrogenic Na/HCO3 cotransporter. from the rabbit renal cortex [18] confirm that a C1 gradient does not influence HC03-gradient-dependent Na influx. Effect of lowering basolateral [HC03] on pH1 and V,,1 Original description of the electrogenic Na/HCO3 cotransporter Ifthere were a simple HC03 conductance (that is, mediated Evidence against involvement of C1 by channel) at the basolateral membrane, then reducing The electrogenic Na/HCO3 cotransporter was first described[HC03}b should produce a fall in pH1 (due to increased exit of in experiments on isolated perfused proximal tubules from theHC03), and a basolateral depolarization (due to increased exit tiger salamander Ambystoma tigrinum [16]. The approach wasof negative charge). Indeed, as illustrated in Figure 2, reducing to use microelectrodes to monitor basolateral membrane poten-pHb from 7.5 to- 6.8 (that is, reducing [HC03]b from 10 to 2 tial (Yb,) as well as either intracellular pH (pH1), intracellularmM) caused pH1 to rapidly fall by more than 0.3, and the Na activity (a"), or intracellular Cl activity (a1dI). A Na-Hbasolateral membrane to depolarize by —30 mV. The effects on exchanger had already been identified at the salamander prox-pH1 and Vb, were largely blocked by SITS, were substantially imal tubule's basolateral membrane [17], and it was expectedreduced in the nominal absence of HC03, but were not that this transporter would be accompanied by either a Cl-significantly altered in the absence of Cl—. HCO3 exchanger (Fig. 1B) or a HC03 channel (Fig. IC). One On the surface, the above data appear to be consistent with should be able to distinguish between these two HC03 trans-the existence of a SITS-sensitive HC03 channel. However, port pathways on the basis of how bI' pH1 and aC respond tocloser analysis [16] revealed an inconsistency. In order for the changes in bath (b) [HC03] and [Cl—I. For example, if thereresting Vbl(thatis, ——56 mY) to be so much larger than the were a Cl-HCO3 exchanger at the basolateral membrane, thencalculated equilibrium potential. for HC03 under resting con- 394 Boron and Boulpaep: Na/HCO3 cotransporter

100-Na be ascribed to stimulation of the electrogenic Na-K pump, Lumen: inasmuch as the voltage changes were unaffected by ouabain. 0-Na 0-Na Bath: ____I Predictions of the model for electrogenic Na/HCO3 cotransport Sits The above results on the salamander proximal tubule led to a model of electrogenic Na/HCO3 cotransport, according to I- which HC03 transport is tightly coupled to the movement in the same direction of Na and net negative charge. Although 7.0 this cotransporter has been identified most frequently at the basolateral membrane of renal tubule and other epithelial cells, a similar transporter has also been found in nonepithelial cells. Or Therefore, in the remainder of this review, we will generalize -20 our discussion so that "membrane voltage" (m) refers to the potential across the membrane in which the cotransporter is —40 located. Similarly, "extracellular" solute concentrations (such —60 as [Na410) refer to those of the external solution facing the 5mm cotransporter. According to the model of electrogenic NaJ HCO3 cotransport, it should be possible to drive the electro- Fig. 3. Effect on pH, and Vbj of removing bath Nat During the two indicated periods, [Na]b was reduced from 100 to 0 m (Na replaced genic NaJHCO3 cotransporter by altering either Vm, [Na]0 or by bis-(2-hydroyxethly)dimethylammonium). 1'C02 was fixed at 1.5% [HC0310 [16]. and [HCO3]b, at 10 mrs (pHb =7.5).In the absence of SITS, the removal of Na caused a large and rapid fall in pH, as well as a large and Predicted effects of altering Vm rapid depolarization. SITS (0.5 mM) largely blocked the pH, changes, and reversed the direction of the VbI changes. Taken from ref. [16], Because the Na/HCO3 cotransporter carries net negative reproduced by permission of Rockefeller University Press. charge in the same direction as Na and HC03, depolarizing the basolateral membrane should reduce the cotransporter- mediated effiux of Na and HC03, or even reverse the transporter and produce an influx. Perhaps the most direct tests ditions (——12 mV), Vb, would have to be dominated by an ionof the predicted effects of voltage changes on electrogenic other than HC03. On the other hand, in order for a reductionNaJHCO3 cotransport have been made on basolateral mem- in [HCO3]b to have produced a —30 mY depolarization whenbrane vesicles derived from rabbit renal cortex. Establishing an the calculated HC03 equilibrium potential changed by only 24inside-positive voltage in these vesicles produces a HC03- mV, bl would have to be dominated by HC03. Unless thedependent uptake of Na [18, 19] that is blocked by SITS [19]. very act of reducing [HCO3]b produced an enormous increase Voltage effects have also been examined in intact cells. in HCO3 permeability, we can conclude that a simple HC03 Alpern [20] found that depolarizing the cells of doubly perfused channel could not have produced the data of Figure 2. rat proximal convoluted tubules with 50 ifiM K or 2 mM Ba ÷ in the basolateral solution caused a reversible increase in pH,. Effect of lowering basolateral [NaJ on pH1 and V,,, This depolarization-induced alkalinization (DIA) was blocked A second difficulty in ascribing the data of Figure 2 to aby removing Na from luminal and basolateral perfusates, simple HC03 channel is that the pH1 changes elicited byconsistent with the hypothesis that the DIA was mediated by altering [HCO3]b are likely to have been complicated by theelectrogenic NaJHCO3 cotransport. Similar results have been activity of the basolateral Na-H exchanger. For example, theobtained on fused giant cells, derived from the frog diluting pHb fall that accompanied the reduction in EHCO3]b doubt-segment, by Wang et al [21]. They found that depolarizing the lessly inhibited Na-H exchange and thereby contributed to thecells either by raising [K]0 or adding Ba caused a pH1 to pH1 decline. Conversely, restoring [HCO3]b and pHb to theirrise, whereas hyperpolarizing the cells by adding furosemide initial values not only removed this inhibition of Na-H ex-caused pH, to fall. Data on the voltage effects of electrogenic change, but also unmasked the stimulation of the exchanger byNaJHCO3 cotransport in a variety of cells are summarized in the intracellular acid load. Thus, the Na-H exchanger wasTable 1. expected to contribute to the pH, recovery (that is, increase) In interpreting DIA data, one should be cautioned that the that follows the return of pHb from 6.8 to 7.5. Indeed, when theelectrogenic NaJHCO3 cotransporter is not the only mechanism experiment of Figure 2 was repeated in the presence of amilo-by which pH, can be affected by voltage changes. When ride (not shown), the pH1 recovery was slowed by about half.salamander proximal tubules are incubated in the nominal However, because the amiloride may have had nonspecificabsence of HC03, depolarization produces a substantial alka- effects or may have failed to completely block the Na-Hlinization, only about one third of which is rapidly blocked by exchanger, an attempt was made to block basolateral Na-Hbath SITS [221. The remainder of the DIA is blocked by the exchange by removing Na from the bath. Amazingly, remov-removal of either luminal Na or luminal lactate [23], and ing bath Na caused large and rapid decreases in both pH1 andappears to be due to the combination of increased luminal bI (Fig. 3). Moreover, the pH1 and VbI changes were revers-lactate influx mediated by a Nallactate cotransporter, and ible, repeatable, and blocked by SITS. The hyperpolarizationincreased basolateral lactate effiux mediated by a H/lactate observed upon returning Na to the basolateral solution cannotcotransporter [24]. Boron and Boulpaep: Na/HCO3 cotransporter 395

Table 1. Data onthe activity, or lackthereof, of electrogenic NaJHCO3cotransport in various preparations Preparation Ref. pH, data a1N orNa4 flux data Voltage data Comments Amphibian renal tubules a a Ambystoma PT 16 [HC03]0—*, pH1 I [HC03]0—*I a1 [HCO ]O—* +Vm a inhibited or , b.c b.c b I,[Na]1—* pH, [Na4] —÷y, blockedby SITS HC03 dependent All a's: Cl independent Necturus PT 30 [HC03]0 —pH [HCO3 lo —+Vm blocked by SITS .[Na10—J, pH1 [Na4] +Vm blocked by SITS frog diluting segment! 56 [HCO3IO —*+Vm blocked by SITS, ACZ fused cells [Na4]0 —+Vm blocked by SITS Mammalian proximal tubules rat PT 20 [HC03]0 —* pH1 blocked by SITS [Na4]0 — pH1 blocked by SITS +Vm L,pH1 blocked by 0 Na4 25 —* b [HC03]0 —* a inhibited or ,[HCO] , pH1 I I a [HC03]0—* +v,,,a b l,[Na4]0—l,pH11' I [Na4]0—I a a [Na4]0 —++Vm blockedby SITS rabbit PCT 28 [HC03]0 inhibited 43% by ACZ & pCO2 . [Na4]—*+Vm] no SITS or ACZ rabbit PST 28 t[HCO310— +Vm inhibited 33% by ACZ [Na4]0 — —blocked by SITS +Vm b 57 . [HCO3]—+ pH,b [HC03]0— [HC03]0 —+Vm a inhibited or b I Ia1 a b , [Na]1—pH, [Na4]0— blockedby SITS rabbit PST/SF 58 [HCO3 lo —+Vm blocked by SITS & ,!.pCO2 [Na4] +Vm blocked by SITS rabbit Si/SF 59 1'[Na4]0—1'pH, blocked by DIDS rabbit S1/JM 59 t[Na4] No DIDS-sensitive pH, f rabbit S2 42 t.[HCO3]0— pH, I [HC03]0—* 'm ,[Na4]0—pH, I [Na4]0 rabbit S2/SF 42 [HCO3]0-+ +Vm 59 t[Na]—*C pH, blockedby DIDS rabbit S2IJM 59 1'[Na4]0 No DIDS-sensitive pH, 1 rabbit S3 45 [HC03]0— pH1 partlyNa4 dependent, and C1 independent [Na] —÷ pH1 C1 independent, and partly SITS sensitive 46 [HC03]0 —*pH, independent of C1 [Na4]0 —* pH, independent of C1, blocked by DIDS a rabbit S3/SF 42 I, [HCO3]O—J pH, a small or bnochange b t[HCO3]0— +Vma [Na4]0 [Na10 +V,,, a 59 C [Na4]—pH1 blocked by DIDS rabbit S3/JM 42 I[Na4]0 +Vm change was very small 59 C [Na4]0 —* pH1 blocked by DIDS blm vesicles/rabbit 18 I [Na4]0 —IpH1 HC03 dependent renal cortex t [HC03]0—22Nainflux HC03 dependent, C1 independent, sensitive to DIDS amiloride insensitive +Vm 22Nainflux HC03 dependent 19 +Vm 22Nainflux HC03 dependent, sensitive to SITS Other epithelial preparations bovine corneal 26 I[HC03]0 Na4 or Li4 dependent endothelium I[Na]0—+Vm HC03 dependent, blocked by SITS/DIDS, Li subst. for Na4 35 22Na uptake HC03 dependent, sensitive to SITS, but ? C1 independence BSC-l cells 36 22Na I [Na4]0 +Vm HC03 dependent, DIDS sensitive, but ? Cl independence gastric oxyntic cells 37 I [HC03]0 —+Vm SITS sensitive, Na4 dependent I [Na4]0 -+Vm SITS sensitive Table I continued on next page. 396 Boronand Boulpaep: Na/HCO3 cotransporter

Table1. Continued Preparation Ref. pH, data a1 or Na flux data Voltage data Comments Nonepithelial cells smooth muscle/ 48,51 t[Na]o—* tpH1 I[Na]o—' Vm guinea pig ureter glial cells/leech 54 acid load —,pH, Na & HC03 dependent, SITS sensitive a.b.c a 54, 55 t[HCO31O—* pH, I [HC03]0 f ,Na t[HCO3]—*—v,,, Alli"s: DIDS sensitive &tpCOz &pCO2 & pCO2 aNadependent bnotSITS or amiloride sensitive not blocked by 0 C1 for 10 mm. oligodendrocytes/ 53 acid load —,I pH, I [NaJ0 —+Vm SITS/DIDS insensitive mouse & C1 independent somites/chick embryo 34 acid load —t pH, HC03 dependent, DIDS sensitive, but? Cl independence Data indicating a lack of electrogenic NaJHCO3 cotransport are indicated by a "!"inthe "Comment" column, and the absence of crucial data, by a"?". Abbreviations are: PT, proximal tubule; PCT, proximal convoluted tubule; PST, proximal straight tubule; SF, superficial nephron; JM, juxtamedullary nephron; Si, S2 and S3, progressively more distal Fl' segments; blm, basolateral membrane; SITS, 4-acetamido-4'-isothiocyano- 2,2'-stilbenedisulfonate; DIDS, 4,4'-diisothiocyano-2,2'-stilbenedisulfonate; ACZ, acetazolamide; ,[HC0310,external [HCO3J is decreased at fixed pCO2 (i.e., pH0 falls); [HC03}0/ pCO2, external [HC03]0 and pCO2 are decreased at fixed pH0; +Vm, cell depolarized (interior more positive); acid load, pH, acutely decreased (e.g., by NH4 prepulse).

pH 7.5 Predicted effects of altering [Na],, Lumen: I The model predicts that reducing [Na]b should promote the coupled exit of HC03, Na and net negative charge, thereby pH 6.8 pH 6.8 pH 6.8 I II decreasing pH, and a1Na, as well as depolarizing the cell. Bath: LJ Moreover, these effects should be independent of C1 and Sits blocked by SITS or DIDS. The predicted effects on pH1 and m 4O are confirmed by the data of Figure 3 [16], and those on a1, by E 30F the data of Yoshitomi, Burckhardt and Fromter on rat proximal .- F tubules [25]. Table 1 summarizes similar data that have been 20L obtained on a variety of preparations. As discussed below, the Vm effect of lowering [Na}0 is probably the characteristic most 0 peculiar to the electrogenic NaJHCO3 cotransporter. Although experiments on salamander proximal tubules have confirmed _20E the Cl independence of the pH1 and Vm effects [161, the Cl independence of the a effect has, to our knowledge, not been demonstrated. -60h Na is not the only cation that functions with the cotrans- 5 mm porter. Data on bovine cornea! endothelial cells indicates that Fig. 4. Effect on a7" of reducing bathpH(i.e.,bath [HC03j).During Li substitutes for Na [261. Work on a variety of preparationsthe three indicated periods, [HCO3]b was reduced from 10 to 2 m at indicates that the cotransporter is inhibited, at least in epitheliala fixed CO2 of 1.5%, causing pHb to fall from 7.5 to 6.8. In the absence cells, by the stilbene derivatives SITS and DIDS. The trans-of SITS, the reduction in HC03 caused a rapid fall in a,• that was porter is also inhibited in epithelial cells by acetazolamide, an followed by a slower recovery. These changes were blocked by 0.5 mas SITS. Taken from ref. [16], reproduced by permission of Rockefeller inhibitor of carbonic anhydrase [27,28].However, work with basolateral membrane vesicles suggests that this inhibition is University Press. due to the effect of acetazolamide on the generation or disposal of HC03, and not a direct effect on the cotransporter [29]. Reducing pH0 by lowering [HC03]0 at a fixed pCO2 causes a Predicted effects of altering [HCO37,, sharp decline in a1Na, followed by a slower a11 recovery. The The model predicts that the coupled exit of HC03, Na andrapid decline probably reflects the coupled uptake of Na and net negative charge should also be promoted by reducingHC03, inasmuch as the rapid a, change is blocked by SITS. [HC03]0. Thus, a reduction in [HC03]0 should produceAs summarized in Table 1, similar pH,, a and Vm data have decreases in pH1 and as well as a depolarization, that arebeen obtained in a variety of cell types. independent of Cl— and blocked by SITS or DIDS. The pH1 and A clear distinction should be drawn between the effects of Ym effects, as well as their sensitivity to SITS, are confirmed inreducing [HC03]0 and pH0 at a constant pCO2 (that is, Figure 2 [16]. The effects on a, are illustrated in Figure 4.metabolic acidosis), and those of reducing [HC03]0 and pCO2 Boron and Boulpaep:Na/HCO3 cotransporter 397 and constant pH0 (that is, isohydric hypocapnia). In both cases,where the subscripts i and o refer to intra- and extracellular, the reduction in [HC03]0 imposes a transmembrane HC01 respectively. The overall change in electrochemical potential gradient that gradually fades as [HCO31 falls. In the case of(iLNHco3) is the sum of the changes in the electrochemical reducing [HCO31O and pH0, there is no CO2 gradient acrosspotentials for Naand HC03, weighted by their molar ratios: the cell membrane, so that the reduction in [HC03]1 is brought [Na]0 about almost exclusively by the effiux of HC03. One would FVm (2a) therefore expect [HC03]1 to fall relatively slowly, and for the NaRT[Na + ] electric current carried by the Na/HCO3 cotransporter to decay = IHrOL 3 Jo slowly. In the case of simultaneously reducing [HC03]0 and nLHCO3 RTIn -+ nFVm (2b) pCO2, the decline in [HC03]1 is brought about not only by the It1.'_3Jj efflux of HC03 per se, but also by the effiux of CO2. The latter promotes the intracellular reaction: HC03 + H —>H20+ rxi +1rLiC'f —1 fl CO2. Thus, [HC03]1 is expected to decline rapidly, so that the 1a Jo".—''3Jo L/.LNa/HCO=RTIn + (n —l)FVm(2c) current carried by the NaJHCO3 cotransporter should also .j -1ii decline rapidly. These predictions are borne out by the data on rabbit PCT's and PST's obtained by Biagi and Sohtell [281. Estimatesof stoichiometry. How large must n be for the They found that simultaneously reducing [HC03]0 and pCO2NaJHCO3 cotransporter model to explain the data? The evi- (at fixed pH0) produced a spiking depolarization that rapidlydence in proximal tubules from amphibians [16, 30] and mam- decayed, whereas reducing [HC03} and pH0 (at fixed pCO2)mals [25] indicates that the NaIHCO3 cotransporter normally produced a rapid depolarization that was relatively stable. mediates the net effiux of Nat, HC03 and net negative charge. For example, blockade of the cotransporter with stilbenes causes a rise in pH1 and a hyperpolarization. Moreover, the Stoichiometry of the electrogenic Na/HCO3 cotransporter cotransporter would have to mediate a net HC03 efflux to There are two general methods for determining the stoichi-account for HC03 reabsorption. In order for the reaction in ometry of a transporter such as the electrogenic NaJHCO3equation 1 to proceed as written (that is, in the direction of cotransporter. The most straightforward approach is to directlyHC03 effiux), the value of Xjs/co3 computed in Equation measure the cotransporter-mediated net fluxes Na and2c must be less than zero. According to the original salamander HC03. The second approach is to use thermodynamics todata, this requirement could be met, though barely, by an n of bracket a range of stoichiometries that is consistent with the2. However, in order to account for data later obtained in rat data. [25] and Necturus [30] proximal tubules, the stoichiometry would have to be 3:1. Flux measurements A more methodical approach for estimating the stoichiometry of the proximal-tubule NaJHCO3 cotransporter was used by In experiments on microperfused rat proximal tubules,Soleimani, Grassl and Aronson [31]. They prepared basolateral Yoshitomi et al [25]measuredthe initial rate of change of pH1membrane vesicles from the rabbit renal cortex, and systemat- and a1ITa following a reduction in [HCO3 10.Theyfound that theically varied either the Nat, HC03 or voltage gradient across initial HC03 flux was 3.1 times larger than the initial Na flux,the membranes while measuring the net Na flux. Thermody- indicating a HC03 : Na stoichiometry of 3: 1. These data havenamics requires that this net flux should approach zero and then the advantage of having been obtained on intact cells. On thereverse as the altered Na, HCO3 or voltage gradient causes other hand, both flux calculations depend critically on the/Na/HCO3 to approach zero and then reverse. Matching the null assumption that the pH1 and changes were mediatedflux to the stoichiometry-dependent sN&HCO3 Soleimani et al entirely by the electrogenic NaJHCO3 cotransporter. Moreover,found that n is between 2.6 and 3.5, consistent with a as is the case for all such calculated HCO3 fluxes, this one isHCO3 : Na stoichiometry of 3. proportional to the assumed intracellular buffering power. Note that the specific examples described above apply only to the electrogenic Na/HCO3 cotransporter at the basolateral Thermodynamics membrane of proximal tubules. The stoichiometry has not been established in non-proximal tubule preparations. The energetics of Na/HCO3 cot ransport. The effects of NaIHCO3 cotransport on membrane voltage indicate that the Kinetics of Na/HCO3 cotransport transporter is electrogenic, moving net negative charge in the same direction as Na and HC03. This can only be achieved Dependence of cotransport on Na and HC03 if more than one HC03 ion moves with each Na ion. The Work on basolateral membrane vesicles isolated from the precise stoichiometry is important not only because it deter-rabbit renal cortex has shown that the HC03-dependent Na mines the amount of charge moving with each turnover of the influx appears to be occur via a single saturable site with a Km cotransporter, but also because it determines the direction of of 9.7 when driven by a HCO3 gradient (pH1 = = net transport. One approach for predicting the direction of net 6.0/pH0 7.5) [18], or a Km of 10.4 m when driven by a voltage gradient in transport is to compute the change electrochemical potential that occurs with each turnover. If n HC03 ions are trans-the presence of 21 mri HC03 [19]. In the same preparation, ported for each Na ion, then the overall reaction is the HC03 dependence of Na uptake ([Na]0 =8mM) was more complex. An apparently high-affinity interaction saturated (Na) + n(HCO31, —*(Na)0+ n(HC03)0, (I)at a [HCO3-]0 of —4 m, whereas a lower affinity interaction 398 Boronand Boulpaep: Na/HCO, cot ransporer saturated at a [HCO3 in the region of 40 m'vi [19]. These datations elicit responses that are more uniquely related to the support the hypothesis that two distinct HC03-related specieselectrogenic Na/HCO3 cotransporter than are other combina- must bind to the carrier before NaJHCO3 cotransport can occur.tions. For example, the depolarization elicited by decreasing [Na]0 is almost diagnostic of the electrogenic NaJHCO3 Evidence for the transport of C03 cotransporter. On the other hand, the depolarization elicited by In work done on basolateral membrane vesicles from thedecreasing [HCO31O and pH0 could be mediated by a pH- rabbit renal cortex, Soleimani and Aronson [32] examined thesensitive K channel, or other pH-sensitive conductances. possibility that the electrogenic NaJHCO3 cotransporter has Perhaps the most difficult distinction to be made is between separate sites for Na, HC03 and C03. They found that Nathe cotransporter and the Na-dependent Cl-HCO3 exchanger. uptake was increased by raising [C0310 at constant [HCO31O.This is illustrated by a recent analysis of acid-base transport in Moreover, DIDS-sensitive Na uptake was increased threefoldrenal mesangial cells. Boyarsky et at [4, 33] found that recovery by the divalent anion sulfite in the presence of HC03. but notof pH1 from an imposed acid load is dependent upon both Na in its absence. However, increasing [C0310 at fixed [HC0310and HCOI, and is blocked by the stilbenes SITS and DIDS. reduced this sulfite-stimulated Na uptake, suggesting compe-The pH1 changes were unaffected by depleting the cells of C1 tition between sulfite and CO3 for a divalent-anion receptor onfor up to 15 minutes, a result that appeared to rule in the the cotransporter. The NaCO3 —ionpair does not seem to be theelectrogenic Na/HCO3 cotransporter. However, when the pe- substrate for the cotransporter inasmuch as harmaline, whichriod of C1 depletion was increased to an hour or more, it competes with Na on many Na-dependent transporters, alsobecame clear that the Na/HCO3 -dependent pH1 recovery competes with Na for the electrogenic NaJHCO3 cotrans-was indeed Cl dependent, and therefore due not to the porter. Thus, the data are consistent with a model in which theelectrogenic NaJHCO3 cotransporter, but to the Na-dependent cotransporter binds one Na, one HCO and one CO3. TheCl-HCO3 exchanger. This experience with mesangial cells calls stoichiometry would thus be one equivalent of Na for eachinto question a recent conclusion that electrogenic NaJHCO3 three equivalents of HC03 or related alkali. cotransport occurs in cells isolated from embryonic chick somites. In that study [34], the authors ruled out a Na- Criteria for identifying the electrogenic Na/HCO3 cotransporterdependent Cl-HCO3 exchanger based on the observation that a As noted in passing in relation to the predictions of thepH1 recovery from an acid load (imposed by pretreating with 30 electrogenic NaJHCO3 cotransporter model, and discussed inmM NHCI) was not blocked by incubating the cells for up to more detail below, a process similar to electrogenic NaJHCO3one hour in a Cl free solution. However, the NH4 solution, cotransport has been identified in a variety of epithelial andapplied and then withdrawn immediately before the assay, nonepithelial cells. This raises the issue of the criteria used toapparently contained 30 mM C1. establish that a candidate process is indeed electrogenic NaJ Even after an electrogenic NaJHCO3 cotransporter has been HCO3 cotransport. For some transporters, establishing suchpositively identified in a cell, caution is still in order for criteria is easy. The Na-K pump is blocked specifically byinterpreting kinetic data. For example, 22Na fluxes cannot be cardiotonic steriods such as ouabain. Thus, the isolated obser-used to study the kinetic properties of the electrogenic NaJ vation of a ouabain-sensitive Na flux can be taken as evidenceHCO3 cotransporter unless contributions from Na-dependent for the Na-K pump, even though this transporter has otherCl-HCO3 exchange have been ruled out. Two cases in point are characteristics (such as, K dependence, electrogenicity). Thekinetic studies of electrogenic NaIHCO3 cotransport in bovine identification of an electrogenic Na/HCO3 cotransporter iscorneal endothelial cells [35] and BSC-l cells [36] in which especially difficult for two reasons. First, no unique inhibitor isHC03-dependent and stilbene-sensitive 22Na fluxes were not known. Second, there are other transport pathways, or combi-shown to be Cl independent. nations thereof, that can easily be confused with the electro- genic Na/HCO3 cotransporter. Thus, identifying a process as Distribution and action of the Na/HCO3 cotransporter electrogenic NaJHCO3 cotransport is not merely a matter of collecting positive evidence; extreme care must be taken in Epithelial cells ruling out alternative explanations. Distribution. As summarized in Table 1, processes similar to In principle, the identification could be made based onthe proximal-tubule electrogenic Na/HCO3 cotransporter have measurements of only one parameter (that is, pH1, a1 or Vm),been identified or tentatively identified in a variety of epithelial while performing only one fundamental maneuver (such as,cells, including several proximal-tubule preparations, bovine reducing [Na10). As summarized in Table 2, variations on thiscorneal endothelial cells [26], and gastric oxyntic cells [371. It is fundamental maneuver (such as, performing it in the presencein the renal proximal tubule where HC03 transport has been of SITS) could allow one to meet what we regard as the minimalstudied most extensively. In this segment of the nephron, the requirements for identifying the cotransporter. Obviously, theelectrogenic NaJHCO3 cotransporter probably plays a key role identification can be made with greater confidence if more thanin the reabsorption of filtered HC03, as summarized in Figure one parameter is measured and/or if more than one fundamental5A. maneuver is executed. However, it is not sufficient to charac- Adaptive changes in proximal-tubule cotransporter activity. terize the process only partially according to each of severalConsistent with the proposed role of the electrogenic NaJHCO3 sets of parameter/maneuver criteria, unless the experimentscotransporter in HCO reabsorption, Akiba, Rocco and War- have been carefully designed to rule out each of the othernock [38] have shown that metabolic acidosis of 24 to 48 hours transport pathways that could yield similar results. duration produces an increase in the Na-H exchange rate in As one might expect, certain parameter/maneuver combina-brush border vesicles, and that this is paralleled by an increase Boron and Boulpaep:Na/HCO3 cotransporter 399

Table 2.Requirements for identifying an electrogenic NaJHCO3cotransporter, based on isolated measurements of pH,, a1Na,orVm Fundamental Transporters to be Observations Parametera maneuver" led outc Necessary Desirable pH1 [Na]0 Na-dependent Cl- pH that is: pH that is: HCO3 exch. -Cl independent -HCO3 dependent Na-H exch. -blocked by stilbenes or -insensitive to amilorides insensitive to amilorides or blocked by stilbenes [HCO]0 & pH0 Cl-HCO3 exch. pH1 that is: pH that is: Na-dependent -Cl independent -Na dependent Cl-HCO3 exch. -HC03 dependent Na-H exch. -blocked by stilbenes or -insensitive to amilorides Other acid-base (i.e., insensitive to amilorides or blocked by stilbenes buffer) transporters -independent of buffers other than HCO3 +Vm HC03 channel j' pH that is: f pH, that is: Monocarboxylate -stilbene sensitive -independent of Cl transport -HCO3 dependent channel blockers -independent of other buffers a1 J. [Na]0 Na-dependent Cl- a that is: that is: HCO3 exch. -Cl independent -HC03 dependent Na-H exch. -blocked by stilbenes -insensitive to amilorides Other Na-depen- -insensitive to inhibitors of transporters "other" transporters -independent of "other" solutes I [HCO3]0 & pH0 Na-dependent I a1 that is: I a1 that is: Cl-HCO3 exch. -blocked by stilbenes -insensitive to amilorides Na-H exch. -HCO3 dependent -insensitive to inhibtors Na/monocarboxylate -C1 independent of "other" transporters cotransport -independent of Other Natdependent monocarboxylates & transport "other" solutes +Vm Monocarboxylate I aTla that is: I a, that is: transport -stilbene sensitive -HCO3 dependent -independent of monocarboxylates

Vm I [Na]0 None +Vm that is: +Vm that is: HCO3 dependent or blocked by stilbenes or blocked by stilbenes HCO3 dependent I [HC03]0 & pH0 HC03 conductance +Vm that is: +Vm that is: Cl-HCO3 exch. & -Na dependent -insensitive to inhibitors C1 channel -HCO3 dependent of K & "other" Na-dependent Cl- -blocked by stilbenes conductances HCO3 exch. & C1 -independent of C1 or Na channel -independent of "other" K channel ions Other pH-senstive conductances It is assumed that only one "parameter" is measured, and only one "fundamental maneuver" is performed. The "necessary observations" are the minimal requirements for ruling Out all transport pathways other than electrogenic NaIHCO3 cotransport. pH1 is intracellular pH; a1 is intracellular Na activity, although concentration could also be measured; Vm is membrane potential, with "+" indicatinga depolarization. b I [Na10 is a decrease in extracellular Na concentration; I [HCO3]0 & pH,, is a decrease in extracellular HCO3 concentration (and thus pH) at a fixed pCO2; +Vm is a depolarization of the membrane (more positive inside cell). Transport pathways, or combinations thereof, that would be expected to respond to the "fundamental maneuver" in the same way as the electrogenic NaIHCO3 cotransporter. Cl-HCO3 exch. is Na-independent Cl-HCO3 exchange. in the NaJHCO3 cotransport rate in basolateral membraneactivity. An adaptive increase in electrogenic NaJHCO3 vesicles. Metabolic alkalosis had the opposite effects. For bothcotransporter activity, assessed as HC03-dependent 22Na transporters, the adaptive effects were due to changes inuptake in basolateral membrane vesicles, also occurs in chronic maximal transport rate, and not to alterations in the affinity forhypercapnia [411. Nat Studies of pH, regulation in rat proximal tubules have Distribution of cotransporter along proximal tubule. In iso- shown that chronic metabolic acidosis [391 as well as hyperfil-lated perfused rabbit proximal tubules, electrophysiological tration [40] each produce an adaptive increase in cotransporterdata indicate that NaJHCO3 cotransporter activity is greater in 400 Boronand Boulpaep:Na/HCO3cotransporier

A Lumen Cell Blood B Lumen Cell Blood

Na H HCOH- H2 CO3 H2C03,

H20 H20

Fig. 5. Proposed roles for the electrogenic Na/HCO3 cotransporter in proximal-tubule cells. (A) Mammalian proximal tubule. Electrogenic NaJHCO3 cotransport would promote the reabsorption of both Na and HC03 by keeping pH sufficiently low to stimulate luminal Na-H exchange. The cotransporter would also directly mediate the basolateral steps of NaHCO3 reabsorption. (B) Amphibian proximal tubule. Electrogenic Na/HCO3 cotransport would promote the reabsorption of monocarboxylates such as lactate by keeping pH1 sufficiently low to stimulate the luminal Na/lactate cotransporter and/or the basolateral H/lactate cotransporter. The basolateral Na effiux would directly contribute to sodium lactate reabsorption. The HC03 equivalents moved by the cotransporter would merely short circuit the acid-base effects of the basolateral H/lactate cotransporter. the S2 than in the more distal S3 segment [42], and pH1The basolateral NaJHCO3 cotransporter would not only provide measurements confirm that cotransporter activity falls in morea means of basolateral Na exit, it would also lower pH1 and distal proximal-tubule segments [43]. Thus, cotransporter ac-therefore be expected to promote lactate transport. tivity correlates well with transepithelial Nat, HC03 and fluid Other epithelia. In the cornea, the NaIHCO3 cotransporter reabsorption, all of which decline in more distal proximal-probably is important for transporting fluid from the cornea to tubule segments [44]. Recent studies of pH1 regulation havethe aqueous humor in the anterior chamber of the eye, and shown that the basolateral membrane of the rabbit S3 segmentthereby maintaining corneal transparency [26]. The role of the contains both a Cl-HCO3 exchanger and an electrogenic NaJcotransporter in the oxyntic cells has not been established [37]. HCO3 cotransporter [45—47]. Inasmuch as both transporters probably mediate a HC03 efflux (that is, reabsorption), it Non-epithelial cells might be asked why it is the NaJHCO3 cotransporter, rather Table 1 also summarizes data from nonepithelial cells that than the Cl-HCO3 exchanger, that is correlated with high ratespossess a transporter that is at least superficially similar to the of NaHCO3 and fluid reabsorption. One reason is that theepithelial NaIHCO3 cotransporter. Perhaps the most striking NaJHCO3 cotransporter directly contributes to Na as well ascharacteristic of these transporters is that they normally func- to HC03 reabsorption. Thus, even though HC03 efflux istion as acid extruders, producing a pH1 recovery from an acute osmotically silent, the Na/HCO3 cotransporter mediates theintracellular acid load. A second peculiarity is that these reabsorption of an osmostically active particle (that is, Nat),cotransporters are often reported to be insensitive to stilbenes and thus directly promotes the reabsorption of water. On thesuch as SITS or DIDS. other hand, the only net osmotic effect of Cl-HCO3 exchange is The first evidence for the nonepithelial NaJHCO3 cotrans- the basolateral uptake of Cl, which retards fluid reabsorption.porter came from Aickin's work on smooth muscle cells from The amphibian proximal tubule. The correlation betweenthe guinea-pig ureter [48]. She found that raising [HCO3]0 and NaJHCO3 cotransport and high rates of HC03 reabsorptionpCO2 at a fixed pH0 causes a sustained increase in pH1 (a breaks down in the amphibian proximal tubule, which has aHC03-induced alkalinization). Furthermore, after cells are potent NaJHCO3 cotransporter [16, 30] but a low rate of HC03 acid loaded by the NH4 prepulse technique [49], the subse- reabsorption. A physiological role for the NaJHCO3 cotrans-quent pH1 recovery requires Na and HC03. Finally, exami- porter in the amphibian proximal tubule was suggested by anation of the Vm recordings reveals that increasing [Na]0 from study, conducted in the nominal absence of HC03, of the1% to 100% of the control value not only caused a rapid pH1 effects of monocarboxylate transport on pH1 regulation in therecovery, but the transient hyperpolarization that is the hall- salamander proximal tubule [24]. As outlined in Figure SB, themark of the epithelial cotransporter. Other work demonstrated salamander proximal tubule has a luminal Na/lactate cotrans-that the pH1 recovery is independent of CY [50], thus pointing porter that mediates the influx of Na and lactate, as well as ato a NaJHCO3 cotransport process. basolateral H/lactate cotransporter that mediates a net efflux of More recently, Aickin [51] has demonstrated that the pH1 H and lactate. The net effect of these two lactate cotransportrecovery from an acid load is insensitive to DIDS. DIDS also processes is expected to be lactate reabsorption and the luminalfails to block comparable NaJHCO cotransporters in the AlO step of Na reabsorption, as well as the substantial intracellularvascular smooth muscle cell line [52], and in cultured mouse alkalinization that has already been observed [24]. However,oligodentrocytes [53]. On the other hand, Deitmer and Schlue the alkalinization probably slows lactate and Na reabsorption.have shown that SITS slows the pH1 recovery in leech glial cells Boron and Boulpaep: Na/HCO3 cotransporter 401 by up to 50% [54], but fails to block the alkalinization induced 8. BORON WF, Dc WEER P: Activeprotontransport stimulated by by the addition of HC03/C02 at fixed pH0. However, the C02/HC03 blocked by cyanide. Nature 259:240—241, 1976 9. RUSSELL JM, BORON WF: Role of chloride transport in regulation HC03-induced alkalinization is blocked by DIDS [55]. The of intracellular pH. Nature 264:73—74, 1976 pH1 recovery from an acid load (applied by an NH4 prepulse)10. THOMAS RC: Ionic mechanism of the H pump in a snail . in chick somites was also blocked by SITS [341. However, Nature 262:54—55, 1976 because there were no Vm data in this study, and because the II.THOMAS RC: The role of , chloride and sodium ions in cells may not have been fully depleted of C1 (see above), it is the regulation of intracellular pH in snail neurones. JPhysiol difficult to rule out the possibility that the pH1 recovery was 273:317—338,1977 12. L'ALLEMAIN G, PARIS S, POUYSSEGUR J: Role of a Na-dependent mediated by a Na-dependent Cl-HCO3 exchanger. C1/HC03 exchange in regulation of intracellular pH1 in fibro- A key question is whether the epithelial and non-epithelial blasts. J Biol Chem 260:4877—4883, 1985 NaJHCO3 cotransporters are fundamentally different. We feel13. R0THENBERG P, GLASER L, SCHLESINGER P, CASSEL D: Activation that differences in stilbene sensitivity, even if they were con- of Na/H4 exchange by epidermal growth factor elevates intracel- lular pH in A431 cells. J Biol Chem 20:12644—12653, 1983 sistent, are not sufficient grounds for concluding that the14. KAILA K, Voiio J: Postsynaptic fall in intracellular pH induced by epithelial and non-epithelial cotransporters are unique. For GABA-activated bicarbonate conductance. Nature 330:163—165, example, the stilbene sensitivity may reflect differences in 1987 post-translational modification of the cotransporters, or subtle 15.KAILAK: GABA-activated movements of formate and acetate: differences in tertiary or quartenary structure. However, the Influence on intracellular pH and surface pH in crayfish skeletal muscle fibres., in Proton Passage Across Cell Membranes. edited difference in the normal directions of transport may be impor- by BOCK G, MARSH J, Chichester, John Wiley & Sons, 1988, pp. tant. As noted above, data indicate that the HC03 : Na 184—186 stoichiometry of the epithelial cotransporter is 3: 1. A thermo-16. BORON WF, BOULPAEP EL: Intracellular pH regulation in the renal dynamic analysis of Na/HCO3 cotransport by leech glial cells proximal tubule of the salamander: Basolateral HC03 transport. J Gen Physiol81:53—94, 1983 (in which pH1, a and Vm have all been measured) indicates17. BORON WF, BOULPAEP EL: Intracellular pH regulation in the renal that in order for the cotransporter to operate in the acid- proximal tubule of the salamander. Na-H exchange. J Gen Physiol extrusion direction, the stoichiometry must be less than 3:1, 81:29—52, 1983 probably 2: 1 [55]. How fundamental is a 3: 1 versus a 2: 1 18. GRASSL SM, ARONSON PS: Na/HCO3 co-transport in basolateral discrepancy in stoichiometry? It has been suggested that the membrane vesicles isolated from rabbit renal cortex. J Biol Chem 261:8778—8783, 1986 epithelial cotransporter achieves a HC03 : Na coupling ratio19. AKIBA T, ALPERN RJ, EvELOFF J, CALAMINA J, WARNOCK DG: of 3: 1 by having three separate binding sites for Nat, C03 and Electrogenic sodium/bicarbonate cotransport in rabbit renal corti- HC03 [29]. If the non-epithelial cotransporter were to achieve cal basolateral membrane vesicles. J Clin Invest 78:1472—1478, 1986 a 2: 1 coupling ratio by having only two binding sites (that is, for20. ALPERN RJ: Mechanism of basolateral membrane H/OH/HCO3 Na and C03), then this would represent a substantial differ- transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process. JGen Physiol86:613—636, 1985 ence in the molecules. On the other hand, if a 2:1 coupling ratio 21. WANG W, DIETL P, SILBERNAGL S. OBERLEITHNER H: Cell mem- were achieved with three binding sites (that is for Na and two brane potential: a signal to control intracellular pH and transepi- separate HC03 ions), then the epithelial and non-epithelial thelial hydrogen ion secretion in frog kidney. Pflugers Arch 409: cotransporters might differ only in the relative affinities of one 289—295, 1987 22. SIEBENS AW, BORON WF: Depolarization-induced alkalinization in of the sites for CO3 versus HCO3. proximal tubules. I. Characteristics and dependence on Na. Am J Physiol 25:F342—F353, 1989 Reprint requests to Dr. Walter F. Boron, Department of Cellular and 23. SIEBENS AW, BORON WF: Depolarization-induced alkalinization in Molecular Physiology, Yale University School of Medicine, New Ha- proximal tubules. II. Effects of lactate and SITS. Am J Physiol ven, Connecticut 06510, USA. 25:F354—F365, 1989 24. 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KOPITO RR, LODIsH HF: Primary structure and transmembrane NaJHCO3 cotransport across the basolateral membrane of the orientation of the murine anion exchange protein. Nature 316: isolated perfused Necturus proximal tubule. Am J Physiol 253: 234—238, 1985 F340—F350, 1987 7. ALPER SL, K0PIT0 RR, LIBRESCO SM, L0DI5H HF: Cloning and 31.SOLEIMANIM, GRASSL SM, ARONSON PS: Stoichiometry of Na- characterization of a munne band 3-related cDNA from kidney and HC03 cotransport in basolateral membrane vesicles isolated from from a lymphoid cell line. J Biol Chem 263:17092—17099, 1988 rabbit renal cortex. JClin Invest79:1276-1280, 1987 402 Boronand Boulpaep: Na/HCO3 cotransporter

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