Spatially distributed alternative splice variants of the renal Na-K-Cl cotransporter exhibit dramatically different affinities for the transported ions.

Three splice variants of the renal Na-K-Cl cotransporter (NKCC2 F, A, and B) are spatially distributed along the thick ascending limb of the mammalian kidney. To test whether NKCC2 splice variants differ in ion transport characteristics we expressed cDNAs encoding rabbit NKCC2 F, A, and B in Xenopus oocytes and determined the ion dependence of bumetanide-sensitive (86)Rb influx. The three splice variants of NKCC2 showed dramatic differences in their kinetic behavior. The medullary variant F exhibited 3-4-fold lower affinity than variants A and B for Na(+) and K(+). Chloride affinities also markedly distinguish the three variants (K(m)F = 111.3, K(m)A = 44.7, and K(m)B = 8.9 mm Cl(-)). Thus, the kinetic properties of the NKCC2 splice variants are consistent with the spatial distribution of the variants along the thick ascending limb as they are involved in reabsorbing Na(+), K(+), and Cl(-) from a progressively diluted fluid in the tubule lumen. Variant B also showed an anomalous inhibition of rubidium influx at high extracellular Na(+) concentrations, possibly important in its highly specialized role in the macula densa. The adaptation of the kinetic characteristics of the NKCC2 variants to the luminal concentrations of substrate represents an excellent example of functional specialization and diversity that can be achieved through alternative mRNA splicing.

The absorptive isoform of the Na-K-Cl cotransporter (NKCC2) 1 is restricted in its distribution to the apical membrane of the thick ascending limb of Henle's loop (TAL) in the vertebrate kidney. Transport of Na ϩ and Cl Ϫ via the NKCC2 comprises a critical component of total renal salt reabsorption (1), which is the central element in whole body fluid and electrolyte balance. Improper function of this process is at the origin of human diseases such as Bartter syndrome (2)(3)(4) and hypertension (5,6). Efficient operation of this transport system must overcome the unusual problem that the luminal concentration of Na ϩ and Cl Ϫ decreases about 5-fold over the length of the TAL as salt is reabsorbed.
Intriguingly three exons encoding a 96-base pair region of NKCC2 are alternatively expressed, giving rise to three different protein sequences in the second transmembrane domain. Each of the three variants (F, A, and B) of NKCC2 shows a specific distribution along the TAL. The F variant is expressed only in the outer medulla, the A variant is found in the outer medulla and cortex, and the B variant is found only in the region of the macula densa (see below, Fig. 1a; Refs. [7][8][9]. Because the membraneembedded regions of transport proteins are most involved in interactions with the transported ions, we have proposed that the three variants of NKCC2 may act with different affinities in transporting ions (7). This hypothesis has been indirectly supported by the finding that the second transmembrane domain plays a major role in determining Na ϩ and K ϩ affinity in the secretory Na-K-Cl cotransporter NKCC1 (10).
Here we show that the three alternatively spliced variants of NKCC2, which are known to be axially distributed along the tubule, are individually specialized for optimal transport. We found that, when expressed in Xenopus oocytes, the three variants differ dramatically in their affinities for Na ϩ , Rb ϩ , and Cl Ϫ . In addition the NKCC2B splice variant that is localized in the macula densa region exhibits unique behavior, consistent with an important role in tubuloglomerular feedback (9,11). The current findings present a remarkable example of molecular specialization underlying physiological specificity in the mammalian kidney. An abstract of these findings has been previously presented (12).

EXPERIMENTAL PROCEDURES
cRNA Preparation-To generate three full-length splice variants of rabbit NKCC2, a 280-bp fragment (SphI-BglII) from the rbNKCC2A was replaced by the same fragment from the rbNKCC2B and rbNKCC2F clones; the three cDNAs are thus identical to one another except for the alternatively spliced 96-bp exon. The cDNAs were subcloned into the EcoRI sites of a modified oocyte expression vector between the untranslated regions of Xenopus ␤-globin (13), and the cRNA for each construct was synthesized with T7 RNA polymerase (mMessage-mMachine, Ambion). After lithium chloride precipitation, cRNA was dissolved in diethyl pyrocarbonate-treated water at a concentration of 1.5-2 g/l.
NKCC2 Functional Expression in Xenopus Oocytes-Xenopus laevis oocytes were injected with 50 nl of cRNA/oocyte and maintained at ϳ17°C in medium containing 96 mM NaCl, 2 mM KCl, 0.9 mM MgCl 2 , 1.8 mM CaCl 2 , 0.1 mM furosemide, and 10 mM HEPES (pH 7.4 at room temperature). Three days after injection, NKCC2 activity was determined in 86 Rb influx assays. Oocytes were preincubated for 20 min at room temperature in a low chloride (3 mM) hypotonic (160 mosM) solution to inhibit the activity of the Xenopus NKCC1 (14) and incubated for 40 min in an influx medium containing 100 mM NaCl, 2 mM RbCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 1 mM Na 2 HPO 4 , 2 mM Na 2 SO 4 , 0.1 mM ouabain, 3 Ci/ml 86 RbCl, 5 mM HEPES (pH 7.4 at room temperature), and optionally 0.25 mM bumetanide in 0.1% Me 2 SO. The dependence of 86 Rb influx on cotransported ions was determined by substitution of Nmethyl glucamine for Na ϩ or K ϩ and of gluconate for Cl Ϫ . Influx was stopped by three rinses with an ice-cold solution containing 100 mM potassium gluconate, 2 mM sodium gluconate, 1 mM MgCl 2 , 1 mM CaCl 2 , * This research was supported by National Institutes of Health Grant DK-17433 and a fellowship (to I. G.) from the Ministerio de Educación y Cultura of Spain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: 1 mM Na 2 HPO 4 , 2 mM Na 2 SO 4 , 5 mM HEPES, 0.25 mM bumetanide, and 0.1 mM ouabain. All solution transfers were executed by transferring oocytes from one well to another in a 48-well plate. 86 Rb in individual oocytes was determined by Cerenkov radiation in a scintillation counter. Data were fit by least square analysis using the Simplex algorithm (PLOT) 2 to a one binding site model (Michaelis-Menton equation) or to a two binding site approximation (Hill equation, n ϭ 2; see Ref. 15). Results are presented as mean Ϯ S.E. of three to seven experiments with four to six oocytes per experimental condition. For Western blot analysis, oocytes (five to eight per group) were solubilized in 100 l of homogenization buffer (1% Triton X-100), and samples containing 50 g of protein were run on SDS gels and transferred to Immobilon-P (Millipore).

RESULTS AND DISCUSSION
Here we report the functional characterization of the three splice variants of NKCC2 in X. laevis oocytes. cRNAs for rabbit NKCC2 were prepared, differing only in the sequence of the 96-bp region encoding the second transmembrane domain (Fig.  1b). Injection of each NKCC2 cRNA led to appropriate synthesis of the transporter protein (Fig. 2b), and the transport activity of each variant, measured as bumetanide-sensitive 86 Rb influx, was at least 6 -7-fold higher than endogenous activity (Fig. 2a). In comparing the behavior of the three variants we found large differences in affinities for the transported ions ( Fig. 3a-c); these differences can be seen to provide a functional rationale for alternative splicing.
Variant F of NKCC2 exhibits the lowest affinity for each of the cotransported ions (Fig. 3, data in blue). This variant is found in the initial segment of the TAL where it is the only form expressed in abundance. In this region of the renal tubule, the inner stripe of the outer medulla, electrolyte concentrations are very much elevated compared with plasma as a result of passive water reabsorption in the thin descending limb (blue bars at the bottom of graphs in Fig. 3). Importantly the K m values for the transported ions are all within the observed range of tubular concentrations, consistent with efficient operation of NKCC2F in this region. It may be noted, however, that if NKCC2F were deployed along the full length of the TAL, it would perform very poorly in removing ions from the dilute tubular fluid present in the cortical regions.
The A variant of NKCC2 was found to have 4-fold higher affinity for Na ϩ and Rb ϩ and 2-fold higher affinity for Cl Ϫ in comparison with NKCC2F (Fig. 3, data in green). NKCC2A is found in the outer stripe of the outer medulla and in the renal cortex. Due to NKCC2 action in reabsorbing Na ϩ , K ϩ , and Cl Ϫ , electrolyte concentrations are reduced 2-10-fold by the time the fluid has reached this region of the tubule (Fig. 3, green  bars). Thus the reduction of ion concentrations is matched by an increase in ion affinities; the structure of the cotransporter appears to have evolved to optimize transport along the diluting segment.
Why is the higher affinity isoform, NKCC2A, not deployed throughout the TAL? One possibility is that the transporter with higher affinity transport has a lower capacity, for example, if NKCC2A were unable to unload ions on the intracellular side as efficiently as NKCC2F. This idea is analogous to a proposal of "dynamic matching" of substrate-and product-containing complexes in evolutionary optimization of the catalytic effectiveness of enzymes (16). In this present case, however, the flux determined for NKCC2F under control conditions in oocytes is lower than that of NKCC2A (Fig. 2a), and from the measured K m values we estimate that at the high salt concentrations of the tubule lumen the two isoforms would exhibit similar transport rates. Thus transport efficiency does not appear to be at the heart of the issue.
An alternative hypothesis is that with a reduced affinity for Na ϩ and Cl Ϫ , the F isoform serves to buffer the luminal concentration of ions in the medullary segment, thus more equally partitioning salt transport between medulla and cortex (17,18). A medullary/cortical distribution of reabsorption can be expected to enhance several features of renal function. 1) NaCl uptake in the medulla takes place against a large gradient; salt recycling and oxygen consumption are minimized by limiting reabsorption in this region. 2) Reduction of medullary diluting power ensures flow-dependent salt delivery to the macula densa and thus enables tubuloglomerular feedback. 3) Medullary/cortical partitioning provides a basis for regional control of regulation of salt reabsorption; as a result, antidiuretic hor-2 Software program by B. Forbush. Functional Specialization of NKCC2 by Alternative Splicing 8768 mone (or vasopressin) is able to enhance the concentration gradient for water reabsorption by altering the ratio of medullary to cortical salt reabsorption in the mouse TAL without substantially changing net salt reabsorption (19).
Variant B is seen to have the highest Cl Ϫ affinity of the variants, ϳ3-fold higher than variant A and 10-fold higher than variant F. The Na ϩ and Rb ϩ affinities are similar to NKCC2A (Fig. 3). Since the distribution of NKCC2B is restricted to the distal-most portion of the TAL (Fig. 1), the splicing phenomenon results again in an apparent adaptation of an NKCC2 variant to the decreasing luminal salt concentrations. On the other hand, the Na ϩ and Rb ϩ affinities are not significantly different between A and B variants except for an anomalous inhibition of Rb ϩ influx seen at higher Na ϩ concentrations.
The punctate distribution of NKCC2B seen in Fig. 1a and in Ref. 8 underscores the specific localization of the B variant to the macula densa cells of the TAL (9). Two important regulatory mechanisms are initiated in these specialized salt-sensing cells, tubuloglomerular feedback and regulation of renin secre-tion; an increase in luminal salt concentration is transduced by macula densa cells to decrease the glomerular filtration rate and to inhibit renin secretion by the juxtaglomerular apparatus (11). NKCC2 plays a central role in the feedback loop by providing the mechanism for the macula densa cells to take up chloride (11,20). Interestingly the shark kidney appears to express only the A and F variants of NKCC2 (21); in conjunction with the fact that the macula densa is absent in lower vertebrates this further suggests a specific relationship between NKCC2B and the macula densa.
It is not immediately obvious why variant B is particularly well suited to a luminal Cl Ϫ -sensing role in the macula densa since its high ion affinities would ensure operation near kinetic saturation, a situation that would decrease its fidelity as a signaling mechanism. We propose that the unique aspect of transport at the level of the macula densa is that net cotransporter ionic distributions are at or near equilibrium across the apical membrane due to the very low luminal ion concentrations (22). Under these conditions, net apical salt transport may be determined by gradient rather than kinetic parameters with the overall transcellular transport limited primarily by Na ϩ pump performance at limiting intracellular [Na ϩ ]. In this circumstance, variant A could perform as well as variant B, and we suggest that variant B significantly differs in another, as yet unidentified, property. One possibility, suggested by the inhibition of Rb ϩ influx at high Na ϩ concentration (Fig. 3a), is that the B variant has a different transport stoichiometry, perhaps allowing Na ϩ to occupy the K ϩ site under some conditions; this would allow uptake of Cl Ϫ against a larger gradient. A second possibility is that NKCC2B varies in some aspect of its regulation, perhaps through a Cl Ϫ -sensing role of the transporter itself.
In this work, we have reported the functional properties of NKCC2 along the TAL. These properties vary with the axial distribution of NKCC2 splice variants in successive regions of that tubule segment. Indeed the variants are shown to have very distinct sets of ion affinities, mirroring the decrease in luminal concentrations of Na ϩ , K ϩ , and Cl Ϫ in the corresponding regions. A similar pattern of axial specialization of the renal Na ϩ pump has been recently reported based on functional differences conferred by four structurally different ␥ subunits of Na,K-ATPase (23). The functional arrangement of renal splice variants is similar to the tonotopic distribution of Ca 2ϩ -activated K ϩ channel splice isoforms in hair cells of the vertebrate cochlea where variants that are more sensitive to Ca 2ϩ have been mapped to the high frequency end of the organ (24,25). These are excellent illustrations of the fine tuning that alternative splicing can confer on the utilization of the limited set of genes in the mammalian genome.