I. INTRODUCTION

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Probably the earliest suggestion of a coupled Na⁺-Cl cotransport process was made by Shanes (318). Evidence for a tightly coupled Na⁺-K⁺-Cl cotransport (NKCC) mechanism, blocked by so-called loop diuretics and unaffected by ouabain, was first presented by Geck et al. (90). At the time Geck et al. (90) presented their findings, there had already been numerous reports of cotransport of all the possible combinations of Na⁺, K⁺, and Cl cotransport processes. For a while it was hoped that all of the reported cation-coupled Cl cotransport (CCC) processes merely represented different modes of a single transport entity. However, as a result of tremendous progress in this field, in the last 5 years we now know that K⁺-Cl (KCC) and Na⁺-Cl (NCC) cotransporters are separate gene products from one another as well as from the NKCC (see sects. II and III). This review focuses almost exclusively on the NKCC.

A by-product of the widespread interest in the NKCC has been the appearance of a substantial number of review articles (e.g., Refs. 43, 50, 86, 109, 110, 114, 163, 171, 172, 239, 255, 266, 277, 281, 297, 311, 320). So why one more? Previous reviews approach the subject from the standpoint of a somewhat special interest within the field, e.g., the properties of the transporter in some particular cell type (red blood cells, Ehrlich ascites tumor cells, and epithelial tissue), some functional aspect (cell volume regulation), and, most recently, the molecular biology and molecular structure of the NKCC.¹ This review draws on data from all these sources in an attempt to distill a focused picture of our current understanding (and ignorance) of this important ion transport mechanism. Perhaps the best reason for a comprehensive review at this time is that the field is poised on the brink of an exciting new era that will undoubtedly yield interesting and unexpected insights into the workings of these CCC processes.

The past 5 years have seen the major energy in this field being directed into molecular characterization of the cotransporter. These efforts have been rewarded in that two isoforms of the NKCC have been cloned as have two isoforms of the KCC and one isoform of the NCC. We now enter a period in which we can extend our understanding of how these cotransporters work using the newly available molecular biological tools. This review will be a success if it serves to help the reader distinguish between what is well understood from what is poorly understood about the NKCC, its transport mechanism, its regulation, and its function. There are several excellent recent reviews that focus on the molecular biological information that has just become available about this cotransporter (110, 114, 163, 277, 281). Therefore, this review does not attempt an exhaustive coverage of that information; rather, a molecular biological framework is presented.

	II. OVERVIEW OF THE CATION-COUPLED CHLORIDE COTRANSPORT FAMILY

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The cotransport of Cl along with Na⁺ and/or K⁺ has been reported for a variety of cells since the 1970s (see sect. III) and was surmised even earlier than that (78). Not long ago, it was difficult for even those working in the field to know with any certainty whether a given K⁺-dependent Cl transport process was a KCC or NKCC or whether a given Na⁺-dependent Cl transport process was NCC or NKCC. In fact, there were reports in the literature which suggested that, in some situations, a single transport moiety could switch "modes" from one to the other given certain stimuli, such as increased osmolarity of bathing solutions (77) or application of vasopressin (334). In addition to an uncertainty about the absolute ion requirements for each of the putative ion cotransporters, there was confusion regarding their pharmacology. Much of this uncertainty resulted from the simultaneous presence of more than one coupled cotransporter in the tissues/cells being studied. We can now be certain that all three of these cotransporters are separate proteins encoded by the same gene family. The gene products of the NCC (80) and the KCC (92, 139, 282) share ~45-50% and 25% identity, respectively, with the gene products of the NKCC (277, 281).

Ion transport studies show all three of these cotransporters share an absolute requirement for Cl as well as at least one cation (either Na⁺ and/or K⁺) and that all three cotransport processes are electrically silent. Haas (110) has proposed that this gene family be termed the cation chloride cotransporter, or CCC, gene family. Pharmacologically, they are somewhat distinguishable. Only the NCC is blocked by thiazide diuretics, whereas only the KCC is blocked by disulfonic acid stilbenes, such as DIDS. The loop diuretics (bumetanide, furosemide) inhibit both the NKCC and the KCC but are much more potent in their action against the NKCC. In addition, there may be an NCC that is inhibited by loop diuretics (e.g., Refs. 196, 241, 334), although there is some disagreement about whether the NCC blocked by bumetanide is the same as that blocked by thiazides (134).

On the basis of a combination of functional and genetic results, both the NKCC and the KCC are found in a wide variety of tissue types. In general, the NCC seems to be largely confined to epithelial tissue such as kidney (especially in the distal nephron), where it participates in the net movement of Na⁺ and Cl across an epithelial barrier. Functionally, the KCC has been best characterized in red blood cells but has also been described in other tissues. The KCC has been associated with regulatory volume decrease in response to cell swelling. In nervous tissue, where it has been recently identified (282), it may participate in maintaining an intracellular Cl concentration ([Cl]_i) at lower than electrochemical equilibrium levels. What follows is a brief description of the properties of the KCC and the NCC. Table 1 summarizes the key similarities and differences among these three CCC.

Of the three members of the CCC family, this is the one for which general recognition and acceptance was perhaps the slowest. This probably relates to the fact that in mammals its most definitive location is the early distal convoluted tubule in the kidney, a region where the NKCC is also prominent. In addition, it was often mistaken for parallel Na⁺/H⁺ and Cl/HCO₃ exchangers. The functional properties of the NCC were first definitively characterized by Stokes et al. (332) using the urinary bladder of the winter flounder, a preparation that apparently lacks the NKCC. They demonstrated 1) a clear interdependency between Na⁺ and Cl effects on net absorption of Cl and Na⁺, respectively; 2) that the transport process did not require K⁺; and 3) that the NCC was inhibited by thiazide diuretics but not by the so-called loop diuretics (which, as we shall see, are the most specific inhibitors of the NKCC) such as furosemide, nor by stilbenedisulfonic acid derivatives (which inhibit the KCC and Cl/HCO₃ exchange, among other anion transport processes). However, there is functional evidence for bumetanide-sensitive K⁺-independent NaCl cotransport in trachea (207, 241) and Necturus gallbladder (196). The later confusion about whether the NCC could be converted into a NKCC probably arose because in some epithelial tissues, as mentioned above, the two transporters spatially coexist (e.g., Ref. 143).

There is some functional evidence for this cotransporter in nonepithelial tissue. In the rabbit heart, treatment with chlorothiazide caused a reduction of cell volume (57). This cell shrinkage effect of thiazide treatment was tentatively attributed to the inhibition of a NCC-mediated uptake process. However, in vascular smooth muscle, thiazides activate a Ca²⁺-activated K⁺ channel (37). It is quite possible the cell shrinkage observed in the heart muscle after thiazide treatment was due to K⁺ (Cl) loss and not to inhibition of an NCC. Also, high-stringency Northern blots failed to reveal any NCC mRNA in rat heart (80). There were early reports of apparent NCC activity in Ehrlich ascites tumor cells (136), but subsequent functional evidence showed that this cell has only the NKCC (89, 155, 202) and the KCC (182). Finally, Northern blot analysis of mammalian tissues (including human tissues) localize it mainly to kidney, but also possibly to small intestine, placenta, prostate, colon, and spleen (in humans, Ref. 45). In rat tissues, high-stringency assays revealed the NCC to be only in the kidney (79). In fact, in situ hybridization probes (79) and mRNA localization (359) reveal that the NCC is exclusively localized to the distal convoluted tubules of the kidney. This result fits very well with what is known about thiazide-sensitive NaCl absorption by the kidney.

The winter flounder urinary bladder behaves functionally much like the mammalian kidney distal tubule and, as we have seen, was the first site of functional characterization of the NCC. The flounder urinary bladder NCC was the first of the CCC to be cloned (80). The human NCC has also been cloned (40) and expressed (225).

Gitelman's syndrome is a human genetic disease associated with a mutation of this thiazide-sensitive cotransporter in the renal distal convoluted tubule (326). The syndrome is characterized by hypokalemic metabolic alkalosis, hypocalcuria, hypomagnesemia, and natriuresis. The mutation, located on chromosome 16, is believed to lead to a loss of function of the NCC.

During the same series of studies that began the linkage between cell volume regulation and NKCC (see sect. IVC), Kregenow (185), also made several critical observations for our understanding of the KCC. The KCC mediates the coupled cotransport of K⁺ and Cl across plasma membranes. Although reversible, it is thermodynamically poised to effect net efflux. Because of this net loss of K⁺ and Cl, it can promote regulatory volume decrease. Thus it is activated by a cell volume increase (135). It may also be involved in "pumping" [Cl]_i to lower than equilibrium levels in neurons (279, 282). At present, there are two known isoforms of the KCC, the "housekeeping" form (KCC1) that is found in a variety of tissues (e.g., Ref. 92) and a neuron-specific isoform (KCC2, Ref. 282). Structurally, both of these isoforms differ more from the NKCC than does the NCC (92, 282). They have an estimated molecular mass of 120-125 kDa (92, 282).

Functional studies have confirmed that the KCC is found in several cell/tissue types, including ascites tumor cells (182) and mammalian kidney epithelial cells (65, 294). However, by far the most detailed functional studies have been performed in the red blood cell (RBC). These properties have been well covered in recent reviews (RBC, Ref. 199; all cells, Ref. 134). I highlight only the key properties that differentiate the KCC from the NKCC. As already mentioned, the KCC is activated by cell swelling, and as we shall see (sects. IIIC and IXC), the NKCC is activated by cell shrinkage. Activation of the KCC by swelling leads to the net loss of K⁺ and Cl along with an osmotic equivalent of water. This results in a reduction of cell volume ("regulatory volume decrease"). Another intriguing functional difference between the KCC and the NKCC is that dephosphorylation activates KCC (e.g., Ref. 154), whereas it inactivates the NKCC (see sect. IXA). The KCC differs from the NKCC in its response to an increase of [Cl]_i as well. Whereas the NKCC is inhibited by such an increase (e.g., Refs. 28, 93; see sect. IXB1), the KCC is stimulated (51, 197, 198). Unlike either the NKCC or the NCC, the KCC can be inhibited by the disulfonic acid stilbenes such as DIDS (52). Because it is unlikely that DIDS crosses the plasmalemma, this property suggests that the DIDS-sensitive site of the KCC is externally accessible.

	III. MOLECULAR CHARACTERIZATION OF THE SODIUM-POTASSIUM-CHLORIDE COTRANSPORTER

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Since 1994, all three members of the CCC family have been cloned from a variety of species (see Refs. 50, 163, 277, 281). A quantitative comparison of the derived amino acid sequences of members of this family with other known proteins suggests that it is a unique family, perhaps distantly related to the amino acid-polyamine-choline family of transporters (277). Full-length clones of two isoforms (see below) of the putative NKCC have been reported from rat kidney (79), shark rectal gland (358), inner medullary collecting duct of the mouse kidney (53), the loop of Henle from rabbit kidney (280), human colon (283), mouse kidney (143), human kidney (325), and bovine aortic endothelium (360). The deduced amino acid sequences of these clones have revealed a protein whose molecular mass varies from ~120 to ~130 kDa. A very similar protein has been reported as encoded by the gene YBR235w of chromosome II of the yeast Saccharomyces cerevisiae (18). Hydropathy profiles of the deduced proteins (using the Kyte-Doolittle algorithm) have shown there are three general regions. A central hydrophobic region of ~50 kDa flanked by an amino- (~20-30 kDa) and a carboxy-terminal (~50 kDa) region. These latter two regions are more hydrophilic than the central region of the molecule. Based on such analyses, Xu et al. (358) suggested that there are 12 -helical membrane-spanning regions. These putative transmembrane regions are highly conserved between isoforms, with 75-90% identity between the NKCC1 and NKCC2 isoforms. It has been suggested that alternative topological models (e.g., 14 membrane-spanning regions) might better conform to other predictors of membrane protein topology (277). Both isoforms have consensus N-linked glycosylation sites on a putative extracellular loop that fits with biochemical evidence that the cotransport protein is significantly glycosylated (293).

As mentioned above, there are two isoforms of the NKCC. The isoform initially found on the basolateral membrane of the shark rectal gland is known as NKCC1 (Ref. 358; also known as SLC12A2). The same isoform was also identified in mouse kidney by Delpire et al. (53) but named BSC2 (for bumetanide-sensitive cotransporter 2). The NKCC nomenclature is used in this review. The other isoform is referred to as NKCC2 (or BSC1; also known as SLC12A1). The NKCC1 isoform is the larger of the two with ~1,200 amino acid residues and a transcript size of ~7.4 kb. It has an overall 58% amino acid identity with NKCC2. The NKCC2 isoform is somewhat smaller than NKCC1, containing ~1,100 amino acid residues with a transcript size of ~5 kb. The difference in molecular size is almost entirely accounted for by an additional 80 amino acids at the amino terminus of the NKCC1. In contrast, the carboxy end of the molecule is relatively well conserved, exhibiting >65% identity among the two isoforms and between the same isoforms from different sources (277). Given the difference in transcript size and the relatively low overall amino acid identity between the two isoforms, it is not surprising that they are products of two different genes. Delpire et al. (53) showed in the mouse that the NKCC1 is localized to chromosome 18, whereas the NKCC2 is localized to chromosme 2 (289). The human NKCC1 is localized to chromosome 5 (283).

The NKCC1 isoform is by far the most widely distributed of the two currently identified isoforms. In addition to being found on the basolateral membrane of secretory epithelia, Northern probe studies have indicated this is the isoform found in the plasmalemma of a wide variety of cell types, including most nonepithelial cells (283, 358). There is some evidence for tissue-specific variants of the NKCC1. For example, skeletal muscle presents a somewhat smaller mRNA transcript than is found in other tissues (6.7 vs. 7-7.5 kb, Ref. 283). Because it is found on the basolateral membrane of epithelial cells, this isoform is often referred to as the "secretory" isoform. However, when it is found in the membrane of nonpolar cells, it may be referred to as the housekeeping isoform.

Figure 1 is a general model based on the hydropathy profile for human colonic NKCC1. As already mentioned, the molecule has three regions. The amino-terminal region has the lowest amino acid identity of the three regions among NKCC1 from different species. There is at least one phosphorylation site in this region. This site has the greatest degree of amino acid identity found in the amino terminus across species (213, 358). On the other hand, the carboxy terminal region is relatively highly conserved among species extending from Cyanobacterium to human (281). The carboxy-terminal end has several consensus phosphorylation sites that have a relatively high degree of identity across species (see sect. IXB2). In addition, there are several hydrophobic regions in the carboxy terminus that might also be embedded in the membrane. It is hypothesized that the central ~500-amino acid residue region is arranged into 12 transmembrane domains with connecting loops. The hydrophobic membrane-spanning region is not only highly conserved between isoforms, but also between the same isoform from different species (e.g., Ref. 281). There are two consensus N-linked glycosylation sites on the extracellular loop between transmembrane (TM) segments 7 and 8. It is currently unknown whether a functional NKCC is a monomer or an oligomer.

View larger version (34K): [in this window] [in a new window]   Fig. 1. Model of Na+-K+-Cl cotransport (NKCC) isoform 1 protein based on its hydropathy profile. NKCC1 protein is ~1,200 amino acids with 12 transmembrane (TM)-spanning domains, numbered TM1-TM12. Transmembrane domains 1, 3, 6, 8, and 10 are rather highly conserved between NKCC1 and NKCC2. In addition, intracellular loop linking transmembrane domains 2 and 3 is highly conserved. Virtually entire difference in molecular mass between NKCC1 (~195 kDa) and NKCC2 (~121 kDa) can be accounted for by an additional 80 amino acids on NH2 terminus of NKCC1. Site-directed mutagenesis studies have shown that changes in cation affinity result from changes in TM2, whereas Cl affinity was affected by changes in TM4 to TM7. Bumetanide binding appears to be associated with transmembrane TM2 to TM7 and TM11 to TM12. [Adapted from Payne and Forbush (281).]

Structure-function studies have just begun. In a recent series of studies, Forbush's group made chimeras from shark and human NKCC1. They also made some point mutations. These studies show that ion binding and transport as well as bumetanide binding depend on the ~500 amino acids believed to comprise the conserved hydrophobic transmembrane regions (147-150). These studies have provided strong evidence that the second transmembrane region (TM2; a region highly conserved across the entire CCC family) is an important site in determining cation and bumetanide binding, but not Cl binding. Chloride binding determinants appear to reside in TM4 and TM7. Bumetanide binding determinants appear to be somewhat more diffuse than those for the ions, being in TM2, -7, -11, and -12.

The NKCC2 (BSC1) isoform has thus far been identified only in the medullary regions of the kidney (rat, Ref. 79; rabbit, Ref. 281). This anatomic location of the NKCC2 corresponds with the location of the loop of Henle and the juxtaglomerular apparatus. Thus NKCC2 is believed to play a critical role in two of the crucial homeostatic functions of the kidneys, namely, regulation of extracellular fluid volume and osmolarity.

In studies on the rabbit kidney NKCC2, Payne and Forbush (280) identified three distinct variants that differed only by an alternately spliced 96-bp exon. With the use of such a hydropathy plot model very similar to that for NKCC1 (refer to Fig. 1), it was shown that these exons coded for the amino acids in TM2 as well as 13 amino acids that are contiguous with TM2, but which extend into the intracellular compartment (280). A particularly interesting addendum to this observation was that high-stringency Northern probes indicated these three variants had very distinct regional localization within the rabbit nephron; one was found only in the renal cortex, one only in the renal medulla, and the third in both the cortex and medulla. Igarashi et al. (143) have made a similar observation for the mouse kidney. This finding is consistent with a proposal made by Knepper and Burg (177) that there are some differences between NKCC function in different regions of the thick ascending limb of the loop of Henle. It is possible that the proposed regional differences in Na⁺ reabsorption along the thick ascending limb might be mediated by the different isoforms of the NKCC2. This interpretation fits well with the finding that TM2 is a critical component of Na⁺ and K⁺ affinities for the NKCC (148, 149).

The NKCC2 has also been identified in the renal juxtaglomerular apparatus (143, 163). Igasrashi et al. (143) showed that one of the alternately spliced isoforms (NKCC2B) is found in macula densa cells. Macula densa cells are involved with two important renal homeostatic processes: tubuloglomerular feedback and secretion of renin. LaPointe's group (194, 195) has presented evidence that strongly suggests a key role for the NKCC in the tubuloglomerular feedback process. LaPointe et al. (195) suggest that the NKCC (NKCC2B?) in the macula densa cells is very near its thermodynamic equilibrium point such that small changes in the NaCl concentration in the nephron tubule lumen could affect the net direction of NKCC transport and thereby change the equilibrium potential of Cl (E_Cl). This in turn is hypothesized to affect the resting membrane potential (V_m) of the basolateral membrane of the macula densa cells which somehow transmit this membrane voltage information to the granule cells of the afferent artery. Evidence for this function of the NKCC2 comes from reports that luminal furosemide inhibits tubuloglomerular feedback (152) and renin secretion (209).

Mutations of NKCC2 have been linked to some forms of Bartter's syndrome, a severe human genetic disease involving hypokalemic alkalosis with hypercalcuria largely attributable to dysfunction of the NKCC2 in the thick ascending limb of the loop of Henle (325). The authors reported a variety of mutations, all in the putative transmembrane region of the molecule. The interested reader is directed to a thoughtful discussion of the roles of the NKCC and NCC in Bartter's and Gitelman's syndromes by Hebert and Gullans (129).

	IV. HISTORY OF THE DISCOVERY OF THE SODIUM-POTASSIUM-CHLORIDE COTRANSPORTER

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The NKCC is distinguished by the fact that it transports Na⁺, K⁺, and Cl stoichiometrically by means of a tightly coupled mechanism that can be blocked by loop diuretics. Although this distinguishing "fingerprint" can be stated briefly, it took nearly 15 years for workers to put the pieces together (90). Three key elements were required: 1) a conceptual breakthrough; 2) a laborious, empirical characterization of Na⁺ and K⁺ fluxes; and 3) a reason to consider that Cl transport might be directly linked to cation transport. The conceptual breakthrough occurred in the early 1960s with Crane's seminal formulation of the sodium gradient hypothesis and the concept of secondary active transport (see Refs. 46, 47) whereby the transmembrane electrochemical gradient (maintained by a primary active transport process) of one solute (usually Na⁺) provides the energy for the thermodynamically uphill movement of a second solute. It is difficult to overemphasize the importance of this concept to the study of cell solute homeostasis. The labor-intensive characterization of Na⁺ and K⁺ fluxes was performed by a variety of workers whose original intent was to characterize further the operation of the Na⁺ pump. The reasons to consider Cl linkage to Na⁺ and K⁺ movements were almost serendipitous. As we shall see, the Cl linkage idea grew out of studies designed to characterize amino acid transport mechanisms.

Hoffman and Kregenow (137) demonstrated in ouabain-treated human RBC that there was a fraction of Na⁺ efflux that was stimulated by intracellular Na⁺, blocked by ethacrynic acid, and which might not use ATP as an energy source. To explain this finding, they hypothesized that in addition to the ouabain-sensitive sodium pump, there was a second, energy-dependent mechanism capable of moving Na⁺ against its electrochemical gradient. They called this mechanism pump II to distinguish it from the ouabain-sensitive Na⁺/K⁺ exchange pump (pump I). Over the next 8 years, the pump II hypothesis was ultimately disproven. However, it served well to stimulate much-needed research into the properties of coupled Na⁺-K⁺ cotransport processes. In the interim, some workers concluded that the ouabain-insensitive Na⁺ efflux was mainly a Na⁺/Na⁺ exchange process (60, 210), whereas others concluded that these fluxes were simply manifestations of different properties of pump I, depending on the particular experimental conditions (309).

By the late 1960s, Na⁺ pump workers began to notice that K⁺ uptake, in the presence of ouabain, was saturable (e.g., Refs. 84, 310). This was a perplexing finding since it was generally believed at that time that K⁺ movements consisted of only two pathways: 1) via the Na⁺ pump and 2) via electrodiffusive leaks (the pump-leak hypothesis). Thus, in the presence of ouabain, K⁺ influx was not expected to be saturable as a function of extracellular K⁺ concentration ([K⁺]_o), unless there was a hitherto unknown mechanism for K⁺ uptake.

Evidence for such an unknown mechanism was provided by Sachs (309). Using human RBC, he performed a critical series of studies that linked K⁺ uptake with Na⁺ uptake via a ouabain-insensitive mechanism. He showed that (in the presence of ouabain) there was a Na⁺ uptake dependent on external K⁺ that could be blocked by 1 mM furosemide. Under the appropriate set of conditions, he could demonstrate a small net efflux of Na⁺ (in the presence of ouabain) that was also blocked by furosemide. This is the first published report of linking K⁺-dependent Na⁺ fluxes to inhibition by a loop diuretic. However, because he and others had demonstrated that furosemide could inhibit pump I (Na⁺-K⁺-ATPase) fluxes, Sachs (309) concluded that the effects seen in the presence of ouabain were probably mediated by pump I, which had different properties under the experimental conditions being used.

Wiley and Cooper (355) were the first to demonstrate the obligatory coupled cotransport of Na⁺ and K⁺. They extended Sach's finding by demonstrating furosemide-inhibitable, mutually dependent Na⁺ and K⁺ fluxes occurring in the presence of ouabain. Figure 2 illustrates their finding that the furosemide-inhibited Na⁺ influx in human RBC required the presence of extracellular K⁺. Figure 3 shows that a portion of the ouabain-insensitive K⁺ uptake required the presence of extracellular Na⁺ and could be inhibited by furosemide. This was the first demonstration that the furosemide-sensitive components of the unidirectional influxes of both Na⁺ and K⁺ required the presence of the other cation. They also noted, as has had Glynn et al. (95), that there was a saturable component of the ouabain-insensitive Na⁺ uptake. This saturable component of Na⁺ uptake was inhibited by furosemide and had a Michaelis constant (K_m) for external Na⁺ of 24 mM. Furthermore, and this was very important at the time, they demonstrated net, furosemide-sensitive fluxes. This observation, coupled with the observation that furosemide inhibited ouabain-insensitive effluxes of K⁺ and Na⁺ in the absence of extracellular Na⁺ and K⁺, showed that the transport process they were studying was not simply isotopic exchange. The conclusion of these workers was that the RBC possessed a cotransport mechanism that moves both Na⁺ and K⁺ and that there was a codependency in their transport. This represented a very critical stage in the development of the idea of the NKCC. It is important to remember that at that time the idea of secondary active transport in general, and cotransporters in particular, was relatively new and by no means universally accepted.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Inhibition by 1 mM furosemide of Na+ influx into human red blood cells bathed either in K+-containing or K+-free media. Each isotonic medium contained 10 mM NaCl plus requisite concentration of indicated cation as chloride salt. Ouabain (0.1 mM) was present in all media. Values are means ± SD. [From Wiley and Cooper (355).]

View larger version (24K): [in this window] [in a new window]   Fig. 3. Inhibition by 1 mM furosemide of ouabain-insensitive K+ influx into human red blood cells bathed in either Na+-rich or Na+-free media. Each isotonic medium contained 6 mM KCl plus requisite concentration of indicated cation as chloride salt. Ouabain (0.1 mM) was present in all media. Values are means ± SD. [From Wiley and Cooper (355).]

The long and continuing association between what has come to be recognized as the NKCC and cell volume regulation that occurs in response to cell shrinkage began with several critical observations by Kregenow (185, 186). He showed that duck RBC, shrunken by exposure to a hypertonic medium, could (in the continued presence of the hypertonic medium) recover toward their original volume. This recovery depended on [K⁺]_o being elevated somewhat above normal levels and the presence of extracellular Na⁺. In the absence of ouabain, cell volume recovery was accompanied by a net uptake mainly of K⁺ and Cl plus the osmotically obligated water. However, Kregenow (186) showed that the volume regulation did not require a functioning Na⁺ pump (Fig. 4). With the Na⁺ pump inhibited, the main increase was seen in the Na⁺ and Cl contents, with a somewhat smaller increase of K⁺. This important observation implied that the primary solute transport mechanism responsible for the volume increase moved Na⁺ (+Cl) in addition to K⁺ (-Cl). Kregenow (186) supported this interpretation by showing that cell shrinkage was accompanied by an increase in the ouabain-insensitive unidirectional isotopic influx and efflux of both Na⁺ and K⁺. However, in keeping with the cationocentric point of view of those times, the increase of Cl content was believed to be "passive," in response to changes in membrane potential caused by the cation movements. Thus Kregenow's findings showed that cell shrinkage activated a ouabain-insensitive Na⁺ and K⁺ transport pathway or pathways that moved these two cations into the cell (K⁺ against its electrochemical gradient) to effect a net uptake of osmotically active particles. For the first time, Cl was also implicated in the process (albeit in an apparently passive role).

View larger version (22K): [in this window] [in a new window]   Fig. 4. Lack of effect of ouabain on response of duck red blood cells to exposure to a hypertonic medium. Duck erythrocytes were incubated in isotonic or moderately hypertonic fluids containing either 2.5 or 19 mM K+. Ouabain (104 M) was added just before beginning experiment. [K+]o, extracellular K+ concentration. [From Kregenow (186).]

D.  Linking Cl to the Coupled Na⁺ and K⁺ Movements

1.  Evidence in RBC

Since the early 1970s, red cell workers were aware that the coupled Na⁺-K⁺ movements they observed were accompanied by Cl movements in the same direction (see above). However, it was not until the late 1970s that investigators directly addressed a critical question: Are the Cl movements obligatorily linked to the cation movements or are they functionally linked via changes in the electrochemical driving forces? Although the question seems straightforward now, at the time there were conceptual and technical roadblocks both to formulating as well as to addressing this question. The conceptual roadblock was the then-current view (based on results from RBC and frog skeletal muscle) that Cl crossed membranes only by electrodiffusive pathways and was distributed at electrochemical equilibrium across all cell membranes. According to this view, Cl simply traversed membranes through very high permeability pathways in response to primary changes in the electrochemical driving forces as set by the movements of cations. Moving beyond this concept required overcoming two technical roadblocks. One was the difficulty of measuring Cl movements. The principal radioisotope of chlorine (³⁶Cl) has a half-life of over 300,000 years, meaning that it can only be produced with a relatively low specific activity, and it is expensive. The measurement of net Cl movements, while possible, was laborious. The second roadblock had to do with a property of the membrane of RBC: it has both a high Cl conductance and a high Cl exchange flux via the band 3 anion exchanger (AE1).

Both the problems outlined above came together to influence the interpretation of the results of an elegant study by Schmidt and McManus (316). Using duck RBC, they examined the role of Cl in the shrinkage-induced volume increase that had first been demonstrated by Kregenow (185, 186). The duck RBC is an excellent subject to study cell-volume regulation and the associated Na⁺ and K⁺ fluxes. However, like other RBC, it is ill-suited for the study of Cl fluxes associated with volume regulation, because the Cl flux via the Cl/HCO₃ exchanger (band 3 or AE1) is orders of magnitude greater than the Cl flux via the NKCC. As a result of this problem, Schmidt and McManus (316) were unable to measure any Cl fluxes directly. Instead, they looked at effects of varying Cl concentrations and concluded that the Cl movements during cell reswelling were in response to the change in the electrochemical driving force on this anion, caused by the net cotransport of Na⁺ and K⁺. There is an additional circumstance that contributed importantly to the conclusion that Cl movements were passive and not obligatorily linked to those of Na⁺ and K⁺. That is the fact that Cl is distributed at thermodynamic equilibrium across the RBC membrane. This means the E_Cl is equal to the V_m. Thus, as long as the process(es) responsible for this passive distribution were operating, measurements of net Cl fluxes could never reveal Cl flux via the NKCC.

In a later publication from the same laboratory (121), the question of whether Cl was directly linked to cation transport was readdressed using duck RBC whose membrane potential was "voltage-clamped" (using valinomycin) and whose band 3 fluxes were blocked by DIDS. Under these conditions, they completely replaced Cl with permeant anions across the duck red cell membrane. Now, with V_m unable to change and band 3 unable to exchange anions, they showed that only Cl and Br could support furosemide-sensitive net Na⁺ transport. In their earlier report (316), they had reported that reducing both [Cl]_i and [Cl]_o to 20 mM (using acetate as the replacement) still permitted a norepinephrine-stimulated uptake of Na⁺ and K⁺ without any change of [Cl]_i. In the earlier work, this observation had been interpreted to mean there was no absolute Cl requirement by the cotransporter. When this experiment was repeated in the presence of DIDS, it was found that only Cl or Br would support the net uptake of the cations (121). Furthermore, in DIDS-treated cells, they were able to show a furosemide-sensitive net uptake of Cl that equaled the furosemide-sensitive net uptake of Na⁺ plus K⁺. In a particularly convincing series of studies summarized in Figure 5, they also showed that the direction of the transmembrane Cl chemical gradient dictated the direction of the furosemide-sensitive net flux of Na⁺ when the membrane potential was "clamped" with valinomycin (142). Thus this study not only demonstrated that Cl was transported by the cotransporter, but also that the chemical gradient for Cl could "energize" net cotransport-mediated fluxes of the cations.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Demonstration in duck red blood cells that Cl chemical gradient can drive furosemide-sensitive Na+ fluxes at a constant membrane potential (calculated to be 12.4 mV over entire range of [Cl]o, where subscript o refers to extracellular). Cells pretreated with 105 M DIDS (to block band 3 anion exchanger) were incubated with 2 × 106 M valinomycin (to "voltage-clamp" membrane potential), ouabain (to block Na+ pump), and norepinephrine (to stimulate NKCC). In addition, media contained 30 mM Na+ and 100 mM K+ ± 1 mM furosemide. Initial intracellular concentrations were as follows (in mmol/l cell water): Na+ = 7.8; K+ = 174.9; Cl = 95.6. Methylsulfate was used to substitute for external Cl. Subscript c refers to cytosolic. [From Haas et al. (121).]

The original misinterpretation of their results was reinforced by an analysis of the thermodynamic equilibrium of the putative Na⁺-K⁺ cotransporter that agreed with their actual results. That one could arrive at the correct final thermodynamic expression starting with incorrect initial assumptions was the result of two pieces of bad luck: 1) Cl is distributed at thermodynamic equilibrium and 2) Cl is a transported species. In the first report (316), they interpreted their results as being due to an electrogenic Na⁺-K⁺ cotransport with Cl following via an electrodiffusive pathway. Starting with this assumption, they derived an equilibrium equation for such a model as follows.

At thermodynamic equilibrium, the net electrochemical potential (_net) is

(1)

where _Na and _K are the electrochemical potentials for Na⁺ and K⁺, respectively. Knowing that Cl is distributed at electrochemical equilibrium (i.e., V_m = E_Cl) allows one to write the following expression for the net electrochemical potential

(2)

Unfortunately, the exact same equation can be derived starting with the assumption that the cotransporter is electroneutral, that is, there are one Na⁺, one K⁺, and two Cl transported per cycle, i.e.

(3)

Thus we see that although the initial assumptions were quite different (Eqs. 1 and 3), the final equation (Eq. 2) is the same. This identity contributed to the initial misinterpretation by Schmidt and McManus (316). It took the development of the voltage-clamped RBC treated with DIDS (to block the band 3 anion exchanger) to distinguish between the two very different models.

Three other groups studying Na⁺ and K⁺ fluxes in human RBC reported a close relationship between ouabain-insensitive cation influxes and the presence of extracellular Cl. In a preliminary note, Kregenow and Caryk (189) reported that Cl was cotransported with Na⁺ and K⁺ during volume regulation in duck RBC. Dunham et al. (59) showed that ~75% of the ouabain-insensitive influx of K⁺ was dependent on [Cl]_o in a concentration-dependent manner. They further showed that 1 mM furosemide abolished this [Cl]_o-dependent K⁺ influx. Similarly, they showed that a portion of the ouabain-insensitive Na⁺ influx required the presence of external Cl and that this fraction of the Na⁺ influx was eliminated by treatment with furosemide. The fact that the magnitude of the [Cl]_o-dependent Na⁺ influx was significantly smaller than that of the same K⁺ influx as well as the fact that Wiley and Cooper (355) had shown that furosemide blocked a substantial portion of K⁺ influx into cells bathed in Na⁺-free media caused them to conclude that they were studying a KCC. At about the same time, Chipperfield (43) also reported a [Cl]_o-dependent K⁺ influx, blocked by furosemide, but noted that "direct evidence for Cl transport by the same system is lacking." Clearly, the inability to measure non-band 3-mediated Cl fluxes in the RBC was a critical impediment to correctly assessing the role of Cl in the RBC cotransporter.

Thus the first definitive experiments showing the direct linkage between the furosemide-sensitive Na⁺ and K⁺ fluxes with Cl fluxes were performed in a different preparation (Ehrlich ascites tumor cells) and for a completely different reason than the series of studies we have just outlined. In the mid 1970s, Heinz et al. (131) were attempting to determine the energy sources for amino acid uptake. In the course of those studies, his group noticed an apparently paradoxical movement of Cl (131). They had demonstrated a strong electrogenic Na⁺ pump component to the V_m of the Ehrlich cells. Thus, as [K⁺]_o was increased from 0 to 15 mM, the V_m became increasingly negative, an effect largely blocked by ouabain. According to the prevailing dogma of those times, Cl is distributed passively and can rapidly move to maintain Cl electrochemical equilibrium (V_m = E_Cl). It was expected that Cl content would decrease in response to the intracellular negativity. In fact, they reported that the Cl content of the Ehrlich cells actually increased as a function of [K⁺]_o, and this response was exaggerated in the presence of ouabain. Furthermore, they showed that this apparently paradoxical Cl uptake could be blocked by furosemide. These findings were followed (87, 88) by a demonstration that furosemide blocked the unidirectional fluxes of Cl and K⁺.

Finally, in a landmark paper, Geck et al. (90) demonstrated virtually all the basic properties of the NKCC (see sect. V). As seen in Figure 6, they first showed that when K⁺-depleted cells were reexposed to external K⁺, they took up K⁺, Na⁺, and Cl in a ratio of 1:1:2. Using a membrane potential-sensitive probe, tetraphenylphosphonium, they showed that these ouabain-insensitive fluxes had no effect on the membrane potential of the cells. Conversely, they also showed that changing the membrane potential had no effect on the apparent cotransport fluxes. Thus the cotransporter was electroneutral. Using a coupling analysis derived from irreversible thermodynamics, they showed that the net movements of all three ions were tightly coupled to one another and to an isosmotic water flow. From these results, they explicitly proposed that there existed a strongly coupled NKCC in the membrane of the Erhlich ascites tumor cell. They further speculated that reports from experiments on RBC and several epithelia could be explained by the existence of a coupled triple cotransporter, the NKCC, rather than either Na⁺-K⁺, K⁺-Cl, or Na⁺-Cl cotransporters as originally postulated.

View larger version (12K): [in this window] [in a new window]   Fig. 6. Furosemide-sensitive net ion movements. Ehrlich ascites tumor cells, previously K+ depleted and Na+ enriched, were incubated for periods up to 5 min in Krebs-Ringer phosphate buffer with 38 mM K+ and 93 mM Na+. Furosemide-inhibitable net ion movement is defined as difference in intracellular ion content (mmol/g dry wt) between cells incubated in presence of 1 mM ouabain and those incubated in 1 mM ouabain and 2 mM furosemide. This furosemide-inhibitable net movement is plotted against incubation times on left panel. Right panel shows ratio of Cl to K+ net movements and ratio of Na+ to K+ movements. [From Geck et al. (90).]

Although for many years the widely accepted view of Cl distribution was that it was at equilibrium and crossed membranes easily and quickly, results from a number of tissues had never been able to fit into that model (e.g., heart muscle, Ref. 191; nerve cells, Ref. 165; muscle, Ref. 238). For technical reasons, data from the squid giant axon were the most convincing in this regard. As early as 1939 chemical analysis of extruded axoplasm had shown [Cl]_i of the giant axon to be very high (~120-150 mM) (23). (It must be remembered that the ionic composition of the squid's extracellular fluid is similar to seawater, i.e., [Cl]_o is ~500 mM.) Keynes (166) made a careful study of the [Cl]_i in the axon to rule out several potential technical problems and very clearly showed that at 120-130 mM, [Cl]_i was at least three times greater than expected if it were distributed at electrochemical equilibrium (i.e., V_m = 70 mV; E_Cl = 35 mV). Furthermore, he made the intriguing observation that 2,3-dinitrophenol, an inhibitor of oxidative phosphorylation, significantly reduced ³⁶Cl influx into axons. In addition, the conductive (electrodiffusive) pathways for Cl transmembrane movement across the squid axolemma are very small. Adelman and Taylor (1) showed that the Cl conductance was so small that it could account for no more than 10% of the "leakage" conductance across the voltage-clamped squid axolemma. Thus it seemed clear that some sort of active transport process must be responsible for this high, nonequilibrium distribution of Cl in squid axoplasm. The search for the mechanistic basis of this nonequilibrium Cl distribution resulted in a series of studies that used the internally dialyzed squid axon preparation (see sect. VA). These studies showed that the mechanism responsible for the high [Cl]_i 1) required intracellular ATP (5, 302), 2) was inhibited at high [Cl]_i (28, 302, 303), 3) involved the cotransport of Na⁺ and was blocked by furosemide (303), and 4) involved the cotransport of K⁺ and was blocked by bumetanide (306).

In the early 1980s, several labs provided evidence that the NKCC process was found in the thick ascending limb of the loop of Henle (101, 103), in the distal tubule of the kidney (245, 246), as well as in the intestine (242). It was demonstrated that these epithelial transport processes required all three co-ions (101, 242, 246) and were blocked by the sulfamoyl benzoic acid diuretics (see sect. VB, Ref. 245).

Thus, by the early 1980s, investigators had reported functional evidence of the NKCC in a wide variety of cell types. This was a period during which much of the functional characterization of this coupled cotransporter was obtained.

	V. FUNDAMENTAL CHARACTERISTICS

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There are three functionally defining and unique characteristics of the NKCC. 1) Ion translocation by the NKCC requires that all three ions (Na⁺, K⁺, and Cl) be simultaneously present on the same side of the membrane. 2) Bumetanide and its congeners (5-sulfamoyl benzoic acid loop diuretics) bind to the cotransporter protein and inhibit ion translocation of all three ions. 3) Under normal ionic conditions, all three ions are translocated, and the translocation process has an electrically silent stoichiometry: 1 Na⁺:1 K⁺:2 Cl for most cells or 2 Na⁺:1 K⁺:3 Cl for at least one cell (the squid giant axon). As a minimum, these three criteria should be met before a given process can be functionally identified as being mediated by the NKCC. In the following section, we consider the evidence for these widely accepted properties and what these properties tell us about the NKCC.

Wherever it has been possible to rigorously examine the individual fluxes of Na⁺, K⁺, and Cl, it has been shown that the NKCC-mediated fluxes of all three ions require the presence of the other two ions on the side of the membrane from which the flux originates (termed the cis-side; e.g., Refs. 8, 90, 121, 229, 303, 306).

Although it would seem to be a simple, straightforward matter to test for this property, using the sensitivity of each flux to bumetanide, it is now clear that complications can arise. Care must be exercised to remove, as much as possible, contributions from other transport pathways for the ion whose flux is being measured. For example, McManus' group initially believed Cl was not directly involved because the band 3 anion exchanger obscured the net Cl fluxes via the NKCC (see sect. IVD1). Prevention of this kind of error requires a preknowledge and understanding of the ion transporters that exist in the particular cell type one is investigating.

Unidirectional flux studies (using radioactive tracers) can be subject to a subtle kind of complicating error: isotopic exchange. In some RBC, the NKCC can mediate isotopic exchange fluxes under the appropriate set of ionic conditions (e.g., Refs. 38, 58, 69, 118, 218; but see Refs. 160, 161, 181; see sect. VIIA for further details). This property can lead to errors when determining the apparent stoichiometry of the cotransport process. It can also lead to misidentification of NKCC as KCC or as NCC. The important thing to remember is that because of this possibility one cannot rule out the presence of NKCC just because the unidirectional flux is not reduced by removal of one of the three cotransported ions. One must strive to arrange conditions that will not permit exchange fluxes of the ion whose flux one is measuring.

Both of these potential problems have been overcome using the internally dialyzed squid giant axon. Because of its large size (~500 µm in diameter and 6-7 cm in length), it has been possible to develop a technique to control the intracellular as well as the extracellular solute composition while measuring unidirectional fluxes. The technique of intracellular dialysis was developed by Brinley and Mullins (31). Briefly, it involves threading a miniature dialysis capillary longitudinally down the axis of the squid axon. The dialysis capillary used has a molecular mass cutoff of ~1,000 kDa, quite satisfactory for the control of small inorganic ions, organic solutes, and ATP. This gives the investigator two powerful advantages for studies on ion transporters. First, it is possible to control the internal milieu, and thereby maintain a steady-state condition in terms of solute concentrations that cannot be maintained in nondialyzed cells. Second, it permits one to directly measure either the unidirectional influx, or the efflux, simply by placing the relevant isotope in the extracellular fluid or the intracellular (dialysis) fluid, respectively, and collecting the fluid from the opposite side of the axolemma.

This technique has been used to examine a number of functional properties of the NKCC, including the requirement for cis-side ions. For the latter studies, the trans-side fluid was designed so that at least one of the necessary co-ions was absent, thereby reducing the possibility of isotopic exchange fluxes (see sect. VIIB). In a series of papers, Russell and co-workers have demonstrated the absolute requirement for the cis-side presence of all three ions both for unidirectional influx (303, 306) as well as for efflux (8). Each of the three co-ions was systematically removed while measuring the bumetanide-sensitive influx of the other two, using a double-label flux approach. The results of three representative experiments are shown in Figure 7. Figure 7A shows the effects on ³⁶Cl and ²⁴Na influx when an axon was sequentially superfused with external fluids that were, in turn, K⁺ free, K⁺ containing, and finally K⁺, 10 µM bumetanide containing. In this axon, it can be seen that providing extracellular K⁺ increased the influx of both Cl (by ~21 pmol·cm²·s¹) and Na⁺ (by ~14 pmol·cm²·s¹) and that both these increases were reversed by the application of extracellular bumetanide. Figure 7, B and C, shows the same general protocol applied to examine the external Na⁺ (Fig. 7B) and Cl requirement. In each case, the increase in the influx of the two co-ions caused by supplying the third one is reversed by bumetanide treatment. These results show that the cis-side absence of any one of the three ions is the same as treating with bumetanide. Thus it is clear that the NKCC requires all three ions be present for transport to occur. In addition, this particular demonstration also shows that after "delivering" the three ions to the inside of the membrane, the cotransporter can return to an outward-facing conformation even though only K⁺ is present on the inside. Because there is no evidence of bumetanide-sensitive K⁺/K⁺ exchange in the squid axon (i.e., K⁺ influx in Fig. 7, B and C, is the same in either the absence of the third co-ion or in the presence of bumetanide), this suggests that the reorientation of the binding sites to the outward-facing conformation must not require intracellular binding, translocation, and extracellular release of ions.

View larger version (19K): [in this window] [in a new window]   Fig. 7. Dependence of cotransport influxes of Na+, K+, and Cl on simultaneous presence of all 3 ions in external fluid. Experiments were carried out on internally dialyzed giant squid axons. Internal fluid in all cases contained the following (in mM): 400 K+, 0 Na+, 0 Cl, 8 Mg2+, and 4 ATP (pH 7.3, osmolality = 970 mosmol/kgH2O). Ouabain (105 M) and tetrodotoxin (107 M) were present at all times. A: effect of external K+ on 24Na and 36Cl influxes. After a control period in 0 mM [K+]o [N-methyl-D-glucamine (NMDG) replacement], 10 mM K+ was applied and both influxes increased; 36Cl influx increased by ~21 pmol·cm2·s1 and 24Na influx by ~14 pmol·cm2·s1. Finally, 10 µM bumetanide was applied causing both fluxes to decrease to levels very near that seen under 0 [K+]o conditions. [Na+]o = 425 mM; [Cl]o = 561 mM. B: effect of external Na+ on 42K and 36 Cl influxes. After a control period in 0 mM [Na+]o (NMDG substitution), 425 mM Na+ was applied leading to increases of both 42K (~8 pmol·cm2·s1) and 36 Cl (~20 pmol·cm2·s1) influxes. Subsequent application of 10 µM bumetanide caused influxes to decrease to levels near those observed in absence of extracellular Na+. [K+]o = 10 mM; [Cl]o = 561 mM. C: effect of external Cl on 42K (~7.5 pmol·cm2·s1) and 22Na (~15 pmol·cm2·s1) influxes. After a control period in 0 mM [Cl]o (gluconate substituted for Cl), 561 mM Cl was applied leading to increases of both 42K and 22Na influxes. Subsequent application of 10 µM bumetanide caused influxes to decrease to levels near those observed in absence of extracellular Cl. [Na+]o = 425 mM; [K+]o = 10 mM. (From A. A. Altamirano, G. E. Breitwieser, and J. M. Russell, unpublished observations.)

Bumetanide is the prototype for the loop diuretics. They are called loop diuretics because the diuresis (increase in urine production) they produce is the result of their action in the thick ascending limb of the loop of Henle. This region of the renal nephron reabsorbs Na⁺ and Cl from the tubular fluid and is responsible for the establishment of the hypertonic interstitium of the renal medulla. Thus, when this isoform of the NKCC (NKCC2) is inhibited, a large increase in urine flow is observed, and the urine is nearly isosmotic with plasma, regardless of the hydration state of the individual, i.e., the individual loses the ability to excrete either a concentrated or a dilute urine. Other representatives of this group (5-sulfamoylbenzoic acid derivatives) are furosemide, an agent used in early studies on the NKCC, but only rarely used experimentally nowadays, and piretanide, a congener that has been used mainly in Europe. Both are chemically closely related to bumetanide but less potent and less specific for the NKCC (e.g., Ref. 313) than bumetanide.

It is difficult to overstate the importance of the loop diuretics to the development of our present level of understanding of the NKCC. They are routinely used to identify fluxes mediated by the NKCC. This latter use has contributed in a major way to our learning that the NKCC is found in a very wide variety of cells (e.g., Ref. 269). They have played an important role in the identification of the protein and the subsequent cloning of the cotransporter, and they have provided information that has been used to interpret ion binding to the NKCC.

Given this importance, it is necessary to emphasize that these agents are not the NKCC's equivalent of the Na⁺ pump's ouabain. In fact, they are far from it. Although these agents inhibit the NKCC with a reasonably high affinity (see below), they can also inhibit other anion transport processes when used in higher concentrations, e.g., Cl/HCO₃ exchange (108, 153), Cl channels (63), and KCC (199). Thus, used alone and/or in high concentrations, not even bumetanide can provide positive proof that a given function is mediated by the NKCC. For example, Kracke et al. (180) showed in human RBC that bumetanide inhibits components of Na⁺ and K⁺ efflux that are neither dependent on cell Cl nor on the mutual pr