Apical K Channels

Electrophysiological experiments in rabbits and rats have identified two types of K+ channels that are involved in the K+ recycling process: a channel with a 30-40 pS conductance (low-conductance channel) and another with a higher conductance of 70-80 pS (moderate conductance channel). High conductance >100 pS), or maxi-K+ channels, have also been identified in apical membranes of rabbit thick ascending limb cells in culture and they are thought to function in cell volume regulation, but not in K+ secretion or recycling. Both the low and the moderate conductance K+ channels are weak inward rectifiers, i.e., they conduct more current in the absorptive than the secretory direction. Although this would seem to imply that these channels migjht not function very well in K+ secretion and K+ recycling, they do in fact have a significant secretory conductance and can effectively transport K+ in the secretory direction. Moreover, these channels exhibit a long open (conducting^) state or high open probability (> 0.9), which means that they are effectively transporting (secreting) K+ almost continuously at the usual intracellular potentials of —30 to —60 mV found in transporting thick ascending limb cells.

Apical membrane K+ channels in thick ascending limbs are metabolically regulated by changes in cellular MgATP concentrations or the MgATP/ADP ratio. Since increases in MgATP or the MgATP/ADP ratio leads to channel inhibition, these channels are sometimes referred to as ATP-sensitive or KATP channels. Like Katp channels in other tissues (e.g., pancreatic /3-cells), the thick ascending limb KATP channels are sensitive to sulfonylureas like glibenclamide (Glyburide), though considerably less sensitive to glibenclamide than those present in /3-cells. This has led to the suggestion that certain sulfonylureas or similar agents may function as diuretics. Furthermore, since similar low conductance Katp channels are thought to mediate K+ secretion in principal cells of the cortical collecting duct these drugs might exert a K+ sparing action. However, to date no K+ channel targeted diuretics have made their way to clinical practice.

The ATP-sensitivity of these K+ channels is thought to provide a system for functionally coupling the rate of Na+ entry mediated by the Na/K/2C1 cotrans-porter with the rate of K+ secretion by apical membrane K+ channels. The model for this "cross-talk" is as follows (see Fig. 7). Increases in Na/K/2C1 cotransporter activity cause enhanced Na+ entry into thick ascending limb cells and a rise in cytoplasmic Na+. The latter stimulates basolateral Na+/K+/ATPase activity, thereby providing for enhanced Na+ exit. The increased ATP utilization by the Na+-pump would lower cytosolic MgATP concentrations or reduce the MgATP/ADP ratio, leading to a rise in apical K+ channel activity. Since increased Na+ entry is matched one-to-one with K+ entry by the Na/K/2C1 co-transporter the rise in apical K+ channel activity ensures that apical Na+ entry matches K+ secretion.

The low conductance K+ channel is activated by PKA-dependent phosphorylation processes, a mechanism that probably accounts for the increase in apical K+ channel activity that accompanies vasopressin-dependent increases in cyclic AMP production. Since Na/K/2C1 cotransporter activity is also increased by vasopressin (as well as other hormones that stimulate adenylyl cyclase), the increase in apical K+ channel activity could help maintain adequate K+ recycling during hormone-enhanced cotransporter activity and NaCl reabsorption. Finally, both the low and the moderate conductance K+ channels are inhibited by reductions in cytosolic pH within the physiological range, an effect that probably contributes to alterations in K+ secretion during acidosis.

Model of the Low Conductance K+ Channel-ROMK

To date only the low conductance K+ channel in the thick ascending limb has been molecularly cloned. Complementary DNA encoding this channel was first isolated from the rat outer medulla and is called ROMK. The biophysical, electrophysiological, and regulatory characteristics of the cloned channel correspond closely to that of the native low conductance, inwardly rectifying (and ATP- and pH-regulated) K+ channels found in the apical membranes of both

FIGURE 9. Molecular model of the low conductance, inwardly rectifying K+ channel present in apical membranes of thick ascending limb cells. This channel is a member of the rapidly expanding family of inward rectifier K+ channels (KIR channels) and the one present in the thick ascending limb is termed ROMK or Kirl.l. KIR channels have a unique topology with only two membrane-spanning helices making them structurally distinct from the voltage- and ligand-gated K+ channels with six membrane helices. This channel is thought to provide a potassium secretory pathway in apical plasma membranes of the thick ascending limb and participate in apical K+ recycling. Mutations in ROMK (Kirl.l) have been found in some patients with antenatal Bartter syndrome, providing strong evidence that this K+ channel plays a significant role in the NaCl transport process in the thick ascending limb. See text for detailed discussion. Also see Fig. 7 for a model of ion transport in the thick ascending limb.

FIGURE 9. Molecular model of the low conductance, inwardly rectifying K+ channel present in apical membranes of thick ascending limb cells. This channel is a member of the rapidly expanding family of inward rectifier K+ channels (KIR channels) and the one present in the thick ascending limb is termed ROMK or Kirl.l. KIR channels have a unique topology with only two membrane-spanning helices making them structurally distinct from the voltage- and ligand-gated K+ channels with six membrane helices. This channel is thought to provide a potassium secretory pathway in apical plasma membranes of the thick ascending limb and participate in apical K+ recycling. Mutations in ROMK (Kirl.l) have been found in some patients with antenatal Bartter syndrome, providing strong evidence that this K+ channel plays a significant role in the NaCl transport process in the thick ascending limb. See text for detailed discussion. Also see Fig. 7 for a model of ion transport in the thick ascending limb.

thick ascending limb and principal cells of the cortical collecting duct. The channel protein is rather small, about 45 kDa (397 amino acids), and exhibits an unusual topology with only two proposed membrane spans (see Fig. 9). This protein model was quite novel since earlier cloned voltage-gated and ligand-gated K+ channels generally had six proposed membrane spans. It has been suggested that the functional ROMK channel may be composed of four identical subunits that form a central pore but this has not been verified. Several alternatively spliced isoforms of ROMK have been identified in the rat and human kidney. These isoforms either alter the initial part of the amino-terminus of the protein or involve only noncoding regions. In the rat, three ROMK proteins with distinct amino-termini have been identified that are differentially expressed along the nephron from medullary thick ascending limb to outer medullary collecting duct. Recent studies using an antibody directed against ROMK

have localized the channel to apical membranes of these nephron segments, including thick ascending limb cells and principal cells of the cortical collecting duct. Structure-function studies of ROMK should add considerably to our understanding of the distal K+ secretory mechanisms and may provide a model for developing K+ channel-specific diuretics.

Lessons from Mutations in the Thick Ascending Limb Na/K/2Cl Cotransporter (Slci2ai) and Apical K+ Channel (KCNJi or ROMK) Found in Human Disease

Richard Lifton and co-workers at Yale University and the International Collaborative Study Group for Bartter-Like Syndromes have identified mutations in both the Slcl2al (Na/K/2C1 cotransporter) and the KCNJI (ROMK apical K+ channel) genes in a number of families with Bartter syndrome, including the antenatal hypercalcemic variant. The syndrome is associated with a hypokalemic alkalosis and in the classic and antenatal variants is accompanied by hypercal-ciuria. Some of these mutations were missense, resulting in alterations in single amino acids, while others resulted in deletions of portions of the transporter proteins. These mutations apparently result in loss of transporter (Na/K/2C1 or K+ channel) function although actual functional studies have not yet been reported.

These exciting discoveries demonstrate that Bartter syndrome is genetically heterogeneous and provide clear evidence that the Slcl2al (BSC1) gene encodes the apical Na/K/2Cl cotransporter in thick ascending limbs. Loss of co-transporter function by gene mutation or inhibition of its activity by loop diuretics results in the same clinical presentation: salt wasting volume depletion, hypokalemic metabolic alkalosis, and hypercalciuria. Likewise, the finding that mutations in KCNJI (ROMK) also leads to an identical clinical syndrome provides strong evidence that ROMK encodes the apical K+ channel in thick ascending limb and that normal function of this channel is vital to maintenance of NaCl reabsorption by the thick ascending limb.

THIAZIDE OR THIAZIDE-LIKE DIURETICS AFFECTING DISTAL CONVOLUTED TUBULE SALT TRANSPORT

General Aspects

The Na/Cl cotransporter represents the major target site for clinically usefulben-zothiadiazine (or thiazide) type diuretics like chlorothiazide (Diuril). The thia-

hydrochlorothiazide (Hydrodiuril) Jj nh polythiazide (Renese)

ciA^A

CH2SCH2CF3

metolazone (Zaroxolyn)

FIGURE 10. Chemical structures of selected thiazide and thiazide-like diuretics.

zide diuretics are analogs of l,2,4-benzothiadiazine-l,l-dioxide and evolved from chemical modification of sulfonamides that were noted to produce diuresis and chloruresis. Many of these thiazide diuretics retain a sulfamyl side-group on the benzene ring (see Fig. 10), which imparts varying carbonic an-hydrase inhibiting activity to these compounds. The order of potency for carbonic anhydrase inhibition by commonly used diuretics is chlorthalidone (67) > benzthiazide (50) > polythiazide (40) > chlorothiazide (14) > hydrochlorothiazide (1) > bendroflumethiazide (0.07). The carbonic anhydrase inhibiting capability of certain thiazides has caused confusion and resulted in incorrect conclusions in some studies. Caution should be exercised when interpreting results of experiments where reductions in NaCl transport were caused by thiazides with the highest carbonic anhydrase inhibiting potencies since the thiazide effect may be due to carbonic anhydrase inhibition rather than a direct action on the Na/Cl cotransporter. For example, some thiazides reduce proximal tubule sodium reabsorption. As detailed below, the thiazide-sensitive Na/ CI cotransporter protein is not expressed in the proximal tubule so that these thiazide effects on proximal tubules likely relate to their carbonic anhydrase inhibiting capacity in a manner identical to that for acetazolamide (see Carbonic Anhydrase Inhibitors, above).

Model of NaCl Transport

The generally accepted mechanism of salt reabsorption by the distal convoluted tubule is shown schematically in Fig. 11. About 5-7% of filtered NaCl is reabsorbed by the distal convoluted tubule. The entry of each sodium ion across the apical membrane of distal convoluted tubule cells is directly and tightly coupled in an electroneutral fashion to one chloride ion by the Na/Cl cotrans-porter. As in the case of NaCl absorption by the thick ascending limb, the entry of these ions is a secondary active transport process because it depends on the favorable electrochemical Na+ gradient that is maintained by the active extrusion of Na' from the cell by the basolateral Na+/K+/ATPase. The specific mechanism of Cl" exit across the basolateral membrane is less clear but probably involves a CI" channel, although other mechanisms such as K/Cl cotransport have not been excluded.

The two most important modulators of the rate of sodium absorption and potassium secretion by the distal convoluted tubule and cortical collecting duct are the amount of sodium delivered and the plasma mineralocorticoid level. The magnitude of NaCl reabsorption increases pari passu with salt delivery. Absolute sodium reabsorption, measured during in vivo microperfusion experiments, was proportional to the distal load. Evidently, the sodium load delivered

Distal

Distal

FIGURE 11. Model of NaCl absorption by the distal convoluted tubule.

to these nephron segments is elevated by diuretics acting on more proximal nephron segments. Both the transepithelial electrical conductance and the passive sodium permeability of these tubular segments are low. Hence, sodium absorption by late distal and cortical collecting tubules may lead to the development of a steep transtubular sodium concentration gradient, which, in turn, diminishes the electrochemical driving force for sodium entry across apical plasma membranes and limits further sodium reabsorption. As the load of sodium presented is raised by increasing the luminal sodium concentration or the tubular urine flow rate (or both), the point at which this limiting gradient is achieved is deflected further downstream with an attendant increase in the absolute amount of sodium that is reabsorbed. Such dynamic actions represent one form of tubular compensatory response to the diminution of NaCl reabsorption at upstream sites. This is the same type of load- or flow-dependence of NaCl reabsorption as described above for the thick ascending limb. Clearly, potassium secretion is also altered secondary to the changes of sodium absorption as described elsewhere.

A recent study showed that the density of the rat Na/Cl cotransporter, estimated by the specific binding of [3H]metolazone, decreased by 70% after adrenalectomy, while selective glucocorticoid or mineralocorticoid replacements increased thiazide receptor density. Thus, both the density of the renal thiazide "receptor" (presumably equivalent to the extent of Na/Cl cotransporter protein) and the quantity of NaCl reabsorbed by the renal Na/Cl cotransporter are under adrenocortical regulation. Moreover, both the gender of the animal and the application of sex hormones appear to regulate the density of thiazide-sensitive cotransporter in rats such that thiazide "receptor" density is higher in females and is decreased by removal of the ovaries.

Other Physiological Consequences of Inhibiting the NaCl Absorption Process in the Distal Convoluted Tubule

One of the most significant effects of changing the rate of NaCl transport by the distal convoluted tubule (as with thiazide diuretics) is an alteration in Ca2+ reabsorption. An inverse relationship has been demonstrated between the rates of NaCl and Ca2+ reabsorption. Thus, thiazide diuretic-mediated reductions in NaCl reabsorption by the distal convoluted tubule result in enhanced rates of Ca2+ reabsorption. This is believed to account for the beneficial effect of thiazides in individuals with calcium nephrolithiasis associated with idiopathic hy-percalciuria. Although the precise origin of this inverse relationship between NaCl and Ca2+ reabsorption remains somewhat controversial, two mechanisms have been proposed to account for it. First, studies using cultured mouse distal convoluted tubule cells have shown that Ca2+ entry is mediated by voltage-gated Ca2+ channels with the unusual characteristic of being activated by membrane hyperpolarization. In these cells, thiazide diuretics inhibit Na+ entry mediated by the Na/Cl cotransporter, which in turn hyperpolarizes the cell and activates apical membrane Ca2+ channels (see Fig. 11). An alternate proposal implicates enhanced Na+/Ca2+ exchange as responsible for the dissociation of sodium and calcium absorption. According to this scheme, by inhibiting apical membrane sodium entry, thiazide diuretics reduce the intracellular Na+ concentration. The decrease of intracellular Na+ increases the electrochemical gradient for basolateral sodium entry, thereby augmenting calcium efflux through the Na+/Ca2+ exchanger.

Increased NaCl delivery to the cortical collecting duct (see Fig. 14) as a consequence of thiazide-mediated inhibition of NaCl reabsorption in the distal convoluted tubule enhances K+ secretion and results in heightened kaliuresis (see Diuretics Affecting Collecting Duct Salt Transport, below, for further details). As with the loop diuretics, the thiazide diuretic-induced kaluresis can lead to significant hypokalemia.

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