Thomas R. Kleyman

Subunit Stoichiometry of the Epithelial Sodium Channel

The epithelial Na+ Channel (ENaC) mediates Na+ reabsorption in a variety of epithelial tissues. ENaC is composed of three homologous subunits, termed α, β, and γ. All three subunits participate in channel formation as the absence of any one subunit results in a significant reduction or complete abrogation of Na+ current expression inXenopus oocytes. To determine the subunit stoichiometry, a biophysical assay was employed utilizing mutant subunits that display significant differences in sensitivity to channel blockers from the wild type channel. Our results indicate that ENaC is a tetrameric channel with an α2βγ stoichiometry, similar to that reported for other cation selective channels, such as Kv, Kir, as well as voltage-gated Na+ and Ca2+ channels that have 4-fold internal symmetry.

A Mechanism for Pentamidine-Induced Hyperkalemia: Inhibition of Distal Nephron Sodium Transport

Pentamidine is an antiparasitic agent used to treat the opportunistic infection Pneumocystis carinii pneumonia [1-6]. Unfortunately, hyperkalemia is an important complication of therapy, observed in as many as 100% of patients with the acquired immunodeficiency syndrome (AIDS) receiving pentamidine for more than 6 days [1-4]. This elevation in the serum potassium level can be seen in the absence of adrenal insufficiency, hyporeninemic hypoaldosteronism, interstitial nephritis, or hyperglycemia (pentamidine-induced pancreatic islet cell dysfunction). Investigators have also reported azotemia in 25% to 95% of patients infected with the human immunodeficiency virus (HIV) who receive pentamidine [6-8]. However, hyperkalemia is usually out of proportion to the degree of coexisting renal insufficiency and is frequently associated with hyperchloremic metabolic acidosis [6, 7, 9]. These findings have led several groups to postulate that pentamidine might directly effect renal tubular K+ secretion [1, 4, 6]. Most renal K+ is excreted through secretory K+ channels located in the apical membrane of principal cells in the cortical collecting tubule [10-12]. The electrochemical driving force for distal nephron K+ secretion (that is, increased urinary lumen negativity and high intracellular K+ levels) is maintained by luminal Na+ entry through apical Na+ channels and serosal K+ uptake through the basolateral Na+/K +-adenosine triphosphatase pump [10-13]. Pentamidine is an aromatic diamidine structurally similar to potassium-sparing diuretics such as amiloride, triamterene, and trimethoprim [5, 13, 14] (Figure 1). We therefore applied transepithelial and single-channel measurement techniques to two well-established models of cortical collecting tubule ion transport (A6 amphibian cell line and primary cultured rabbit cortical collecting tubules) to investigate the effects of pentamidine on renal tubular Na+ reabsorption and, therefore, K+ secretion. Figure 1. Structures of pentamidine and potassium-sparing diuretics such as amiloride, triamterene, and trimethoprim. Methods Transepithelial Measurements on A6 Distal-Nephron Cell Line Cultures The A6 cell (American Type Culture Collection, Rockville, Maryland) subpassages 91 to 96 were grown to confluency on collagen-coated polycarbonate filters (Costar; Cambridge, Massachusetts) in the presence of 1.5 M of aldosterone. Filters were mounted in a modified Ussing chamber, and macroscopic current was measured at room temperature with a DVC-1000 voltage clamp (World Precision Instruments, Sarasota, Florida) [14-16]. We measured short-circuit current under voltage-clamp conditions and added 10 M of amiloride to the luminal bath at the end of each experiment to determine the amiloride-sensitive component of the short-circuit current. Bath solutions contained the following: 100 mM of NaCl, 4 mM of KCl, 2.5 mM of NaHCO3, 1 mM of KPo 4, 1 mM of CaCl2, 11 mM of glucose, and 10 mM of n-2-hydroxyethylpiperazine-n-2-ethanesulfonic (HEPES) (pH, 7.4). Patch Clamp Measurements on Primary Cultures of Rabbit Cortical Collecting Tubules As previously described [17], renal cortices from New Zealand white rabbits (body weight, 1 to 2 kg) were collagenase digested and subjected to Percoll density centrifugation. Rabbit cortical collecting tubule fragments were separated and grown to confluency on permeable, collagen-coated Millipore-CM filters (Millipore Corp., Bedford, Massachusetts) in the presence of 1.5 M of aldosterone. Unitary channel events were measured at 37 C with a List EPC-7 Patch Clamp (Medical Systems Corp., Greenvale, New York). Data were digitized, recorded, and analyzed as previously described [10, 14, 17]. Patch pipette and extracellular bath solutions consisted of a physiologic saline solution containing the following: 140 mM of NaCl (final NaCl concentration after titration to a pH of 7.4 with NaOH), 5 mM of KCl, 1 mM of CaCl2, 1 mM of MgCl2, and 10 mM of HEPES (pH, 7.4). Chemicals Pentamidine (1,5-bis[p-Amidinophenoxyl]-pentane bis[2-hydoxyethane-sulfonate salt]) was of the highest commercial grade available (Sigma Chemical, St. Louis, Missouri). We added appropriate solvent vehicles to control baths that by themselves did not change the Na (+) channel activity. Results The amiloride-sensitive component of the short-circuit current is a measure of the net Na+ transport across the renal epithelium [14, 16, 18]. When we applied various concentrations of pentamidine to the solution, bathing the luminal or urinary surface of A6 distal nephron cell monolayers, we observed a dose-dependent inhibition of the amiloride-sensitive, short-circuit current (five experiments) (Figure 2). This inhibition developed rapidly, and 50% blockage of the amiloride-sensitive, short-circuit current (50% inhibitory concentration) occurred with 700 M of pentamidine at a pH of 7.4. Figure 2. Effect of pentamidine on short-circuit current (Isc) in A6 distal nephron cells. In previous studies, we have characterized the amiloride-sensitive, 4-picosiemen Na+ channel that is responsible for physiologic, mineralocorticoid-dependent Na+ reabsorption in the mammalian distal nephron [17]. We therefore examined the effect of pentamidine on 4-picosiemen Na+ channel activity in apical, cell-attached patches on principal cells of primary cultured rabbit cortical collecting tubules. The results, summarized in Table 1, show that addition of 1.0 M of pentamidine to the serosal bath or luminal bath (outside the cell-attached pipette) did not significantly affect Na+ channel activity within the unexposed patch membrane (no pentamidine was added to the pipette solution). In contrast, when pentamidine was placed in direct contact with the luminal surface of the patch membrane (1.0 M of pentamidine in the pipette solution), Na+ channel activity (measured as the number of channels times the open probability) decreased to 40% of control values. Table 1. Effects of Pentamidine on Principal-Cell Na+ Channels The pentamidine-induced inhibition of Na+ channel activity appeared to be primarily caused by a decrease in open probability (percentage of time an individual channel is open) rather than the number of channels per patch (membrane channel density) (Table 1). We confirmed this impression in four cell-attached patches that contained only one Na+ channel and therefore permitted us to directly calculate open probability (Figure 3). In these latter experiments, we could also show reversibility of the inhibitory effects of luminal pentamidine on Na+ channel kinetics. Figure 3. Effect of intrapipette pentamidine on Na+ channel activity in rabbit cortical collecting tubule primary cultures. Top. Bottom. Discussion The electrochemical driving force for K+ secretion in the cortical collecting tubule is maintained by luminal Na+ entry through apical Na+ channels and by serosal K+ uptake through the basolateral Na+/K+-adenosine triphosphatase pump [10-12]. We have previously shown that the mechanism of action for potassium-sparing diuretics, such as amiloride, trimethoprim, and triamterene, is direct blockade of these apical Na+ channels [13, 14, 17, 18]. Therefore, unlike most diuretics, which promote both natriuresis and kaliuresis, these latter drugs actually inhibit kaliuresis [13, 16]. In this study, we showed that luminal exposure to pentamidine inhibits Na+ reabsorption in both mammalian and amphibian distal nephron cells. Luminal pentamidine inhibited both amiloride-sensitive, macroscopic short-circuit currents and individual Na+ channels at concentrations greater than 50 M and 1.0 M, respectively. Differences in the apparent inhibitory constant of pentamidine for the Na+ channel in rabbit principal cells and in amphibian A6 cells may reflect either subtle variabilities within the structure of Na+ channels expressed in mammalian and amphibian renal cells or the different techniques used to examine Na+ transport [14, 19]. Little information is available on the pharmacokinetics of pentamidine in humans [5]. Pentamidine has a serum half-life of 5 to 6 hours after one parenteral dose, but the half-life increases to a mean of 52 89 hours after multiple doses [20]. Only 15% to 20% of a single intramuscular dose is excreted daily in the urine, yielding urinary concentrations of 20 to 25 g/mL (1 g/mL M) [5]. In patients with impaired renal function, parenteral pentamidine is excreted in the urine at a rate of 1.85 to 7.13 mg/d [20]. Urinary concentrations after daily administration of aerosolized pentamidine range from 1.3 to 778 ng/mg of creatinine per milliliter [21]. Therefore, most of the administered pentamidine becomes protein- and tissue-bound, with an estimated volume of distribution of 3 L/kg body weight and the greatest accumulation occurring in the kidney [5]. Urinary levels are detectable for months after therapy is discontinued. In patients with AIDS, autopsy studies have found detectable levels of pentamidine in renal tissue for as long as 1 year after the last dose [1]. Tissue depot release probably accounts for the wide range of reported urinary concentrations and the observation that pentamidine-induced hyperkalemia can persist for days after the drug is withdrawn [2]. In conclusion, pentamidine therapy for treating HIV-infected patients with P. carinii pneumonia can be associated with life-threatening hyperkalemia. We have shown that pentamidine, at concentrations found clinically in the urine, directly and reversibly blocks apical Na+ channels in a manner similar to potassium-sparing diuretics. The result is a decrease in the electrochemical driving force for both K+ and H+ secretion in the cortical collecting tubule. It is therefore not surprising that hyperkalemia and hyperchloremic metabolic acidosis are observed in patients treated with pentamidine, amiloride, triamterene, or trimethoprim [6, 7, 9, 14, 22]. These renal tubular effects provide a mechanism for pentamidine-induced hyperkalemia in patients without sever

Subunit Stoichiometry of a Core Conduction Element in a Cloned Epithelial Amiloride-Sensitive Na+ Channel

The molecular composition of a core conduction element formed by the alpha-subunit of cloned epithelial Na+ channels (ENaC) was studied in planar lipid bilayers. Two pairs of in vitro translated proteins were employed in combinatorial experiments: 1) wild-type (WT) and an N-terminally truncated alphaDeltaN-rENaC that displays accelerated kinetics (tauo = 32 +/- 13 ms, tauc = 42 +/- 11 ms), as compared with the WT channel (tauc1 = 18 +/- 8 ms, tauc2 = 252 +/- 31 ms, and tauo = 157 +/- 43 ms); and 2) WT and an amiloride binding mutant, alphaDelta278-283-rENaC. The channels that formed in a alphaWT:alphaDeltaN mixture fell into two groups: one with tauo and tauc that corresponded to those exhibited by the alphaDeltaN-rENaC alone, and another with a double-exponentially distributed closed time and a single-exponentially distributed open time that corresponded to the alphaWT-rENaC alone. Five channel subtypes with distinct sensitivities to amiloride were found in a 1alphaWT:1alphaDelta278-283 protein mixture. Statistical analyses of the distributions of channel phenotypes observed for either set of the WT:mutant combinations suggest a tetrameric organization of alpha-subunits as a minimal model for the core conduction element in ENaCs.

Renal Epithelial Protein (Apx) Is an Actin Cytoskeleton-regulated Na+ Channel

Apx, the amphibian protein associated with renal amiloride-sensitive Na+ channel activity and with properties consistent with the pore-forming 150-kDa subunit of an epithelial Na+ channel complex initially purified by Benos et al. (Benos, D. J., Saccomani, G., and Sariban-Sohraby, S. (1987) J. Biol. Chem. 262, 10613-10618), has previously failed to generate amiloride-sensitive Na+ currents (Staub, O., Verrey, F., Kleyman, T. R., Benos, D. J., Rossier, B. C., and Kraehenbuhl, J.-P. (1992) J. Cell Biol. 119, 1497-1506). Renal epithelial Na+ channel activity is tonically inhibited by endogenous actin filaments (Cantiello, H. F., Stow, J., Prat, A. G., and Ausiello, D. A. (1991) Am. J. Physiol. 261, C882-C888). Thus, Apx was expressed and its function examined in human melanoma cells with a defective actin-based cytoskeleton. Apx-transfection was associated with a 60-900% increase in amiloride-sensitive (Ki = 3 μM) Na+ currents. Single channel Na+ currents had a similar functional fingerprint to the vasopressin-sensitive, and actin-regulated epithelial Na+ channel of A6 cells, including a 6-7 pS single channel conductance and a perm-selectivity of Na+:K+ of 4:1. Na+ channel activity was either spontaneous, or induced by addition of actin or protein kinase A plus ATP to the bathing solution of excised inside-out patches. Therefore, Apx may be responsible for the ionic conductance involved in the vasopressin-activated Na+ reabsorption in the amphibian kidney.

ANTIIDIOTYPIC ANTIBODY RECOGNIZES AN AMILORIDE BINDING DOMAIN WITHIN THE ALPHA SUBUNIT OF THE EPITHELIAL NA+ CHANNEL

We previously raised an antibody (RA6.3) by an antiidiotypic approach which was designed to be directed against an amiloride binding domain on the epithelial Na+channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na+ transport across A6 cell monolayers. RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with tunicamycin, consistent with the size of nonglycosylated Xenopus laevis αENaC. RA6.3 specifically recognized an amiloride binding domain within the α-subunit of mouse and bovine ENaC. The deduced amino acid sequence of RA6.3 was used to generate a three-dimensional model structure of the antibody. The combining site of RA6.3 was epitope mapped using a novel computer-based strategy. Organic residues that potentially interact with the RA6.3 combining site were identified by data base screening using the program LUDI. Selected residues docked to the antibody in a manner corresponding to the ordered linear array of amino acid residues within an amiloride binding domain on the α-subunit of ENaC. A synthetic peptide spanning this domain inhibited the binding of RA6.3 to αENaC. This analysis provided a novel approach to develop models of antibody-antigen interaction as well as a molecular perspective of RA6.3 binding to an amiloride binding domain within αENaC.

Differential arrival of newly synthesized apical and basolateral plasma membrane proteins in the epithelial cell line A6

The labeling of specific cell surface proteins with biotin was used to examine both protein distribution and delivery of newly synthesized proteins to the apical and basolateral cell surface in A6 cells. Steady-state metabolic labeling with [35S]methionine followed by specific cell surface biotinylation demonstrated polarization of membrane proteins. The delivery of newly synthesized proteins to the apical or basolateral cell surface was examined by metabolic labeling with [35S]methionine using a pulse-chase protocol in combination with specific cell surface biotinylation. Newly synthesized biotinylated proteins at the apical cell surface reached a maximum after a 5 min chase, and then fell over the remainder of a 2 hr chase. The bulk flow of newly synthesized proteins to the basolateral membrane slowly rose to a maximum after 90 min. The detergent Triton X-114 was used to examine delivery of hydrophilic and hydrophobic proteins to the cell surface. Delivery of both hydrophilic and hydrophobic proteins to the apical cell surface reached a maximum 5 to 10 min into the chase period. The arrival of hydrophilic proteins at the basolateral surface showed early delivery and a maximum peak delivery at 120 min into the chase period. In contrast, only an early peak of delivery of newly synthesized hydrophobic proteins to the basolateral membrane was observed.

Chapter 3 Subunit Stoichiometry of Heterooligomeric and Homooligomeric Epithelial Sodium Channels

Publisher Summary The chapter presents a study on subunit stoichiometry of hetero-oligomeric and homo-oligomeric epithelial sodium channels. Epithelial Na + channels (ENaCs) have a key role in the regulation of urinary Na + reabsorption, extracellular fluid volume homeostasis, and blood pressure, and are a major site of action of volume regulatory hormones. ENaCs are composed of three homologous subunits, termed α, β, and γ . One of the fundamental challenges pertaining to the structure of the Na + channel is the determination of its subunit stoichiometry. To determine the subunit stoichiometry of ENaC, a biophysical assay utilizing mutant subunits is used that display significant differences in sensitivity to channel blockers from the wild-type channel. The results suggest that ENaCs are a tetrameric channel, with a α 2 βγ stoichiometry. The α -subunit itself is sufficient to form functional channels. The results suggest that similar to heterooligomeric αβγ Na + channels, homooligomeric α -subunit Na + channels are also tetrameric in structure. The chapter presents an example of a biophysical approach, considering the situation in which an ion channel is formed by a single polypeptide “X,” it is assumed that the channel has been cloned, can be functionally expressed in Xenopus oocytes , and is inhibited by the compound “Y.”

Application of the Amiloride Series in the Study of Ion Transport

Amiloride and amiloride analogs have been extensively used as inhibitors of Na+-selective transport proteins. The interaction of amiloride analogs with the epithelial Na+ channel, Na+/H+ exchanger, and Na+/Ca2+ exchanger is summarized. The potential use of amiloride analogs as biochemical probes for amiloride-sensitive transport proteins, and as immunologic tools for development of antibodies directed against these proteins is discussed, as are some of the limitations and problems encountered using amiloride analogs as “specific” inhibitors of defined transport proteins.