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Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853
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Abstract |
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Introduction |
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 Top Introduction Energizing the membranes and...Evidence for energizing...Two-electrode voltage-clamp...The Ussing paradigm and...Primary and secondary...References |
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Electron micrographs of Malpighian tubules reveal two morphological features that are not commonly observed in other transporting epithelia: 1) an abundance of intracellular concretions and 2) the location of mitochondria in microvilli of the apical brush border (Fig. 1, A and B
). The intracellular concretions consist mainly of Ca2+ and Mg2+ (type I) or K+ (type II) in an organic matrix of glycosaminoglycans and proteoglycans (14), but how these concretions take part in transepithelial ion transport is unknown. In contrast, the high density of mitochondria in the brush border indicates a region of high metabolic activity associated with the apical membrane, as further suggested by the movement of mitochondria into and out of microvilli with increasing and decreasing epithelial transport rates (3).
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). What uses ATP is the vacuolar (V-type) H+-ATPase in the microvillus plasma membrane. V-type H+-ATPases appear to prefer ATP-resplendent environments in general, since they are also found in mitochondria-rich cells of frog skin and in intercalated cells of the distal nephron. Where they occur, V-type H+-ATPases are often highly expressed relative to other transporters, which may reflect their slow transport rate, between 10 and 100 ions/s. Cotransporters move between 300 and 10,000 ions/s, and channels allow the passage of 1,000,000–100,000,000 ions/s (13).
In eukaryotic cells, the V-type H+-ATPase is thought to function exclusively as proton pump, transporting up to three H+ ions per ATP hydrolyzed (9, 13). Since H+ ions are extruded without balancing charge, the V-type H+-ATPase is purely electrogenic, generating apical membrane voltages in excess of 100 mV in Malpighian tubules (Fig. 1).
Because the V-type H+-ATPase secretes H+ ions into the lumen of Malpighian tubules, secreted fluid should turn acidic. However, the pH of fluid secreted by Malpighian tubules of the yellow fever mosquito remains remarkably neutral (pH = 7.2) under control and diuretic conditions (12). Perhaps protons secreted by the V-type H+-ATPase are confined to the microenvironment (glycocalyx?) of the brush border, where the pH may be substantially lower than in the bulk fluid of the tubule lumen (Fig. 1). If this is so, then the proton chemical potential in the microenvironment of the brush border could power the efflux of Na+ and K+ via Na+/H+ and K+/H+ antiporters that have yet to be isolated (Fig. 1C). If one H+ ion returns to its cytoplasmic origin in exchange for one Na+ (or K+), the antiporter is electroneutral and not affected by voltage. A stoichiometry other than 1:1 would make the antiporter conductive and responsive to membrane voltage.
Regardless of stoichiometry, antiporter(s) and V-type H+-ATPases constitute a bioenergetic H+ cycle of continuous H+ efflux and influx catalyzed by these two membrane-bound enzymes (Fig. 1C). The cycle begins with the efflux of H+ via primary active transport powered by the V-type H+-ATPase at the expense of ATP. The cycle continues with the return influx of H+ that powers the extrusion of Na+ and K+ via secondary active transport (Fig. 1C).
On the opposite side of the epithelial cell, Na+ entry across the basolateral membrane is electrically silent under control conditions (Fig. 2). However, Na+ entry is conductive during and after the blood meal, when the release of mosquito natriuretic peptide has opened Na+ channels via cAMP (Fig. 2B). K+ enters the cell via Ba2+-blockable K+ channels that dominate the electrical conductance of the basolateral membrane under control conditions (8). Overall, transepithelial secretion of Na+ and K+ through principal cells is active, which is reflected in lumen-positive transepithelial voltages between 20 and 60 mV (Fig. 2, A and C). The counter ion of transepithelial Na+ and K+ secretion is Cl–, which follows passively through the epithelial shunt pathway (Fig. 2, B and C). In Malpighian tubules of the yellow fever mosquito, Cl– appears to pass through the paracellular pathway, especially under conditions of diuresis stimulated by the neuropeptide leukokinin (1). Cl– may also take a transcellular route through stellate cells, as in Malpighian tubules of the fruit fly (10).
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Energizing the membranes and epithelial transport pathways |
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TopIntroductionEnergizing the membranes and...Evidence for energizing...Two-electrode voltage-clamp...The Ussing paradigm and...Primary and secondary...References |
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and Fig. 2, B and C). Two routes may be taken: a short return pathway via channels and/or electrogenic carriers located in the microvillus apical membrane and a long route via the epithelial shunt and the basolateral membrane (Fig. 2, B and C). As to the short pathway, transepithelial Na+, K+, and Cl– secretion can be explained without involving ion channels in the microvillus apical membrane, where, so far, none have been conclusively demonstrated. This leaves electrogenic Na+/H+ and K+/H+ antiport with a stoichiometry of 1:2 or greater as a viable hypothesis for current returning across the apical membrane.
As to the long route, there is good agreement that current passing through the epithelial shunt (paracellular pathway and/or stellate cells) is carried by Cl– moving from the hemolymph (peritubular side) to the tubule lumen, which is the mechanism of transepithelial Cl– secretion (1, 10). Current across the basolateral membrane is carried by cations, mostly K+ under control conditions but also Na+ in the presence of the mosquito natriuretic peptide or its second messenger cAMP (Fig. 2, B and C). Thus current is carried by H+ across the apical membrane, by Cl– across the shunt pathway, and by K+ (and Na+) across the basolateral membrane (Fig. 2C). It follows that the transepithelial voltage is lumen positive and largely the product of current and shunt resistance as well as that the basolateral membrane voltage is cell negative and largely the product of current and basolateral membrane resistance (Fig. 2C).
Since current is the same at any point in the intraepithelial current loop (Fig. 2C), transcellular secretion of cations (Na+, K+) equals transepithelial secretion of anions (Cl–), preserving electroneutrality of the solutions on both sides of the epithelium, since NaCl and KCl are transported from hemolymph to tubule lumen. Furthermore, electrical coupling matches entry and exit rates of cations across basolateral and apical membranes, respectively, thereby preserving intracellular steady states in the face of large ionic throughputs, especially during diuresis. Thus the ready transmission of electrical imbalances along conductive materials serves not only saltatory conduction in nerves, it also serves energy transfers in epithelia by electrically coupling transport pathways near to and far from the voltage source, the V-type H+-ATPase in the case of the insect Malpighian tubule.
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Evidence for energizing membranes and epithelial transport pathways with the V-type H+-ATPase |
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TopIntroductionEnergizing the membranes and...Evidence for energizing...Two-electrode voltage-clamp...The Ussing paradigm and...Primary and secondary...References |
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).
A more definitive case for energizing epithelial transport sites by the V-type H+-ATPase is made by bafilomycin, an inhibitor of V-type H+-ATPases. Bafilomycin completely inhibits transepithelial electrolyte and fluid secretion in Malpighian tubules (2). At the same time, apical and basolateral membrane voltages and the transepithelial voltage decrease to zero in parallel, confirming electrical coupling of the three transport sites. Furthermore, bafilomycin increases the transepithelial resistance by ~50% (from 7.8 to 11.6 k
cm), indicating the block of a conductive transport pathway (2). The blocked pathway appears to be located in the apical membrane, because the fractional resistance of this membrane more than doubles, increasing from 0.27 to 0.57. The increase in both transepithelial resistance and fractional membrane resistance is consistent with the known mechanism of action of bafilomycin, the block of the H+ channel of the V0 sector of the proton pump embedded in the plasma membrane of the apical brush border.
In cell systems supported by the V-type H+-ATPase, blocking the proton channel of the electrogenic pump is one way to stop electrogenesis and membrane and transepithelial transport. Withholding ATP is another way to inhibit the proton pump. As soon as dinitrophenol (DNP, 10–4 M) enters the peritubular bath of the isolated perfused Malpighian tubule, all membrane and transepithelial voltages go to zero, as in the presence of bafilomycin (Fig. 3A). In parallel, the transepithelial resistance increases, again by ~50%, from 11.4 to 16.8 kcm, and the fractional resistance of the apical membrane significantly rises, again from 0.32 to 0.57 (11). Thus the inhibition of metabolism duplicates the effects of bafilomycin, indicating tight coupling between oxidative metabolism and the function of the V-type H+-ATPase that is already suggested by the anatomic proximity of ATP synthesis and utilization (Fig. 1). ATP is the obvious link between metabolism and the V-type H+-ATPase, because other inhibitors of ATP synthesis, cyanide and fluoride, also inhibit membrane and epithelial voltages with striking resemblance to the effects of DNP (8). Although the three inhibitors (DNP, cyanide, and fluoride) disrupt the chain of oxidative metabolism at different sites, their effects converge on the inhibition of ATP synthesis. Thus inhibition of ATP synthesis stops membrane and epithelial transport and abolishes membrane and transepithelial voltages, confirming 1) the central role of the V-type H+-ATPase in electrogenesis and epithelial transport and 2) electrical coupling of transport through the paracellular pathway and across the basolateral membrane to the transport activity of the V-type H+-ATPase at the apical membrane (Figs. 1 and 2). The prompt depolarization and repolarization of all voltages with the speed of the bath exchange, as DNP washes into and out of the peritubular bath, suggest high turnover rates of ATP in Malpighian tubules (Fig. 3A).
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), the Na+-K+ pump is not essential to transepithelial transport. In fact, inhibition of the pump with ouabain actually increases rates of transepithelial Na+ and fluid secretion and hyperpolarizes the transepithelial voltage, documenting the stimulation of transepithelial transport (7). Maddrell has argued that stimulation of transepithelial Na+ secretion is exactly the expected response to inhibition of the Na+-K+-ATPase at the basolateral membrane: the inhibition blocks the removal of Na+ across the basolateral membrane, thereby increasing the availability of intracellular Na+ for extrusion into the tubule lumen via the Na+/H+ antiporter (Figs. 1 and 2). Thus stimulation of transepithelial Na+ and fluid secretion after inhibition of the Na+-K+ pump confirms that the primary energizer of epithelial transport in Malpighian tubules is the V-type H+-ATPase. |
Two-electrode voltage-clamp studies in principal cells of the tubule |
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TopIntroductionEnergizing the membranes and...Evidence for energizing...Two-electrode voltage-clamp...The Ussing paradigm and...Primary and secondary...References |
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). With both current and voltage electrodes located in the cytoplasm of a single principal cell, the voltage measured with respect to ground in the peritubular bath is still the basolateral membrane voltage, but the input resistance is the basolateral membrane resistance in parallel with the series resistances of the apical membrane and the shunt (Fig. 2C). In the experiment illustrated in Fig. 3C, the basolateral membrane voltage was –85 mV and the cell input resistance was 322 k under control conditions. Bafilomycin reduced the basolateral membrane voltage to –7 mV as in previous studies of isolated perfused tubules (2), and it increased cell input resistance to 561 k, a 74% increase, consistent with the inhibition of the V-type H+-ATPase in the apical membrane (Figs. 2 and 3). The subsequent inhibition of ATP synthesis with DNP had little effect on voltage because electrogenesis was already inhibited by bafilomycin, but it increased cell input resistance by another 265%, from 561 k to 2,048 k. This large increase in cell input resistance points to the presence of other conductive pathways in principal cells in addition to the H+ pump conductance already inhibited by bafilomycin. These conductive pathways may include ATP-dependent channels and electrogenic carriers that we have yet to identify. Furthermore, principal cells could also be electrically coupled via gap junctions that may close in the absence of ATP, thereby increasing cell input resistance. |
The Ussing paradigm and the Wieczorek/Harvey paradigm |
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TopIntroductionEnergizing the membranes and...Evidence for energizing...Two-electrode voltage-clamp...The Ussing paradigm and...Primary and secondary...References |
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In epithelia, the Na+-K+-ATPase plays a central role in the Ussing model that, for more than 50 years, has provided the conceptual foundation for investigating and understanding transport across a variety of epithelia (Fig. 4A). Originally conceived to explain Na+ absorption across the frog skin, the model has been widely applied to explain transepithelial transport of a large number of solutes by the selective polarization of the Na+-K+-ATPase and Na+- or K+-dependent transport systems in specific basolateral and apical membrane domains. The intellectual appeal of the Ussing model lies in the unity it brings to the diversity of epithelial transport activities supported by the Na+-K+-ATPase and associated Na+- and K+-dependent carriers and channels.
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In the last 20 years, the V-type H+-ATPase has been increasingly found in animal cells, first in endosomal membranes and then in plasma membranes (15). In endosomal membranes, the V-type H+-ATPase participates in the acidification of lysosomes and the processing of glycoproteins, in the uptake of glutamate and 
-aminobutyric acid by synaptic vesicles, in the exocytosis of insulin and glucagon, in the delay of apoptosis, and in the process of "degradative endocytosis" of filtered proteins in the kidney. Macrophages use the V-type H+-ATPase to kill phagocytosed bacteria. In plasma membranes of animal cells, the V-type H+-ATPase takes part in the deposition of Ca2+ in the shell of clams, in the resorption of bone by osteoclasts, in K+ secretion by the insect midgut, in the uptake of Na+ from fresh water by the fish gill, in transepithelial secretion of Na+ and K+ in insect Malpighian tubules, in the maturation of mammalian sperm, in the acidification of urine in turtle bladder and mammalian collecting duct, and in the placental implantation of the trophoblast (15). Tumor cells are thought to acquire multidrug resistance by virtue of colocalizing in their plasma membranes the V-type H+-ATPase and P-glycoprotein, the "drug efflux pump."
Goblet cells of the caterpillar midgut, which are devoid of detectable traces of the Na+-K+-ATPase, figure prominently in our present understanding of the central role of V-type H+-ATPases in electrogenesis and epithelial transport (5, 15). Antibodies against the V-type H+-ATPase have conclusively localized this proton pump in the apical membrane of midgut epithelial cells, where it is responsible for membrane voltages as high as 240 mV and for powering transepithelial K+ secretion via K+/nH+ antiport located in the same membrane. These observations have led Wieczorek and Harvey (5, 15) to propose an epithelial transport model founded entirely on H+ transport mediated by a proton pump and proton-driven transporters (Fig. 4B). The model was immediately accepted for the insect midgut and later extended to Malpighian tubules and insect ovarian follicle cells (5, 6, 10, 12, 14, 15). The good fit of this model to data from transporting epithelia devoid of the Na+-K+-ATPase and the ready adaptation of this insect model to vertebrate and mammalian epithelia powered by H+ pumps suggest the qualities of a new transport paradigm that joins rather then replaces the Ussing paradigm.
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Primary and secondary electrogenesis |
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TopIntroductionEnergizing the membranes and...Evidence for energizing...Two-electrode voltage-clamp...The Ussing paradigm and...Primary and secondary...References |
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). In contrast, electrogenesis is primary in plasma membranes energized by the V-type H+-ATPase (Fig. 4). Here the energy of ATP hydrolysis is transformed directly into voltage through the pumping of protons, yielding membrane voltages >100 mV in apical membranes of Malpighian tubules and >200 mV in goblet cells of lepidopteron midgut (1, 15). The difference between primary electrogenesis by an ATP-driven proton pump and secondary electrogenesis via the diffusion of ions is so fundamental as to suggest differences in the functional design of the cells and the mechanism of homeostasis.
As to intracellular homeostasis, the regulation of intracellular Na+ and K+ concentrations may not figure as importantly in cells supported by the V-type H+-ATPase as it does in cells supported by the Na+-K+-ATPase. Instead, the regulation of intracellular H+ concentration may receive first homeostatic priority. Widely ranging intracellular K+ concentrations (20–90 mM) that so far have been measured in Malpighian tubules and voltage-driven K+ entry into the cell via basolateral membrane K+ channels (Fig. 2) do not indicate the kind of intracellular K+ regulation expected in cells that depend on well-controlled intracellular K+ concentrations to support membrane voltage and K+-mediated transport systems.
As to functional design, electrogenic carriers have an obvious advantage in environments of high voltage and current. For example, an antiporter with a stoichiometry of Na+/nH+ can make use of the proton gradient but, more importantly, can capitalize on the high voltage associated with primary electrogenesis. An electrogenic K+/nH+ antiporter has already been proposed for extruding K+ across the apical membrane in lepidopteron midgut (Fig. 4B), and related electrogenic K+/nH+ and Na+/nH+ antiporters may be operating in apical membranes of Malpighian tubules (Fig. 2). Differences in functional design may also be reflected in the structural and functional properties of channels. In membranes energized by Na+-K+ or Ca2+ pumps, Na+, K+, and Ca2+ channels mediate the transformation of a concentration difference into a voltage difference as ions diffuse down their concentration gradients. In membranes energized by a purely electrogenic pump such as the V-type H+-ATPase, the reverse may be the case, i.e., channels may mediate voltage-to-concentration transformations, in which the newly gained concentration difference can be used again to drive electroneutral transport systems. However, channels are not perfectly reversible. Thus ion channels serving voltage-concentration transformations may require structural and functional adaptations different from those serving concentration-voltage transformations. For example, the electrical potential generated by the V-type H+-ATPase is the driving force for the uptake of ionic malate through malate-selective anion channels in some plant vacuoles (4). The malate potential thus established is then available to drive malate-dependent symport and antiport systems.
The Na+-K+-ATPase is likely to retain its standing as the main energizer of plasma membranes, particularly in membranes of nerve and muscle, but it cannot make this claim for all cells. The growing list of plasma membranes that accommodate the V-type H+-ATPase documents that energizing plasma membranes is not the exclusive domain of the Na+-K+ pump. In some tissues, most notably insect (lepidopteron) midgut, the V-type H+-ATPase is the sole energizer of the apical plasma membrane, and other examples of plasma membranes devoid of the Na+-K+ pump and powered exclusively by the V-type proton pump may be found. Malpighian tubules house both Na+-K+-ATPase and V-type H+-ATPase, but the latter dominates and powers transport through transcellular and paracellular pathways. The application of the TEVC method in single cells of the Malpighian tubule along with the intracellular injection of transport stimulators and inhibitors provide an intracellular vantage point for observing membrane and epithelial transport in an intact, natural epithelial cell engaged in transepithelial transport. In view of the high electrogenic capacity of the apical membrane, it will be possible to measure pump currents emanating from the V-type H+-ATPase in its normal environment of the cell. Furthermore, TEVC methods may uncover new isoforms and families of carriers and transporters that take advantage of the large electrical potentials generated by V-type H+-ATPases. And finally, in principal cells such as those found in Malpighian tubules of A. aegypti, TEVC methods will permit a glimpse at the interactions between the Na+-K+-ATPase and the V-type H+-ATPase, revealing how these two ion pumps integrate with each other and with channels and carriers to bring about transepithelial secretion of electrolytes and water.
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Acknowledgments |
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Because of the journal limit on references, many citations were omitted that would otherwise have been included and that I would like to acknowledge. For a full list of references please write to kwb1{at}cornell.edu.
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References |
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TopIntroductionEnergizing the membranes and...Evidence for energizing...Two-electrode voltage-clamp...The Ussing paradigm and...Primary and secondary...References |
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